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https://openalex.org/W4322757019	https://zenodo.org/records/7692656/files/Metal%20catalyst%20free%20gas.Nature%20Communications_2021.pdf	English	null	Metal-catalyst-free gas-phase synthesis of longchain hydrocarbons	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	8,013	1 ESISNA group. Instituto de Ciencia de Materiales de Madrid (ICMM-CSIC), c/ Sor Juana Inés de la Cruz 3, 28049 Madrid, Spain. 2 Institute for Mechanical Systems, ETH Zurich, Leonhardstrasse 21, 8092 Zurich, Switzerland. 3 School of Engineering, Brown University, Providence, RI 02912, USA. 4 Instituto de Nanociencia y Materiales de Aragon (INMA), Spanish National Research Council (CSIC), University of Zaragoza, 50009 Zaragoza, Spain. 5 Laboratorio de Microscopias Avanzadas (LMA), University of Zaragoza, 50009 Zaragoza, Spain. 6 Center for High-Resolution Electron Microscopy (CћEM), School of Physical Science and Technology, ShanghaiTech University, 393 Middle Huaxia Road, Pudong, Shanghai 201210, China. 7 Catania Astrophysical Observatory (INAF), Via Santa Soﬁa, 78, 95123 Catania, Italy. 8 Institute of Chemical Physics, Université Paris-Saclay, 91400 Orsay, France. 9 Synchrotron Soleil, L’Orme des Merisiers, Saint-Aubin - BP 48, 91192 Gif-sur-Yvette, France. 10 Instituto de Ciencia y Tecnología de Polímeros, ICTP-CSIC, c/ Juan de la Cierva 3, 28006 Madrid, Spain. 11 Department of Chemistry, Ben Gurion University of the Negev, Beer Sheva 84105, Israel. 12 Instituto de Química-Física Rocasolano (IQFR- CSIC), c/ Serrano 119, 28006 Madrid, Spain. 13 Instituto de Física Fundamental (IFF-CSIC), c/ Serrano 123, 28006 Madrid, Spain. 14 Department of Mechanical and Aerospace Engineering, Princeton University, Princeton, NJ 08544, USA. 15 Photoactivated Processes Unit IMDEA Energía, Av. Ramón de la Sagra, 3 28935 Móstoles, Spain. 16 Institute of Physics of the CAS, Cukrovarnicka 10, Prague, Czech Republic. 17These authors contributed equally: Lidia Martínez, Pablo Merino, Gonzalo Santoro. ✉email: pedro.deandres@csic.es; georgehaller@ethz.ch; gary@ictp.csic.es; gago@icmm.csic.es ARTICLE NATURE COMMUNICATIONS \| (2021) 12:5937 \| https://doi.org/10.1038/s41467-021-26184-0 \| www.nature.com/naturecommunications Metal-catalyst-free gas-phase synthesis of long- chain hydrocarbons In our approach, gas-phase reaction occurs to produce alkane chains and ﬁbres, avoiding surface diffusion and reactor-wall adsorption, usually occurring in arc-discharge or physical– chemical vapour-deposition techniques19–21. Here, in contrast to other procedures, the long alkane chains and subsequent ﬁbres develop in the gas phase and do not form by surface diffusion. Chemical analysis performed by X-ray photoelectron spectro- scopy (XPS) on the ﬁbres conﬁrms that no metal traces from the walls or the magnetron head can be found in the system (see Supplementary Notes 3 and 5). Here, we describe an alternative route to the generation of long hydrocarbon chains under radically different conditions to cur- rent approaches, employing carbon atoms and molecular hydrogen as precursors in a gas-phase process at moderate temperature (<450 K) and low pressure (<0.5 bars) without the use of metal catalysts. While alkane formation under these con- ditions is theoretically unexpected, we demostrate experimentally and we rationalise the results using theoretical calculations to uncover the important role played by CH2 and H radical and carbon nanoparticles (C–NP) in the overall process showing that, albeit kinetically unfavourable, C–C chain growth is energetically possible on the surface of carbon nanoparticles. This process is schematically represented in Fig. 1a. Thermodynamic mechanism for gas-phase C–C-bond forma- tion. Theoretical studies indicate that the stable formation of CH2 radicals in the gas phase from C and H2, albeit energetically favoured (ΔG = −3 eV) and virtually barrierless (B = 0 eV), requires the removal of excess energy by a third body. Molecular dynamics simulations of collisions between C and H2 show the formation of a metastable CH2* intermediate. The average life- time, where the three atoms remain bonded at distances closer than 1.9 Å, yields CH2* lifetimes of about τ = 10 ps, which agrees with previous ab initio calculations22. Locally, we observe tem- perature ﬂuctuations due to collisions, reaching values up to 103 K. The excess energy (about 3 eV) is observed to be efﬁciently equally shared between all the vibrational degrees of freedom immediately after the formation of the radical, preventing instant dissociation and increasing the CH2* lifetime. Metal-catalyst-free gas-phase synthesis of long- chain hydrocarbons Lidia Martínez 1,17, Pablo Merino1,13,17, Gonzalo Santoro 1,17, José I. Martínez 1, Stergios Katsanoulis 2, Jesse Ault3, Álvaro Mayoral 4,5,6, Luis Vázquez 1, Mario Accolla1,7, Alexandre Dazzi8, Jeremie Mathurin8, Ferenc Borondics 9, Enrique Blázquez-Blázquez 10, Nitzan Shauloff11, Rosa Lebrón-Aguilar 12, Jesús E. Quintanilla-López 12, Raz Jelinek 11, José Cernicharo 13, Howard A. Stone 14, Victor A. de la Peña O’Shea 15, Pedro L. de Andres1✉, George Haller 2✉, Gary J. Ellis 10✉& José A. Martín-Gago 1,16✉ Development of sustainable processes for hydrocarbons synthesis is a fundamental challenge in chemistry since these are of unquestionable importance for the production of many essential synthetic chemicals, materials and carbon-based fuels. Current industrial processes rely on non-abundant metal catalysts, temperatures of hundreds of Celsius and pressures of tens of bars. We propose an alternative gas phase process under mild reaction conditions using only atomic carbon, molecular hydrogen and an inert carrier gas. We demonstrate that the presence of CH2 and H radicals leads to efﬁcient C-C chain growth, producing micron- length ﬁbres of unbranched alkanes with an average length distribution between C23-C33. Ab- initio calculations uncover a thermodynamically favourable methylene coupling process on the surface of carbonaceous nanoparticles, which is kinematically facilitated by a trap-and- release mechanism of the reactants and nanoparticles that is conﬁrmed by a steady incompressible ﬂow simulation. This work could lead to future alternative sustainable synthetic routes to critical alkane-based chemicals or fuels. 1 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-26184-0 C arbon–carbon (C–C)-bond formation is at the root of organic chemistry and is a fundamental building block for many essential materials in modern society, from phar- maceuticals to polymers. In particular, alkanes are important raw materials for the chemical industry and form the main con- stituent of fuels and lubricating oils, their predominant sources being natural gas and crude oil. In recent years, extensive efforts have been devoted to developing sustainable methods for the generation of alkanes and their derivatives that impact many scientiﬁc disciplines, including chemistry, materials science, energy, and health. Many technologies have been established, including transition metal–catalysed processes such as oleﬁn metathesis and cross-coupling1–3 or the production of clean fuels and chemical feedstocks via advanced catalytic approaches in, for example, Fischer–Tropsch synthesis (FTS)4–6. FTS is a mature technology reaching an average conversion of 45–50% and in some speciﬁc cases as high as 90%4,7. Metal-catalyst-free gas-phase synthesis of long- chain hydrocarbons However, the control of selectivity to long-chain hydrocarbons is highly challenging and cannot exceed 45%, as predicted by the Anderson–Schulz–Flory (ASF) law5,8. It is also important to highlight the recent devel- opments in alkane production via microbial bioreﬁneries6, and photocatalytic production of hydrocarbon from fatty acids9, but these are still at low-technology development levels. It is clear that all of these processes play vital roles in the drive toward cleaner and energy-efﬁcient technologies for future sustainable chemicals and fuels that are critical to counteract the harmful effects of global warming and fulﬁl future societal needs1,10. However, at the present time, most industrial processes leading to C–C chains inevitably depend on expensive or critical raw materials, such as metal catalysts, non-environmentally friendly or toxic solvents, or require harsh reaction conditions in terms of temperature or pressure. Thus, there is certainly room for new approaches that can lead to groundbreaking technologies to provide for our future materials and energy needs. in length and exhibit diameters between 6 and 20 nm (Fig. 1b, c). More detailed HR-TEM images show atomic features of 2.2 Å with an intermolecular spacing of 3.6 Å (Fig. 1d, e, Supplemen- tary Note 2 and Supplementary Fig. 2), which are compatible with semicrystalline alkane structures (see also Supplementary Note 6)13,14. Interestingly, when no hydrogen is used in the process, we form only amorphous C-NPs (see supplementary Note 2)15. Some of these are visible in Fig. 1c. Real-time mass spectrometry analysis of the reaction products in the gas phase shows that they are mainly composed of short aliphatic molecules in the C1–C4 range (Fig. 2a), although the electron-impact dissociation pattern prevents quantiﬁcation of these species. Gas chromatography–mass spectrometry (GC–MS) of the ﬁbres reveals that they are essentially composed of unbranched alkanes, with a relatively narrow distribution of C23–C33 (Fig. 2b), with a probability chain growth close to unity according to an ASF analysis6. Raman spectroscopy (Fig. 2c) conﬁrms the alkane structure of the ﬁbres (see Supplementary Note 6), revealing characteristic spectral ﬁngerprints of solid-state semicrystalline long-chain n-alkanes16–18. Further, AFM-coupled infra-red photothermal nano-IR spectra recorded from an individual ﬁbre (spatial resolution is 10 nm, Fig. 2d) clearly show methylene and methyl vibrational deformation modes (Fig. 2e) comparable to those obtained from n-alkanes with intermediate chain lengths in the C23–C33 range (see Supplementary Notes 6 and 7). Metal-catalyst-free gas-phase synthesis of long- chain hydrocarbons Assuming that the reaction occurs in an Ar atmosphere with an estimated density greater than 1018 atoms/cm3, corresponding to our experimental conditions, collisions with a third body are expected during the lifetime of the metastable CH2, helping to remove the excess energy and stabilising the methylene radical (see Supplementary movie 1 and supplementary Note 10). As the chain grows, more degrees of freedom are accumulated, and the lifetime increases accordingly. MMUNICATIONS \| (2021) 12:5937 \| https://doi.org/10.1038/s41467-021-26184-0 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-26184-0 3c) expands with the length of the chain, until it saturates at 1.48 Å for 6 C units, Fig. 3b (a typical value for a single C–C bond), Fig. 3b. Therefore, this bond becomes susceptible to electrophilic attack by a H atom24, which could arise either from diffusion on the C-NP surface or, more likely, from the gas phase. Given the barriers, we have estimated that such a process would ideally take place in the order of seconds (see Supplementary Note 13). The subsequent agglomeration and gas-phase entangle- ment of the detached alkane chains results in the formation of the parafﬁn ﬁbres shown in Fig. 1. CH2 þ C2H4 ! C3H6ðΔG ¼ 3:3 eV; B ¼ 3 eVÞ; ð2Þ ð2Þ which is very effective in preventing hydrocarbon growth (see Supplementary Note 11). A catalyst is generally needed to avoid the aforementioned bottleneck, to assume the role of weakening the strong C=C double bond, which lowers the barrier for CH2 addition and facilitates the formation of new C–C single bonds. g In our experiments, we do not take advantage of a catalyst as such, but the reaction C CH2 þ CH2 ! C C2H4ðΔG 3 eV; B 0 eVÞ; ð3Þ proceeds without a noticeable kinetic energy barrier because we exploit the fact that bonding to a contiguous C atom prevents the formation of the strong double bond in reaction (1). Such additional C atom in reaction (3) is provided in our system by a low-coordinated atom on the surface of an amorphous C-NP, also produced in the same reaction chamber. Thus, on adsorption of a CH2 intermediate, the highly reactive low-coordinated atoms of the C-NPs hinder the formation of the strong double bond, inhibiting barrier formation and facilitating subsequent growth (Fig. 3a and supplementary Note 12). Trap and release in the ﬂow. As commented above, such a mechanism is thermodynamically favourable but kinetically controlled, since the sputtered C atoms are ejected at high speed from the C target and dragged by the Ar circulation. In the vicinity of the surface, there will be insufﬁcient time for reaction with hydrogen. Furthermore, the growth of nanoparticles using sputter-gas aggregation sources is a thermodynamic none- quilibrium problemout-of-eqeulibrium process25,26. Therefore, although our predictions indicate that growth of long The ﬁnal steps are chain termination and detachment of the as- grown hydrocarbon from the C-NP surface. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-26184-0 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-26184- e 5 nm 1 nm d = 3.6 Å d = 2.2 Å d 5 nm c 500 nm b 500 nm a C-nanoparticle 1 2 3 4 5 6 7 Reaction path CH2 CH2 attack point CH2 CH2 CH2 CH2 CH2 H H ... C23 - C33 Chain termination and detachment Fig. 1 General schematic of the C–C coupling mechanism leading to long ﬁbres. a Pictorial views of the DFT-optimised ﬁrst steps for the growth of an alkane on a model (random generated) amorphous C-NPs by sequential addition of CH2 groups. b SEM image of a mat of ﬁbres collected on a silicon surface. c ex situ AFM image of a typical individual ﬁbre collected on a silicon surface. The bright spots correspond to amorphous C-NPs that are also produced (colour scale 0 nm, 30 nm). d Cs-corrected HR-TEM image of an individual ﬁbre where locally ordered regions can be easily distinguished. e High-magniﬁcation image of those regions. a 7 d 5 nm 5 nm Fig. 1 General schematic of the C–C coupling mechanism leading to long ﬁbres. a Pictorial views of the DFT-optimised ﬁrst steps for the growth of an alkane on a model (random generated) amorphous C-NPs by sequential addition of CH2 groups. b SEM image of a mat of ﬁbres collected on a silicon surface. c ex situ AFM image of a typical individual ﬁbre collected on a silicon surface. The bright spots correspond to amorphous C-NPs that are also produced (colour scale 0 nm, 30 nm). d Cs-corrected HR-TEM image of an individual ﬁbre where locally ordered regions can be easily distinguished. e High-magniﬁcation image of those regions. as the strong C=C double bond creates a barrier of about 3 eV for the addition of the next monomer as the strong C=C double bond creates a barrier of about 3 eV for the addition of the next monomer process or via chemical reactions, such as C2 + H2 →C2H + H12,15,23. The process to saturate the chain presents a negligible energy barrier, if any, and its occurrence will be proportional to the atomic hydrogen density (Fig. 3b). With regard to detachment after the alkane chain has grown on the C- NP, this can be rationalised as follows: the length of the bond between the ﬁrst carbon in the alkane to the C-NPs (dC1–C2 in Fig. Results h Synthesis of hydrocarbon chains. The synthesis is achieved by using a sputter gas-aggregation source11 (see supplementary Note 1), especially developed to produce gas-phase C clusters and C-NPs12. In this system, the sputtering gas (Argon) ﬂow also serves as dragging gas. The careful design of the reactor condi- tions facilitates, via a ﬂuid dynamics-based trapping and release mechanism, a large increase in the residence time of the reacting species, which leads to the efﬁcient formation of long hydro- carbon chains with a signiﬁcant reaction yield. These chains are entangled to form long micron-length ﬁbres. Figure 1b shows a mat of ﬁbres collected on a silicon oxide surface during around 100 min, with a conversion >50% and a C24+ selectivity >70% (see more details about efﬁciency of the process and scaling possibi- lities in the Supplementary Note 2) with an average density estimated from the images of about 1010 ﬁbres/cm2. Images obtained from single ﬁbres show that they can reach up to 3 μm g y In the presence of such a high third-body density, the main impediment to the growth of the hydrocarbons by continuous aggregation of CH2 is the formation of ethylene CH2 þ CH2 ! C2H4ðΔG ¼ 6:3 eVÞ: ð1Þ ð1Þ NATURE COMMUNICATIONS \| (2021) 12:5937 \| https://doi.org/10.1038/s41467-021-26184-0 \| www.nature.com/naturecommunicatio 2 ARTICLE NATURE COMMUNICATIONS \| (2021) 12:5937 \| https://doi.org/10.1038/s41467-021-26184-0 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-26184-0 2 Spectroscopic composition of the ﬁbres. a Mass spectrum recorded with a mass quadrupole spectrometer with hydrogen introduced into the system (black line) and without hydrogen introduced into the system (red line—shifted down for clarity). The more intense unlabelled peaks, at 40 and 20 m/z, correspond to Ar (the sputtering gas) and its double ionisation, respectively, whereas the peaks at 36 and 38 m/z are due to stable Ar isotopes. b GC–MS chromatogram obtained from a very dilute solution of the ﬁbres in acetonitrile collected from an Au substrate (red), showing a distribution of chain lengths centered around C27 (peak marked corresponds to a known line from Irganox additive from the micropipette tip). A reference spectrum of a C28 linear alkane standard (green) was included for comparison. c Representative Raman spectrum recorded from a highly covered sample deposited on a SiOx substrate, with the most characteristic vibrational modes indicated. d AFM topography image of a ﬁbre-rich area on an Au substrate recorded with AFM-IR (spatial resolution 10 nm) and (e) IR spectrum obtained at the marked position in the previous image, from a single nanoﬁber at 10 nm resolution, with methyl- and methylene-group vibrations indicated. Fig. 3 DFT energy and bond-length calculations. a Computed enthalpy-variation diagram for each step in Fig. 1a along the reaction pathway. Enthalpy is given in eV, and all the subreactions are produced spontaneously with no thermokinetic reaction barrier. b Proposed mechanisms for interruption of the growth process after C6H12 formation on a C-NP by capture of a H to form C7H15, and (c) DFT-resulting C–C-bond length (in Å) between the carbon atoms C1 and C2 binding the C-NP and the growing alkane as a function of the alkane length up to n = 30. C–C-bond lengths for the gas-phase C2H4 and C2H6 are also shown for comparative purposes. Electrophilic H attack at the C1–C2 bond leads to gas-phase CnH2n+2. Fig. 3 DFT energy and bond-length calculations. a Computed enthalpy-variation diagram for each step in Fig. 1a along the reaction pathway. Enthalpy is given in eV, and all the subreactions are produced spontaneously with no thermokinetic reaction barrier. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-26184-0 For both processes, atomic H is required, which can be obtained from direct dissociation of H2 by the electrons generated in the sputtering 3 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-26184-0 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-26184-0 Fig. 2 Spectroscopic composition of the ﬁbres. a Mass spectrum recorded with a mass quadrupole spectrometer with hydrogen introduced into the system (black line) and without hydrogen introduced into the system (red line—shifted down for clarity). The more intense unlabelled peaks, at 40 and 20 m/z, correspond to Ar (the sputtering gas) and its double ionisation, respectively, whereas the peaks at 36 and 38 m/z are due to stable Ar isotopes. b GC–MS chromatogram obtained from a very dilute solution of the ﬁbres in acetonitrile collected from an Au substrate (red), showing a distribution of chain lengths centered around C27 (peak marked * corresponds to a known line from Irganox additive from the micropipette tip). A reference spectrum of a C28 linear alkane standard (green) was included for comparison. c Representative Raman spectrum recorded from a highly covered sample deposited on a SiOx substrate, with the most characteristic vibrational modes indicated. d AFM topography image of a ﬁbre-rich area on an Au substrate recorded with AFM-IR (spatial resolution 10 nm) and (e) IR spectrum obtained at the marked position in the previous image, from a single nanoﬁber at 10 nm resolution, with methyl- and methylene-group vibrations indicated. Fig. 2 Spectroscopic composition of the ﬁbres. a Mass spectrum recorded with a mass quadrupole spectrometer with hydrogen introduced into the system (black line) and without hydrogen introduced into the system (red line—shifted down for clarity). The more intense unlabelled peaks, at 40 and 20 m/z, correspond to Ar (the sputtering gas) and its double ionisation, respectively, whereas the peaks at 36 and 38 m/z are due to stable Ar isotopes. b GC–MS chromatogram obtained from a very dilute solution of the ﬁbres in acetonitrile collected from an Au substrate (red), showing a distribution of chain lengths centered around C27 (peak marked * corresponds to a known line from Irganox additive from the micropipette tip). A reference spectrum of a C28 linear alkane standard (green) was included for comparison. c Representative Raman spectrum recorded from a highly covered sample deposited on a SiOx substrate, with the most characteristic vibrational modes indicated. d AFM topography image of a ﬁbre-rich area on an Au substrate recorded with AFM-IR (spatial resolution 10 nm) and (e) IR spectrum obtained at the marked position in the previous image, from a single nanoﬁber at 10 nm resolution, with methyl- and methylene-group vibrations indicated. Fig. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-26184-0 b Proposed mechanisms for interruption of the growth process after C6H12 formation on a C-NP by capture of a H to form C7H15, and (c) DFT-resulting C–C-bond length (in Å) between the carbon atoms C1 and C2 binding the C-NP and the growing alkane as a function of the alkane length up to n = 30. C–C-bond lengths for the gas-phase C2H4 and C2H6 are also shown for comparative purposes. Electrophilic H attack at the C1–C2 bond leads to gas-phase CnH2n+2. NATURE COMMUNICATIONS \| (2021) 12:5937 \| https://doi.org/10.1038/s41467-021-26184-0 \| www.nature.com/naturecommunicatio 4 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-26184-0 a Graphite target c Dome-shaped surface saddle point b Graphite target Ar inlet Type-1 Type-2 Main stream saddle-focus points 1 2 3 4 50 100 150 200 250 300 350 d Time (s) (deg.) θ Fig. 4 Trap-and-release ﬂow mechanism at the magnetron head. a General schematic of the magnetron head: orange lines represent the main Ar stream. Detail of the Ar+ ion impact (red arrows) toward the graphite target and the possible trajectory of knock out C atoms ejected toward the attraction areas (blue arrow), where gas-phase C–C coupling occurs to form C-NPs (blue spheres) and alkane chains (not at scale). b, c Torodial sectional streamlines ( b) and the stable manifold of the saddle point (c) in the region close to the graphite target. Trap-and-release events for C atoms released in the ﬂow are shown by red streamlines. d Residence-time distribution as a function of azimuth along the injection ring. This distribution is computed for ﬂuid trajectories originating from a circular ring of initial conditions with a uniform grid spacing along the azimuthal and radial directions, respectively. c Dome-shaped surface saddle point b Graphite target Ar inlet Type-1 Type-2 Main stream saddle-focus points 1 2 3 4 50 100 150 200 250 300 350 d Time (s) (deg.) θ a Graphite target d d Fig. 4 Trap-and-release ﬂow mechanism at the magnetron head. a General schematic of the magnetron head: orange lines represent the main Ar stream. Detail of the Ar+ ion impact (red arrows) toward the graphite target and the possible trajectory of knock out C atoms ejected toward the attraction areas (blue arrow), where gas-phase C–C coupling occurs to form C-NPs (blue spheres) and alkane chains (not at scale). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-26184-0 b, c Torodial sectional streamlines ( b) and the stable manifold of the saddle point (c) in the region close to the graphite target. Trap-and-release events for C atoms released in the ﬂow are shown by red streamlines. d Residence-time distribution as a function of azimuth along the injection ring. This distribution is computed for ﬂuid trajectories originating from a circular ring of initial conditions with a uniform grid spacing along the azimuthal and radial directions, respectively. accumulate onto a 2D-separation surface, pass by the top of the dome, and enter the main Ar stream whereby they are released from the vicinity of the target. The whole process can be visualised in Supplementary Movie 2. hydrocarbon chains is an energetically favourable process, increased residence time of the sputtered material in the region close to the magnetron is necessary27,28. It is clear that special kinetic factors are required to allow the reactants and products to be temporarily conﬁned together. This interplay between type-1 and type-2 planes provides the necessary trap-and-release mechanism. In fact, arbitrarily long trapping times can arise for atoms starting arbitrarily close to type-1 planes. The expected value of the carbon atom’s residence times is about 0.83 seconds (see Fig. 4d), which enables reactions that otherwise could not take place for trajectories without trapping. Thus, the experiments were undertaken in a reaction chamber designed to increase the residence time (Fig. 4a and Supplemen- tary Note 1). To fully understand the nature of the process, we performed direct numerical simulations of the Ar ﬂow dynamics using our reaction-chamber geometry in order to uncover possible regions where the reacting species can be trapped in the ﬂow and the reaction products subsequently released. From the experimental geometry, a Reynolds number Re ∼20–30 and a Mach number, Ma <0.007, were estimated. At these values, unsteadiness and compressibility in the ﬂow are not expected to play a signiﬁcant role, allowing us to use a steady, incompressible simulation of the gas-phase ﬂow. We found that these conditions give rise to a temporary trap-and-release mechanism that is already present in the steady, 3D streamline geometry of ﬂuid particles. This work demonstrates the efﬁcient generation of long alkane chains entangled into ﬁbres without the need for metal nanoparticle catalysts. Ab initio calculations reveal the role of C-NP and CHx and H radicals in the reaction pathways responsible for hydrocarbon growth. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-26184-0 On the other hand, ﬂuid- dynamics simulations reveal a trap-and-release mechanism demonstrating the feasibility of the process. The moderate temperatures involved (see Supplementary Note 3) and the high efﬁciency of the process suggest that after further development and optimisation, this methodology could lead toradically alternative routes to the clean and efﬁcient production of valuable alkane-based chemicals. The understanding of the C–C coupling mechanisms and the dynamics of the process have a multi- disciplinary impact and offers a powerful tool for the design and optimisation of long-chain hydrocarbons based on simple and abundant building blocks. Despite the toroidal recirculation zone suggested by different sectional streamline plots (Fig. 4b), the fully 3D analysis yields a solution with a fourfold broken symmetry. Speciﬁcally, a detailed tracking of judiciously chosen streamlines reveals the presence of four almost planar, invariant vertical surfaces, along with a dome- shaped surface. The vertical surfaces are 2D stable (type-1) and unstable (type-2) manifolds of saddle-focus-type points (Fig. 4b), whereas the dome-shaped surface (Fig. 4c) serves as the 2D-stable manifold of a saddle-type stagnation point29. Near the type-1 plane, the dome-shaped surface runs into the oriﬁce in backward time, preventing the ﬂuid particles under it from escaping into the main Ar jet (see Supplementary Note 14). Instead, they are forced to spiral toward the saddle-focus ﬁxed point and then are ejected toward an adjacent type-2 plane, where the dome- shape surface does not form a barrier to the outward-spiralling particles (see Supplementary Note 14). Rather, the ﬂuid particles NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-26184-0 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-26184-0 heptacosane (C27), nonacosane (C29), hentriacontane (C31), and dotriacontane (C32) were obtained from Sigma-Aldrich. heptacosane (C27), nonacosane (C29), hentriacontane (C31), and dotriacontane (C32) were obtained from Sigma-Aldrich. the experiments with hydrogen, extrapure H2 (99.99% purity) was injected through the lateral entrances of the aggregation zone (named Reaction chamber in Sup- plementary Fig. 1) at ﬂow rates of 1 sccm during fabrication. The typical power applied to the magnetron was 75 W. The magnetron and aggregation zone were water-cooled at 10 °C throughout the experiments. Nano-infra-red (AFM-IR). Nano-Infra-red (AFM-IR)30 performed using a NanoIR2-s (Anasys Instruments) based at the SMIS-3 Beamline of Synchrotron Soleil (L’Orme Les Merisiers, Paris, France) was used incorporating a tuneable mid- IR multichip MIRcat quantum cascade laser (QCL) source (Daylight Solutions). Typical experimental conditions employed were tapping frequency of the 1st mode = 395.6 kHz (detecting topography), QCL pulse rate of 356 kHz, pulse widths 300–170 ns, tapping frequency of the 2nd mode = 61.9 kHz (IR detection), and spectra scanned between 1325 and 1725 cm−1 with a step size of 1 cm−1. Instrument control and data collection and handling were performed using Ana- lysis Studio v3.14.6549 software (Anasys Instruments). During operation, the Reaction chamber pressure reaches about 0.1 mbar, and in the Collection chamber, 9 × 10−4 mbar was measured. The substrates employed for collecting the nanostructures are boron-doped Si(100) with its native oxide (SiOx), polycrystalline Au, and highly oriented pyrolytic graphite (HOPG) for AFM analysis; polycrystalline Au for nano-IR; a single-crystal Au(111) surface for XPS; holey carbon TEM grids for TEM microscopy; and boron-doped Si(100) with its native oxide (SiOx) and polycrystalline Au for Raman spectroscopy. The fabricated structures were either collected in the Collection chamber (See Supplementary Fig. 1), and for some particular experiments, inside the reaction chamber. In order to compare the AFM-IR spectra obtained from the ﬁbres, Fourier transform-infra-red (FTIR) attenuated total reﬂectance (ATR) spectra from standard n-alkanes (detailed in the Raman section above) were recorded at a spectral resolution of 4 cm−1 using a Specac Golden Gate horizontal ATR incorporating a heating stage in a Perkin Elmer System 2000 FT-IR equipped with a nitrogen-cooled HgCdTe detector. Scanning electron microscopy (SEM). SEM was performed in two different instruments. SEM images of Fig. 1 and Supplementary Fig. 2 were obtained at the chemistry department of the Ben-Gurion University of the Negev, Israel. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-26184-0 The carrier gas used was helium with a ﬂow rate of 1 mL min−1. The sample extract was obtained by carefully washing the surface of Au slides containing ﬁbre deposits from our experimental set-up machine, and a volume of 1 μL was injected in pulsed splitless mode at 1.5 bar during 2 min at 290 °C. The chosen chromatographic method lasted 37.5 min. The initial oven temperature was 80 °C, which was increased up to 340 °C at a controlled rate of 8 °C min−1, then maintained at that temperature for 5 min. This chromatographic method is suitable for the optimal separation of linear hydrocarbons between around C12 (dodecane) and C49 (nonatetracontane). Electron impact (70 eV) was the selected ionisation type for the mass spectrometer, and the m/z = 57 identiﬁcation ion was selected for determination of the n-alkanes in the sample. We investigate the lifetime of the metastable compound CH2* by ab initio- extended Lagrangian Born–Oppenheimer molecular dynamics simulations in the microcanonical ensemble. To produce realistic simulations, which at the same time could span over timescales longer than 10 ps, we utilise a plane-waves basis with on-the-ﬂy generation (OTFG) ultrasoft pseudopotentials34, energy cutoff of 325 eV, 10−6-eV energy threshold to reach self-consistency, and the same GGA-PBE XC functional and MP wave-function sampling35. Spin-polarised energy bands and a dispersion correction to include long-range van der Waals interactions have been used36. Finally, the reaction is simulated inside a 10 × 10 × 10-Å3 periodic cell box. Fluid-dynamics numerical calculations. Steady-state, three-dimensional numer- ical simulations were performed using the open-source computational ﬂuid- dynamics toolbox OpenFOAM37. The incompressible, steady-state Navier–Stokes equations were solved using the SIMPLE (Semi-Implicit Method for Pressure Linked Equations) algorithm as implemented in SimpleFoam solver38. Spatial derivatives are accurate to second order, and the solver is iterated, until the changes in the pressure and all three velocity components are less than 10−6 Pa and 10−6 m/s, respectively. The system was simulated for Reynolds numbers of 10, 20, and 30 based on the average inlet velocity and inlet width. Above this value, the results failed to converge due to transitioning to unsteady ﬂow. The simulation domain was generated using the snappyHexMesh utility and has around 35-million grid cells with local reﬁnement at the region of interest. The grid-size resolution in this region was approximately 1 µm, which is about three orders of magnitude smaller than the main dimensions on the magnetron. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-26184-0 Due to the pressure during operation in this chamber (9 × 10−4 mbar), the detection was carried out with a Faraday cup instead of using a secondary electron multiplier. Molecular dynamics. We investigate the assembly of CH2 from two-body colli- sions between H2 and C. Alternative routes, such as collisions between CH and H, have also been explored with similar results. Such two-body reactions eventually need to dissipate their excess energy to become a viable seed eventually, e.g., through collisions with a third body, which to be effective must occur within the compound’s lifetime. The experiment takes place in a dense atmosphere of Ar, which implies that collisions must occur in a timescale of the order of 10 ps. We investigate the lifetime of the metastable compound CH2* by ab initio- extended Lagrangian Born–Oppenheimer molecular dynamics simulations in the microcanonical ensemble. To produce realistic simulations, which at the same time could span over timescales longer than 10 ps, we utilise a plane-waves basis with on-the-ﬂy generation (OTFG) ultrasoft pseudopotentials34, energy cutoff of 325 eV, 10−6-eV energy threshold to reach self-consistency, and the same GGA-PBE XC functional and MP wave-function sampling35. Spin-polarised energy bands and a dispersion correction to include long-range van der Waals interactions have been used36. Finally, the reaction is simulated inside a 10 × 10 × 10-Å3 periodic cell box. Molecular dynamics. We investigate the assembly of CH2 from two-body colli- sions between H2 and C. Alternative routes, such as collisions between CH and H, have also been explored with similar results. Such two-body reactions eventually need to dissipate their excess energy to become a viable seed eventually, e.g., through collisions with a third body, which to be effective must occur within the compound’s lifetime. The experiment takes place in a dense atmosphere of Ar, which implies that collisions must occur in a timescale of the order of 10 ps. W i i h lif i f h bl d CH * b b i i i Gas chromatography – mass spectrometry (GC–MS). A Hewlett Packard 6890 GC gas chromatograph coupled to an Agilent Technologies model 5973 single- quadrupole mass spectrometer was employed for ex situ analysis of surface deposits. Chromatographic separation was performed on an Agilent DB-5ht poly(5% phenyl–95% methylsiloxane) fused-silica capillary column of 15 m length × 250 μm internal diameter and 0.1 μm wall thickness. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-26184-0 Fibres were collected on a silicon wafer, then air-dried overnight. The dried sample was sputter-coated with 5 nm. Measurements were conducted on a FEI Verios, SEM (Thermo Fisher Scientiﬁc, XHR 460 L). The images were viewed at 65,000X and 12,000X magniﬁcation, respectively, with an acceleration voltage of 3 kV. The rest of the images were taken at the Microscopy Laboratory of the Characterization Service in the Institute of Polymer Science & Technology, (ICTP-CSIC, Madrid, Spain) using a Field-Emission Hitachi SU 8000 microscope with 0.8kV accelerating voltage and 2.5 mm working distance. Atomic force microscopy (AFM). AFM images were performed using the Cer- vantes AFM System equipped with the Dulcinea electronics from Nanotec Elec- tronica S.L. and a Nanoscope IIIa system (Veeco) and Agilent 5500 PicoPlus (Agilent). All images were analysed using WSxM software31. X-ray photoelectron spectroscopy (XPS). XPS was carried out in situ using a PHOIBOS 100 1D electron/ion analyser with a one-dimensional delay-line detector. X-ray photons of 1486.6 eV (Al Kα emission line) were supplied by a water-cooled commercial X-ray gun XR 50 M (Specs) coupled to a Focus 500 X-ray monochromator (Specs). Transmission electron microscopy (TEM). TEM observations were carried out using a cold-ﬁeld emission gun in a JEOL GrandARM 300 Atomic Resolution Electron Microscope that was operated at 80 kV, conditions at which we could verify that there was no graphitisation induced by electron-beam damage to the samples. The column was ﬁtted with a JEOL double-spherical Cs aberration cor- rector. The ﬁbres were directly collected in-ﬂight by deposition onto holey TEM grids in the Collection chamber in such a way that they are self-supported, thus minimising the inﬂuence of any support. Ab initio calculations. To benchmark different mechanisms, we have computed formation and reaction enthalpies using ab initio plane-wave density functional theory. Details of the calculations are given in Supplementary Note 9. Furthermore, transition-state (TS) geometries have been obtained by the climbing-image nudged elastic band method32 complemented by the sequential application of the linear synchronous and quadratic synchronous transit (LST/QST) methods33. Mass spectrometry (MS). MS was performed in situ in the Collection chamber with a quadrupole mass spectrometer (QMS) PrismaPlus® QMG 220 M2 (Pfeiffer) with a range of 1–100 atomic mass units. The acquisition of the data was carried out at about 30 cm after the MICS exit. Methods l sample growth. Fabrication of carbon structures was undertaken using a scaled-up and customised multiple-ion-cluster source with three magnetrons, MICS3, from Oxford Applied Research Ltd. working in ultrahigh vacuum (UHV) (base pressure <5 × 10−10 mbar)11. The schematic of the experimental magnetron head and aggregation chambers is provided in Supplementary Note 1. The 2 magnetron used for these experiments was loaded with a graphite target of 99.95% purity. The sputtering gas (Ar, 99.999% purity) was kept constant for all the experiments at 150 sccm (50 sccm injected through each of the 3 magnetrons of the MICS3). For NATURE COMMUNICATIONS \| (2021) 12:5937 \| https://doi.org/10.1038/s41467-021-26184-0 \| www.nature.com/naturecommunications 5 References 1. Grela, K. Oleﬁn Metathesis: Theory and Practice https://doi.org/10.1002/97811 18711613 (Wiley Online Library, 2014). 30. Dazzi, A., Prazeres, R., Glotin, F. & Ortega, J. M. Local infrared microspectroscopy with subwavelength spatial resolution with an atomic force microscope tip used as a photothermal sensor. Opt. Lett. 30, 2388–2390 (2005). y y 2. de Meijere A., Bräse S., Oestreich, M. Metal-Catalyzed Cross-Coupling Reactions and More https://doi.org/10.1002/9783527655588 (Wiley Online Library, 2013). 31. Horcas, I. et al. WSXM: A software for scanning probe microscopy and a tool for nanotechnology. Rev. Sci. Instrum. 78, 13705 (2007). y 3. Choi, J. & Fu, G. C. 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AM acknowledges to the Spanish Ministry of Science (RYC2018-024561-I), to the Regional Government of Aragon (DGA E13_20R), to the National Natural Science Foundation of China (NFSC-21850410448, NSFC- 21835002), and to the The Centre for High- resolution Electron Microscopy (CħEM), supported by SPST of ShanghaiTech University under contract No. EM02161943. S.K. and G.H. acknowledge funding from the Tur- bulent Superstructures Program of the German National Science Foundation (DFG) 13. Lolla, D. et al. Polyvinylidene ﬂuoride molecules in nanoﬁbers, imaged at atomic scale by aberration corrected electron microscopy. Nanoscale 8, 120–128 (2016). y py 14. Kuei, B. et al. New opportunities in transmission electron microscopy of polymers. Mater. Sci. Eng. R Rep. 139, 100516 (2020). y g p 15. Santoro, G. et al. The chemistry of cosmic dust analogs from C, C2, and C2H2 in C-rich circumstellar envelopes. Astrophys. J. 895, 97 (2020). 16. Gall, M. J., Hendra, P. J., Peacock, O. J., Cudby, M. E. 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A., Cernicharo, J. & de Andres, P. L. Etching of graphene in a hydrogen-rich atmosphere toward the formation of hydrocarbons in circumstellar clouds. J. Phys. Chem. C 118, 26882–26886 (2014). ARTICLE ARTICLE Code availability 25. Mukherjee, P., Balamurugan, B., Shield, J. E. & Sellmyer, D. J. Direct gas-phase formation of complex core–shell and three-layer Mn–Bi nanoparticles. RSC Adv. 6, 92765–92770 (2016). Simulation and rendering packages QUANTUM ESPRESSO, CASTEP, OpenFOAM, and Houdini Apprentice availability is guaranteed since they are free open-source codes, which can be found in https://www.quantum-espresso.org/, http://www.castep.org/, https://www.openfoam.com/, and https://www.sidefx.com/products/houdini-apprentice/, respectively. 26. Koten, M. A., Mukherjee, P. & Shield, J. E. Core–shell nanoparticles driven by surface energy differences in the Co–Ag, W–Fe, and Mo–Co systems. Part. Part. Syst. Charact. 32, 848–853 (2015). respectively. y 27. Kousal, J. et al. 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S.K., J.A., H.S., and G.H. made the ﬂow calculations, L.M. and L.V. performed AFM experiments, A.M. performed HR-TEM characterisation, G.S., N.S., and R.J. performed SEM characterisation, A.D., J.M. F.B., G.J.E. and J.A.M-G conducted the nano-IR experiments, V.d.P.O. contributed to the catalytic concepts and ADF analysis, G.S. and G.J.E. performed the Raman measure- ments on the ﬁbres, and G.J.E. made complimentary IR and Raman measurements. E. B-B., G.J.E., R.L.-A., and J.Q.-L. made GC–MS measurements and analysed the data. J.C. con- tributed to the general idea of the machine design and to the choice of the reactants. P.d.A., G.H., G.J.E. coordinated the ab initio, ﬂow calculations, and optical spectroscopy measure- ments, respectively. All authors discussed the results and participated in the paper preparation and editing. The whole research was coordinated by J.A.M.-G. p 18. Abbate, S., Zerbi, G. & Wunder, S. L. 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NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-26184-0 The inlet conditions of the simulation were uniform inlet velocity and zero normal-pressure gradient (which introduces a trivial amount of error near the inlet). The outlet conditions were uniform ﬁxed outlet pressure and zero normal gradient of velocity (which assumes fully developed ﬂow at the outlet). The boundary conditions on the walls were no slip for the velocity and zero-normal gradient for the pressure. Raman spectra. Raman spectra were recorded using a Renishaw InVia Reﬂex Raman Microspectroscopy System (Renishaw plc., Wotton-under-Edge, UK) using two different laser sources: an Ar+ laser (λ0 = 514.5 nm, E = 2.41 eV) for the samples grown in our experimental setup and a diode-pumped solid-state (DPSS) laser (λ0 = 785 nm, E = 1.58 eV) for the standard n-alkanes. The laser was focused on the sample placed on the microscope stage using a 100× objective (NA = 0.85), with laser powers of <2 mW (for the 514.5 nm laser) and <20 mW (for the 785 nm laser) at the sample and a lateral spatial resolution (spot size) of between 1 and 1.5 μm. The spectral resolution was better than 4 cm−1 and several accumulations were coadded with an acquisition time of 10 s. The spectral data were analysed using Renishaw WiRE 5.0 software. More than seventy spectra were obtained from many different grown samples with varying hydrogen-ﬂow rates, from 0.15 to 5 sccm, and on different substrates, including SiOx and polycrystalline Au. Band- peak frequencies were obtained by curve deconvolution of the spectra using the WiRE software employing a combined Gaussian/Lorentzian line shape (20% Gaussian). The n-alkanes tricosane (C23), tetracosane (C24), hexacosane (C26), NATURE COMMUNICATIONS \| (2021) 12:5937 \| https://doi.org/10.1038/s41467-021-26184-0 \| www.nature.com/naturecommunications 6 Competing interests Competing interests The authors declare no competing interests. p g The authors declare no competing interests. 7 NATURE COMMUNICATIONS \| (2021) 12:5937 \| https://doi.org/10.1038/s41467-021-26184-0 \| www.nature.com/naturecommunicatio © The Author(s) 2021 Additional information Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41467-021-26184-0. Correspondence and requests for materials should be addressed to Pedro L.de Andres, George Haller, Gary J. Ellis or José A. Martín-Gago. Peer review information Nature Communications thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available. Reprints and permission information is available at http://www.nature.com/reprints Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. NATURE COMMUNICATIONS \| (2021) 12:5937 \| https://doi.org/10.1038/s41467-021-26184-0 \| www.nature.com/naturecommunications 8 8 NATURE COMMUNICATIONS \| (2021) 12:5937 \| https://doi.org/10.1038/s41467-021-26184-0 \| www.nature.com/naturecommunicatio
https://openalex.org/W3201732814	https://inscience.uz/index.php/socinov/article/download/1222/1424	Russian	null	Сурункали сил касаллиги билан оғриган болалар оиласининг тиббий-ижтимоий тавсифи	Society and innovations	2,021	cc-by	5,631	Guli SHAIKHOVA,1 Laziz AZIMOV2, Shahzoda ZOKIRKHONOVA3 Tashkent Medical Academy Tashkent Dental Institute Tashkent Medical Academy Tashkent Dental Institute Academy, Tashkent, Uzbekistan. E-mail: guli.shayhova@gmail.com. 1 Professor, Doctor of Medicine, Department of Hygiene children, teenager and Nutrition hygiene Tashkent Medical Academy, Tashkent, Uzbekistan. 2181-1415/© 2021 in Science LLC. This is an open access article under the Attribution 4.0 International (CC BY 4.0) license (https://creativecommons.org/licenses/by/4.0/deed.ru) 2 Independent applicant for the Department of Hygiene children, teenager and Nutrition hygiene Tashkent Medical Academy, Tashkent, Uzbekistan. 3 Associate Professor at the Department of Pediatric Dentistry Tashkent Dental Institute, Tashkent, Uzbekistan. y, , 3 Associate Professor at the Department of Pediatric Dentistry Tashkent Dental Institute, Tashk 1 Professor, Doctor of Medicine, Department of Hygiene children, teenager and Nutrition hygiene Tashkent Medical Academy, Tashkent, Uzbekistan. E-mail: guli.shayhova@gmail.com. 2 Independent applicant for the Department of Hygiene children, teenager and Nutrition hygiene Tashkent Medical Academy, Tashkent, Uzbekistan. 3 Associate Professor at the Department of Pediatric Dentistry Tashkent Dental Institute, Tashkent, Uzbekistan. Жамият ва инновациялар – Общество и инновации – Society and innovations Special Issue – 8 (2021) / ISSN 2181-1415 АННОТАЦИЯ Калит сўзлар: касалланиш, тиббий ва ижтимоий омиллар. Анкета картаси ёрдамида сил касаллиги билан оғриган болаларнинг ижтимоий шароитлари ўрганилди, шу билан бирга 92 фоизи ижтимоий аҳволда, уларнинг кўп қисми яшаш шароитлари оғир, таълим даражаси паст, тўйиб овқатланмаган ва сил касаллиги бўйича саводхонлиги пастлиги қайд этилди. Тадқиқот усуллари. Иш жараёнида қўйилган вазифа- ларга қараб методлар мажмуасидан фойдаланилди: анкета методи, тиббий-социологик тадқиқот, гигиеник, тиббий- статистик, клиник. Тадқиқотнинг мақсади. Сил касаллигига чалинган болаларнинг тиббий-ижтимоий хусусиятларини ўрганиш. Тадқиқот натижалари: сўровнома харитасидан фойда- ланиб, сил касаллигига чалинган болаларнинг ижтимоий шароитлари ўрганилди, бунда 92% ижтимоий жиҳатдан оғир турмуш шароити, таълим даражаси пастлиги, тўйиб овқатланмаслик ва силга қарши масалалар бўйича санитария саводхонлиги пастлиги билан боғлиқ эканлиги маълум бўлди. ARTICLE INFO ARTICLE INFO Article history: Received July 2021 Received in revised form 20 July 2021 Accepted 15 August 2021 Available online 15 September 2021 Keywords: morbidity, medical and social factors. ARTICLE INFO Article history: Received July 2021 Received in revised form 20 July 2021 Accepted 15 August 2021 Available online 15 September 2021 Keywords: morbidity, medical and social factors. Using the questionnaire card, the social conditions of children suffering from tuberculosis were studied, and it was revealed that 92% are socially with a large proportion of people with difficult living conditions, a low level of education, malnutrition and low health literacy on tuberculosis. Article history: Received July 2021 Received in revised form 20 July 2021 Accepted 15 August 2021 Available online 15 September 2021 Research methods. In the process of work, a complex of methods was used depending on the tasks set: the method of questioning, medical and sociological research, hygienic, medical and statistical, clinical. Purpose of the study. Study of medical and social characteristics of children suffering from tuberculosis. Research results: the study of the influence of social factors on the quality of life of various groups of the population showed that persons suffering from tuberculosis differed from the opposite group: 92.0% are socially unprotected segments of the population with a monthly income lower than the national average subsistence minimum by almost 10 times, unemployed, pensioners and workers with a lower income per family member, a low level of education (67%). 2181-1415/© 2021 in Science LLC. This is an open access article under the Attribution 4.0 International (CC BY 4.0) license (https://creativecommons.org/licenses/by/4.0/deed.ru) E mail: guli.shayhova@gmail.com. 2 Independent applicant for the Department of Hygiene children, teenager and Nutrition hygiene Tashkent Medical Academy, Tashkent, Uzbekistan. 3 Associate Professor at the Department of Pediatric Dentistry Tashkent Dental Institute, Tashkent, Uzbekistan. КИРИШ ҚИСМИ Тошкент вилояти соғлиқни сақлаш тизимининг бутун тарихи давомида болалар ахолисини сил билан касалланишининг энг паст кўрсаткичлари 1991– 1992 йилларда 100 минг ахолига 5,6-6,0ни ташкил этиш билан қайд этилган. Бироқ 1993 йилда касалланиш кескин ўсди –ўсиш суръати 195%ни ташкил этди. Кейинчалик, 1994-1995 йиллардаги бир қадар пасайишдан сўнг, ўсиш тенденцияси давом этди. 1991-1999 йиллар давомида кўрсаткичларни ўсиш суръати 512,5% ни ташкил этиб, 1999 йилларда – 100 минг ахолига нисбатан ўхшаши йўқ бўлган энг юқори кўрсаткич – 34,3 ни ташкил этди. 2000 йилда касалланиш пасайди, бироқ пасайишни суръати сезиларсиз -12,8% бўлди. 2000 йилдан бошлаб, касалланиш кўрсаткичи етарли даражада юқорилигича қолиб, доимий равишда 100 000 ахолига нисбатан 24,4-30,0 чегарасида ўзгариб турди [1, 2, 3, 24]. Ҳозирги вақтда Тошкент вилояти болалардаги сил касаллиги бўйича ноқулай эпидемиологик вазиятли минтақага киради. 2006–2008 йиллардаги касалланиш- нинг ўртача кўрсаткичи 100 минг ахолига 29,1%ни ташкил этди, бу мамлакат бўйича ўртача кўрсаткичдан 1,6-1,8 мартага юқоридир. 2008 йилда МБТ болаларини бирламчи юқтириши эса -32,7%ни ташкил этди, шунингдек бу ҳам Ўзбекистон Республикаси бўйича ўртача кўрсаткичлардан юқори бўлди. Болалардаги сил билан касалланиш вилоят ҳудуди бўйича нотекис тақсимланган. (13,14). Касалланиш тузилмасида сезиларли даражадги ажралиш билан инфиль- трация ва зичлашиш фазасидаги кўкрак қафаси ичи лимфатик тугунларининг сили устунликка эга, унинг улушига эса барча холатларнинг 62,3%и тўғри келади. Сўнгги йилларда 0-14 ёшли болалар ўртасида сил микобактерияларини ажратувчи беморлар аниқланмади, 15-17 ёшли болалар ўртасида эса улар барча беморларнинг 17,3%ини ташкил этди. 2006-2008 йилларда вилоятнинг болалар ахолисида сил билан касалланишни ўртача даражаси 100 минг ахолига 33,9%ни ташкил этди, бу Ўзбекистон Республикаси бўйича ўртача 1,5 марта юқоридир. 1985–1990 йиллар даврида кўрсаткичларни аста секин пасайиш тенденцияси ўз ўрнига эга бўлди, пасайиш суръати -2,3–11,4% чегарасида ўзгариб турди, 1991–1992 йилларда оғриқлилик 1,7 мартага пасайди ва ўрганилган даврда 100 минг ахолига нисбатан энг кичик -12,5–12,6 га етди. Бироқ 1993 йилдаёқ у 2 мартадн кўпроққа ошди (ўсиш суръати 100,8%ни ташкил этди) ва 1999 йилгача ўсишнинг турли суръатларида давом этди. 1999 йилда кузатишлар даври мобойнидаги энг юқори кўрсаткичлар – 100 минг ахолига нисбатан 72,2 га етганлиги қайд этилди. 2000 йилдан бошлаб унча катта бўлмаган (2000–2003 йиллар) оғриқлиликни янада аҳамиятли пасайиши қайд этилиши кузатилди, аммо 2006–2007 йилларда кўрсаткичлар яна ошиб, 1995– 1996 йиллардаги кўрсаткичларга етиб олди. 2008 йилда оғриқлилик яна пасайди, аммо пасайиш суръати унча катта эмас – бор йўғи 18,4%ни ташкил этди. ( 21,24 ). 2002 йилдан бошлаб бирламчи ногиронлик кўрсаткичини сезиларли ўсиш тенденцияси қайд этилиб, 2002 йилда ўсиш суръати 178,9% нит ташкил этди. 2004 йилда бирламчи ногиронлик даражаси деярли 2000 йил даражасигача пасайди. Жамият ва инновациялар – Общество и инновации – Society and innovations Special Issue – 8 (2021) / ISSN 2181-1415 Жамият ва инновациялар – Общество и инновации – Society and innovations Special Issue – 8 (2021) / ISSN 2181-1415 АННОТАЦИЯ Ключевые слова: заболеваемость, медико-социальные факторы. С помощью карты анкеты изучены социальные условия у детей, страдающих туберкулезом, при этом было выявлено, что 92% – это социально с большой долей лица с тяжелыми бытовыми условиями, низким уровнем образования, неполноценным питанием и низкой санитарной грамотность по вопросам туберкулеза. Методы исследования. В процессе работы использованы комплекс методов в зависимости от поставленных задач: метод анкетирования, медико-социологические исследования, гигиенические, медико-статистические, клинические. Цель исследования. Изучение медико – социальной характеристики детей, страдающих туберкулезом. Результаты исследований: С помощью карты анкеты изучены социальные условия у детей, страдающих туберкулезом, при этом было выявлено, что 92 % – это социально с большой долей лица с тяжелыми бытовыми условиями, низким уровнем образования, неполноценным питанием и низкой санитарной грамотьность по вопросам туберкулеза. 558 КИРИШ ҚИСМИ Сўнгра 2007 йилгача кузатишларнинг бутун даври мобойнида 100 минг ахолига нисбтан энг юқори даражагача етиб – 11,5%га эришди ҳамда оғишмай ўсишда давом этди. 2008 йилда бирламчи ногиронлик даражаси пасайди, бироқ пасайиш суръати бор йўғи 36,5%ни ташкил этди, 2008 йилдаги кўрсаткичлар 2001 йилдаги кўрсаткичлардан 3,8 мартага ошди ва жуда юқорилигича қолди. 559 Жамият ва инновациялар – Общество и инновации – Society and innovations Special Issue – 8 (2021) / ISSN 2181-1415 2008 йилдаги умумий ногиронлик даражаси 2001 йилдаги кўрсаткичлардан 4,5 мартага ошди. Сезиларли узилишлар билан бирламчи ва умумий ногиронлик тузилмасида инфильтратив сил етакчиликка эга, унинг улушига бирламчи ногиронликнинг 57,7%, умумийнинг -41,3% тўғри келади. Сил оқибатида болалар ахолиси ногиронлигининг энг мухим сабаби бўлиб, фон касалликлар ҳисобланади. 2007-2008 йилларда 64,3% ногирон болалар йўлдош касалликларга эга бўлдилар, бунда 43,2% болаларда 2-3 йўлдош касалликлар ташхисланди [9, 14, 24]. Тадқиқотнинг мақсади: сурункали сил касаллиги билан оғриган болалар ва уларнинг оиласининг тиббий-ижтимой тавсифи. Тадқиқотнинг усуллари: аналитик, клиник, сўровнома, тиббий-ижтимоий тадқиқотлар, ҳисоб-китоб ва статистик таҳлил усуллари қўлланилган. Тадқиқот объекти. Тошкент шаҳри ва Тошкент вилоятида жойлашган Зангиота тумани болалар сил касалликлар даволаш сиҳатгоҳида тарбияланувчи мактабгача ёшдаги 3-7 ёшли 670 (436та ўғил бола ва 234 та қиз бола) болалар олинди. Тадқиқотнинг асосий мазмуни Тошкент шаҳри ва Тошкент вилояти болалари орасидан ўпка сили билан касалланган 3 ёшдан 7 ёшгача бўлган болалар ва уларнинг оила аъзолари орасида ўтказилган анкета сўров натижаларига кўра, 67,0% қишлоқ аҳолиси, 33,0% шаҳар аҳолиси болаларига тўғри келади. Болаларнинг жинси бўйича таҳлили шуни кўрсатдики, 69,5%, ўғил болалар 30,5% қиз болалар ташкил қилди. 94,5%ида сил микобактериялари аниқланган. (1-расм). 1-Расм. Беморларнинг яшаш жойи ва жинси бўйича тақсимланиши 33 67 шаҳар қишлоқ 69 31 ўғил болалар қиз болалар 1-Расм. Беморларнинг яшаш жойи ва жинси бўйича тақсимланиши Касалликнинг давомийлигига кўра, биринчи марта аниқланганлар 75,1%, силнинг сурункали формалари – 16,8%, касалликнинг рецидиви – 8,2%. Касалликнинг давомийлигига кўра, биринчи марта аниқланганлар 75,1%, силнинг сурункали формалари – 16,8%, касалликнинг рецидиви – 8,2%. 560 Жамият ва инновациялар – Общество и инновации – Society and innovations Special Issue – 8 (2021) / ISSN 2181-1415 Беморларнинг касаллик давомийлигига кўра тақсимланиши 75 16,8 8,2 биринчи марта касалланганлар силнинг сурункали формаси касалликнинг рецидиви Жамият ва инновациялар – Общество и инновации – Society and innovations Special Issue – 8 (2021) / ISSN 2181-1415 биринчи марта касалланганлар 75 Беморларнинг касаллик давомийлигига кўра тақсимланиши Эпидемиологик тадқиқотларда сил инфекциясининг тарқалишида инсон- нинг инфекцияга чалинувчанлиги (иммунитети) муҳим ахамиятга эга. Жумладан, сил инфекцияга чалинувчанлик инсоннинг ёшига, жинсига, иммунитети, хамрох касалликларга боғлиқ. [16, 17]. Сил билан зарарланиш айниқса, эрта болалик ёшида (1 ёшгача ва 1 ёшдан 5 ёшгача) хавфли хисобланади. Бу даврда иммунитет тизими хали етарли даражада ривожланмаган [5, 6, 18]. Наслий мойиллик ёки сил инфекциясига мойиллик юқори бўлган организмларда касаллик ривожланишининг хавфи юқоридир. (12 ). Жамият ва инновациялар – Общество и инновации – Society and innovations Special Issue – 8 (2021) / ISSN 2181-1415 нлардир. Анкета сўровнома натижалари кўрсатдики, 15,6% оналар ва 62,2% оталар тамаки чекади, бунда 8,8% респондентлар, яъни мактаб ёшидаги болаларнинг 13,8% ота-оналари болалар бўладиган хоналарда чекадилар. Касал бўлган болаларнинг 21,4% оилаларида спиртли ичимликларни мунтазам равишда истеъмол қиладилар, 14,0% оталар ва 3,9% оналар боланинг касал бўлган вақтида ёки ундан олдин спиртли ичимликларни истеъмол қилганлар. 14,5% респондентлар оиланинг ички муҳитини бефарқлик каби, 6,2% – жуда таранг, низоли сифатида баҳолайдилар. Бемор бўлган болаларни аксарият оилаларини моддий холати паст, ҳатто жуда паст (41,0%), фақат 21,1% эса яхши моддий таъминотга эга бўлади. Фақат 24,1% оилалар 1 оила аъзосига 6 м2 дан ортиқ алохида яшаш майдонига ва барча коммунал қулайликларга эга бўлган турар жой майдонига эга, 34,7% бемор болалар маҳаллаларда (ховлили уй), кўп қаватли уй хонадонида истиқомат қиладилар, 5,6% ижара уйларида яшайдилар. 26,3% оилаларда болалар учун алоҳида хона ажратиш имконияти мавжуд, бироқ 33,2% оилаларнинг болаларига умумий хонадан фақат бурчак ажратилган, 12,4% бемор болалар учун ҳеч қандай алохида шароитлар яратилмаган. 26,3% оилаларда болалар учун алоҳида хона ажратиш имконияти мавжуд, бироқ 33,2% оилаларнинг болаларига умумий хонадан фақат бурчак ажратилган, 12,4% бемор болалар учун ҳеч қандай алохида шароитлар яратилмаган. Умуман олганда фақат 59,2% респондентлар ўз оиласи шароитини осойишта сифатида баҳолаган бўлса, бу вақтда 34,0% - бола саломатлиги учун алохида ҳавф омилларини мавжудлигини кўрсатдилар, 6,8% ўз оиласини нохуш, осойишта бўлмаганлар қаторига киритган. Ёпиқ муассаса тарбияланувчилари кўпинча овқатланишнинг ёмонлиги (50,4%) ва тоза ҳавода кам бўлишни (49,2%) кўрсатдилар. Бундан ташқари, 12,9% жисмоний фаоллик пастлиги ва қўшимча машғулотларни узоқ вақт давом этишини, 14,9% – уйқуни етишмаслигини кўрсатдилар. Жисмоний фаолликнинг пастлигини 14,9% уйқуни етишмаслигини – 5,8%, компьютер олдида узоқ ўтиришни – 0,8% ота-оналар қайд этдилар. Мактаб ўқувчиларининг 4,1% доимий бўлмаган, ёмон оқатланишни кўрсатдилар, бунда ота-оналарнинг фикрича 19,0% болалар ёмон ёки жуда ёмон овқатланганлар. Ота- оналар ва болаларнинг ўзлари томонидан ҳавф омилларини қиёсий баҳолаш қиёсий ёндошувини талаб этади. Шу билан бир вақтда бир қатор субъектив омилларга болалар ва ота-оналари- нинг муносабати турлича: демак, масалан, болалар учун қўшимча машғулотлар жуда узоқ этаётгандек қабул қилиниши мумкин, ота-оналар учун – бу меъёрдек, бола унга уйқу етишмаётгандек, кам ухлаётгандек ҳисоблаш мумкин, ота-оналар эса ўзини уйқу вақтини боланинг уйқу вақти билан солиштириб, у учун ажратилаёт- гандек уйқу соатларини етарли деб таҳмин қилинади. Равшанки, ушбу омилларни, саломатликка таъсирини баҳолашдаги мазкур холатда боларнинг ўзларини субъектив хисларини ҳисобга олиш керак. Алохида ҳавф омиллари бўйича ота- оналар одатда кам маълумотга эга бўлиши мумкин. Ёш катталашиб бориши билан иммун тизим ривожланиб боради, организм- нинг силга чидамлилиги ҳам ортиб боради, лекин касалликларга чалинишда турмуш тарзи омилларининг аҳамияти ҳам жуда мухимдир. Етарлича овқатлан- маслик, тез тез касалликларга чалиниш, асаб рухий зўриқишлар (стресслар) шулар жумласидандир [10, 12, 15]. Ўрганилган анкета маълумотлари ёрдамида аниқланган бемор болалар оиласидаги болаларнинг ўртача сони 2,28 ни ташкил этди, битта болали оилалар улуши 21,4%, иккитали – 55,5%, учта – 10,7%, тўртта ва ундан кўп бўлган болалилар эса – 12,4%ни ташкил этди. Шундай қилиб кўп болалали оилалар улушига 23,1% тўғри келди. Бемор болаларнинг фақат 78,8% қисми тўлақонли оилада истиқомат қилади ва тарбияланади, бу вақтда 12,8%-отасиз, фақат онаси томони- дан тарбияланади, 3,5% болалар бувиси ва буваси ёки улардан бири билан истиқомат қилади, 1,2% опа-синглиси ва ака-укаси билан истиқомат қилади, 1% эса бошқалар томонидан (васийлар, оталиқққа олганлар, яқин қариндошлари билан истиқомат қилади). Касалликка чалинган болаларнинг ота-оналарнинг аксариятини маълумот- лилик даражаси жуда паст: 58,2% оналар ва 51,0% оталар ҳатто ўрта махсус маълумотга ҳам эга эмаслар ва фақат 22,0% оналар ва 21,5% оталар олий ёки тугалланмаган олий маълумотга эгалар. Бемор болалар оналарининг кўпчилиги уй бекаси (37,1%), хизматчи (21,4%), ишбилармонлик билан шуғулланганлар (17,6%), ёҳуд ўз мутахассислиги бўлмаган (24,9%) иш билан машғулдирлар. Оталарнинг ижтимоий ҳолати оналарнинг ижтимоий холатидан сезиларли фарқ қилади: улар орасида аксарият қисмини хизматчилар (27,2%), ҳарбий хизматчилар (26,4%) ва ишсизлар (12,0%) ташкил этади. 4,8% оналар ва 5,6% оталар илгари судланга- 561 Жамият ва инновациялар – Общество и инновации – Society and innovations Special Issue – 8 (2021) / ISSN 2181-1415 олдидаги саломатлик холатини баҳолаши сезиларли даражада юқори бўлди: 40,6% болалар ўзларининг аъло даражадаги, 32,3% – яхши, 26,1%и қониқарли даражадаги саломатликка эга деб ҳисоблайдилар ва фақат 1,0% касалликка нисбатан уларнинг саломатлиги ёмон даражада деб таҳмин қиладилар. Равшанки жавоблардаги бундай фарқ шу билан боғлиқки, ўз болаларини саломатлигини баҳолашда оналар одатда саломатлиги устида жуда қайғурувчи шахснинг умумий дезадаптациясини гипертрофирланган холатини кўрадилар. Саломатликни баҳолашдан фарқли равишда, оналар ва болаларнинг ўзлари томонидан жисмоний тайёргарликни баҳолаш янада яқинроқ бўлди: 5,1% ота – оналар ва 6,0% мактаб ўқувчилари уларни ёмон деб, мос холда 49,8% ва 47,5% – яхши деб баҳоладидар. Ёпиқ муассаса тарбияланувчилари оилада истиқомат қилувчи болаларга нисбатан касаллик олдида ўзининг саломатлиги ва жисмоний тайёргарлик холатини сезиларли даражада паст баҳоладилар. Демак, ёпиқ муассаса болаларининг фақат 42,4% ўз саломатлигини аъло ва яхши деб баҳолаган бўлса, фақат 4,0% болалар ўзларида аъло даражадаги жисмоний тайёргарлик бор деб таҳмин қиладилар, ёмон ва қониқарсиз кўрсаткичлар – 46,5% болаларда аниқланди. Ўз оиласида тарбияланаётган ва ижтимоий дезадаптацияланган мактаб ўқувчилари, вояга етмаганларнинг ўзини саломатлиги ва жисмоний тайёргар- лигини қиёсий баҳолаш, ижтимоий мослаштирилган мактаб ўқувчиларида ўзига баҳо бериш бошқа ижтимоий гуруҳ болаларига нисбатан солиштирилганда ўзларига юқори баҳо беришини кўрсатди. Демак дезадаптацияланган мактаб ўқувчиларининг 73,7% касалликка нисбатан ўз саломатлигини, – 83,9% жисмоний тайёргарлигини аъло ва яхши деб баҳоладилар. Ўз болаларининг саломатлик холати тўғрисида оналарнинг ҳавотирланиш даражасининг таҳлили кўрсатдики, болалари касалликка чалингунча фақат 34,8% респондентлар болаларининг саломатлиги тўғрисида қайғурганлар, тез-тез хавотир олганлар эса – 49,5% ни ташкил этади, шу билан бирга 4,7% оналар ўз фарзандларининг саломатлиги билан умуман қизиқмаган бўлсалар, 11,0% – эса баъзи холатлардагина қизиқганлар. Оналарнинг бир қисмини ўз болаларининг саломатлик холати бўйича қайд этилган хавотирланиш даражаси кўпинча мос бўлмайди ва сезиларли пасаяди, бу эса бола ҳаётининг кундалик саломатлигини сақлаш фаолиятига ва профессионал тиббий ёрдамга эҳтиётсизлик билан муносабатига нисбатан руҳий асос бўлиб хизмат қилади. Демак, фақат 15,4% болалар касалланган вақтда мактабга бормадилар, 84,6% болалар эса бемор бўлган вақтида машғулотларга қатнаган, жумладан 8,1% болалар касаллик белгилари кучли намоён бўлган вақтида ҳам машғулотларга қатнашганлар. Беморларни касалланиши вақтида мактаб ўқувчиларининг фақат 11,7% и шифокорларга мурожаат қилдилар, 77,9% фақат бола кучли касал бўлганда тиббий ёрдамга мурожаат қилдилар, 10,4% эса амалий жиҳатдан тиббий ёрдамга умуман мурожаат қилмаганлар. Тиббий ёрдамга мурожаат қилишнинг ўзи касалликдан даволанишни кафолатламайди. Болани шифокор тавсияларини қатъий бажариши ўта мухимдир. Бироқ, 57,4% болалар шифокор тавсияларини бажармайдилар, 33,5% – мунтазам равишда бажармайдилар ва фақат 9,1% ушбу тавсияларга доим риоя қилганлар. Ўз оиласида истиқомат қилувчи мактаб ўқувчиларининг фақат 9,4% ва ёпиқ муассасаларнинг 4,0% тарбияланув- чилари ўз саломатлигини мустаҳкамлаш учун мунтазам равишда жисмоний тарбия билан (тонгги бадантарбия, чиниқтириш муалажалари) шуғулланганлар. Аниқки дезадаптацияланган болаларда бундай имкониятнинг ўзи бўлмаган. Сўровномада иштирок этган ялпи оналарнинг 34,9% респондентлари касалликка нисбатан ўз болаларининг саломатлик ҳолатини аъло деб, 25,9% – яхши, худди шунчаси (25,9%) – қониқарли, 14,5% – ёмон деб баҳоладилар. Болаларнинг ўзларини касаллик 562 Инфекцион жараён кечишига сил билан зарарланган одамларда ортирилган иммунитетни мавжудлиги ёки мавжуд бўлмаслиги жуда катта таъсир кўрсатади. 563 Жамият ва инновациялар – Общество и инновации – Society and innovations Special Issue – 8 (2021) / ISSN 2181-1415 Жамият ва инновациялар – Общество и инновации – Society and innovations Special Issue – 8 (2021) / ISSN 2181-1415 Жамият ва инновациялар – Общество и инновации – Society and innovations Special Issue – 8 (2021) / ISSN 2181-1415 Жамият ва инновациялар – Общество и инновации – Society and innovations Special Issue – 8 (2021) / ISSN 2181-1415 Ҳар бир одамнинг индивидуал ўзига хослиги қанчалик мухим бўлишига қарамасдан сил эпидемиологияси ахоли ёки унинг алохида гуруҳлари орасида силни тарқалишини аниқлаб берувчи умумий қонуниятларни ўрганади. Бу билан сил муаммосига бўлган эпидемиологик ёндошув, индивидуумни ўрганувчи клиникадан фарқ қилади. Шу нуқтаи назардан ахоли ўртасида силга энг таъсирчан бўлган гуруҳни ажратиб олиш ўта мухимдир. Бундай гуруҳларни силни ривожланишини юқори ҳавфи бўлган ахоли гуруҳлари деб номлаш қабул қилинган. Сил патогенезини эътиборга олган холда, силнинг бирламчи шаклларини ривожланиш ҳавфини ортишига касалликни юқтирмаган шахслар, болалар, ўсмирлар ва 30 ёшгача бўлган катталар эга бўлади, чунки ахоли ўртасида ёшини ортиши билан сил билан зарарланганлар сони ортиб боради: 40 ёшга келиб бундай холатлар сони 70–90% ни ташкил этади [4-8]. Шунингдек зарарланган одамлар сони эпидемиологик кўрсаткич сифатида қаралади – касалликни юқтириш уларда 100 минг ахолига нисбатан фоизларла ифодаланади. Кўпинч касалликни юқтириш турли ёш гуруҳларга қўлланилиши билан аниқланади.. Чақалоқларни силга қарши эмлаш, болалар ва ўсмирларнинг қайта эмлаш ўтказиладиган мамлакатларда, ахолини сил билан касалланиш кўрсаткичларини аниқлаш, эмлаш ва қайта эмлашдан сўнг мусбат тубиркулин синамаларини пайдо бўлиши сабабли катта қийинчиликни туғдиради. Шунга қарамасдан “зарарланиш ҳавфи” ва ушбу кўрсаткичлар билан силни юқадиган шакли билан оғриган бемор- лар сони ўртасидаги ўзаро алоқа ўрнатишга имкон берувчи ҳисоблаш усуллари ишлаб чиқилди. Оилаларнинг турмуш тарзи ва шароити, ота оналарнинг ижтимоий холати хар қандай инфекциянинг тарқалишида мухим омиллардан биридир. Ижтимоий холати бўйича сил билан касалланганлар: хизматчилар (5,8%); ишчилар 441 нафар (21,7%); ишламайдиган 1071 нафар (52,8%); нафақа ёшидагилар 321 (15,8%); ишбилармонлар 76 (3,7%) ни ташкил этди. Улардан сўровномада иштирок этган умумий сондан, фаол силли энг меҳнатга яроқли бўлган (15 ёшдан 50 ёшгача) 78,5% ни ташкил этди.(3-расм). 3-расм. Беморларнинг ижтимоий ҳолатига кўра тақсимланиши 0,00% 50,00% 100,00% 150,00% 200,00% 250,00% 300,00% 350,00% 400,00% 5,80% 21,70% 50,50% 15,80% 3,7 2,3 3-расм. Беморларнинг ижтимоий ҳолатига кўра тақсимланиши 564 Жамият ва инновациялар – Общество и инновации – Society and innovations Special Issue – 8 (2021) / ISSN 2181-1415 Битта ишловчига ўндан ортиқ одамни қарам бўлиш миқдори беморлар орасида 27,4% ни ташкил этади, бу асосан оиланинг меҳнатга яроқли аъзолари ёки оила бошлиғи ўз фарзандларини ташлаб, республика ташқарисидан тасодифий мардикорлик билан пул топишга кетган оилалардир. Битта меҳнатга яроқли шахсга боқиманданинг 1 дан 8 гача бўлганлари асосан 3%ни, 4 дан 6 гача бўлганлар – 3,9%ни ва 3 гача бўлганлар эса – 6,4% беморларга тўғри келади. Ўз вақтида ташхислаш ва мос бўлган даволашни олганлигини аниқлаш мақсадида 500 нафар респондентларга берилган: “Бошқа шифокорда қанча вақт давомида даволандингиз?”, саволига қуйидагича: 32,2% – 10-15 кун давомида; 23,0%- бир ой давомида; 21% – 3 ойгача; 8,0% – 6 ойгача; 9,6%-бир йилдан ортиқ вақт давомида деган жавоблар олинди. (4-расм). Бошқа шифокорларда қанча вақт давомида даволандингиз? Сил билан оғриган сўровномада иштирок этганлар миқдоридан 61,4% бемор- лар фтизиатрга ўз вақтида мурожаат қилганлар, 14,5% ўзларини соғлом деб ҳисоблаганлар; 15,1% беморлар ўзларида мавжуд бўлган клиник белгилар тезда ўтиб кетади деб ўйлаганлар; 4,8% қон туплаганларидан сўнг шифокорга мурожаат қилганлар ва 3,2% таниш шифокорда даволанганлар. “Касаллик бошлангандан сўнг қанча вақт ўтиб сил ташхиси қўйилган?”, деган саволга қуйидагича жавоблар олинди: – 61,4% бир ой давомида; 20,9%-уч ойдан сўнг; 8,0%-4, 6 ойдан сўнг; 9,6% – бир йлдан сўнг, беморлар асосан силнинг ўпкадан ташқари шакллари билан оғриганлар.(5-расм) 0 5 10 15 20 25 30 35 32,2 23 21 8 Бошқа шифокорларда қанча вақт давомида даволандингиз? 0 5 10 15 20 25 30 35 32,2 23 21 8 Бошқа шифокорларда қанча вақт давомида даволандингиз? Сил билан оғриган сўровномада иштирок этганлар миқдоридан 61,4% бемор- лар фтизиатрга ўз вақтида мурожаат қилганлар, 14,5% ўзларини соғлом деб ҳисоблаганлар; 15,1% беморлар ўзларида мавжуд бўлган клиник белгилар тезда ўтиб кетади деб ўйлаганлар; 4,8% қон туплаганларидан сўнг шифокорга мурожаат қилганлар ва 3,2% таниш шифокорда даволанганлар. “Касаллик бошлангандан сўнг қанча вақт ўтиб сил ташхиси қўйилган?”, деган саволга қуйидагича жавоблар олинди: – 61,4% бир ой давомида; 20,9%-уч ойдан сўнг; 8,0%-4, 6 ойдан сўнг; 9,6% – бир йлдан сўнг, беморлар асосан силнинг ўпкадан ташқари шакллари билан оғриганлар.(5-расм) “Касаллик бошлангандан сўнг қанча вақт ўтиб сил ташхиси қўйилган?”, деган саволга қуйидагича жавоблар олинди: – 61,4% бир ой давомида; 20,9%-уч ойдан сўнг; 8,0%-4, 6 ойдан сўнг; 9,6% – бир йлдан сўнг, беморлар асосан силнинг ўпкадан ташқари шакллари билан оғриганлар.(5-расм) 565 Жамият ва инновациялар – Общество и инновации – Society and innovations Special Issue – 8 (2021) / ISSN 2181-1415 5-расм. Касаллик бошланишидан қанча вақт ўтиб сил ташхиси қўйилган? “Сизнинг яшаш майдонингиз ободонлаштирилганми?” деган саволга 2,8% “ҳа” ва 97,2% “йўқ” жавоби олинди. Оиланинг бир аъзоси учун 9м2дан кичик бўлган яшаш майдони билан таъминланиши 87% беморларда қайд этилди, бу одамларни жуда зич яшашидан гувоҳлик беради бу эса сил инфекциясини тез 0 10 20 30 40 50 60 70 80 90 100 61,4 20,9 8 9,6 Жамият ва инновациялар – Общество и инновации – Society and innovations Special Issue – 8 (2021) / ISSN 2181-1415 5-расм. Касаллик бошланишидан қанча вақт ўтиб сил ташхиси қўйилган? “Сизнинг яшаш майдонингиз ободонлаштирилганми?” деган саволга 2,8% “ҳа” ва 97,2% “йўқ” жавоби олинди. Оиланинг бир аъзоси учун 9м2дан кичик бўлган яшаш майдони билан таъминланиши 87% беморларда қайд этилди, бу одамларни жуда зич яшашидан гувоҳлик беради, бу эса сил инфекциясини тез тарқалишига сабаб бўлади. “Сизнинг яшаш майдонингиз ободонлаштирилганми?” деган саволга 2,8% “ҳа” ва 97,2% “йўқ” жавоби олинди. Оиланинг бир аъзоси учун 9м2дан кичик бўлган яшаш майдони билан таъминланиши 87% беморларда қайд этилди, бу одамларни жуда зич яшашидан гувоҳлик беради, бу эса сил инфекциясини тез тарқалишига сабаб бўлади. 6-расм. Касалликни давомийлик муддатига боғлиқ холда бемларни тақсимланиши 2,8 97,2 6-расм. Касалликни давомийлик муддатига боғлиқ холда бемларни тақсимланиши Мазкур кўрсаткич ишсизлар орасида юқорилигича қолмоқда ва 49,1%ни, ишчиларда – 19,6%, нафақадагиларда – 14,5%, хизматчиларда – 3,5% ва ишбилар- монларда – 0,3%ни ташкил этади. Сил билан оғриган беморларнинг фақат 10% дагина оиланинг ҳар бир аъзоси учун ажратилган яшаш майдони 10-12 м2ни ташкил этади. Шунингдек битта оиладаги сил билан оғриганлар миқдори ўрганилди. Бунда қуйидаги натижалар олинди: 76,7% – оилада битта одам, 19,3% – оилада иккита одам, 1,8% – оилада учта одам, 0,1% – оилада тўртта одам.(7-расм). Шундай қилиб, битта оилада иккита ва ундан ортиқ кишини сил билан касалланишлари миқдори 22,5% ни ташкил этади, бу эса силни дориларга чидамли шаклларини ўсганлигидан гувоҳлик беради, 70,5% сил билан 1-2 йил давомида касалланганлар; 21,3% 3-4 йил давомида; 8,2% – 5-6 йил давомида касалланганларни ташкил этади. 2,8 97,2 6-расм. Касалликни давомийлик муддатига боғлиқ холда бемларни тақсимланиши Мазкур кўрсаткич ишсизлар орасида юқорилигича қолмоқда ва 49,1%ни, ишчиларда – 19,6%, нафақадагиларда – 14,5%, хизматчиларда – 3,5% ва ишбилар- монларда – 0,3%ни ташкил этади. Сил билан оғриган беморларнинг фақат 10% дагина оиланинг ҳар бир аъзоси учун ажратилган яшаш майдони 10-12 м2ни ташкил этади. Шунингдек битта оиладаги сил билан оғриганлар миқдори ўрганилди. Бунда қуйидаги натижалар олинди: 76,7% – оилада битта одам, 19,3% – оилада иккита одам, 1,8% – оилада учта одам, 0,1% – оилада тўртта одам.(7-расм). Шундай қилиб, битта оилада иккита ва ундан ортиқ кишини сил билан касалланишлари миқдори 22,5% ни ташкил этади, бу эса силни дориларга чидамли шаклларини ўсганлигидан гувоҳлик беради, 70,5% сил билан 1-2 йил давомида касалланганлар; 21,3% 3-4 йил давомида; 8,2% – 5-6 йил давомида касалланганларни ташкил этади. 566 Жамият ва инновациялар – Общество и инновации – Society and innovations Special Issue – 8 (2021) / ISSN 2181-1415 7-расм. Битта оилада сил билан касалланганлар миқдорини ўрганиш. 0 10 20 30 40 50 60 70 80 90 100 76,7 19,3 1,8 Жамият ва инновациялар – Общество и инновации – Society and innovations Special Issue – 8 (2021) / ISSN 2181-1415 7-расм. Битта оилада сил билан касалланганлар миқдорини ўрганиш. Қолган ахолига нисбатан сил билан касалланиш ҳавф юқори бўлган шахслар контингенти маълум. Ҳозирги кунда сил билан оғриганлар – кўпинч наркоманлар, АИЖБлар, қамоқдагилар ёки қамоқ муддатини ўтаганлар, спиртли ичимликларни сурункали истеъмол қилувчилар, ишсизлар, сил билан касалланиш юқори бўлган минтақалардан келган мигрантлар – бу сил бўйича ижтимоий ҳавф гуруҳини ташкил этувчилардир. Бироқ, ахоли ўртасида тиббий ҳавф гуруҳи деб номланувчи саломатлик холати бўйича юқори даражадаги ҳавфга эга бўлган шахслар гуруҳи мавжуд. Мазкур ҳавф гуруҳига ўпканинг сурункали яллиғланиш касалликлари билан оғриган беморларни, қайталанувчи атипик зотилжам, юқори нафас йўлларининг кўп марта қайталанувчи касалликлари билан оғриганлар, экссудатив плевритни бошидан ўтказганлар, ўпканинг касб касалликлари билан оғриганлар, меъда ва ўн икки бармоқ ичакнинг яра касалликлари билан оғриганлар, меъда-ичакларда жарроҳлик амалиётини ўтказганлар, қандли диабет ёки буйрак усти безларининг сурункали етишмаслигидан азият чекувчилар, узоқ вақт давомида гормонал дори воситаси билан даволагганлар, сил билан касаллангандан сўнг даволанган ва ҳисобдан чиқарилган, рентген мусбат шахсларни киритиш лозим. Кўрсатилган гуруҳларни ажратиб олиш ва уларни тизимли равишда текшириб бориш силни ўз вақтида аниқлашнинг бевосита шартларидан бири бўлиб ҳисобланади. Умумий даволаш тармоғи шифокорлари тажоввузкор контингентлар тўғрисида маълу- мотга эга бўлишлари ва поликлиника шароитида уларн ҳар йили мунтазам равишда флюорографик текширувларини ўтказишлари лозим (8-расм). Шунингдек шифокор фтизиатрлар томонидан кўрсатилган силга қарши ёрдам сифати ўрганилди. “Сиз шифокор-фтизиатр ишидан қониқасизми?”, деган саволга асосан силнинг сурункали шакли билан оғриган беморларнинг сўров- номада иштирок этган 92% “ҳа” -8% “йўқ” – деб жавоб берган. Жамият ва инновациялар – Общество и инновации – Society and innovations Special Issue – 8 (2021) / ISSN 2181-1415 Жамият ва инновациялар – Общество и инновации – Society and innovations Special Issue – 8 (2021) / ISSN 2181-1415 88% 90% 92% 94% 96% 98% 100% 92% 8% Шундай қилиб ахолининг турли гуруҳларини ҳаёт сифатига ижтимоий омиллар таъсирини ўрганиш шуни кўрсатдики, силдан азият чекувчи шахслар қарши қўйилган гуруҳдан қуйидагича фарқ қилади: 92,0% – бу ойлик даромади республика бўйича тирикчилик учун энг кам миқдордан деярли ўн марта паст бўлган ахолининг ижтимоий ҳимояланмаган қатлами, ишсизлар, нафақадагилар ва оиланинг битта аъзосини энг паст даромадига эга бўлган ишчилар, маълумотининг паст даражаси (67%), катта сондаги ишсизлар (2 мартадан юқори), истиқомат қилишнинг қониқарсиз турар жой ва маиший, ҳамда санитар шароитлари (4 марта), кўп сондаги одамларнинг зич яшаши (87% беморлар 9м2 дан кам жойда истиқомат қиладилар), битта ишловчи қарамоғида кўп сонли одамларни бўлиши (10 одамдан кўп – 29 марта), оғир маиший шароитга эга бўлганларнинг энг катта улуши, тўлақонли бўлмаган овқатланиш (рационда оқсил ва витаминлар етишмаслиги), сил масалалари бўйича санитар билимларининг пастлигидир. 567 Жамият ва инновациялар – Общество и инновации – Society and innovations Special Issue – 8 (2021) / ISSN 2181-1415 Шундай қилиб ахолининг турли гуруҳларини ҳаёт сифатига ижтимоий миллар таъсирини ўрганиш шуни кўрсатдики, силдан азият чекувчи шахслар арши қўйилган гуруҳдан қуйидагича фарқ қилади: 92,0% – бу ойлик даромади еспублика бўйича тирикчилик учун энг кам миқдордан деярли ўн марта паст ўлган ахолининг ижтимоий ҳимояланмаган қатлами, ишсизлар, нафақадагилар ва иланинг битта аъзосини энг паст даромадига эга бўлган ишчилар, маълумотининг аст даражаси (67%), катта сондаги ишсизлар (2 мартадан юқори), истиқомат илишнинг қониқарсиз турар жой ва маиший, ҳамда санитар шароитлари (4 марта), ўп сондаги одамларнинг зич яшаши (87% беморлар 9м2 дан кам жойда истиқомат иладилар), битта ишловчи қарамоғида кўп сонли одамларни бўлиши (10 одамдан ўп – 29 марта), оғир маиший шароитга эга бўлганларнинг энг катта улуши, ўлақонли бўлмаган овқатланиш (рационда оқсил ва витаминлар етишмаслиги), ил масалалари бўйича санитар билимларининг пастлигидир. 88% 90% 92% 94% 96% 98% 100% 92% 8% 8. 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Туберкулез, выявление, лечение и мониторинг по К. Томену. Второе издание. Т.: 2007. – С. 304–307. 5. Кучма В.Р., Ушаков И.Б., Соколова Н.В. и др. - Методы оценки качества жизни школьников. М.: Издательство ГУ НЦЗД РАМН, 2006. – С. 98. 5. Кучма В.Р., Ушаков И.Б., Соколова Н.В. и др. - Методы оценки качества жизни школьников. М.: Издательство ГУ НЦЗД РАМН, 2006. – С. 98. 6. Король О.И. Туберкулез у детей и возможности его предупреждения // Туберкулез: Проблемы диагностики, лечения и профилактики: Тр. Всерос. науч.- практ. Конф. / Под ред. Ю.Н. Левешева. –СПб., 2003. –С. 100–103. р ф / р 7. Лозовская М.Е. Эффективность использования ламинария у подростков при комплексном лечении туберкулеза легких. // Вопросы питания. – 1,2005. – С. 40–43. 7. Лозовская М.Е. Эффективность использования ламинария у подростков при комплексном лечении туберкулеза легких. // Вопросы питания. – 1,2005. – С. 40–43. 568 Оценка динамики качества жизни детей как критерий эффективности стационарного лечения // Вопросы современной педиатрии. – 2009. – № 6. – C. 7–12. д р 17. Baussano I., Nunn P., Williams B., Pivetta E., Bugiani M., Scano F. Tuberculosis among health care workers. Emerg. Infect. Dis. 2011; 17(3): 488–94. р 17. Baussano I., Nunn P., Williams B., Pivetta E., Bugiani M., Scano F. Tuberculosis among health care workers. Emerg. Infect. Dis. 2011; 17(3): 488–94. 18. Kuchma V.R., Ushakov I.B., Sokolova N.V. – Methods for assessing quality of pupils’ life. Moscow, CA SCCH RAMS Publ., 2006. – P. 98. (In Russian). 18. Kuchma V.R., Ushakov I.B., Sokolova N.V. – Methods for assessing quality of pupils’ life. Moscow, CA SCCH RAMS Publ., 2006. – P. 98. (In Russian). 19. Moiseeva O.V. The quality of teenagers’ life from Udmurt families during their tuberculosis chemoprophylaxis. Voprosy sovremennoi pediatrii. [Questions of modern pediatrics], 2009, no. 6. – PP. 12–13 (In Russian). 19. Moiseeva O.V. The quality of teenagers’ life from Udmurt families during their tuberculosis chemoprophylaxis. Voprosy sovremennoi pediatrii. [Questions of modern pediatrics], 2009, no. 6. – PP. 12–13 (In Russian). 20. Nienhaus A., Schablon A., Preisser A.M., Ringshausen F.C., Diel R. Tuberculosis in healthcare workers – a narrative review from a German perspective. J. Occup. Med. Toxicol. 2014; 9(1): 9. 20. Nienhaus A., Schablon A., Preisser A.M., Ringshausen F.C., Diel R. Tuberculosis in healthcare workers – a narrative review from a German perspective. J. Occup. Med. Toxicol. 2014; 9(1): 9. 21. Parpieva N.N., Belotserkovets V.G., Iakubbekov T.Iu. Medical and social characteristics of patients with co-infection HIV/Tuberculosis. 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https://openalex.org/W4387908485	https://tc.copernicus.org/preprints/tc-2023-102/tc-2023-102.pdf	English	null	Reply on RC1	null	2,023	cc-by	12,106	ERROR: type should be string, got "https://doi.org/10.5194/tc-2023-102\nPreprint. Discussion started: 30 June 2023\nc⃝Author(s) 2023. CC BY 4.0 License. Evaluating different geothermal heat flow maps as basal boundary conditions during\n1\nspin up of the Greenland ice sheet\n2\n3\nTong ZHANG1, William COLGAN2, Agnes WANSING3, Anja LØKKEGAARD2, Gunter LEGUY4\n4\nWilliam LIPSCOMB4, and Cunde XIAO1\n5\n6\n1State Key Laboratory of Earth Surface Processes and Resource Ecology, Beijing Normal\n7\nUniversity, Beijing, CHINA\n8\n2Geological Survey of Denmark and Greenland, DENMARK\n9\n3Kiel University, Kiel, GERMANY\n10\n4National Center for Atmospheric Research, UNITED STATES\n11\n12\nCorresponding author: Tong Zhang, tzhang@bnu.edu.cn\n13\n14\nABSTRACT\n15\nThere is currently poor scientific agreement whether the ice-bed interface is frozen or\n16\nthawed beneath approximately one-third of the Greenland ice sheet. This disagreement in basal\n17\nthermal state results, at least partly, from a diversity of opinion in the subglacial geothermal heat\n18\nflow basal boundary condition employed in different ice-flow models. Here, we employ seven\n19\nGreenland geothermal heat flow maps in widespread use to 10,000-year spin ups of the\n20\nCommunity Ice Sheet Model (CISM). We perform both a fully unconstrained transient spin up,\n21\nas well as a nudged spin up that conforms to Ice Sheet Model Intercomparison Project for\n22\nCMIP6 (ISMIP6) protocol. Across the seven heat flow maps, and regardless of unconstrained or\n23\nnudged spin up, the spread in basal ice temperatures exceeds 10°C over large areas of the ice-\n24\nbed interface. For a given heat flow map, thawed-bedded ice-sheet area is consistently larger\n25\nunder unconstrained spin ups than nudged spin ups. Under the unconstrained spin up, thawed-\n26\nbedded area ranges from 33.5 to 60.0% across the seven heat flow maps. Perhaps\n27\ncounterintuitively, the highest iceberg calving fluxes are associated with the lowest heat flows\n28\n(and vice versa) for both unconstrained and nudged spin ups. This highlights the direct, and\n29\nnon-trivial, influence of choice of heat flow boundary condition on the simulated equilibrium\n30\nthermal state of the ice sheet. We suggest that future ice-flow model intercomparisons should\n31\nemploy a range of basal heat flow maps, and limit direct intercomparisons to simulations\n32\nemploying a common heat flow map. 33\n34 The Cryosphere INTRODUCTION\n35\nThere is presently a tremendous diversity of opinion regarding the geothermal heat flow\n36\nbeneath the Greenland ice sheet due to a paucity of direct measurements of geothermal heat\n37\nflow beneath the ice-sheet interior. While many subaerial, submarine and shallow subglacial\n38\nmeasurements have been made around the ice-sheet periphery, deep subglacial\n39\nmeasurements have only been made at six deep ice coring sites within the ice-sheet interior\n40\n(Camp Century, DYE-3, GRIP, GISP2, NGRIP and NEEM). Consequently, the magnitude and\n41\nspatial distribution of Greenland’s subglacial geothermal heat flow remains poorly constrained\n42\nacross the seven unique Greenland heat flow models presently in widespread use (Figure 1)\n43\n[Shapiro and Ritzwoller, 2004; Rezvanbehbahani et al., 2017; Martos et al., 2018; Greve, 2019;\n44\nLucazeau, 2019; Artemieva, 2019; Colgan et al., 2022]. These individual geothermal heat flow\n45\nmodels are derived from a variety of techniques that interpret a variety of geophysical variables\n46\n(Table 1). We briefly discuss broad differences in the methodology and geophysical input\n47\nvariables of these existing heat flow maps. 48 The Rezvanbehbahani et al. [2017], Lucazeau [2019] and Colgan et al. [2022] heat flow\n49\nmaps are perhaps methodologically most similar. These three maps use machine learning or\n50\ngeostatistics to predict heat flow as a function of diverse geophysical variables such as\n51\ntopography, tectonic age, observed gravity and magnetic field etc. They differ not only in the\n52\napplied method but also in the utilized set of geophysical variables and their domains. Whereas\n53\nRezvanbehbahani et al. [2017] and Lucazeau [2019] only used global data, Colgan et al. [2022]\n54\nsubstituted global datasets with Greenland specific local data. In contrast, the Shapiro and\n55\nRitzwoller [2004], Martos et al. [2018] and Artemieva [2019] heat flow maps all employ\n56\nlithospheric models of varying complexity and more specific geophysical variables to infer heat\n57\nflow. Shapiro and Ritzwoller [2004] correlate the seismic shear wave velocities of the upper 300\n58\nkm with heat flow observations and use this connection to predict heat flow from tomography\n59\ndata in areas without heat flow observations. Martos et al. [2018] use magnetic data to infer the\n60\nCurie temperature depth. Artemieva [2019] assumes an isostatic equilibrium and translates the\n61\ncorresponding topographic residuals to temperature anomalies which are then converted to a\n62\nlithosphere-asthenosphere boundary undulation. ABSTRACT There is currently poor scientific agreement whether the ice-bed interface is frozen or\n16\nthawed beneath approximately one-third of the Greenland ice sheet. This disagreement in basal\n17\nthermal state results, at least partly, from a diversity of opinion in the subglacial geothermal heat\n18\nflow basal boundary condition employed in different ice-flow models. Here, we employ seven\n19\nGreenland geothermal heat flow maps in widespread use to 10,000-year spin ups of the\n20\nCommunity Ice Sheet Model (CISM). We perform both a fully unconstrained transient spin up,\n21\nas well as a nudged spin up that conforms to Ice Sheet Model Intercomparison Project for\n22\nCMIP6 (ISMIP6) protocol. Across the seven heat flow maps, and regardless of unconstrained or\n23\nnudged spin up, the spread in basal ice temperatures exceeds 10°C over large areas of the ice-\n24\nbed interface. For a given heat flow map, thawed-bedded ice-sheet area is consistently larger\n25\nunder unconstrained spin ups than nudged spin ups. Under the unconstrained spin up, thawed-\n26\nbedded area ranges from 33.5 to 60.0% across the seven heat flow maps. Perhaps\n27\ncounterintuitively, the highest iceberg calving fluxes are associated with the lowest heat flows\n28\n(and vice versa) for both unconstrained and nudged spin ups. This highlights the direct, and\n29\nnon-trivial, influence of choice of heat flow boundary condition on the simulated equilibrium\n30\nthermal state of the ice sheet. We suggest that future ice-flow model intercomparisons should\n31\nemploy a range of basal heat flow maps, and limit direct intercomparisons to simulations\n32\nemploying a common heat flow map. 33 34 1 https://doi.org/10.5194/tc-2023-102\nPreprint. Discussion started: 30 June 2023\nc⃝Author(s) 2023. CC BY 4.0 License. The Cryosphere The Cryosphere Of the 21\n87\nparticipating models submissions within ISMIP6, twelve prescribed geothermal heat flow\n88\naccording to Shapiro and Ritzwoller [2004], five prescribed it according to Greve [2019], two\n89\nprescribed it as a hybrid assimilation of four older geothermal heat flow models [Pollack et al.,\n90\n1993; Tarasov and Peltier, 2003; Fox Maule et al., 2009; Rogozhina et al., 2016], and one\n91\nprescribed a spatially uniform geothermal heat flow. 92\nFor Greenland, the ISMIP6 ensemble suggests that ~40% of the ice-sheet bed is frozen,\n93\nmeaning basal ice temperatures below the pressure-melting-point temperature, and ~33% of\n94\nthe ice-sheet bed is thawed, meaning basal ice temperatures at the pressure-melting-point\n95\n[MacGregor et al., 2022]. The ISMIP6 ensemble disagrees on whether the basal thermal state is\n96\nfrozen or thawed beneath the remaining ~28% of the ice sheet. It is unclear what portion of this\n97\ndisagreement is associated with the use of differing geothermal heat flow boundary conditions\n98\nacross ISMIP6 ensemble members. The potential influence of geothermal heat flow boundary\n99\ncondition on basal ice temperature also remains unclear. For example, basal ice that is 1°C\n100\nbelow pressure-melting-point temperature deforms approximately ten times more than ice 10°C\n101\nbelow the pressure-melting-point temperature at the same driving stress [Hooke, 2019]. 102 Despite the clear links between geothermal heat flow and ice dynamics, a standardized\n85\ngeothermal heat flow as the basal thermal boundary condition was not prescribed in the Ice\n86\nSheet Model Intercomparison Project for CMIP6 (ISMIP6) [Goelzer et al., 2020]. Of the 21\n87\nparticipating models submissions within ISMIP6, twelve prescribed geothermal heat flow\n88\naccording to Shapiro and Ritzwoller [2004], five prescribed it according to Greve [2019], two\n89\nprescribed it as a hybrid assimilation of four older geothermal heat flow models [Pollack et al.,\n90\n1993; Tarasov and Peltier, 2003; Fox Maule et al., 2009; Rogozhina et al., 2016], and one\n91\nprescribed a spatially uniform geothermal heat flow. 92 For Greenland, the ISMIP6 ensemble suggests that ~40% of the ice-sheet bed is frozen,\n93\nmeaning basal ice temperatures below the pressure-melting-point temperature, and ~33% of\n94\nthe ice-sheet bed is thawed, meaning basal ice temperatures at the pressure-melting-point\n95\n[MacGregor et al., 2022]. The ISMIP6 ensemble disagrees on whether the basal thermal state is\n96\nfrozen or thawed beneath the remaining ~28% of the ice sheet. The Cryosphere Both latter methods then infer heat flow from\n63\nthe respective isotherms by applying a thermal model. The Greve [2019] heat flow map is rather\n64\nunique in using paleoclimatic forcing of an ice-flow model to infer heat flow with a minimum of\n65\ngeophysical variables. 66\nIn North Greenland, there is especially poor agreement among the present generation of\n67\ngeothermal heat flow models. Some models infer a widespread North Greenland high heat-flow\n68 In North Greenland, there is especially poor agreement among the present generation of\n67\ngeothermal heat flow models. Some models infer a widespread North Greenland high heat-flow\n68 2 https://doi.org/10.5194/tc-2023-102\nPreprint. Discussion started: 30 June 2023\nc⃝Author(s) 2023. CC BY 4.0 License. The Cryosphere The Cryosphere anomaly (e.g. [Greve, 2019]), some do not (e.g. [Lucazeau, 2019]). Other models offer products\n69\nwith and without this high heat-flow anomaly (e.g. [Rezvanbehbahani et al., 2017]). There are\n70\nnumerous secondary disagreements as well, including if a model infers traces of the Iceland\n71\nHotspot Track transiting from West to East Greenland [Martos et al., 2018], or if a model infers\n72\nelevated heat flow in East Greenland in closer proximity to the Mid-Atlantic Ridge [Artemieva,\n73\n2019], or if a model infers a low heat-flow anomaly associated with the North Atlantic Craton in\n74\nSouth Greenland [Colgan et al., 2022]. 75\nGeothermal heat flow comprises a critical basal thermal boundary condition in\n76\nGreenland ice sheet models. It can significantly influence basal ice temperature and rheology,\n77\nwhich in turn influences basal meltwater production and friction [Karlsson et al., 2021]. Given\n78\nthe nonlinear relation between ice temperature and rheology, and that most ice deformation\n79\noccurs in the deepest ice layers, relatively small changes in basal ice temperature can result in\n80\nrelatively large changes in ice velocity [Hooke, 2019]. In extreme cases, diminished geothermal\n81\nheat flow along subglacial ridges may contribute to the formation of massive refrozen basal ice\n82\nmasses [Colgan et al., 2021], or sharply enhanced geothermal heat flow may contribute to the\n83\nonset of major ice-flow features [Smith-Johnsen et al., 2020]. 84\nDespite the clear links between geothermal heat flow and ice dynamics, a standardized\n85\ngeothermal heat flow as the basal thermal boundary condition was not prescribed in the Ice\n86\nSheet Model Intercomparison Project for CMIP6 (ISMIP6) [Goelzer et al., 2020]. The Cryosphere It is unclear what portion of this\n97\ndisagreement is associated with the use of differing geothermal heat flow boundary conditions\n98\nacross ISMIP6 ensemble members. The potential influence of geothermal heat flow boundary\n99\ncondition on basal ice temperature also remains unclear. For example, basal ice that is 1°C\n100\nbelow pressure-melting-point temperature deforms approximately ten times more than ice 10°C\n101\nbelow the pressure-melting-point temperature at the same driving stress [Hooke, 2019]. 102 3 https://doi.org/10.5194/tc-2023-102\nPreprint. Discussion started: 30 June 2023\nc⃝Author(s) 2023. CC BY 4.0 License. The Cryosphere The Cryosphere 4\nIn preparation for ISMIP7, there is a clear motivation to more fully explore the choice of\n103\ngeothermal heat flow boundary condition on modeled basal ice temperatures. Here, we spin up\n104\nan ice-flow model with seven different geothermal heat flow boundary conditions. This allows us\n105\nto isolate the influence of choice of geothermal heat flow boundary condition on simulated\n106\nthermal state and ice flow. We also discuss the pros and cons of these seven Greenland\n107\ngeothermal heat flow products in the specific context of potential utility for ISMIP7 Greenland ice\n108\nflow simulations. 109\n110\nMETHODS\n111\nWe use the Community Ice Sheet Model (CISM) [Lipscomb et al., 2019; Goelzer et al.,\n112\n2020]. These simulations were run on a regular 4 km grid with ten vertical layers, using a\n113\nhigher-order velocity solver with a depth-integrated viscosity approximation based on Goldberg\n114\n[2011]. There is no dependence of basal sliding on basal temperature or water pressure. All\n115\nfloating ice is assumed to calve immediately. For partly grounded cells at the marine margin,\n116\nbasal shear stress is weighted using a grounding-line parameterization. 117\nWe perform two types of ice-sheet spin ups that we denote Case 1 and Case 2. The\n118\nCase 1 spin up iteratively nudges the friction coefficients in the basal-sliding power law to\n119\nminimize misfit against observed present-day ice thickness. In this spin up, we use a classic\n120\nWeertman-type nonlinear basal friction law [Weertman, 1979]:\n121\nـǆ = ƭ Ǚǆ\n1/m−1Ǚǆ\n(1)\n122\nWhere ـǆ is the basal traction, Ǚǆ is the basal velocity, and m is a dimensionless constant that\n123\nwe adopt as 3. C is the friction coefficient, in units of Pa yr m-1, that is nudged during spin-up. The Cryosphere 124\nThe Case 1 spin up directly conforms to ISMIP6 protocol [Goelzer et al., 2020; Nowicki et al.,\n125\n2020]. 126\nIn contrast, the Case 2 spin up is fully transient, meaning that it does not constrain or\n127\nnudge the basal sliding parameters towards observed present-day ice thickness. In this spin up,\n128\nwe use a pseudo-plastic sliding law [Aschwanden et al., 2016]:\n129\nـǆ =−ـǇ\nǙǆ\n\|Ǚǆ\|1−qǙ0\nǕ\n(2)\n130\nwhere ـǇ is the transient yield stress in Pa, q is a dimensionless pseudo-plastic exponent\n131\nthat we adopt as 0.5, and Ǚ0 is a threshold speed that we adopt as 100 m/a. We assume a\n132\nspatially and temporally constant friction coefficient, which allows ice thickness to evolve away\n133\nfrom present-day observations. While the Case 1 spin up ice geometry matches present-day,\n134 In preparation for ISMIP7, there is a clear motivation to more fully explore the choice of\n103\ngeothermal heat flow boundary condition on modeled basal ice temperatures. Here, we spin up\n104\nan ice-flow model with seven different geothermal heat flow boundary conditions. This allows us\n105\nto isolate the influence of choice of geothermal heat flow boundary condition on simulated\n106\nthermal state and ice flow. We also discuss the pros and cons of these seven Greenland\n107\ngeothermal heat flow products in the specific context of potential utility for ISMIP7 Greenland ice\n108\nflow simulations. 109 We use the Community Ice Sheet Model (CISM) [Lipscomb et al., 2019; Goelzer et al.,\n2\n2020]. These simulations were run on a regular 4 km grid with ten vertical layers, using a\n3\nhigher-order velocity solver with a depth-integrated viscosity approximation based on Goldberg\n4\n[2011]. There is no dependence of basal sliding on basal temperature or water pressure. All\n5\nfloating ice is assumed to calve immediately. For partly grounded cells at the marine margin,\n6\nbasal shear stress is weighted using a grounding-line parameterization. 7 We perform two types of ice-sheet spin ups that we denote Case 1 and Case 2. The\n118\nCase 1 spin up iteratively nudges the friction coefficients in the basal-sliding power law to\n119\nminimize misfit against observed present-day ice thickness. In this spin up, we use a classic\n120\nWeertman-type nonlinear basal friction law [Weertman, 1979]:\n121 ـǆ = ƭ Ǚǆ\n1/m−1Ǚǆ\n(1) ǆ\n,\nǆ\ny,\nwe adopt as 3. The Cryosphere 139 Under both Case 1 and 2 spin-ups, the ice sheet was initialized with present-day\n140\nthickness and bed topography [Morlighem et al., 2017] and an idealized vertical englacial\n141\ntemperature profile. The ice sheet was then spun up for 10,000 years under surface mass\n142\nbalance and surface temperature forcing from a 1980–1999 climatology provided by the MAR\n143\nregional climate model [Fettweis et al., 2017]. By the end of spin-up, the ice sheet is assumed to\n144\nhave achieved a transient equilibrium, with transient englacial ice temperatures no longer\n145\ninfluenced by the initial englacial temperature assumption. Here, we use the CISM bed interface\n146\ntemperature field (‘btemp’) to represent the ice-bed temperature. We assume this field is at\n147\ntransient equilibrium following both Case 1 and 2 spin ups (Figure 2). 148 We repeat the Case 1 and Case 2 spin ups seven times each without modification in\n149\ntheir configuration and execution, only substituting the prescribed geothermal heat flow serving\n150\nas the basal boundary condition each time (Table 1) . Each of the seven heat flow maps is re-\n151\ngridded from their native grid to the CISM grid using bilinear interpolation. For heat flow maps\n152\nthat are only available onshore, meaning they omit offshore, or submarine, areas of the CISM\n153\ndomain, we similarly infill fjord heat flow values using bilinear interpolation. 154 These seven maps provide a diverse representation of the magnitude and spatial\n155\ndistribution of Greenland heat flow, with the mean heat flow within the CISM ice-sheet domain\n156\nranging from ~42 mW m-2 in the Colgan et al. [2022] map to ~64 mW m-2 in the Lucazeau [2019]\n157\nmap. For Rezvanbehbahani et al. [2017] we use the middle range scenario of NGRIP = 135 mW\n158\nm-2. For Artemieva [2019], we use the “model 1” scenario, which adopts a deeper continental\n159\nMoho depth than the “model 2”. For Colgan et al. [2022] we use their recommended “without\n160\nNGRIP” scenario. 161 Of the seven heat flow maps that we consider, only two are global maps [Shapiro and\n162\nRitzwoller, 2004; Lucazeau, 2019], the remaining five are Greenland-specific maps. Of these\n163\nfive Greenland-specific maps, all but Colgan et al. [2022] are limited to the onshore domain,\n164\nexcluding the offshore domain (Figure 1; Table 1). The Cryosphere C is the friction coefficient, in units of Pa yr m-1, that is nudged during spin-up. 124\nThe Case 1 spin up directly conforms to ISMIP6 protocol [Goelzer et al., 2020; Nowicki et al.,\n125\n2020]. 126 In contrast, the Case 2 spin up is fully transient, meaning that it does not constrain or\n127\nnudge the basal sliding parameters towards observed present-day ice thickness. In this spin up,\n128\nwe use a pseudo-plastic sliding law [Aschwanden et al., 2016]:\n129 ـǆ =−ـǇ\nǙǆ\n\|Ǚǆ\|1−qǙ0\nǕ\n(2) ـǆ =−ـǇ\nǙǆ\n\|Ǚǆ\|1−qǙ0\nǕ\n(2) ـǆ =−ـǇ\nǙǆ\n\|Ǚǆ\|1−qǙ0\nǕ\n(2) where ـǇ is the transient yield stress in Pa, q is a dimensionless pseudo-plastic exponent\n131 where ـǇ is the transient yield stress in Pa, q is a dimensionless pseudo-plastic exponent\n131 where ـǇ is the transient yield stress in Pa, q is a dimensionless pseudo-plastic exponent\n131\nthat we adopt as 0 5 and Ǚ0 is a threshold speed that we adopt as 100 m/a We assume a\n132 where ـǇ is the transient yield stress in Pa, q is a dimensionless pseudo-plastic exponent\n131\nthat we adopt as 0.5, and Ǚ0 is a threshold speed that we adopt as 100 m/a. We assume a\n132\nspatially and temporally constant friction coefficient, which allows ice thickness to evolve away\n133\nfrom present-day observations. While the Case 1 spin up ice geometry matches present-day,\n134 that we adopt as 0.5, and Ǚ0 is a threshold speed that we adopt as 100 m/a. We assume a\n132\nspatially and temporally constant friction coefficient, which allows ice thickness to evolve away\n133\nfrom present-day observations. While the Case 1 spin up ice geometry matches present-day,\n134 4 https://doi.org/10.5194/tc-2023-102\nPreprint. Discussion started: 30 June 2023\nc⃝Author(s) 2023. CC BY 4.0 License. The Cryosphere The Cryosphere there can be appreciable biases in ice thickness under the non-nudged Case 2 spin up. The\n135\nCase 2 spin up does not conform to ISMIP6 protocol. It is foreseeable, however, that the\n136\nforthcoming ISMIP7 protocol will encourage fully transient spin ups. Transient spin ups are\n137\narguably more physically-based than nudged spin ups, but it is more challenging to reproduce a\n138\nspecific (present-day) ice-sheet configuration with them. The Cryosphere The seven heat flow maps are evaluated\n165\nagainst differing numbers of in-situ heat flow observations within a Greenland domain defined\n166\nas <500 km from Greenlandic shores. The Rezvanbehbahani et al. [2017], Martos et al. [2018]\n167\nand Greve[2019] heat flow maps employed ≤9 primarily subglacial in-situ observations from\n168 5 https://doi.org/10.5194/tc-2023-102\nPreprint. Discussion started: 30 June 2023\nc⃝Author(s) 2023. CC BY 4.0 License. The Cryosphere deep boreholes in the ice-sheet interior. The remaining four maps employed significantly more\n169\nin-situ heat flow observations (≥278), including more subaerial, submarine and shallow\n170\nsubglacial measurements, associated with progressively improving versions of the International\n171\nHeat Flow Database [Jessop et al., 1976; Fuchs et al., 2021]. 172\n173\nRESULTS\n174\nCase 1 spin up\n175\nThe Colgan et al. [2022] heat flow map, which has the lowest mean geothermal heat\n176\nflow of all seven products, yields the smallest area of thawed basal temperatures (21.8%) and\n177\nthe coldest basal temperature anomaly relative to ensemble mean (Figure 3; Table 2). 178\nConversely, the relatively high Martos et al. [2018] heat flow map, which has the third highest\n179\nmean heat flow of all seven products, yields twice the area of thawed basal temperatures\n180\n(54.4%) and one of the warmest basal temperature anomalies relative to ensemble mean. 181\nAcross the seven-member ensemble, however, there is considerable variation in magnitude and\n182\nspatial distribution of ensemble spread in basal ice temperatures (Figure 4). The seven heat\n183\nflow maps yield broadly similar modeled basal ice temperatures RMSEs of between 1.0 and\n184\n2.8 °C in comparison to observed basal ice temperatures at 27 Greenland ice sheet boreholes\n185\n(Figure 5) [Løkkegaard et al., 2022]. 186\nGenerally, ensemble spread in modeled ice-bed temperature approaches zero in the\n187\nablation area, especially in Central West Greenland, where basal thermal state is thawed\n188\nregardless of choice of heat flow map. Ensemble spread is generally largest along the main flow\n189\ndivide of the ice sheet. At South Dome, the ensemble spread exceeds 10°C over an ~105 km2\n190\narea. This highlights that choice of heat flow map has a substantial influence on simulated basal\n191\nthermal state over the North Atlantic Craton. Case 2 spin up\n208 Similar to the Case 1 spin up, the Case 2 spin up also yields the smallest area of thawed\n209\nbasal temperatures (33.5%) with the Colgan et al. [2022] lowest mean geothermal heat flow\n210\nmap and the largest area of thawed basal temperatures (60.0%) with the Martos et al. [2018]\n211\nrelatively high mean geothermal heat flow map (Figure 9). Critically, the thawed-bedded area for\n212\na given heat flow map is consistently larger under the Case 2 (transient) spin up than Case 1\n213\n(nudged) spin up (Table 2). Basal ice temperatures are accordingly warmer under Case 2 spin\n214\nup than Case 1 spin up (Figure 10). As ice-sheet sensitivity generally increases with the\n215\nthawed-bedded area over which basal movement and subglacial hydrology can occur, this\n216\nsuggests that transient ice-sheet spin ups may be regarded as more sensitive than nudged\n217\nones. The apparent ice-temperature warming effect of a transient spin up appears to increase\n218\nwith decreasing heat flow. The shift towards warmer basal temperatures under Case 2 spin up\n219\nis most apparent in the Colgan et al. [2022] lowest mean geothermal heat flow map, where the\n220\ntemperature difference is >5 °C beneath a large portion of Central Greenland. All heat flow\n221\nmaps present large differences in basal ice temperature between Case 1 and Case 2 spin ups\n222\nin regions of fast ice flow around the ice sheet periphery. 223 The spatial pattern of Case 2 ensemble agreement broadly follows that of Case 1,\n224\nalthough the Case 2 agreement is generally poorer. This is attributable to the unconstrained\n225\nnature of the Case 2 spin up. The magnitude and spatial distribution of ensemble spread in\n226\nbasal ice temperatures under Case 2 spin up largely reflects that of Case 1 spin up, the Case 2\n227\nensemble spread is smaller in Central East Greenland, and larger for peripheral ice caps,\n228\nespecially Flade Isblink in Northeast Greenland (Figure 4). The Case 2 spin up reproduces the\n229\nobserved basal ice temperatures at 27 Greenland ice sheet boreholes with an RMSE of\n230\nbetween 1.5 and 2.8 °C (Figure 5) [Løkkegaard et al., 2022]. This is not significantly different\n231\nfrom the RMSE range of the Case 1 spin up. The Cryosphere Accordingly, iceberg calving\n203\nis highest in the lowest heat flow simulations (Figure 8). The relatively narrow ensemble spread\n204\nin iceberg calving (~1%; 2 Gt yr-1 ensemble range against 322 Gt yr-1 ensemble mean) is\n205\nultimately constrained to surface mass balance forcing at transient equilibrium. 206\n207 Lucazeau [2019] and Colgan et al. [2022] maps yield, respectively, low and high ice-velocity end\n201\nmembers. Similarly, within the Rezvanbehbahani et al. [2017] simulation, the low heat-flow\n202\nanomaly in southeast Greenland yields a high ice-velocity anomaly. Accordingly, iceberg calving\n203\nis highest in the lowest heat flow simulations (Figure 8). The relatively narrow ensemble spread\n204\nin iceberg calving (~1%; 2 Gt yr-1 ensemble range against 322 Gt yr-1 ensemble mean) is\n205\nultimately constrained to surface mass balance forcing at transient equilibrium. 206\n207 The Cryosphere While the Northeast Greenland Ice Stream is\n192\nthawed regardless of choice of heat flow map, there is also an ~105 km2 area in Central East\n193\nGreenland where ensemble spread exceeds 10°C. Finally, choice of heat flow map appears to\n194\ninfluence whether the North Greenland ablation area is thawed or frozen. 195\nThe Case 1 spin up nudges the ice-flow model towards present-day ice thickness by\n196\niteratively adjusting basal friction coefficients. The ensemble differences in adjusted basal\n197\nfriction coefficient generally reaches a maximum where ice velocities reach a minimum (Figure\n198\n6). Perhaps counterintuitively, the highest surface ice velocities are associated with the lowest\n199\ngeothermal heat flows (Figure 7) For example the high and low heat flow end members of the\n200 Generally, ensemble spread in modeled ice-bed temperature approaches zero in the\n187\nablation area, especially in Central West Greenland, where basal thermal state is thawed\n188\nregardless of choice of heat flow map. Ensemble spread is generally largest along the main flow\n189\ndivide of the ice sheet. At South Dome, the ensemble spread exceeds 10°C over an ~105 km2\n190\narea. This highlights that choice of heat flow map has a substantial influence on simulated basal\n191\nthermal state over the North Atlantic Craton. While the Northeast Greenland Ice Stream is\n192\nthawed regardless of choice of heat flow map, there is also an ~105 km2 area in Central East\n193\nGreenland where ensemble spread exceeds 10°C. Finally, choice of heat flow map appears to\n194\ninfluence whether the North Greenland ablation area is thawed or frozen. 195 The Case 1 spin up nudges the ice-flow model towards present-day ice thickness by\n196\niteratively adjusting basal friction coefficients. The ensemble differences in adjusted basal\n197\nfriction coefficient generally reaches a maximum where ice velocities reach a minimum (Figure\n198\n6). Perhaps counterintuitively, the highest surface ice velocities are associated with the lowest\n199\ngeothermal heat flows (Figure 7). For example, the high and low heat flow end members of the\n200 6 https://doi.org/10.5194/tc-2023-102\nPreprint. Discussion started: 30 June 2023\nc⃝Author(s) 2023. CC BY 4.0 License. The Cryosphere Lucazeau [2019] and Colgan et al. [2022] maps yield, respectively, low and high ice-velocity end\n201\nmembers. Similarly, within the Rezvanbehbahani et al. [2017] simulation, the low heat-flow\n202\nanomaly in southeast Greenland yields a high ice-velocity anomaly. Case 2 spin up\n208 [2022] heat flow maps that\n250\nyield substantially thicker ice in North Greenland also yield lower ice temperatures there. 251\nSimilarly, the Greve [2019] and Lucazeau [2019] heat flow maps that yield substantially thinner\n252\nice in North Greenland also yield faster velocities there. While relative velocity differences in the\n253\nice-sheet interior can appear striking in both magnitude and extent, there are also velocity\n254\ndifferences around the ice-sheet periphery, which strongly influences the iceberg calving from\n255\ntidewater glaciers. Iceberg calving under Case 2 (transient) spin up has a greater ensemble\n256\nspread (~5%; 18 Gt yr-1 ensemble range against 365 Gt yr-1 ensemble mean) than under Case 1\n257\n(nudged) spin up (Figure 8). Similar to the Case 1 spin up, however, the Colgan et al. [2022]\n258\nlowest heat flow map again has the highest iceberg calving flux, while the relatively high Martos\n259\net al. [2018] and Greve [2019] heat flow maps have substantially lower iceberg calving fluxes at\n260\nequilibrium. 261\n262\nDISCUSSION\n263\nThe apparent association of higher ice velocities with lower geothermal heat flows under\n264\nCase 1 spin up outwardly appears to be a clear artifact of nudging the basal friction coefficient\n265\nduring spin up. This effect has previously been described as the surface velocity paradox,\n266\nwhereby constraining an ice flow model to match observed ice thickness results in\n267\nunderestimating deformational velocities where basal sliding is present, and overestimating\n268 Empirical temperature observations therefore justify neither the Case 1 nor Case 2 spin up\n235\napproach. 236\nIn comparison to the Case 1 spin ups, the Case 2 spin ups generally result in thicker ice\n237\nin East Greenland and thinner ice in West Greenland (Figure 11). These substantial differences\n238\nin ice thickness (i.e. ±100 m) are clearly attributable to the fully transient nature of Case 2 spin\n239\nups in comparison to the nudging of Case 1 spin ups towards observed present-day ice\n240\ngeometry. Specific Case 2 spin ups can yield very different ice thicknesses. For example, the\n241\nShapiro and Ritzwoller [2004] and Colgan et al. [2022] heat flow maps yield substantially thicker\n242\nthan observed ice in North Greenland, while the Greve [2019] and Lucazeau [2019] heat flow\n243\nmaps yield substantially thinner than observed ice in North Greenland. Case 2 spin up\n208 Basal ice temperatures are better resolved by\n232\nCase 1 spin up for three heat flow maps, and better resolved by Case 2 spin up for two heat\n233\nflow maps, with the remaining two heat flow maps yielding the same RMSE under both spin ups. 234 7 https://doi.org/10.5194/tc-2023-102\nPreprint. Discussion started: 30 June 2023\nc⃝Author(s) 2023. CC BY 4.0 License. The Cryosphere Empirical temperature observations therefore justify neither the Case 1 nor Case 2 spin up\n235\napproach. 236\nIn comparison to the Case 1 spin ups, the Case 2 spin ups generally result in thicker ice\n237\nin East Greenland and thinner ice in West Greenland (Figure 11). These substantial differences\n238\nin ice thickness (i.e. ±100 m) are clearly attributable to the fully transient nature of Case 2 spin\n239\nups in comparison to the nudging of Case 1 spin ups towards observed present-day ice\n240\ngeometry. Specific Case 2 spin ups can yield very different ice thicknesses. For example, the\n241\nShapiro and Ritzwoller [2004] and Colgan et al. [2022] heat flow maps yield substantially thicker\n242\nthan observed ice in North Greenland, while the Greve [2019] and Lucazeau [2019] heat flow\n243\nmaps yield substantially thinner than observed ice in North Greenland. Similarly, the ice\n244\nthickness at South Dome varies considerably across the seven heat flow map simulations. The\n245\nmagnitude of ice thickness differences associated with heat flow maps is non-trivial, and the\n246\nspatial distribution is complex. 247\nThere are considerable velocity differences across the seven Case 2 spin up simulations. 248\nGenerally, these velocity differences are negatively correlated with the ice thickness differences. 249\nFor example, the Shapiro and Ritzwoller [2004] and Colgan et al. [2022] heat flow maps that\n250\nyield substantially thicker ice in North Greenland also yield lower ice temperatures there. 251\nSimilarly, the Greve [2019] and Lucazeau [2019] heat flow maps that yield substantially thinner\n252\nice in North Greenland also yield faster velocities there. While relative velocity differences in the\n253\nice-sheet interior can appear striking in both magnitude and extent, there are also velocity\n254\ndifferences around the ice-sheet periphery, which strongly influences the iceberg calving from\n255\ntidewater glaciers. Case 2 spin up\n208 Similarly, the ice\n244\nthickness at South Dome varies considerably across the seven heat flow map simulations. The\n245\nmagnitude of ice thickness differences associated with heat flow maps is non-trivial, and the\n246\nspatial distribution is complex. 247 Empirical temperature observations therefore justify neither the Case 1 nor Case 2 spin up\n235\napproach\n236 There are considerable velocity differences across the seven Case 2 spin up simulations. 248\nGenerally, these velocity differences are negatively correlated with the ice thickness differences. 249\nFor example, the Shapiro and Ritzwoller [2004] and Colgan et al. [2022] heat flow maps that\n250\nyield substantially thicker ice in North Greenland also yield lower ice temperatures there. 251\nSimilarly, the Greve [2019] and Lucazeau [2019] heat flow maps that yield substantially thinner\n252\nice in North Greenland also yield faster velocities there. While relative velocity differences in the\n253\nice-sheet interior can appear striking in both magnitude and extent, there are also velocity\n254 There are considerable velocity differences across the seven Case 2 spin up simulations. 248\nGenerally, these velocity differences are negatively correlated with the ice thickness differences. 249\nFor example, the Shapiro and Ritzwoller [2004] and Colgan et al. [2022] heat flow maps that\n250\nyield substantially thicker ice in North Greenland also yield lower ice temperatures there. 251 Case 2 spin up\n208 Iceberg calving under Case 2 (transient) spin up has a greater ensemble\n256\nspread (~5%; 18 Gt yr-1 ensemble range against 365 Gt yr-1 ensemble mean) than under Case 1\n257\n(nudged) spin up (Figure 8). Similar to the Case 1 spin up, however, the Colgan et al. [2022]\n258\nlowest heat flow map again has the highest iceberg calving flux, while the relatively high Martos\n259\net al. [2018] and Greve [2019] heat flow maps have substantially lower iceberg calving fluxes at\n260\nequilibrium. 261\n262\nDISCUSSION\n263\nThe apparent association of higher ice velocities with lower geothermal heat flows under\n264\nCase 1 spin up outwardly appears to be a clear artifact of nudging the basal friction coefficient\n265\nduring spin up. This effect has previously been described as the surface velocity paradox,\n266\nwhereby constraining an ice flow model to match observed ice thickness results in\n267\nunderestimating deformational velocities where basal sliding is present, and overestimating\n268 Empirical temperature observations therefore justify neither the Case 1 nor Case 2 spin up\n235\napproach. 236\nIn comparison to the Case 1 spin ups, the Case 2 spin ups generally result in thicker ice\n237\nin East Greenland and thinner ice in West Greenland (Figure 11). These substantial differences\n238\nin ice thickness (i.e. ±100 m) are clearly attributable to the fully transient nature of Case 2 spin\n239\nups in comparison to the nudging of Case 1 spin ups towards observed present-day ice\n240\ngeometry. Specific Case 2 spin ups can yield very different ice thicknesses. For example, the\n241\nShapiro and Ritzwoller [2004] and Colgan et al. [2022] heat flow maps yield substantially thicker\n242\nthan observed ice in North Greenland, while the Greve [2019] and Lucazeau [2019] heat flow\n243\nmaps yield substantially thinner than observed ice in North Greenland. Similarly, the ice\n244\nthickness at South Dome varies considerably across the seven heat flow map simulations. The\n245\nmagnitude of ice thickness differences associated with heat flow maps is non-trivial, and the\n246\nspatial distribution is complex. 247\nThere are considerable velocity differences across the seven Case 2 spin up simulations. 248\nGenerally, these velocity differences are negatively correlated with the ice thickness differences. 249\nFor example, the Shapiro and Ritzwoller [2004] and Colgan et al. DISCUSSION\n263 8 https://doi.org/10.5194/tc-2023-102\nPreprint. Discussion started: 30 June 2023\nc⃝Author(s) 2023. CC BY 4.0 License. The Cryosphere deformational velocities where basal sliding is absent [Ryser et al., 2014]. Avoiding this surface\n269\nvelocity paradox is the main motivation for undertaking the Case 2 spin up, in which basal\n270\nfriction coefficients are not nudged. Under Case 2 spin up, during which ice thicknesses are not\n271\nconstrained, there is clearly more variation in the geometry, velocity and thermal state of the ice\n272\nsheet at the end of the 10,000-year fully transient spin up. Perhaps counterintuitively, however,\n273\nthe highest iceberg calving fluxes remain associated with the lowest heat flow maps (and vice\n274\nversa for lowest iceberg calving fluxes). In fully transient Case 2 simulations, this behavior\n275\ncannot be attributed to a model artifact from the surface velocity paradox associated with\n276\nnudging in Case 1 spin up. We instead speculate that a substantial portion of this variability\n277\nsimply reflects increased ice thicknesses under decreased heat flow. 278 The potential influence of anomalously high geothermal heat flow on contemporary local\n279\nice-sheet form and flow has been previously highlighted, with suggestions including: the onset\n280\nof the Northeast Greenland ice stream may be associated with elevated geothermal heat flow\n281\n[Fahnestock et al., 2001]; there may be a feedback between deeply-incised glaciers and\n282\ntopographic enhancement of local geothermal heat flow [van der Veen et al., 2007]; and that the\n283\ntransit of the Iceland hotspot may have deposited anomalous heat into the subglacial\n284\nlithosphere that influences ice flow today [Alley et al., 2019]. Our evaluation suggests\n285\nknowledge of where anomalously low geothermal heat flow may be influencing contemporary\n286\nregional ice-sheet form and flow can help constrain choice of heat flow map. For example, the\n287\nwidespread presence of Last Glacial Period ice in the ablation area across North Greenland\n288\nsuggests that heat flow must be sufficiently low to prevent basal melt across the region\n289\n[MacGregor et al., 2020]. This broad condition is only characteristic of a minority of the heat flow\n290\nmaps we evaluate, specifically the Shapiro and Ritzwoller [2004], Rezvanbehbahani et al. [2017]\n291\nand Colgan et al. [2022] maps. 292 South Dome appears to be the most sensitive portion of the ice sheet to choice of\n293\ngeothermal heat flow basal boundary condition. DISCUSSION\n263 Here, we evaluate simulated basal temperature against observed\n322\nbasal temperature at 27 selected Greenland boreholes. This evaluation appears to provide\n323\nsome insight on which heat flow map or spin up approach is most locally suitable. Rather than\n324\nquantitative comparisons against point temperature observations, however, there seems to be\n325\nvalue in qualitative comparisons between heat flow map and large-scale ice sheet features,\n326\nsuch as evaluating which heat flow map can yield widespread frozen-bedded in North\n327\nGreenland under contemporary conditions. Naturally, evaluation of these seven heat flow maps\n328\nwould be strengthened by using more than a single community ice flow model, as we do here. 329\nWithin our simulation ensemble, the unconstrained spin ups may generally be regarded\n330\nas simulating more sensitive ice sheets than the nudged spin ups, as the unconstrained spin\n331\nups yield greater thawed-bedded area and higher iceberg calving flux. While most recent ice-\n332\nsheet simulations projecting Greenland's future sea-level contribution have largely focused on\n333\nnudged spin ups, our simulation ensemble unsurprisingly suggests that unconstrained transient\n334\nspin up is required to fully resolve the choice of geothermal heat flow boundary condition on ice-\n335\nsheet geometry and velocity. Given the strong influence of choice of geothermal heat flow on ice\n336 beneath South Dome characteristic of the Rezvanbehbahani et al. [2017] map are more likely to\n303\nyield an Eemian-persistent South Dome than paleo-ice-sheet simulations that adopt the high\n304\nheat flow beneath South Dome characteristic of the Lucazeau [2019] map. Simply put, choice of\n305\nheat flow map influences not only contemporary simulations of ice-sheet form and flow, but also\n306\npaleo-ice-sheet simulations as well. 307 Given the non-linear dependence of deformational velocity on ice temperature, properly\n310\nresolving the thermal state of the Greenland ice sheet is critical for generating reliable ice-flow\n311\nsimulations. We have performed both nudged and unconstrained, transient ice-sheet spin ups of\n312\n10,000 years in duration employing seven geothermal heat flow models. Under a nudged spin\n313\nup, we find that the thawed-bedded ice-sheet area ranges from 21.8 to 54.4% across these heat\n314\nflow models. Under a fully unconstrained, transient spin up, the thawed-bedded ice-sheet area\n315\nis consistently larger, ranging from 33.5 to 60.0%. The transient spin up also yields inter-\n316\nsimulation differences in both ice thickness and velocity that are large in magnitude and extent. DISCUSSION\n263 There, choice of heat flow map results in an\n294\nensemble spread in ice-bed temperature of >10°C over an area the size of Iceland. There is\n295\ncurrently a poor level of scientific understanding whether South Dome persisted through the\n296\nEemian interglacial, with some ice-sheet reconstructions suggesting persistence of the ice\n297\nsheet’s southern lobe [Quiquet et al., 2013; Stone et al., 2013] and others suggesting local\n298\ndeglaciation [Otto-Bliesner et al., 2006; Helsen et al., 2013]. Our evaluation specifically\n299\nhighlights substantial disagreement over geothermal heat flow within the North Atlantic Craton\n300\nthat underlies South Dome. Similar to the contemporary persistence of Last Glacial Period ice in\n301\nNorth Greenland, we speculate that paleo-ice-sheet simulations that adopt the low heat flow\n302 9 https://doi.org/10.5194/tc-2023-102\nPreprint. Discussion started: 30 June 2023\nc⃝Author(s) 2023. CC BY 4.0 License. The Cryosphere beneath South Dome characteristic of the Rezvanbehbahani et al. [2017] map are more likely to\n303\nyield an Eemian-persistent South Dome than paleo-ice-sheet simulations that adopt the high\n304\nheat flow beneath South Dome characteristic of the Lucazeau [2019] map. Simply put, choice of\n305\nheat flow map influences not only contemporary simulations of ice-sheet form and flow, but also\n306\npaleo-ice-sheet simulations as well. 307\n308\nSUMMARY REMARKS\n309\nGiven the non-linear dependence of deformational velocity on ice temperature, properly\n310\nresolving the thermal state of the Greenland ice sheet is critical for generating reliable ice-flow\n311\nsimulations. We have performed both nudged and unconstrained, transient ice-sheet spin ups of\n312\n10,000 years in duration employing seven geothermal heat flow models. Under a nudged spin\n313\nup, we find that the thawed-bedded ice-sheet area ranges from 21.8 to 54.4% across these heat\n314\nflow models. Under a fully unconstrained, transient spin up, the thawed-bedded ice-sheet area\n315\nis consistently larger, ranging from 33.5 to 60.0%. The transient spin up also yields inter-\n316\nsimulation differences in both ice thickness and velocity that are large in magnitude and extent. 317\nThis ensemble of simulations highlights that sector-scale ice flow, both peripheral and interior,\n318\ncan be described as at least moderately sensitive to choice of heat flow. 319\nThe recent effort to compile all Greenland englacial temperature observations into a\n320\nstandardized database now permits the thermal state of ice-sheet simulations to be evaluated\n321\nagainst all empirical data. DISCUSSION\n263 317\nThis ensemble of simulations highlights that sector-scale ice flow, both peripheral and interior,\n318\ncan be described as at least moderately sensitive to choice of heat flow. 319 10 https://doi.org/10.5194/tc-2023-102\nPreprint. Discussion started: 30 June 2023\nc⃝Author(s) 2023. CC BY 4.0 License. The Cryosphere The Cryosphere dynamics that we document, it seems prudent to limit the direct intercomparison of ice-sheet\n337\nsimulations to those using a common heat flow map. Similar to employing a range of commonly\n338\nprescribed climate forcing scenarios, it would be ideal for future ISMIP ensembles to employ a\n339\nrange of commonly prescribed basal forcing conditions. 340\n341\nACKNOWLEDGEMENTS\n342\nT.Z. and C.X. thank the Natural Science Foundation of China grant (42271133), Faculty of\n343\nGeographical Sciences, Beijing Normal University (2022-GJTD-01) and the State Key\n344\nLaboratory of Earth Surface Processes and Resource Ecology (2022-ZD-05) for financial\n345\nsupport. A.L. and W.C. thank the Independent Research Fund Denmark (Sapere Aude 8049-\n346\n00003) and the Novo Nordisk Foundation (Center for Sea-Level and Ice-Sheet Prediction) for\n347\nfinancial support. A.W. thanks the European Space Agency and the German Research Council\n348\n(DFG) for their financial support through the projects 4D-Greenland and GreenCrust. G.L. and\n349\nW.L. were supported by the National Center for Atmospheric Research, which is a major facility\n350\nsponsored by the National Science Foundation under Cooperative Agreement no. 1852977. 351\nComputing and data storage resources for CISM simulations, including the Cheyenne\n352\nsupercomputer (https://doi.org/10.5065/D6RX99HX), were provided by the Computational and\n353\nInformation Systems Laboratory (CISL) at NCAR. 354\n355\nDATA AVAILABILITY\n356\nTo help accelerate community efforts towards exploring the influence of geothermal heat flow on\n357\nice-sheet simulations, we have deposited a copy of the seven geothermal heat flow maps that\n358\nwe evaluate here at Zenodo (https://doi.org/10.5281/zenodo.7891577). Interpolated versions of\n359\nthese seven geothermal heat flow datasets are provided on a common coarse-resolution .nc\n360\ngrid that conforms with CISM standards. 361\n362\nAUTHOR CONTRIBUTIONS\n363\nT.Z. and W.C. conceptualized this study and were responsible for formal analysis. A.L. and A.W. 364\nprovided data curation. T.Z., C.X., W.L. and G.L. provided funding, resources, and software. All\n365\nauthors participated in interpretation of the data and writing of the manuscript. 366\n367\nCOMPETING INTERESTS\n368\nThe contact author has declared that none of the authors has any competing interests. REFERENCES\n371 Artemieva, I. Lithosphere structure in Europe from thermal isostasy. Earth-Science Reviews,\n372\n188, 454–468, https://doi.org/10.1016/j.earscirev.2018.11.004, 2019. 373 Alley, R., D. Pollard, B. Parizek, S. Anandakrishnan, M. Pourpoint, N. Stevens, J. MacGregor,\n374\nK. Christianson, A. Muto and N. Holschuh. 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Similar to employing a range of commonly\n338\nprescribed climate forcing scenarios, it would be ideal for future ISMIP ensembles to employ a\n339\nrange of commonly prescribed basal forcing conditions. 340 dynamics that we document, it seems prudent to limit the direct intercomparison of ice-sheet\n337\nsimulations to those using a common heat flow map. Similar to employing a range of commonly\n338\nprescribed climate forcing scenarios, it would be ideal for future ISMIP ensembles to employ a\n339\nrange of commonly prescribed basal forcing conditions. 340 T.Z. and C.X. thank the Natural Science Foundation of China grant (42271133), Faculty of\n343\nGeographical Sciences, Beijing Normal University (2022-GJTD-01) and the State Key\n344\nLaboratory of Earth Surface Processes and Resource Ecology (2022-ZD-05) for financial\n345\nsupport. 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The Cryosphere The Cryosphere TABLES\n518\n519\nTable 1 - Characteristics of the seven geothermal heat flow models we explore as basal thermal\n520\nboundary conditions: methodology used to derive each model, number of geophysical datasets\n521\nemployed by each model, number of in-situ heat flow observations considered by each model,\n522\naverage heat flow (± standard deviation) within a common CISM Greenland ice sheet area, and\n523\nthe domain coverage of each model. Adopted from Colgan et al. [2022] and arranged from\n524\nlowest to highest average geothermal heat flow beneath the ice sheet. The Cryosphere 525\n526\nModel\nMethodology\nGeophysical\ndatasets\n[unitless]\nGreenland\nobservations\n[unitless]\nGeothermal\nheat flow\n[mW m-2]\nDomain\ncoverage\nColgan et al. [2022]\nMachine\nlearning model\n12\n419\n41.8 ± 5.3\nGreenland;\noceanic and\ncontinental\nRezvanbehba\nhani et al. [2017]\nMachine\nlearning model\n20\n9\n54.1 ± 20.4\nGreenland;\ncontinental\nonly\nShapiro and\nRitzwoller\n[2004]\nSeismic\nsimilarity\nmodel\n4\n278\n55.7 ± 9.4\nGlobal;\noceanic and\ncontinental\nArtemieva\n[2019]\nThermal\nisostasy\nmodel\n8\n290\n56.4 ± 12.6\nGreenland;\ncontinental\nonly\nMartos et al. [2018]\nForward\nlithospheric\nmodel\n5\n8\n60.1 ± 6.6\nGreenland;\ncontinental\nonly\nGreve [2019]\nPaleoclimate\nand ice flow\nmodel\n3\n8\n63.3 ± 19.1\nGreenland;\ncontinental\nonly\nLucazeau\n[2019]\nGeostatistical\nmodel\n14\n314\n63.8 ± 7.1\nGlobal;\noceanic and\ncontinental\n527\n528 526\nModel\nMethodology\nGeophysical\ndatasets\n[unitless]\nGreenland\nobservations\n[unitless]\nGeothermal\nheat flow\n[mW m-2]\nDomain\ncoverage\nColgan et al. [2022]\nMachine\nlearning model\n12\n419\n41.8 ± 5.3\nGreenland;\noceanic and\ncontinental\nRezvanbehba\nhani et al. [2017]\nMachine\nlearning model\n20\n9\n54.1 ± 20.4\nGreenland;\ncontinental\nonly\nShapiro and\nRitzwoller\n[2004]\nSeismic\nsimilarity\nmodel\n4\n278\n55.7 ± 9.4\nGlobal;\noceanic and\ncontinental\nArtemieva\n[2019]\nThermal\nisostasy\nmodel\n8\n290\n56.4 ± 12.6\nGreenland;\ncontinental\nonly\nMartos et al. [2018]\nForward\nlithospheric\nmodel\n5\n8\n60.1 ± 6.6\nGreenland;\ncontinental\nonly\nGreve [2019]\nPaleoclimate\nand ice flow\nmodel\n3\n8\n63.3 ± 19.1\nGreenland;\ncontinental\nonly\nLucazeau\n[2019]\nGeostatistical\nmodel\n14\n314\n63.8 ± 7.1\nGlobal;\noceanic and\ncontinental\n527 527\n528 16 https://doi.org/10.5194/tc-2023-102\nPreprint. Discussion started: 30 June 2023\nc⃝Author(s) 2023. CC BY 4.0 License. 529\nTable 2 - Thawed-bedded ice-sheet area associated with Case 1 (nudged) and\n530\n(unconstrained) spin-ups of 10,000-years duration for the seven geothermal hea\n531\n532\nModel\nCase 1\nCase 2\nColgan et al. [2022]\n21.8%\n33.5%\nRezvanbehbahani et al. [2017]\n43.0%\n48.0%\nShapiro and Ritzwoller [2004]\n35.5%\n44.3%\nArtemieva [2019]\n50.2%\n52.8%\nMartos et al. [2018]\n54.4%\n60.0%\nGreve [2019]\n53.6%\n57.4%\nLucazeau [2019]\n52.5%\n59.7%\n533 529\nTable 2 - Thawed-bedded ice-sheet area associated with Case 1 (nudged) and Case 2\n530\n(unconstrained) spin-ups of 10,000-years duration for the seven geothermal heat flow da\n531 533 17 17 https://doi.org/10.5194/tc-2023-102\nPreprint. Discussion started: 30 June 2023\nc⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/tc-2023-102\nPreprint. Discussion started: 30 June 2023\nc⃝Author(s) 2023. CC BY 4.0 License. The Cryosphere The Cryosphere 534\n535 FIGURES FIGURES\n534\n535\n536\nFigure 1 - (a-g): The seven geothermal heat flow maps considered as basal thermal boundary\n537\nconditions, expressed as anomalies from their ensemble mean. Colorbars saturate about 10\n538\nand 100 mW m-2. (i): Ensemble mean. Units for all plots mW m-2. 539 Figure 1 - (a-g): The seven geothermal heat flow maps considered as basal thermal boundary\n537\nconditions, expressed as anomalies from their ensemble mean. Colorbars saturate about 10\n538\nand 100 mW m-2. (i): Ensemble mean. Units for all plots mW m-2. 539 18 https://doi.org/10.5194/tc-2023-102\nPreprint. Discussion started: 30 June 2023\nc⃝Author(s) 2023. CC BY 4.0 License. The Cryosphere\n540\nFigure 2 - Case 1: (a-g) Ice-bed temperature relative to pressure melting point at transient\n541\nequilibrium using the seven geothermal heat flow maps. (i) Ensemble mean ice-bed\n542\ntemperature. Units in all plots °C below pressure-melting-point temperature. (Compare against\n543\nCase 2 in Figure 9.)\n544\n545 The Cryosphere 540\nFigure 2 - Case 1: (a-g) Ice-bed temperature relative to pressure melting point at transient\n541\nequilibrium using the seven geothermal heat flow maps. (i) Ensemble mean ice-bed\n542\ntemperature. Units in all plots °C below pressure-melting-point temperature. (Compare agains\n543\nCase 2 in Figure 9.)\n544 Figure 2 - Case 1: (a-g) Ice-bed temperature relative to pressure melting point at transient\n541\nequilibrium using the seven geothermal heat flow maps. (i) Ensemble mean ice-bed\n542\ntemperature. Units in all plots °C below pressure-melting-point temperature. (Compare against\n543\nCase 2 in Figure 9.)\n544 19 19 https://doi.org/10.5194/tc-2023-102\nPreprint. Discussion started: 30 June 2023\nc⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/tc-2023-102\nPreprint. Discussion started: 30 June 2023\nc⃝Author(s) 2023. CC BY 4.0 License. The Cryosphere 546\nFigure 3 - Case 1: (a-g) Relative anomaly from ensemble mean in ice-bed temperature at\n547\ntransient equilibrium using the seven geothermal heat flow maps. (i) Ensemble mean ice-bed\n548\ntemperature. Units in all plots °C below pressure-melting-point temperature. (Compare against\n549\nCase 2 in Figure 10.)\n550\n551 5 6\nFigure 3 - Case 1: (a-g) Relative anomaly from ensemble mean in ice-bed temperature at\n547\ntransient equilibrium using the seven geothermal heat flow maps. (i) Ensemble mean ice-bed\n548\ntemperature. Units in all plots °C below pressure-melting-point temperature. (Compare against\n549\nCase 2 in Figure 10.)\n550 20 https://doi.org/10.5194/tc-2023-102\nPreprint. Discussion started: 30 June 2023\nc⃝Author(s) 2023. The Cryosphere CC BY 4.0 License. The Cryosphere 552\n553\nFigure 4 - (a) and (b): Ensemble agreement in basal thermal state (frozen or thawed) across\n554\nthe seven heat flow maps (a: Case 1, b: Case 2). Units are the fraction of simulations that\n555\nsuggest thawed bed. (c) and (d): Ensemble spread (the difference between maximum and\n556\nminimum values for different experiments) in basal ice temperature across the seven heat flow\n557\nmaps (c: Case 1, d: Case 2). Units are °C. 558\n559 552 52 553\nFigure 4 - (a) and (b): Ensemble agreement in basal thermal state (frozen or thawed) across\n554\nthe seven heat flow maps (a: Case 1, b: Case 2). Units are the fraction of simulations that\n555\nsuggest thawed bed. (c) and (d): Ensemble spread (the difference between maximum and\n556\nminimum values for different experiments) in basal ice temperature across the seven heat flow\n557\nmaps (c: Case 1, d: Case 2). Units are °C. 558\n559 21 The Cryosphere\n560\nFigure 5 - Modeled ice-bed temperature across the seven heat flow maps versus observed ice-\n561\nbed temperature at 27 Greenland ice sheet boreholes where ice temperatures have been\n562\nobserved. (a-g) Modeled versus observed comparison across the seven geothermal heat flow\n563\nmaps. Case 1 spin ups shown in blue. Case 2 spin ups shown in red. 564\n565 The Cryosphere 560\nFigure 5 - Modeled ice-bed temperature across the seven heat flow maps versus observed ice-\n561\nbed temperature at 27 Greenland ice sheet boreholes where ice temperatures have been\n562\nobserved. (a-g) Modeled versus observed comparison across the seven geothermal heat flow\n563\nmaps. Case 1 spin ups shown in blue. Case 2 spin ups shown in red. 564\n565 22 https://doi.org/10.5194/tc-2023-102\nPreprint. Discussion started: 30 June 2023\nc⃝Author(s) 2023. CC BY 4.0 License. The Cryosphere The Cryosphere\n566\nFigure 6 - Case 1: (a-g) The basal friction coefficient at transient equilibrium using the seven\n567\ngeothermal heat flow maps, expressed as anomalies from the ensemble mean. Units are % and\n568\ncolorbars saturate at ±100%. (i) Ensemble mean basal friction coefficient at transient equilibrium. 569\nUnits are Pa yr m-1, with the colorbar saturating at 106 Pa yr m-1. The Cryosphere 570\n571 566\nFigure 6 - Case 1: (a-g) The basal friction coefficient at transient equilibrium using the seven\n567\ngeothermal heat flow maps, expressed as anomalies from the ensemble mean. Units are % and\n568\ncolorbars saturate at ±100%. (i) Ensemble mean basal friction coefficient at transient equilibrium. 569\nUnits are Pa yr m-1, with the colorbar saturating at 106 Pa yr m-1. 570\n571 23 https://doi.org/10.5194/tc-2023-102\nPreprint. Discussion started: 30 June 2023\nc⃝Author(s) 2023. CC BY 4.0 License. The Cryosphere\n572\nFigure 7 - Case 2: (a-g) Surface ice velocity at transient equilibrium using the seven geothermal\n573\nheat flow maps, expressed as anomalies from their ensemble mean. Units are % and colorbars\n574\nsaturate at ±100%. (i) Ensemble mean surface ice velocity at transient equilibrium. Units are m\n575\nyr-1. 576\n577 The Cryosphere 572\nFigure 7 - Case 2: (a-g) Surface ice velocity at transient equilibrium using the seven geothermal\n573\nheat flow maps, expressed as anomalies from their ensemble mean. Units are % and colorbars\n574\nsaturate at ±100%. (i) Ensemble mean surface ice velocity at transient equilibrium. Units are m\n575\nyr-1. 576\n577 Figure 7 - Case 2: (a-g) Surface ice velocity at transient equilibrium using the seven geothermal\n573\nheat flow maps, expressed as anomalies from their ensemble mean. Units are % and colorbars\n574\nsaturate at ±100%. (i) Ensemble mean surface ice velocity at transient equilibrium. Units are m\n575\nyr-1. 576\n577 24 24 The Cryosphere\n578\n579\nFigure 8 - Total Greenland ice sheet calving flux over the 10,000-year spin up using the seven\n580\ngeothermal heat flow maps for Case 1 (a) and Case 2 (b). Units are Gt yr-1. The first 500 years\n581\nof the simulations are not shown due to artifacts associated with model initialization. 582\n583\nhttps://doi.org/10.5194/tc-2023-102\nPreprint. Discussion started: 30 June 2023\nc⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/tc-2023-102\nPreprint. Discussion started: 30 June 2023\nc⃝Author(s) 2023. CC BY 4.0 License. The Cryosphere 578\n579\nFigure 8 - Total Greenland ice sheet calving flux over the 10,000-year spin up using the seven\n580\ngeothermal heat flow maps for Case 1 (a) and Case 2 (b). Units are Gt yr-1. The first 500 years\n581\nof the simulations are not shown due to artifacts associated with model initialization. The Cryosphere 582\n583 9 579\nFigure 8 - Total Greenland ice sheet calving flux over the 10,000-year spin up using the seven\n580\ngeothermal heat flow maps for Case 1 (a) and Case 2 (b). Units are Gt yr-1. The first 500 years\n581\nof the simulations are not shown due to artifacts associated with model initialization. 582\n583 25 25 https://doi.org/10.5194/tc-2023-102\nPreprint. Discussion started: 30 June 2023\nc⃝Author(s) 2023. CC BY 4.0 License. The Cryosphere\n584\n585\nFigure 9 - Case 2: (a-g) Ice-bed temperature relative to pressure melting point at transient\n586\nequilibrium using the seven geothermal heat flow maps. (i) Ensemble mean ice-bed\n587\ntemperature. Units in all plots °C below pressure-melting-point temperature. (compare against\n588\nCase 2 in Figure 2). 589\n590 The Cryosphere 584\n585 585\nFigure 9 - Case 2: (a-g) Ice-bed temperature relative to pressure melting point at transient\n586\nequilibrium using the seven geothermal heat flow maps. (i) Ensemble mean ice-bed\n587\ntemperature. Units in all plots °C below pressure-melting-point temperature. (compare against\n588\nCase 2 in Figure 2). 589\n590 26 26 https://doi.org/10.5194/tc-2023-102\nPreprint. Discussion started: 30 June 2023\nc⃝Author(s) 2023. CC BY 4.0 License. The Cryosphere\n591\nFigure 10 - Case 2: (a-g) Relative anomaly from ensemble mean in ice-bed temperature at\n592\ntransient equilibrium using the seven geothermal heat flow maps. (i) Ensemble mean ice-bed\n593\ntemperature. Units in all plots °C below pressure-melting-point temperature. (Compare against\n594\nCase 1 in Figure 3.)\n595\n596 The Cryosphere Figure 10 - Case 2: (a-g) Relative anomaly from ensemble mean in ice-bed temperature at\n592\ntransient equilibrium using the seven geothermal heat flow maps. (i) Ensemble mean ice-bed\n593\ntemperature. Units in all plots °C below pressure-melting-point temperature. (Compare against\n594\nCase 1 in Figure 3.)\n595 27 27 https://doi.org/10.5194/tc-2023-102\nPreprint. Discussion started: 30 June 2023\nc⃝Author(s) 2023. CC BY 4.0 License. The Cryosphere\n599\n600\nFigure 11 - Case 2: (a-g) Anomaly in ice thickness at Case 2 transient spin up, in comparison to\n601\nCase 1 nudged spin up, using the seven geothermal heat flow maps. Units in all plots m and\n602\nexpressed as Case 2 minus Case 1. 603\n604 The Cryosphere 599\n600 600\nFigure 11 - Case 2: (a-g) Anomaly in ice thickness at Case 2 transient spin up, in comparison to\n601\nCase 1 nudged spin up, using the seven geothermal heat flow maps. The Cryosphere Units in all plots m and\n602\nexpressed as Case 2 minus Case 1. 603\n604 28 28"
W4214551863.txt	https://downloads.hindawi.com/journals/jspec/2022/7416046.pdf	en		A New Rule-Based Classification Method Using Shape-Based Properties of Spectral Curves	Journal of spectroscopy	2,022	cc-by	9,188	Hindawi Journal of Spectroscopy Volume 2022, Article ID 7416046, 17 pages https://doi.org/10.1155/2022/7416046 Research Article A New Rule-Based Classification Method Using Shape-Based Properties of Spectral Curves Songuel Polat ,1,2 Alain Tremeau ,2 and Frank Boochs 1 1 i3mainz, Institute for Spatial Information and Surveying Technology, Mainz University of Applied Sciences, 55128 Mainz, Germany 2 Hubert Curien Laboratory, University Jean Monnet, 42100 Saint-Etienne, France Correspondence should be addressed to Songuel Polat; songuel.polat@gmx.de Received 10 August 2021; Revised 15 September 2021; Accepted 15 January 2022; Published 24 February 2022 Academic Editor: Arnaud Cuisset Copyright © 2022 Songuel Polat et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Due to its high spatial and spectral information content, hyperspectral imaging opens up new possibilities for a better understanding of data and scenes in a wide variety of applications. An essential part of this process of understanding is the classiﬁcation part. However, the high spatial and spectral resolution also leads to enormous amounts of data. The eﬀective handling and use of such datasets for classiﬁcation requires processing steps (dimensionality reduction through feature selection or feature extraction) that are not always goal-oriented. In this article, a new general classiﬁcation approach is presented that uses the geometric shape of spectral signatures instead of purely statistical methods. In contrast to classical classiﬁcation approaches (e.g., SVM, KNN), not only are reﬂectance values taken into account, but also parameters such as curvature points, curvature values, and the curvature behavior of spectral signatures are used to develop shape-describing rules in order to use them for classiﬁcation by a rule-based procedure with IF-THEN queries. The ﬂexibility and eﬃciency of the methodology are demonstrated on datasets from two diﬀerent application domains and lead to convincing results with good performance. 1. Introduction Optical technology developments are extending the possibilities to better understand the world and its resources. Starting with images consisting of three color channels covering the visual electromagnetic spectrum, the developments since the late 1960s opened up the possibility of using spectral properties for identiﬁcation of materials by using multispectral images with tens of channels. Especially with the developments in the last two decades, another enormous step forward was made and the low spectral resolution of multispectral images was overcome. With several hundred narrow channels, hyperspectral imaging (HSI) opens up completely new possibilities for analysis in a wide variety of application ﬁelds [1]. As examples, [2–4] use HSI to maintain and increase crop yields in precision agriculture. The evaluation of food quality and safety through the use of HSI is part of the research of [5–7]. Other applications of HSI can be found in medicine to diagnose diseases or to monitor wound healing [8–10], in the art market to verify the authenticity of artworks [11–13], and in forensics to analyze crime scenes [14–16]. The key technology behind those applications is the HSI with detailed spectral and spatial information, which makes the HSI a powerful information source for advanced classiﬁcation methods like k-nearest neighbor, support vector machines, random forests, neural networks, and deep learning approaches. A comparison of these mentioned classiﬁcation methods is conducted in [17] and shows that there is no classiﬁer that consistently provides the best performance and that the quality of the classiﬁcation result mainly depends on factors such as the availability of training samples, processing requirements, tuning parameters, and speed of the algorithm. Another aspect to be considered in the context of mentioned classiﬁers is the Curse of Dimensionality. If high dimensional HSI are directly used as input, the classiﬁcation accuracy decreases, while the computational eﬀort of the model tends to increase 2 exponentially. To avoid this problem, a dimensionality reduction is essential [18]. The reduction of dimensions implies that algorithms automatically have to extract a set of characteristic spectral values, which have to represent the deciding features of the objects in question, from the entire course of a spectral signature. The automatic determination of the optimal number of relevant features [19] or the time required for example in the case of graph-based methods [20] is among the main problems. The amount of advanced existing studies (e.g., [19–27]) proves that there is no existing satisfactory, robust, and reliable methodology and that this topic is one of the main open issues in spectral imaging. As a consequence, this recommended step leads to loss of information in the spectral space, because the selected bands do not give an accurate description of the original spectral signature. Other classiﬁcation approaches based on indices [28–30] also arbitrarily select a subset of spectral values. One of the most popular is the Normalized Diﬀerence Vegetation Index (NDVI) from the ﬁeld of remote sensing [31], which allows the classiﬁcation of vegetation covered areas on the Earth’s surface by combining only a few bands of NIR and Red wavelengths. Another example of such an index is the Normalized Diﬀerence Plastic Index (NDPI), which combines shortwave infrared bands for the classiﬁcation of plastic materials in urban areas [32]. By limiting to a small number of spectral bands in a restricted spectral range, broad classiﬁcations such as separation between plants, water, or urbanized areas can be made, but ﬁner separation (e.g., between plant species or plastic materials) would be diﬃcult due to the reduced number of spectral bands and contradicts the use of HSI, which oﬀers the enormous advantage of high-resolution spectral information. Both band selection methods and indices consider only a small set of characteristic values, while the shape of the spectra is not taken into account. A consideration of the shape of spectral curves as important information source can be found in [33–35]. The work of [34] consists of a classiﬁcation approach that fully utilizes the shape of spectral curves by using a code to parameterize the spectral curve shape. The research of [35] deals with the deﬁnition of analysis rules based on spectral features like band position, band depth, band width, and band asymmetry. These key parameters are used to describe the absorption features of spectra. Disadvantage of both works is the time required to develop the description parameters of the curve shapes. Both approaches use tables to store the parameters and a subsequent matching process between the parameters of the reference data and the spectra that need to be classiﬁed, which also makes the classiﬁcation process a time-consuming task. The advantage of considering shapes, especially in combination with high-resolution spectral data, is clear: spectra express the mixed reﬂectivity of the elements that make up an object (e.g., molecules, pigments, cell structure, and water content), which is why each individual component has only a proportional inﬂuence on a spectrum. Changes in the composition of the elements then mainly change the mixture of all spectral contributions, resulting in local or regional variations and changing the shape of a spectrum. Journal of Spectroscopy In this article we follow the shape-based works of [33–35] and propose a new rule-based classiﬁcation method using shape-based properties of spectral curves like curvature points, curvature values, curvature direction, and spectral values. Particular attention should be paid to the following points: (i) The formulation of the rules should not become complex (ii) The classiﬁcation process should not be timeconsuming Keeping these points in mind, an approach was developed that allows establishing rules, using any kind of logical elements, in a straightforward manner. The establishment of rules implies existing knowledge. A prior analysis of the data and the acquisition of knowledge enable a better understanding of the data and allow structuring and simplifying a problem. Research papers from the ﬁeld of remote sensing that use the advantage of knowledge can be found in [36–41]. The experimental part for the evaluation of the method was performed on two diﬀerent datasets. A detailed description of the method and the used datasets are part of the following section. 2. Materials and Methods 2.1. Spectral Data Set Acquisition. The hyperspectral systems used in this work are two pushbroom cameras from Specim Ltd. (Oulu, Finland). The Specim FX10 captures the spectral signature from 400 nm to 1000 nm (233 bands), while the Specim FX17 captures the spectral signature from 900 nm to 1700 nm (229 bands). All spectral images acquired by these cameras are radiometrically normalized by using dark reference images for dark-current (closed shutter) and a white reference image to reduce the inﬂuence of the intensity variability. For the white calibration a 99% reﬂectance tile was used. As shown in Figure 1, a set of diﬀerent HSI consisting of two diﬀerent object types are used to demonstrate the proposed method. The ﬁrst dataset was captured with the Specim FX17 and shows an image of seven classes of diﬀerent plastic types. It has a resolution of 661 × 500 pixels and 229 bands with a spectral range from 900 nm to 1700 nm. The second scene was captured with the Specim FX10 and the Specim FX17 and shows ten classes of diﬀerent plant types. The HSI of both cameras were combined and have a resolution of 1220 × 640 pixels and 462 bands covering the spectral range from 400 nm to 1700 nm. The HSI with diﬀerent plastic types is used to demonstrate the basic functionality of the approach and gets more attention due to existing ground truth (the datasets can be found on https://doi.org/10.5281/zenodo.5068201). As part of a waste sorting application, the demonstration also covers experiments with real waste consisting of plastics and electronic waste like printed circuit boards (PCB). The plantbased dataset, on the other hand, is used to demonstrate the potential and ﬂexibility of the approach regarding diﬀerent kind of applications. Journal of Spectroscopy 3 3 UP PE 5 PS PE UP PMMA PA PS 1 10 7 PVC PS PF PA MA PM 8 2 PE PF MA C PV PM PA PVC 4 6 9 UP (a) (b) Figure 1: Images of the captured scenes. (a) Seven classes of diﬀerent plastics (plastic samples were kindly provided by PlasticsEurope Deutschland e.V (https://www.plasticseurope.org/de)) consisting of phenolic resin (PF), polyamide (PA), polyvinylchloride (PVC), polyethylene (PE), polymethylmethacrylate (PMMA), polyester resins (UP), and polystyrene (PS); (b) ten classes of diﬀerent vegetation types consisting of Taraxacum (1), Inula conyza (2), Campanula rotundifolia (3), moss (4), Geranium robertianum (5), Asplenium trichomanes (6), green sedum (7), moss and red sedum (8), moss (9), moss and lichen (10). 2.2. Methodology. Spectral signatures oﬀer the ability to distinguish between materials and are the result of reﬂected light from the surface, which is captured within a broad electromagnetic spectrum. The reason for diﬀerent signatures is a combination of molecules in materials and the morphological structure. Diﬀerent molecules result in diﬀerent spectral signatures. The morphological structure, on the other hand, is important because of the resulting light path that is created by reﬂection, absorption, transmission, and deﬂection from the diﬀerent components of an object. That is why plants show diﬀerent spectra when the cell structure changes, e.g., due to stress or aging. Both diﬀerences in molecules and structure result in diﬀerent shaped spectral curves. The idea of our work is to describe the spectral curve shape by a combination of spectral values and shape-based parameters, to use this knowledge for the formation of rules. Changes in the material composition of objects lead to local or regional changes in the course of spectral signatures. These changes inevitably lead to changes in the curvature behavior, what makes the curvature κ to a signiﬁcant parameter for the shape description and the modelling of spectral changes. Mathematically, the curvature is the change of a curve that occurs when the curve is traversed and can be expressed in parametric form for each point P(x(t), y(t)) using equation (1), where points refer to derivatives. € 8 � \|x_ y€ − y_ x\| 3/2 2 2 􏼐x_ + y_ 􏼑 . (1) While the curvature of a straight line is zero everywhere and the curvature of a circle is equal at all points, the curvature for all other curves changes from point to point and indicates how strongly the curve at a point P deviates from a straight line. Thus, for the description of spectral shapes, we use the following properties of curvature: (i) The dimension of the current rate of change of the direction of a point moving on the curve: the greater the curvature, the greater the dimension of change (ii) The behavior of the curvature: if the curvature value is positive, it is called a convex curve, and in the case of a negative curvature value, it is considered a concave curve. As shown in Figure 2, extreme values of the second derivative are used to select signiﬁcant parameters. The combination of these parameters with selected spectral values allows a precise description of the shape of spectral curves using a few selective spectral bands. The base for the calculation of the curvatures is preprocessed spectral curves. The preprocessing consists of a smoothing and a subsequent step of Continuum Removal. A schematic representation of all essential steps can be found in Figure 3. Continuum removal is a normalization procedure which allows a better quantiﬁcation of absorption peaks after removing the overall concave shape of spectral curves [42, 43] and is illustrated in Figure 4. Due to the particularly highlighted absorption bands, rules based on curvature values can be developed much more eﬃciently. An example for continuum removed spectra and the calculated curvatures is shown in Figure 5. Depicted are continuum removed spectra for two diﬀerent plastic types (PE, PS). In addition to the course of the spectral signature, the calculated curvature values are shown as positive and negative vertical lines in green and red color. The longer the line, the stronger the curvature at the respective band. The curve behavior is represented by red and magenta colored dots. These points are maximum and minimum points and are automatically determined by the local maxima and minima of the second derivative. It helps to distinguish between concave and convex curve behavior, providing an important source of information for describing the shape. For the subsequent formation of rules, mainly the red lines are used, since these reﬂect both the concave and convex behavior and a signiﬁcant change in curvature. All other curvature values (green lines) are not considered in the rule formation. To ensure that only signiﬁcant changes in the curve are captured, a threshold is set for the selection of the relevant curvatures (red lines). The setting of the threshold Journal of Spectroscopy Continuum Removed Reflectance 4 max min min min 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 2nd derivative max Wavelength (nm) Figure 2: Use of minimum and maximum positions of 2nd derivative to select signiﬁcant parameters for shape description. Parameters are the location (min/max), the curvature values at these locations (red lines), and the direction of curvature value (up for convex and down for concave behavior). Spectral Signature Pre-processing using Continuum Removal Calculation of curvature values Determination of extreme values (min/max) of 2nd derivative Signiﬁcant band selection: curvature value> threshold AND band == extreme value threshold is suﬃcient. The examples in Figure 5 show the result of selected red lines for a threshold of 0.1. Building on these extracted parameters regarding the spectra for each material, a collection of conditions is formulated and used for the classiﬁcation. It should be mentioned here that, in addition to the used local shaped values, other rules can also be integrated. For example, rules are describing global shape eﬀects (e.g., the expressivity of the green peak for plants) or adding relations of averaged reﬂectivity in diﬀerent regions as indices like NDVI do. One of the main advantages of rule-based classiﬁer is the simplicity. Once the knowledge on which rules are based has been worked out, conditions can be easily set up. Further advantages are the performance, the ability to handle redundant and irrelevant attributes, and the ﬂexible extensibility of rule sets [44]. Acquiring knowledge can seem eﬀortful, but in view of the resulting advantages, it should be seen as a clear beneﬁt which allows structuring and simplifying problems by using expert knowledge. A comparison of deep learning and a knowledge-based method can be found in [45] and show that a rule-based method can even be better as machine learning-based methods. For better illustration of rule formation, we refer to the spectral curves of PE and PS from Figure 5 and express them as shown in Table 1. The parameter CV stands for the curvature value at a speciﬁc spectral band and a positive or negative threshold is used to distinguish between convex and concave curve behavior. All conditions with a negative threshold will capture the downward red lines (concave behavior), while all conditions with a positive threshold will capture upward red lines (convex behavior). As already mentioned, for rule formation mainly the bands with high curvature are taken into account (red lines). Nevertheless, this does not mean that all bands are really necessary for the rule formation. By analyzing the data in advance, it is possible to achieve a clean classiﬁcation even with a smaller number of selected bands. Therefore, only six conditions are deﬁned for sample PS in Table 1, while in Figure 5 a total of seven red lines are present. The continuum removed spectra and rules for all other existing plastic types in the used dataset are listed in the Appendix (Figures 7–15). The corresponding spectral signatures are also shown in Figure 6. As an additional condition the continuum removed reﬂectance value (CRRV) could be used, if it is not possible to suﬃciently distinguish the shape. For instance, to separate PF-black from all other available plastic types. 3. Results and Discussion Figure 3: Schematic representation of proposed method. Once the rules are established, the next step is to apply them. This involves a pixel-by-pixel processing of the corresponding dataset and a check of the rule conditions for each individual pixel. If a condition applies, this pixel will be assigned to the appropriate class. mainly depends on the curve shape. The smaller the threshold the more detailed the description of the curves. However, for highly variant spectra (Figure 6) higher 3.1. Classiﬁcation Results for Plastic Samples. Applying the developed rules in the Appendix to the dataset consisting of plastic samples in Figure 1, results shown in Figure 16 were Rule formation using signiﬁcant bands Journal of Spectroscopy 5 0.8 1 0.7 Reﬂectance 0.6 0.5 0.4 original spectra 0.3 Continuum Removed Reﬂectance convex hull 0.2 0.9 0.8 0.7 0.6 0.5 continuum removed spectra 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 0.4 Wavelength (nm) Wavelength (nm) Figure 4: The use of continuum removal for quantiﬁcation of absorption peaks by connecting the maxima of spectra with straight lines (left), setting the convex hull to 100%, and subtracting the original spectra (right). PS 2 1.8 Continuum Removed Reflectance 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 0 Wavelength (nm) (a) 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 0.2 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 Continuum Removed Reflectance PE 2 Wavelength (nm) (b) Figure 5: Continuum Removed Reﬂectance spectra (black line), calculated curvature values (green lines), maximum points with concave behavior (red dots), and minimum points with convex behavior (magenta dots) for (a) polystyrene (PS); (b) polyethylene (PE). obtained. The result of the classiﬁcation shows that the individual samples were classiﬁed correctly. A closer look at the border areas of the samples shows that a shadow eﬀect occurs and that this eﬀect can inﬂuence the quality of the classiﬁcation. This shadow eﬀect occurs especially with samples (e.g., PS) that do not lie in a planar position. Another point to consider here is the classiﬁcation of PF samples. The continuum removed reﬂectance spectrum illustrated in the Appendix does not correspond to the real spectrum of the material PF. The PF samples used here are composed of black colorants. A well-known problem is the strong absorption of black colorants like carbon black. Due to the strong absorption of light from the UV to the NIR there is no reﬂected light that can be detected by the sensor and thus no spectral information that can be used for a classiﬁcation [46, 47]. The rule-based approach presented here oﬀers the advantage that even in the absence of reﬂectance rules can be established based on the existing limited information, which permit such a classiﬁcation. In the case of these samples, it means all black plastics will be classiﬁed as PF-black. It must also be noted in this example that the samples used have clean, homogeneous surfaces and the resulting spectra have a correspondingly high degree of shape similarity. However, considering applications in the ﬁeld of waste sorting, it is more common to work with dirty and damaged materials. Therefore, for the evaluation of the methodology, a dataset (1253 × 578 pixels and 229 bands) with real waste consisting of plastic parts (objects 1–19) and circuit boards (objects 20–27) was processed additionally. The classiﬁcation result for the real waste dataset is shown in Figure 17. The objects in the dataset were chosen randomly from a collection of diﬀerent plastic parts. Therefore, it is not surprising that certain materials (e.g., PMMA, PVC, PE, and 6 Journal of Spectroscopy 1 2 0.9 0.4 0.3 0.2 0.1 0 900 1000 1100 1200 1300 1400 Wavelength (nm) ABS PA PE PF-Black 1500 1600 1700 PMMA PP PS PVC Figure 6: Reﬂectance spectra of available plastics. Each category of plastic material corresponds with a speciﬁc spectral shape; likewise the shapes of Continuum Removed Reﬂectance Spectra are diﬀerent. This means the rule deﬁned for a category is speciﬁc to this category and there is no possible confusion as the shapes are distinct enough. So, such rules can be used only if spectral curve shapes are all suﬃciently distinct. Table 1: Shape-based rule for PS and PE. IF CV1108 < − 0.1 AND CV1174 < − 0.1 AND CV1608 < − 0.1 AND CV1143 > + 0.1 AND CV1204 > + 0.1 AND CV1677 > + 0.1 THEN class ⟶ PS IF CV1139 < − 0.1 AND CV1253 < − 0.1 AND CV1357 < − 0.1 AND CV1215 > + 0.1 AND CV1394 > + 0.1 AND THEN class ⟶ PE 1.4 1.2 1 0.8 0.6 0.4 0.2 0 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 0.5 1.6 Wavelength (nm) Figure 8: Continuum removed reﬂectance spectra and shapebased rule for PE. 2 PF-Black 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 0.6 Continuum Removed Reﬂectance 0.7 Continuum Removed Reﬂectance 1.8 0.8 Reflectance PE Wavelength (nm) Figure 9: Continuum removed reﬂectance spectra and shapebased rule for PF-black. 2 PA 2 1.8 Continuum Removed Reﬂectance 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 0 Wavelength (nm) Figure 7: Continuum removed reﬂectance spectra and shapebased rule for PA. 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 0.2 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 Continuum Removed Reﬂectance PMMA Wavelength (nm) Figure 10: Continuum removed reﬂectance spectra and shapebased rule for PMMA. Journal of Spectroscopy 2 7 PVC 2 1.8 Continuum Removed Reﬂectance 1.6 1.4 1.2 1 0.8 0.6 0.4 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0.2 0 0 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 Continuum Removed Reﬂectance 1.8 Wavelength (nm) Wavelength (nm) Figure 11: Continuum removed reﬂectance spectra and shapebased rule for PVC. 2 Figure 14: Continuum removed reﬂectance spectra and shapebased rule for PP. PS 2 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 Wavelength (nm) Figure 12: Continuum removed reﬂectance spectra and shapebased rule for PS. UP 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 0 Wavelength (nm) Figure 13: Continuum removed reﬂectance spectra and shapebased rule for UP. 0 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 0 Continuum Removed Reﬂectance ABS 1.8 Continuum Removed Reﬂectance Continuum Removed Reﬂectance 1.8 2 PP Wavelength (nm) Figure 15: Continuum removed reﬂectance spectra and shapebased rule for ABS. UP) are not present. In addition to the plastic types mentioned so far, polypropylene (PP) and acrylonitrile butadiene styrene (ABS) were identiﬁed on the basis of spectra from [48, 49] and formulated as rules. Furthermore, an additional rule was established for the classiﬁcation of printed circuit boards. A look at the classiﬁed plastic parts shows that the nonhomogeneity of the surfaces is partly reﬂected in the results in form of unclassiﬁed or misclassiﬁed pixels. Nevertheless, it can be stated that the classiﬁcation was generally successful by using the developed ten rules. The black plastic parts (objects 1 and 17 in Figure 17) that are missing were not modelled in this example, because the used background also consists of black plastic and a separation in this special case proves to be diﬃcult. Also, since the spectra of object number 5 in Figure 17 could not be assigned to any material, no rule for classiﬁcation was established. An important step in the recycling process is the separation of plastics and electronic waste. In particular, the high variation of diﬀerent material compositions from 8 Journal of Spectroscopy UP PVC PS PMMA PF-Black PE PA Background Figure 16: Classiﬁcation result using developed rules. 2 PCB 1 20 3 5 4 ABS 6 PP 21 22 7 UP 23 24 9 PVC 25 PS 8 11 12 10 PMMA PF 13 26 15 14 18 16 17 19 PE PA 27 Background Figure 17: Classiﬁcation result for real waste (plastics and printed circuit boards) using developed rules. Journal of Spectroscopy 0,8 0,7 0,6 0,5 Reﬂectance which PCBs are made makes separation diﬃcult. However, the example shown here also demonstrates that prior analysis of data and the use of rules based on acquired knowledge can lead to a more eﬃcient recycling process. Due to the already mentioned high variation of PCBs and the composition of diﬀerent materials (e.g., board, conductors, solder joints, resistors, and capacitors), a holistic assessment of a PCB is diﬃcult to implement. Nevertheless, it is evident in this example that the board has been identiﬁed in all cases and, as expected, only the areas with conductive tracks and metallic or electronic components have not been assigned and consequently ended up in a category with black plastics and the background. A signiﬁcant diﬀerence to the samples shown in Figure 16 is the heterogeneity of the surfaces. Diﬀerences in depth, shadow eﬀects on the surface, and dirt lead to a very high variability of spectral signatures. As an example, a region of object number 12 in Figure 17 was selected. In this area, diﬀerences in depth, shadow eﬀects, and soiling can be found. A representation of the high variability in form of spectral shifts is shown in Figure 18. The shape-based classiﬁcation result for object number 13, however, shows that a satisfactory result was achieved despite the high variability. The reason for the robustness of the proposed method is that even if the spectral values change and result in spectral shifts due to certain factors like dirt or shadow, the geometric shape remains stable. The processing time using MATLAB on a machine with an Intel(R) Core(TM) i7-10700 CPU @ 2.90 GHz and 16 GB RAM was 136 seconds for the real waste dataset and 44 seconds for the plastic dataset from Figure 16. In order to compare the quality of the proposed method to standard methods, we decided to use one of the most commonly used pixel-based classiﬁers. Therefore, the dataset shown in Figure 16 (plastic samples) and the dataset shown in Figure 17 (real waste samples) were processed using a supervised multiclass SVM classiﬁer (C-SVC, One-VersusAll), which is based on the selection of a training set to obtain a model and the subsequent application of this model for the prediction of classes. The dataset with the real waste was limited to the plastic objects. As already described in [17], the quality of the results strongly depends on the chosen training samples and parameterization. For this reason, three diﬀerent kernels, namely, Radial Basis Function (RBF), linear, and polynomial, with diﬀerent parameters have been tested on the datasets. Due to the multicategory classiﬁcation, a classiﬁcation threshold is used, which is the maximum distance to the hypersurface for conducted classiﬁcation to a speciﬁc class. The parameters for the kernels and the classiﬁcation threshold were obtained by testing and checking the results. Because of the homogeneity of the plastic samples, one material sample of each material class was manually labelled and used as training set. A description of the datasets can be found in Table 2. The best results for the plastic dataset using diﬀerent kernels and classiﬁcation thresholds are shown in Figure 19. The accuracy of each classiﬁcation method compared is reported in Table 3. In principle, the results of this homogeneous and optimal dataset are comparable for most of 9 0,4 0,3 0,2 0,1 0 1.000 1.100 1.200 1.300 1.400 1.500 1.600 1.700 Wavelength [nm] Figure 18: Spectral signature variability for object 12 of real waste dataset (spectra of all pixels inside the red box). Table 2: Number of samples and training samples for each class. Number of Number of training samples samples PA 16276 3783 PE 16212 3659 PF-Black 11157 4930 PMMA 15779 4071 Plastic dataset PS 16877 4123 PVC 16645 4869 UP 16098 4613 PA 13343 2114 PS 81238 10444 Real waste dataset PP 116620 11936 ABS 11703 1638 Class classiﬁcation metrics used. Nevertheless, signiﬁcant differences can be seen in the resulting images, especially at the edge of objects, in form of unclassiﬁed or misclassiﬁed pixels. As already mentioned, this is due to a shadow eﬀect that locally occurs in some areas, which impacts mainly the quality of the SVM methods. While the rule-based approach also classiﬁes these edge areas and some shadow parts as corresponding material, the SVM results in misclassiﬁcations, especially using linear and polynomial kernels. Compared to the plastic dataset, the real waste dataset has a typical situation consisting of objects with a high degree of variety. This not only increases the eﬀort required to train the model, but also makes the training process more diﬃcult. Small subsets of the individual samples were manually selected as training set. This strategy was considered to be appropriate due to the strong diﬀerences between the plastic samples. Thus, when selecting the subsets, special care was taken to ensure that particularly critical areas, such as shadow areas caused by depth, dirty areas, and damaged surfaces, were also covered in the training set. The best resulting class-labels are presented in Figure 20 and show also the eﬀect of diﬀerent classiﬁcation thresholds. The corresponding numerical values are listed in Table 4. 10 Journal of Spectroscopy (a) (b) (c) (d) Figure 19: Classiﬁcation results using the (a) proposed shape-based method; SVM with (b) RBF kernel, C-value of 1, and classiﬁcation threshold of 0.5; (c) linear kernel, C-value of 1, and classiﬁcation threshold of 0.3; (d) polynomial kernel, C-value of 1, and classiﬁcation threshold of 0.3. In principle, also for this dataset it can be stated that, despite the challenging objects of the real waste dataset, a high degree of accuracy can be achieved for both methods. The best SVM result was obtained with an RBF kernel consisting of a C-Value of 0.2 and a classiﬁcation threshold of 0.3. In comparison, the rule-based method produces slightly better values for the diﬀerent metrics. Also noticeable is the more homogeneous representation of objects in the resulting class-labels in Figure 20. The reason for this is again the robustness against spectral shifts. While SVM is based on pure reﬂectance values, the shape-based approach only needs to consider the geometric shape of spectral Journal of Spectroscopy 11 Table 3: Classiﬁcation accuracies for plastic dataset. Metrics Overall accuracy Precision Sensitivity False positive Rate F1-score Kappa Rule-based method 0.9694 0.9604 0.9551 0.0077 0.9564 0.8603 SVM (a) 0.9647 0.9731 0.9272 0.0114 0.9487 0.8384 SVM (b) 0.9641 0.9403 0.9623 0.0076 0.9503 0.8358 SVM (c) 0.9641 0.9403 0.9623 0.0076 0.9503 0.8359 (a) Solver: C-SVC; Classiﬁcation type: one-versus-all; kernel: RBF; C-value: 1; threshold: 0.5. (b) Solver: C-SVC; classiﬁcation type: one-versus-all; kernel: linear; C-value: 1; threshold: 0.3. (c) Solver: C-SVC; classiﬁcation type: one-versus-all; kernel: polynomial; C-value: 1; threshold: 0.3. signatures. One of the advantages of the shape-based approach is that, theoretically, only one spectrum per material is required for the rule formation. When using an SVM, it is necessary to ensure that, in case of high variability datasets, all data representing the spectral variations of one material class are included in the training set. Fulﬁlling this requirement can be a challenging task. 3.2. Classiﬁcation Results for Plants. Compared to the dataset consisting of plastics and electronic waste, spectral signatures of plants often show a very similar curve shape. This fact generally complicates the classiﬁcation process. To illustrate the ability of classifying even in such diﬃcult cases and the ﬂexibility of the methodology presented in this work, rules were developed to distinguish between plant species based on the spectral signatures shown in Figure 21, which are coherent with spectral signatures provided in [50]. Due to the similarity of the shape, in this particular case, rules have been formulated using mainly Continuum Removed Reﬂectance Values (CRRV) as well as curvature parameters. This involved using the entire spectrum from 500 nm to 1700 nm to work out ﬁne diﬀerences regarding the water content, the cell structure, and the pigments of used plants. In this context, it must be taken into account that the spectral properties of a plant species can also vary, as spectral signatures will be aﬀected by factors such as species, variety, age, internal cell structure, environmental conditions, chemical composition, and nutrient content [51]. An example for such a variation is given in Figure 22 for moss. Although not implemented in this work, one possible way to deal with such heterogeneous spectra could be the addition of rules based on texture features or other local image features [52–54]. A total of six rules were developed to separate the ten samples in groups of moss, lichen, red sedum, green sedum, Geranium robertianum, and green leaves. The results in Figure 23 show that, in principle, a classiﬁcation between diﬀerent plant species is possible. For example, it can be clearly seen that red sedum is distinctly diﬀerent from the other plant species due to its coloring. Green sedum also diﬀers based on the cell structure, whereby the properties of this plant seem also partially reﬂected in moss, which can be relatively well distinguished from the other plants due to the spectral signatures in the NIR and the low reﬂectance around 550 nm. Furthermore, it was possible to classify the lichens in object 10. The very strong spectral similarity between the leaves does not allow a clean separation between the individual species. Only Geranium robertianum diﬀers due to its low reﬂectance in the range around 550 nm. In this context, it is important to note that the spectral signatures shown in Figure 21 only reﬂect the spectral signature of one pixel and that the recognizable diﬀerences, which refer to reﬂectance values or shape, are not consistently present due to the high variability within a plant species. Nevertheless, it can be stated also for this example that a rule-based approach is a ﬂexible method and, depending on prior analytical eﬀorts, has the potential to provide useful results. 4. Discussion Results of a rule-based classiﬁcation approach were presented, based on the shape of spectral signatures. The rules are established in a supervised way and are based not only on spectral values, but especially on parameters that describe the geometric form of a spectral signature. These parameters include the automatic determined curvature points (i.e., speciﬁc spectral values obtained through the 2nd derivative), curvature values, and curvature behavior. The eﬀectiveness of this method was demonstrated with diﬀerent datasets from completely diﬀerent ﬁelds of application. In particular, the separation of materials with signiﬁcant geometric diﬀerences in the course of the curve leads to convincing results, which is reﬂected in the Overall Accuracy (OA) of the plastic dataset with 96.94% and the real waste dataset with 98.42%. An essential advantage over classical classiﬁcation approaches is the possibility of describing the course of a spectrum in detail on the basis of a few selective parameters. Classic approaches do not require a previous analysis of the data and oﬀer the advantage of automated processing based on the actual data. The prerequisite for this is, on the one hand, the availability of a large number of training data, which is not always readily available and could be a very time-consuming task (e.g., annotation), and preprocessing in the form of a dimensional reduction. This reduction is a selection of informative bands and often leads to a loss of information within the spectral signatures, because it is based on pure statistical reduction. With the method presented here, the ﬁnest changes in the course of the curve can be identiﬁed and modelled by using theoretically only one 12 Journal of Spectroscopy (a) (b) (c) (d) (e) (f ) Figure 20: Classiﬁcation results using (a) proposed shape-based method; SVM with (b) RBF kernel, C-value of 0.2, and classiﬁcation threshold of 0.2; (c) RBF kernel, C-value of 0.2, and classiﬁcation threshold of 0.3; (d) RBF kernel, C-value of 0.2, and classiﬁcation threshold of 0.4; (e) RBF kernel, C-value of 0.2, and classiﬁcation threshold of 0.5; (f ) RBF kernel, C-value of 0.2, and classiﬁcation threshold of 0.6. Journal of Spectroscopy 13 Table 4: Classiﬁcation results for real waste dataset. Metrics Overall accuracy Precision Sensitivity False positive Rate F1-score Kappa Rule-based method 0.9842 0.9842 0.9498 0.0075 0.9661 0.9506 SVM (a) 0.9770 0.9398 0.9477 0.0093 0.9396 0.9283 SVM (b) 0.9826 0.9698 0.9434 0.0098 0.9540 0.9456 SVM (c) 0.9805 0.9807 0.9353 0.0119 0.9556 0.9392 SVM (d) 0.9760 0.9856 0.9208 0.0152 0.9504 0.9249 SVM (e) 0.9586 0.9854 0.8831 0.0267 0.9290 0.8707 (a) Solver: C-SVC; classiﬁcation type: one-versus-all; kernel: RBF; C-value: 0.2; threshold: 0.2. (b) Solver: C-SVC; classiﬁcation type: one-versus-all; kernel: RBF; C-value: 0.2; threshold: 0.3. (c) Solver: C-SVC; classiﬁcation type: one-versus-all; kernel: RBF; C-value: 0.2; threshold: 0.4. (d) Solver: C-SVC; classiﬁcation type: one-versus-all; kernel: RBF; C-value: 0.2; threshold: 0.5. (e) Solver: C-SVC; classiﬁcation type: one-versus-all; kernel: RBF; C-value: 0.2; threshold: 0.6. pigments cell structure water content 0.9 0.8 0.7 Reﬂectance 0.6 0.5 0.4 0.3 0.2 0.1 0 -0.1 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 1 2 3 4 5 6 7 8 9 10 Figure 21: Spectra of measured plants. Two diﬀerent cameras were used to acquire the spectra (Specim FX10 and Specim FX17). The discontinuities in the range of 900 nm and the greater noise in the VIS range can be explained by the diﬀerences in spectral sensitivity of the cameras. In particular, the range below 500 nm exhibits strong noise eﬀects and was not considered in the rule formation. Taraxacum (1), Inula conyza (2), Campanula rotundifolia (3), moss (4), Geranium robertianum (5), Asplenium trichomanes (6), green sedum (7), red sedum (8), moss (9), and lichen (10). spectrum instead of a large amount of training data. The prerequisite for this is a previous analysis of the spectral signatures and the establishment of the rules based on expert knowledge. This process may be considered time-consuming, but considering the parameters used, it does not require a particularly high investment of time. An automated generation of the rules would also be conceivable, since the essentially used parameters such as curvature value, curvature point, and curvature behavior are determined by simple mathematical methods. A disadvantage that can arise with the automatic formulation of rules is that the number of rules increases with the number of categories. This can lead to complex rules that conﬂict with each other. Modelling the rules based on expert knowledge can avoid this by understanding the interaction of the factors, which makes it possible to formulate the simplest concept that gives the best results. Basically, when dealing with spectral data, it can be stated that the number of categories that can be classiﬁed from spectral bands is small enough that a limited number of rules can be used. The analysis of spectral signatures in advance has the further advantage that there is no need to use the entire spectrum for classiﬁcation. If signiﬁcant shape diﬀerences are detected within a limited spectral range, it is suﬃcient to consider only this range in order to establish the rules based on it, which leads to an increase in performance. While spectral values can vary strongly depending on diﬀerent factors, diﬀerences in the shape of spectral signatures are only due to diﬀerences in material composition. This fact is the reason for the promising results and conﬁrm our expectations of the proposed analytical method, which is robust to spectral variations and allows an unambiguous description of the spectra based on the basic idea of using the shape of the individual ﬁngerprint of diﬀerent materials or objects. 14 Journal of Spectroscopy 0,5 0,4 Reﬂectance 0,3 0,2 0,1 0 -0,1 500 600 700 800 Wavelength [nm] 900 1.000 Figure 22: Spectral variations in VIS for one type of moss regarding all pixels in the red box. 1 3 2 inula conyza taraxacum 4 campanula rotundifolia geranium robertianum moss asplenium trichomanes 5 6 green sedum 7 red sedum 8 10 9 moss moss lichen moss Figure 23: Classiﬁcation result for plants using VIS and NIR (500 nm–1700 nm). Furthermore, the processing time must be mentioned. The pixel-by-pixel processing and checking for conditions using MATLAB on a machine with an Intel(R) Core(TM) i710700 CPU @ 2.90 GHz and 16 GB RAM takes 113.42 seconds for the plant dataset (1220 × 640 pixels and 462 bands) shown in Figure 23. In comparison, the shape-based classiﬁcation in [34] for a dataset of 512 × 512 pixels and 6 bands takes about 9 minutes (computer conﬁguration: CPU 2.93 GHz and installed memory 4.00 GB). Another comparison with a research using a classic classiﬁcation method like SVM also shows a better performance. The processing time for a comparable dataset of 1168 × 696 pixels and 520 Journal of Spectroscopy bands takes 2980.21 seconds using 30 bands and 9381.37 seconds using all bands of the dataset (computer conﬁguration: Intel Core i7-6800k 3.40-GHZ CPU and installed memory 64 GB) [55]. 15 [6] 5. Conclusions Considering the shape of spectral signatures and describing them by curvature parameters and spectral values proves to be a convincing supervised method for classifying diverse groups of materials from diﬀerent ﬁelds of application. Further evaluation on publicly available datasets, e.g., from the ﬁeld of remote sensing, also with respect to other classiﬁcation methods, would be of interest. A possible disadvantage of this methodology could be the necessary prior analysis of the data. This circumstance could be eliminated by automation. Especially in the case of significantly diﬀerent spectra, like the plastic samples, automation is certainly feasible and would bring the beneﬁt of an unsupervised method. [7] [8] [9] [10] Data Availability [11] The HSI datasets used to support the ﬁndings of this study have been deposited in the Zenodo repository (https://doi. org/10.5281/zenodo.5068201). 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https://openalex.org/W3215357798	https://www.frontiersin.org/articles/10.3389/fmicb.2021.749760/pdf	English	null	Alone Yet Not Alone: Frankia Lives Under the Same Roof With Other Bacteria in Actinorhizal Nodules	Frontiers in microbiology	2,021	cc-by	13,683	ORIGINAL RESEARCH published: 02 December 2021 doi: 10.3389/fmicb.2021.749760 Alone Yet Not Alone: Frankia Lives Under the Same Roof With Other Bacteria in Actinorhizal Nodules Faten Ghodhbane-Gtari1,2,3, Timothy D’Angelo4†, Abdellatif Gueddou3, Sabrine Ghazouani3, Maher Gtari1,3 and Louis S. Tisa4* 1 Laboratoire Microorganismes et Biomolécules Actives, Faculté des Sciences de Tunis, Université de Tunis El Manar, Tunis, Tunisia, 2 Institut Supérieur de Biotechnologie de Sidi Thabet, Université de la Manouba, Sidi Thabet, Tunisia, 3 Unité de Bactériologie Moléculaire et Génomique, Centre Urbain Nord, Institut National des Sciences Appliquées et de Technologie, Université de Carthage, Tunis, Tunisia, 4 Department of Molecular, Cellular, and Biomedical Sciences, University of New Hampshire, Durham, NH, United States Actinorhizal plants host mutualistic symbionts of the nitrogen-ﬁxing actinobacterial genus Frankia within nodule structures formed on their roots. Several plant-growth- promoting bacteria have also been isolated from actinorhizal root nodules, but little is known about them. We were interested investigating the in planta microbial community composition of actinorhizal root nodules using culture-independent techniques. To address this knowledge gap, 16S rRNA gene amplicon and shotgun metagenomic sequencing was performed on DNA from the nodules of Casuarina glauca. DNA was extracted from C. glauca nodules collected in three different sampling sites in Tunisia, along a gradient of aridity ranging from humid to arid. Sequencing libraries were prepared using Illumina NextEra technology and the Illumina HiSeq 2500 platform. Genome bins extracted from the metagenome were taxonomically and functionally proﬁled. Community structure based off preliminary 16S rRNA gene amplicon data was analyzed via the QIIME pipeline. Reconstructed genomes were comprised of members of Frankia, Micromonospora, Bacillus, Paenibacillus, Phyllobacterium, and Aﬁpia. Frankia dominated the nodule community at the humid sampling site, while the absolute and relative prevalence of Frankia decreased at the semi-arid and arid sampling locations. Actinorhizal plants harbor similar non-Frankia plant-growth- promoting-bacteria as legumes and other plants. The data suggests that the prevalence of Frankia in the nodule community is inﬂuenced by environmental factors, with being less abundant under more arid environments. Edited by: F. L. Consoli, University of São Paulo, Brazil Reviewed by: Monali C. Rahalkar, Agharkar Research Institute, India Luis Wall, National University of Quilmes, Argentina *Correspondence: Louis S. Tisa louis.tisa@unh.edu ††† Present address: Timothy D’Angelo, Bigelow Laboratory for Ocean Sciences, East Boothbay, ME, United States Specialty section: This article was submitted to Systems Microbiology, a section of the journal Frontiers in Microbiology Received: 29 July 2021 Accepted: 08 November 2021 Published: 02 December 2021 Keywords: actinorhizal symbiosis, microbiome, endophyte, symbiont, plant-growth-promoting bacteria INTRODUCTION Ghodhbane-Gtari F, D’Angelo T, Gueddou A, Ghazouani S, Gtari M and Tisa LS (2021) Alone Yet Not Alone: Frankia Lives Under the Same Roof With Other Bacteria in Actinorhizal Nodules. Front. Microbiol. 12:749760. doi: 10.3389/fmicb.2021.749760 Casuarinaceae family comprises four plant genera (Allocasuarina, Casuarina, Ceuthostoma, and Gymnostoma) and approximately 86 species and 13 subspecies indigenous to Australia (Steane et al., 2003; Gtari and Dawson, 2011; Diagne et al., 2013; Normand et al., 2014) that are frequently introduced into approximately 150 tropical and subtropical countries where they play an important role for land reclamation, crop protection and as windbreaks (Diagne et al., 2013; December 2021 \| Volume 12 \| Article 749760 Frontiers in Microbiology \| www.frontiersin.org Metagenomic Analysis of Actinorhizal Root Nodule Ghodhbane-Gtari et al. Potgieter et al., 2014). Large-scale planting of casuarinas has proven to have a strong impact especially in China, Senegal, Egypt, and Tunisia (Gtari and Dawson, 2011). In arid and semi- arid areas, salinization of soils and groundwater is a serious problem causing a drastic reduction in agricultural production (Munns, 2005; Rengasamy, 2006)1. Over 800 million hectares of land throughout the world are salt aﬀected. A common method for dealing with salt stress problems is to reclaim saline soils with multipurpose, fast-growing salt-tolerant tree species such as Casuarina tree. Casuarina are notable for high salt tolerance (Tani and Sasakawa, 2003) and have been used as a green barrier. Despite the applied interests, substantial global invasion consequences for Casuarina has noted in Florida and the Mascarene Islands (Potgieter et al., 2014). Potgieter et al., 2014). Large-scale planting of casuarinas has proven to have a strong impact especially in China, Senegal, Egypt, and Tunisia (Gtari and Dawson, 2011). In arid and semi- arid areas, salinization of soils and groundwater is a serious problem causing a drastic reduction in agricultural production (Munns, 2005; Rengasamy, 2006)1. Over 800 million hectares of land throughout the world are salt aﬀected. A common method for dealing with salt stress problems is to reclaim saline soils with multipurpose, fast-growing salt-tolerant tree species such as Casuarina tree. Casuarina are notable for high salt tolerance (Tani and Sasakawa, 2003) and have been used as a green barrier. Despite the applied interests, substantial global invasion consequences for Casuarina has noted in Florida and the Mascarene Islands (Potgieter et al., 2014). found between cells, while others are associated in an intimate relation with the plant. INTRODUCTION For that intimate association to occur, complex relationships are required during the colonization of plant tissues. Plant endophytes are described as having the ability to restrain pathogen infection, to accelerate seedling emergence, and to promote plant growth. Thus, they can be considered as biocontrol agents (Ryan et al., 2008; Eljounaidi et al., 2016; Santoyo et al., 2016). In this study, we investigated the structure and function of bacterial communities found in Casuarina glauca nodules and associated rhizosphere soils and bulk soils from three diﬀerent bioclimates in Tunisia. We used both high-throughput amplicon (targeting of the 16S rRNA gene) and whole metagenome shotgun sequencing of C. glauca nodules found from the diﬀerent bioclimates: humid, semi-arid and arid condition. For comparative purposes, high-throughput amplicon sequencing was performed on rhizosphere soils, and bulk soils for these three sites. Actinorhizal plants, like Casuarina, form a nitrogen-ﬁxing symbiosis with the Actinobacteria Frankia that results in the formation of root nodule structures where the bacteria are located (Chaia et al., 2010; Normand et al., 2014). Besides Frankia, actinorhizal plants are known to associate with mycorrhizae including both ectomycorrhizal or endomycorrhizal fungi (Zhong et al., 1995; Duponnois et al., 2003; Diagne et al., 2013; Zhou et al., 2017). The symbioses with Frankia and mycorrhizae allow actinorhizal host plants to colonize harsh environmental terrains including highly contaminated, dry, poorly drained, nutrient-poor, and salinized soils (Diagne et al., 2013, 2015). Sampling Samples of Casuarina glauca root nodules, rhizosphere, and bulk soils. Samples were collected in February 2015 from three sites in Tunisia that represented diﬀerent bioclimatic regions (Figure 1). Sample sites were in Tamra (37◦3′26′′ N 9◦14′17′′ E), Sidi Bouzid (35◦2′ 7.58′′ N 9◦29′2.18′′ E), and Medenine (33◦21′17′′ N 10◦30′19′′ E), representing Mediterranean-humid, semi-arid and arid bioclimatic regions, respectively. Three to four root nodules were used for each replicate and three replicate samples were taken for each site. For the soil and rhizosphere samples, triplicate samples of 5 g soil were collected from each site. Bulk soil was soil that was not in contact with the root system of any plant. Rhizosphere soil was collected by removing organic litter and 15 cm of the surrounding soil from around the C. glauca roots. Soil that was attached to roots after removal of the surrounding soil was collected. The samples were placed in sterile plastic tubes and stored at −20◦C until analysis. DNA extractions were performed within 24 h of sampling. p g Besides Frankia, several other non-Frankia bacteria have been isolated as a by-product of Frankia isolation attempts (Ghodhbane-Gtari and Tisa, 2014). Large numbers of other bacteria were collected from these actinorhizal nodules, occupying the same microniche as Frankia. Most of these isolates were ignored or discarded as being irrelevant to the plants. However, these non-Frankia Actinobacteria have consistently been isolated from several actinorhizal plants including Casuarina (Guillén et al., 1993; Niner et al., 1996; Valdes et al., 2005; Ghodhbane-Gtari et al., 2010), and consist of Actinobacteria from Micromonospora (Valdes et al., 2005; Ghodhbane-Gtari et al., 2010), Nocardia (Ghodhbane-Gtari et al., 2010, 2019) and Streptomyces genera (Ghodhbane-Gtari et al., 2010). Similarly, non-symbiotic bacteria, such as Azospirillum, Bacillus, Pseudomonas, and Streptomyces, are known to be auxiliary bacteria in several Rhizobium-legume symbioses (Iruthayathas et al., 1983; Grimes and Mount, 1984; Li and Alexander, 1990). In this case, both wild and cultivated legume nodules are not exclusively inhabited by rhizobia, but contain diverse assemblages of non-rhizobial bacteria (Dudeja et al., 2012; Aserse et al., 2013; Martínez-Hidalgo and Hirsch, 2017). These nodule non-rhizobial communities may be inﬂuenced more by soil type rather plant genotype (Leite et al., 2017). 1http://fao.org DNA Extractions For the rhizosphere and bulk soil samples, genomic DNA (gDNA) was extracted from 0.5 g of soil using the Qiagen DNA extraction kit as described previously (Ghodhbane-Gtari et al., 2010, 2014). For the C. glauca root nodule samples, three to four nodules were used per gDNA extraction and nodules were surface sterilized as described previously (Ghodhbane-Gtari et al., 2019). Root nodules were thoroughly rinsed with water. Surface sterilization conducted by shaking in 30% (v/v) H2O2 for 30 min. Sterilized nodules were rinsed thoroughly several times with sterile water. The ﬁnal wash liquid was tested for microbial growth and only nodules resulting in a sterile ﬁnal washing liquid were further considered for DNA extraction. The nodule was then aseptically powdered in liquid nitrogen using a sterile mortar and the resulting powder was used for DNA extraction using Qiagen DNA Plant Mini-kit following the manufacturer’s protocol. During the last decade, culture-dependent and –independent approaches have been used to intensively investigate these eﬀects. For legume nodules, rhizobial, and non-rhizobial endophytes communities have been initiated (Martínez-Hidalgo and Hirsch, 2017; Zhang et al., 2018; Mayhood and Mirza, 2021), but very little is known about microbial communities and their structure associated with actinorhizal nodules. Endophytic bacteria can be December 2021 \| Volume 12 \| Article 749760 Frontiers in Microbiology \| www.frontiersin.org 2 Metagenomic Analysis of Actinorhizal Root Nodule Ghodhbane-Gtari et al. FIGURE 1 \| Map and schematic of amplicon survey sampling design: For each sample type from each site, 3 replicates were sampled. Modiﬁed from Hénia (2008). FIGURE 1 \| Map and schematic of amplicon survey sampling design: For each sample type from each site, 3 replicates were sampled. Modiﬁed from Hénia (2008). mplicon survey sampling design: For each sample type from each site, 3 replicates were sampled. Modiﬁed from Hénia (2008). Frontiers in Microbiology \| www.frontiersin.org Amplicon and Whole Genome Shotgun Sequencing used for both the metabarcoding and metagenome shotgun sequencing methods, and metagenome libraries for both methods were sequenced at 150 bp read lengths at the Hubbard Center for Genome Studies (University of New Hampshire, Durham, NH, United States). To investigate the prokaryotic community proﬁles of diﬀerent DNA extracted, sequences corresponding to the V4 hypervariable region the 16S subunit rRNA gene were ampliﬁed by PCR using the Earth Microbiome Project 515F/806R universal primers under conditions described previously (Caporaso et al., 2012). For each sample, triplicate ampliﬁcations were performed. The triplicates were pooled, and all samples were normalized to equal DNA concentrations. Paired-end sequencing of the ampliﬁcation product was performed using the Illumina HiSeq 2500 platform at the Hubbard Center for Genome Studies (University of New Hampshire, Durham, NH, United States). Read lengths of 250 bp were obtained and processed as described below. Amplicon Sequence Data Processing The 16S rRNA gene amplicon sequences for each sample were initially processed with Quantitative Insights into Microbial Ecology (QIIME) pipeline (Caporaso et al., 2010b). Paired-end reads were joined using Fastq-join (Aronesty, 2011). Paired-end reads were truncated from positions where nucleotides received a Phred score of 20 or less. Reads with more than three consecutive low-quality nucleotides were discarded. Reads with less than 75% high-quality base-call scores were also discarded. This processing resulted in a total of 176,319 reads with an average length of 253 base pairs. To generate metagenomes for the C. glauca root nodule samples, whole genome shotgun sequencing was performed. Sequencing libraries for the samples were prepared using the Illumina NextEra Library Preparation protocol according to the manufacturer’s instructions and were sequenced on an Illumina HiSeq 2500. The same triplicate nodule DNA extractions were The reads were clustered as described in the open reference- based protocol provided in QIIME (version 1.9.0-dev) (Rideout et al., 2014). OTUs were clustered by UCLUST, using a 97% December 2021 \| Volume 12 \| Article 749760 Frontiers in Microbiology \| www.frontiersin.org 3 Metagenomic Analysis of Actinorhizal Root Nodule Ghodhbane-Gtari et al. of times, out of the 100 jackknifed trees, that the consensus conﬁguration was observed. percent identity threshold to deﬁne an OTU (Edgar, 2010). Taxonomy was assigned to each OTU cluster using the UCLUST consensus taxonomy assigner by aligning the centroid sequence of each cluster to the Green Genes 13_8 rRNA database with PyNAST (DeSantis et al., 2006; Caporaso et al., 2010a; Edgar, 2010). OTUs that were unable to be aligned to the Green Genes 13_8 database were excluded from downstream analysis. OTUs that only contained one sequence (singletons) were also excluded from downstream analysis. The resulting OTU tables were ﬁltered to exclude all unassigned sequences, and sequences that assigned to mitochondrial or chloroplast taxonomy. Nodule samples were analyzed by two-way cluster analysis to investigate the diﬀerence in nodule community structure across the environmental gradient in the sampling design. An OTU table was constructed that was corrected for 16S operon copy number abundance by PICRUSt and summarized to relative abundance at the genus level. The samples were grouped by hierarchical clustering using the average linkage method. The Bray-Curtis distance between each sample based on taxonomic composition was used for sample clustering. The taxa were clustered by their abundance patterns across samples using the Bray-Curtis distance between metric. Amplicon Sequence Data Processing Clustering was performed using the data for all genera present. However, for visualization purposes, only taxa that made up greater than 1% of the community were displayed. Frontiers in Microbiology \| www.frontiersin.org Amplicon Sequence Data Analysis Amplicon Sequence Data Analysis Alpha diversity for each sample was calculated based on the Shannon Diversity Index (Shannon, 1948) and signiﬁcant diﬀerences in diversity based on sample type (Nodule, Rhizosphere, or Soil) were determined using ANOVA and the t-test in Microsoft Excel. Alpha diversity was also calculated in the similar manner for nodule samples from individual sampling locations. Community beta diversity among samples was measured by the UniFrac distance (Lozupone and Knight, 2005). Both the weighted (quantitative) and unweighted (qualitative) versions of the distance metric were used. Both of these metrics can yield diﬀerent but complementary results from the same dataset (Lozupone et al., 2007). Distance matrices produced using these metrics were ordinated using Principle Coordinates Analysis (PCoA). Ordinations were created for distance matrices produced by CSS (for both 16S copy number corrected and not corrected), for raw OTU tables with 16S operon copy number correction and for evenly rareﬁed OTU tables. Soil and Rhizosphere samples were analyzed to test the hypothesis that C. glauca maintains a unique microbial community within the rhizosphere at the diﬀerent sampling locations. Soil (n = 9) and Rhizosphere (n = 9) samples from all sites were pooled together and analyzed independently of nodule samples in the same fashion as described above. To determine if there were signiﬁcant groupings between soil and rhizosphere by climate type, the soil and rhizosphere samples were grouped into a humid group (n = 6) and non-humid group (n = 18). MRPP was performed as above with this grouping using the unweighted UniFrac distance matrix. This metric calculates phylogenetic distance between samples based oﬀof the presence or absence of taxa and is thus useful for identifying diﬀerences between samples that come from environments that host distinct communities. Data Normalization The OTU table resulting from the process above was rariﬁed to an equal sampling depth based on the smallest library size (1,762 reads) by random sub-sampling of all samples. Alternatively, Cumulative Sum Scaling (CSS), available through the metagenomeSeq R package and facilitated through QIIME, was used to normalize the OTU table (Paulson et al., 2013). Because choice of normalization method has been shown to eﬀect downstream analysis (McMurdie and Holmes, 2014), both methods were used to compare results of normalization techniques. For Alpha diversity analysis, the original OTU table was converted to closed-reference format and corrected for variation in 16S rRNA copy number using the online Galaxy server version of PICRUSt (Langille et al., 2013). This correction was also performed on CSS normalized OTU tables and raw OTU tables in order to assess eﬀects on ordination results. y p y Multi-Response Permutation Procedures (MRPP) were performed to determine if there were signiﬁcant diﬀerences in phylogenetic distances based on the sample type (Nodule, Soil, or Rhizosphere). MRPP analysis was performed in R (Version 3.2.1) using the Vegan Library (Oksanen et al., 2016). MRPP To determine if nodules formed a statistically signiﬁcant group based on the weighted and unweighted UniFrac distance matrices, the samples were partitioned into two categories, nodule (n = 9) and non-nodule (n = 18). Signiﬁcance was calculated by permutation of the distance matrices 999 times to determine if the groups had within-group distances that were diﬀerent to what would be expected by chance. All MRPP analysis was performed on the weighted and unweighted UniFrac distance matrices produced by the diﬀerent normalization methods described previously (Lozupone et al., 2007; McMurdie and Holmes, 2014). Taxonomic Classiﬁcation The read sets, with plant-derived reads removed, were aligned to the Frankia genome using Bowtie2. The percentage of the reads that aligned to the Frankia genome bin were recorded from the standard output statistics in order to compare the relative abundance of Frankia in the shotgun data in comparison to the replicated amplicon data. Taxonomic information was assigned to each ORF identiﬁed as essential gene by searching against the RefSeq protein database using BlastP with an E value cutoﬀof 1e−5, with ﬁve maximum target sequences. The resulting BlastP output in XML format was uploaded into MEGAN (Version 5) (Huson et al., 2007). The Lowest Common Ancestor (LCA) algorithm was used to make a consensus taxonomic assignment for each ORF based oﬀof the top ﬁve BlastP hits using the following settings: Top Percent = 5, Minimum Support = 1, Rank = Species. The resulting ﬁle was parsed using the hmm.majority.vote.pl script to derive a consensus taxonomic assignment for each assembled contig based oﬀof the taxonomy of the ORFs that it contains. 4https://bioconductor.org/packages/Biostrings 5http://broadinstitute.github.io/picard/ Metagenome Assembly and Analysis Sequencing Reads Preparation and Assembly Metagenome Assembly and Analysis Sequencing Reads Preparation and Assembly Trimmomatic was used to remove Nextera adapters and ﬁlter the raw reads based on quality (Bolger et al., 2014). The following parameters were used. Three base-pairs were trimmed from the tailing and leading ends of each read. The reads were scanned with a sliding window of four base-pairs, and sections of reads were discarded when the average quality score of the base-pairs was below 15. After this process, only reads that retained a minimum length of 36 base pairs were used for downstream Jackkniﬁng, which is the repeated subsampling of a dataset, was used to determine the conﬁdence level in Beta diversity analysis. This technique was performed by randomly subsampling 100 sequences per sample from the rareﬁed OTU table. For each subsampling, an Unweighted Pair Group Method with Arithmetic Mean (UPGMA) tree was created using the unweighted UniFrac distance matric between samples, based on those 100 randomly subsampled reads. This process was repeated 100 times. A consensus tree was built from the 100 trees with jackknife values for each node representing the number December 2021 \| Volume 12 \| Article 749760 4 Metagenomic Analysis of Actinorhizal Root Nodule Ghodhbane-Gtari et al. processes. Of these resulting reads, only reads that retained both forward and reverse pairs were used for assembly. processes. Of these resulting reads, only reads that retained both forward and reverse pairs were used for assembly. The coverage and taxonomic information produced above was loaded into the Rstudio environment along with the composite metagenomic co-assembly using BioStrings4. The GC content and tetranucleotide frequency distribution of each assembled contig was calculated using the mmgenome R package. A principle component analysis (PCA) of normalized tetranucleotide frequencies of all contigs was performed in R using the mmgenome package. The composite co-assembly was visualized in Rstudio and binning was performed using the tools available in the mmgenome R package. The data that was produced as described above and accompanying rmarkdown ﬁles allow the reproduction of the binned draft genomes. In order to perform diﬀerential coverage binning of draft genomes, the read sets from each of the three sampling locations were combined in silico and a composite co-assembly was created. SPAdes (version 3.6.0) was used to assemble the combined reads using the metagenomic setting, without error correction (Bankevich et al., 2012). Functional Annotation Reads from each site were assembled individually using SPAdes- 3.6.0 using the metagenomic setting (Bankevich et al., 2012). The resulting assembled contigs were annotated using the Prokka annotation pipeline using the metagenomic settings (Seemann, 2014). Calculation of Coverage and Relative Abundance Coverage proﬁles of individual genomes with PCR duplicates removed were calculated to compare genome coverages at diﬀerent sites. Reads were aligned to the metagenome assembly using the Bowtie2 alignment tool (Version 2.2.5) (Langmead and Salzberg, 2012). The SAM ﬁle resulting from alignment with Bowtie2 was converted to BAM format. The aligned reads were sorted to the left-most coordinate on the indexed metagenome assembly using SAMtools (Version: 0.1.19-96b5f2294a) (Li et al., 2009). Duplicate reads were removed using Picard Tools MarkDuplicates5. Coverage for the contigs was calculated from the resulting SAM ﬁle using Bedtools (v2.17.0) (Quinlan and Hall, 2010). The coverage calculations per-contig were averaged for all the contigs within a given genome bin. The average coverage for the entire genome was normalized by the amount of reads per data set corresponding to the diﬀerent sampling sites. The percentage of the given read sets that aligned to plant- derived DNA was used to normalize each genome coverage value based on the diﬀering amounts of plant-derived DNA in the diﬀerent read sets. Additional information for assembled contigs was generated following the workﬂow as described by the mmgenome package developers (Albertsen et al., 2013). Open Reading Frames (ORFs) within the assembled metagenomic contigs were predicted and translated to amino acid sequence using the metagenomic setting of Prodigal (Version 2.6.2) (Hyatt et al., 2010). The translated amino acid fasta ﬁle of predicted ORFs was searched against Hidden Markov Models (HMMs) of 111 essential prokaryotic single copy genes provided in the mmgenome package using hmmsearch (Eddy, 1998). ORFs identiﬁed as essential genes were extracted from the co-assembly using Perl scripts provided by the mmgenome package. Metagenome Assembly and Analysis Sequencing Reads Preparation and Assembly The assembly process used Khmer lengths of 22, 33, and 55 to produce the ﬁnal assembled contigs. Binning Metagenomic Assembled Genomes (MAGs) of individual members of the metagenomic community were binned using the mmgenome package (Albertsen et al., 2013) for R2 using the Rstudio IDE3. This package facilitates distinguishing individual genomes from metagenomes primarily by the unique coverage values of assembled sequences belonging to organisms present in multiple samples, but at diﬀerent abundances. Coverage values for each contig per-site was performed by individually aligning each read-set to the co-assembled contigs using the Bowtie2 alignment tool (Version 2.2.5) (Langmead and Salzberg, 2012). The resulting Sequence Alignment Map (SAM) ﬁles were converted to binary format and sorted to the left- most coordinates on the assembled sequences and depth of coverage per-base was calculated using SAMtools (Version: 0.1.19-96b5f2294a) (Li et al., 2009). The resulting ﬁles containing coverage per-base per-contig were converted to average coverage per-contig scripts in the Mmgenome package. 2http://r-project.org 3http://Rstudio.com Mining of Metagenome for Plant-Growth-Promoting Genes and Secondary Metabolites The Genome Feature File (GFF) produced by Prokka was parsed using a custom python script to evaluate the presence and December 2021 \| Volume 12 \| Article 749760 Frontiers in Microbiology \| www.frontiersin.org 5 Metagenomic Analysis of Actinorhizal Root Nodule Ghodhbane-Gtari et al. taxonomy of plant-growth-promoting genes and genes involved in cellulose, chitin, and pectin degradation within the nodule community. Genes known for plant auxin production (Indole acetic acid and Phenyl acetic acid), phosphate solubilization (alkaline phosphatases and phytases), ACC Deaminase activity and degradation of chitin, cellulose and pectin were identiﬁed for each of the three data sets. The presence of these enzymes was identiﬁed by parsing the GFF ﬁle for the Enzyme Commission (EC) number assigned to a given ORF by the Prokka pipeline. Enzymes that were identiﬁed by their EC numbers were annotated by aligning the amino acid sequence of the entry against the RefSeq protein database using BlastP with an E value cutoﬀof 1e−5 and a maximum output of ﬁve entries. Cladograms of the BlastP output were constructed using the Lowest Common Ancestor (LCA) algorithm in MEGAN (Version 5.0) using the following settings: Top Percent = 5, Minimum Support = 1, Rank = Genus. A summary of the EC numbers used in this analysis is in Supplementary Tables 1–3. The antiSMASH pipeline (version 3.0) was used to identify secondary metabolites in the binned genomes (Weber et al., 2015). antiSMASH was run using the metagenomic setting to improve gene prediction for highly fragmented metagenomic assemblies. FIGURE 2 \| Photograph of Casuarina tree stands at the sampling and representative nodules for those trees. FIGURE 2 \| Photograph of Casuarina tree stands at the sampling and representative nodules for those trees. 9.52 ± 0.69 (SD), displayed in Supplementary Figure 2A. The alpha diversity was signiﬁcantly less for nodules than for rhizosphere and soil samples (Students t-test, p = 0.00001). Alpha diversity was calculated for the nodule samples at the three diﬀerent samples locations. Nodules from Tamra had an average SDI of 2.28 (SD = 0.584), nodules from Sidi Bouzid had an average SDI of 7.32 (SD = 0.389) and nodules from Medenine had an SDI of 3.82 (SD = 0.394) (Supplementary Figure 2B). Beta Diversity Beta diversity was analyzed by measuring the weighted and unweighted UniFrac distances (Lozupone and Knight, 2005). An Unweighted Pair Ground Method with Arithmetic Mean (UPGMA) consensus tree was built from the unweighted UniFrac distance matrix (Figure 4). Numbers on the nodes of the tree represent the percentage of trees out of 100 bootstrapped trees that had the conﬁguration of the ﬁnal consensus tree. The grouping on the tree shows that nodule samples from all sites form one clade when analyzed by the presence or absence of taxa in the samples, after removing one outlier (Sample ID: N3SIDI). Diversity of the Casuarina Nodule Community and Surrounding Soil Three distinct sites in Tunisia (Figure 1) were chosen to assess the eﬀect of environmental conditions on the microbial community structure and diversity for Casuarina nodules, rhizosphere and surrounding soil. These sites represented a humid well- developed forest at Tamra, a semi-arid, poorly evolved saline soils of Sidi Bouzid and an arid, poorly evolved saline soils of Medenine. Figure 2 shows representative Casuarina stands from each of these sites and nodules from those stands. There were no obvious diﬀerences in nodule shape or size among the three sites and the plants exhibited the same level of general health. After correction for 16S operon copy number, the relative abundance of taxa present in the root nodules was analyzed. At the humid sampling site, the relative abundance of Frankia was an average of 80% of the nodule community. This prevalence dropped to 1.5% and 0.5% and the semi-arid and arid sampling sites, respectively. These data are like the shotgun sequencing data (Supplementary Figure 3), where reads mapping to the binned Frankia genome comprised 60% of the humid site read set and that level dropped to 1.71% and 1.63% for the semi-arid and arid sampling sites, respectively. Supplementary Figure 3 shows the relative abundance of Frankia at the three sampling sites using the shotgun and amplicon data. Figure 3A gives a taxonomic summary of the nodule samples at the class level. The taxonomic summary of the rhizosphere and bulk soils is shown in Figure 3B. Preprocessing of the 16S rRNA gene amplicon sequences resulted in a total of 7,855 OTUs from the 149,879 paired reads. Individual library sizes ranged from 1,762 to 11,542 reads per sample, with a mean of 5,551 reads per sample. Rarefaction plots were performed in order to estimate the completeness of community sampling at the sequencing depth performed. The rarefaction plots for average observed OTUs per sample type is shown in Supplementary Figure 1. These plots show that the nodule samples were sequenced close to saturation, while increased sequencing depth could improve the documentation of rare OTUs for rhizosphere and soil samples. Alpha Diversity The Shannon Diversity Index (SDI) was used to measure alpha diversity. This metric was measured on the relative abundance after correction for 16S operon copy number. Diversity measures were grouped by sample type and are as follows; Nodules: 4.47 ± 2.29 (SD), Rhizosphere: 9.33 ± 0.233 (SD) and Soil: December 2021 \| Volume 12 \| Article 749760 Frontiers in Microbiology \| www.frontiersin.org 6 Metagenomic Analysis of Actinorhizal Root Nodule Ghodhbane-Gtari et al. FIGURE 3 \| Class level taxonomic summary of nodule, Rhizosphere and soil samples. (A) Nodules grouped by site. The relative abundance of the nodule samples was summarized at the class level after corrected for 16S operon copy number variation. The humid and arid samples are signiﬁcantly less diverse than the semi-arid samples, as can be seen in (A). This can be seen here, as the humid site is dominated by Actinobacteria (the genus Frankia), and the arid site is dominated by Acinetobacter and Pseudomonas. (B) Rhizosphere and soil samples grouped by sampling site. These charts show relative abundance of taxa at the class level after correction for 16S operon copy number FIGURE 3 \| Class level taxonomic summary of nodule, Rhizosphere and soil samples. (A) Nodules grouped by site. The relative abundance of the nodule samples was summarized at the class level after corrected for 16S operon copy number variation. The humid and arid samples are signiﬁcantly less diverse than the semi-arid samples, as can be seen in (A). This can be seen here, as the humid site is dominated by Actinobacteria (the genus Frankia), and the arid site is dominated by Acinetobacter and Pseudomonas. (B) Rhizosphere and soil samples grouped by sampling site. These charts show relative abundance of taxa at the class level after correction for 16S operon copy number. vel taxonomic summary of nodule, Rhizosphere and soil samples. (A) Nodules grouped by site. The relative abundance of the nodule samples he class level after corrected for 16S operon copy number variation. The humid and arid samples are signiﬁcantly less diverse than the semi-arid seen in (A). This can be seen here, as the humid site is dominated by Actinobacteria (the genus Frankia), and the arid site is dominated by seudomonas. (B) Rhizosphere and soil samples grouped by sampling site. These charts show relative abundance of taxa at the class level after FIGURE 3 \| Class level taxonomic summary of nodule, Rhizosphere and soil samples. Alpha Diversity (A) Nodules grouped by site. The relative abundance of the nodule samples was summarized at the class level after corrected for 16S operon copy number variation. The humid and arid samples are signiﬁcantly less diverse than the semi-arid samples, as can be seen in (A). This can be seen here, as the humid site is dominated by Actinobacteria (the genus Frankia), and the arid site is dominated by Acinetobacter and Pseudomonas. (B) Rhizosphere and soil samples grouped by sampling site. These charts show relative abundance of taxa at the class level after correction for 16S operon copy number. what taxa they host when compared to the soil and rhizosphere samples. The rhizosphere and soil primarily cluster based oﬀ the sampling location, this trend is also evident in ordinations This result shows that although the relative abundance of taxa within the nodule diﬀered from sampling location (Figure 3 and Supplementary Figure 3), the nodules are distinct based on December 2021 \| Volume 12 \| Article 749760 Frontiers in Microbiology \| www.frontiersin.org 7 Metagenomic Analysis of Actinorhizal Root Nodule Ghodhbane-Gtari et al. FIGURE 4 \| Unweighted pair group method with Arithmetic Mean (UPGMA) Consensus Tree. The tree was constructed from the rareﬁed unweighted UniFrac distance Matrix. The consensus tree was built by creating 100 trees from 100 randomly subsampled reads from each sample. The numbers on the nodes represent the percentage of the 100 trees that conformed to this conﬁguration. The red box outlines that all nodule samples form one cluster on the tree. The other clades on the tree show that the soil and rhizosphere communities cluster mostly by sampling site. FIGURE 4 \| Unweighted pair group method with Arithmetic Mean (UPGMA) Consensus Tree. The tree was constructed from the rareﬁed unweighted UniFrac distance Matrix. The consensus tree was built by creating 100 trees from 100 randomly subsampled reads from each sample. The numbers on the nodes represent the percentage of the 100 trees that conformed to this conﬁguration. The red box outlines that all nodule samples form one cluster on the tree. The other clades on the tree show that the soil and rhizosphere communities cluster mostly by sampling site. of the data using the unweighted UniFrac distance metric (Supplementary Figure 4). type using the both forms of the UniFrac distance metric. Supplementary Table 4 summarizes the chance-corrected results of the MRPP analysis. Alpha Diversity The A Statistics, the chance-corrected within-group agreement, is a value between 1 and 0 that describes the within-group heterogeneity of a given group. If all samples in a group are identical A = 1, if the heterogeneity in a group is equal to what would be expected by chance A = 0 (Mielke and Kenneth, 2007). The beta diversity distance matrices were ordinated using PCoA. Distance matrices were constructed using the weighted and unweighted UniFrac distances on OTU tables that had been normalized by rarefaction, a raw OTU table with ribosomal operon copy number correction and by cumulative sum-scaling (with and without ribosomal operon copy number correction). PCoA ordinations of these distance matrixes can be seen in Figure 5 and Supplementary Figures 4, 5. For the sake of clarity, Figure 5 represents weighted UniFrac distances normalized by cumulative sum-scaling without ribosomal operon copy number correction. All of the methods gave similar results (Supplementary Figure 4). In all ordinations, nodule samples are separated from the soil and rhizosphere samples along the ﬁrst Principal Coordinate, which explains the most phylogenetic variation in the data. Multi-response Permutation Procedures (MRPP) agreed with the results seen in the UPGMA tree, with signiﬁcant grouping of nodules for every normalization Frontiers in Microbiology \| www.frontiersin.org Metagenomic Assembled Genomes Reconstructed Using Differential Coverage Binning FIGURE 5 \| Principal coordinates ordination of CSS normalized UniFrac distance matrices: Ordinations where normalized using Cumulative Sum Scaling. Ordinations was made using the weighted UniFrac distance. Symbols represent: nodules (•), rhizosphere (■), and soil (▲). For all ordinations MRPP showed a statistically signiﬁcant grouping of the nodule samples, as outlined in Supplementary Table 4. Seven predominant genomes were binned using the mmgenome R package. A plot of the metagenomic assembly can be seen in Figure 6A. The binned genomes were identiﬁed being in the genera Frankia, Micromonospora, Bacillus (two isolates), Phyllobacterium, Paenibacillus, and Aﬁpia. Figure 5, the ordinations show the grouping of nodule samples by location along the second principal coordinate, which explains the second largest amount of taxonomic variation in the dataset. Phyllobacterium, Paenibacillus, and Aﬁpia. Two of the binned genomes annotated to the phylum Actinobacteria. The ﬁrst genome bin contained 4,111 ORFs that annotated to the genus Frankia. The annotations for the ORFs in this bin had top hits on many of the Frankia clade 1c Casuarina infective strains. The most top annotations that hit on a single sequenced strain was 775 of the 4,111 ORFs annotated to Frankia sp. BMG5.23, a salt-tolerant Tunisian isolate (Ghodhbane-Gtari et al., 2014). Reads mapping to the binned Frankia draft genome comprised 60% of the reads in from the humid sampling site, Tamra. Of the reads from semiarid Sidi Bouzid and arid Gabes, 1.7% and 1.6% of the reads mapped to the Frankia genome bin, respectively. Genome bin two resulted in an assembly that contained 6,522 ORFs with 6,053 annotating to the genus Micromonospora. The ORFs that annotated to Micromonospora were mainly comprised of annotations to Micromonospora purpureochromogenes, with 4,565 ORFs. Three genomes that were binned annotated to the phylum Firmicutes. The ﬁrst contained 6,268 ORFs with 5,866 of them annotating to the genus Paenibacillus. The second Firmicutes genome bin contained 4,275 ORFs, 4,060 of which annotated to the genus Bacillus. Of those ORFs annotating to the genus Bacillus, 3,416 of them annotated to B. simplex. The last Firmicutes genome had 4,215 ORFs that also annotated to the genus Bacillus with 3,313 of them annotating to B. aquimaris and 569 annotating to B. vietnamensis. Two of the binned genomes annotated to the phylum proteobacteria. The ﬁrst binned genome contained 4,370 ORFs that annotated to the genus Aﬁpia. The second proteobacteria genome bin contained 3,068 ORFs that annotated to the genus Phyllobacterium. Shotgun Metagenomic Sequencing Quality ﬁltering of shotgun metagenomic reads resulted in a total of 83,027,118 paired end reads. The library for Sites 1, 2, and 3 were comprised of 33,051,352, 34,239,886, and 15,735,880 reads, respectively. In silico co-assembly of the reads resulted in an assembly contained 3,153,711 contigs totaling 56,964,841 base pairs in length. The statistics for the assembly produced by Quast (Gurevich et al., 2013) are outlined in Supplementary Table 7. Metagenomic Assembled Genomes Reconstructed Using Differential Coverage Binning General genome characteristics of the binned genomes are summarized in Table 1. Nodule Analysis The results of the UPGMA Tree (Figure 4) and the PCoA ordinations (Figure 5 and Supplementary Figures 4, 5) show that nodules host a distinct prokaryotic community compared to the soil and rhizosphere samples. Ordination and MRPP results show that this grouping is statistically signiﬁcant whether the unweighted UniFrac or weighted UniFrac is used. Nodules form a distinct group based on the taxa they host, but the relative composition of this group of taxa changes drastically along the environmental gradient sampled (Figure 3A). In December 2021 \| Volume 12 \| Article 749760 Frontiers in Microbiology \| www.frontiersin.org 8 Metagenomic Analysis of Actinorhizal Root Nodule Ghodhbane-Gtari et al. FIGURE 5 \| Principal coordinates ordination of CSS normalized UniFrac distance matrices: Ordinations where normalized using Cumulative Sum Scaling. Ordinations was made using the weighted UniFrac distance. Symbols represent: nodules (•), rhizosphere (■), and soil (▲). For all ordinations MRPP showed a statistically signiﬁcant grouping of the nodule samples, as outlined in Supplementary Table 4. sites (Sidi Bouzid and Medenine). For all normalization types, a signiﬁcant grouping was identiﬁed by MRPP (Supplementary Table 6), showing that the environmental conditions signiﬁcantly change the taxa present between the humid and arid soils that were sampled. A taxonomic summary of these samples at the class level are presented in Figure 3B. Frontiers in Microbiology \| www.frontiersin.org Coverage Values for Genome Bins A remarkable change in microbial community dominance was observed across climate stages. In the humid climate zone, represented by Tamra site, Actinobacteria was the most dominant class, while the semiarid (Sidi Bouzid) and arid (Medenine) environments had Alphaproteobacteria and Gammaproteobacteria, respectively, as the most prominent class. This result was surprising because Frankia is Actinobacteria and would be expected to dominate the nodule environment. Coverage Values for Genome Bins Genome coverage for binned genomes was calculated in order to compare coverage patterns across sites to the replicated 16S rRNA gene amplicon survey. The only binned genome which followed the same patterns as the replicated analysis was the genome bin that annotated as a member of Frankia (Figure 6A) compared to the relative abundance of Frankia between the two datasets. For those calculations, reads that aligned to plant- derived DNA was ﬁrst removed. The resulting read sets for each site were aligned to the reassembled Frankia genome bin using Bowtie2. The percentage of those reads that aligned to the genome bin was recorded. The Frankia genome bin was the dominant member of the humid site, but coverage values were much lower for the semi-arid and arid sampling sites (Figure 6B). However, the relative abundance of Frankia was drastically aﬀected by the environmental gradient. Nodule occupancy was highest (80%) in plants at the Tamra (humid zone); but was drastically reduced in nodules from the semi-arid Sidi Bouzid (1.5%) and arid Medenine (0.5%). One possible explanation of this phenomenon is that the physical and chemical characteristics of semi-arid and arid environments are not beneﬁcial for the maintenance of the actinorhizal symbiosis or Frankia persistence. Soils in the semi-arid and arid regions of Tunisia are poorly evolved calcareous soils (Mtimet, 2001). In these soils, phosphorous is known to be immobilized by formation of complexes with calcium cations (Tunesi et al., 1999). Low phosphorus levels decrease the frequency of C. cunninghamiana nodulation by Frankia (Yang, 1995) and nodule weight of Frankia-C. equisetifolia (Sanginga et al., 1989). Similarly, drying and heat are known to decrease the ability of some Frankia strains to infect C. equisetifolia (Sayed, 2011). Dryness, high temperature and low levels of available phosphorous are characteristics of soils in arid environments and these factors could be inﬂuencing the low abundance and absence of Frankia in nodules at these environments. Coverage Values for Genome Bins Detecting Casuarina infective clades of Frankia through plant-trapping assays have proven diﬃcult (Gtari et al., 2002, 2007) and the scarcity of detection of Casuarinaceae- infective Frankia outside of the plants native range has been demonstrated (Mirza et al., 2007). One possible explanation is that Casuarina-infective Frankia are more persistent in humid environments that are more similar to the riparian habitat that is preferred by Casuarina within their host range. These results are congruent with the need for intentional inoculation with a compatible Frankia strain necessary for nodulation in some land reclamation projects in Africa (Gauthier et al., 1985). However, this reasoning does not explain the absence of Frankia within mature nodules. The absence of detected Frankia in four of the nine nodule samples leads to one hypothesis that another member of the community may be responsible for the formation of nodules on C. glauca. Agrobacterium rhizogenes will induce pseudonodules on Elaeagnus angustifolia that are indistinguishable from Frankia – induced nodules (Berg et al., 1992). Our results show an OTU of genus Agrobacterium that was as diﬀerentially abundant in nodule samples. The genus Agrobacterium was present in eight of the nine nodule samples ranging from 0.01% to 2.8% relative abundance. This suggests that a member of this genus could play a role in nodule formation when Frankia is in low abundance. Another hypothesis is that this absence is a seasonal variation and part of the life cycle of Frankia under semi-arid and arid conditions. As the levels of Frankia decreases, other microbes could establish more dominance within the nodule. A seasonal study of the changes in community structure could address this hypothesis. Functional Mining of Metagenomes The shotgun metagenomic data was mined for genes of interest with potential functional importance to the actinorhizal symbiosis and C. glauca health. Genes potentially responsible for plant–growth-promoting traits were mined from GFF ﬁles produced by the Prokka Annotation pipeline (Seemann, 2014). Figures 7–9 show the results of this analysis as outlined by site. Cladograms for the taxonomy of the genes identiﬁed are presented. The number of genes for each function is reported in each ﬁgure. The sampling sites 1, 2, and 3 had 159, 183, and 202 genes of interest, respectively. Since shotgun sequencing was not performed with replicates, these results are presented qualitatively as presence or absence data. Secondary Metabolites Identiﬁed in Binned Genomes The MAGs reconstructed from the metagenomic dataset were analyzed for the presence of secondary metabolite biosynthetic pathways using AnitSMASH (Weber et al., 2015). The secondary metabolite biosynthetic clusters that were identiﬁed in the binned genomes are outlined in Table 2. Predicted biosynthetic clusters from the binned genomes were predicted to produce siderophores, terpenes, non-ribosomal peptide synthases and other secondary metabolites. The genome Frankia and Micromonospora genome bins contained the most secondary metabolite biosynthetic clusters about 25 for each bin genome, although only clusters which have a predicted product are listed here. Rhizosphere and Soil Analysis To understand diﬀerences in the rhizosphere and soil communities across the sampling sites, these samples were analyzed separately from the nodule samples. Rhizosphere soils from all sites were analyzed as one sample category to test the hypothesis that C. glauca hosts a distinct microbial community within the rhizosphere across environmental gradients. When soil and rhizosphere samples from all sites were grouped together by type, the MRPP results were inconsistent in their signiﬁcance, depending on the OTU table normalization strategy. A summary of these results is found in Supplementary Table 5. Statistical signiﬁcance (P < 0.05) was only observed with the evenly rareﬁed OTU table and the raw OUT table that corrected for 16S operon copy number Variation. Ordinations of these data (Supplementary Figures 4, 5) show that the rhizosphere and soil samples formed two clusters only on the raw OTU table and the evenly rareﬁed OTU table. CSS normalization produces diﬀerent results with the soil and rhizosphere samples not forming distinct groups. The ordinations using the unweighted UniFrac distance, which considers the presence and absence of taxa, show that soil and rhizosphere samples clustered based on climate. When the data is ordinated using this metric, two distinct clusters of rhizosphere and soil samples formed Supplementary Figure 6. These clusters correspond to the humid sampling site (Tamra) and the two arid sampling December 2021 \| Volume 12 \| Article 749760 Frontiers in Microbiology \| www.frontiersin.org 9 Metagenomic Analysis of Actinorhizal Root Nodule Ghodhbane-Gtari et al. Prominent Microbial Groups of Nodules, Rhizosphere, and Soil Associated to Casuarina glauca The 16S rRNA gene amplicon analysis of the taxonomic community in nodules showed that they were largely dominated by Actinobacteria, Alphaproteobacteria, and Gammaproteobacteria (Figure 3). The remaining sequences matched with Bacilli, Planctomycetia and Deltaproteobacteria. December 2021 \| Volume 12 \| Article 749760 Frontiers in Microbiology \| www.frontiersin.org 10 Metagenomic Analysis of Actinorhizal Root Nodule Ghodhbane-Gtari et al. FIGURE 6 \| (A) Plot of metagenomic contigs larger than 5 kbp produced by the mmgenome R Package. The seven dominant genomes that were binned using the mmgenome R package are labeled with their annotated genus. The purple and black cluster in the center of the image are contigs derived from C. glauca DNA. (B) Normalized genome coverage of genome bins. Reads from each of the three sampling sites were aligned to the co-assembled contigs. SAMtools was used to sort and index the coordinates of the aligned reads and Picard MarkDuplicates was used to remove duplicate reads with identical coordinates. Bedtools was then used to calculate coverage per contig per site. Coverage values were normalized by millions of reads per data set and the relative amount of plant DNA that comprised each data set. FIGURE 6 \| (A) Plot of metagenomic contigs larger than 5 kbp produced by the mmgenome R Package. The seven dominant genomes that were binned using the mmgenome R package are labeled with their annotated genus. The purple and black cluster in the center of the image are contigs derived from C. glauca DNA. (B) Normalized genome coverage of genome bins. Reads from each of the three sampling sites were aligned to the co-assembled contigs. SAMtools was used to sort and index the coordinates of the aligned reads and Picard MarkDuplicates was used to remove duplicate reads with identical coordinates. Bedtools was then used to calculate coverage per contig per site. Coverage values were normalized by millions of reads per data set and the relative amount of plant DNA that comprised each data set. TABLE 1 \| General assembly characteristics of each binned genome. Isolate genus Closest annotation Assembly length Open reading frames GC% Frankia Frankia sp. BMG5.23 4.92 Mbp 4,111 70.25% Micromonospora M. purpureochromogenes 7.28 Mbp 6,522 72.26% Bacillus (1) B. simplex 4.64 Mbp 4,060 40.0% Bacillus (2) B. aquimaris/vietnamensis 4.7 Mbp 4,215 41.53% Phyllobacterium Phyllobacterium sp. UNC302MFCol5.2 4.8 Mbp 3,068 59.42% Aﬁpia A. broomeae 5.3 Mbp 4,370 61.64% Paenibacillus Paenibacillus sp. Prominent Microbial Groups of Nodules, Rhizosphere, and Soil Associated to Casuarina glauca HGF5 7.0 Mbp 5,866 49.56% The assembly length and GC content were calculated by Quast. Open reading frames were predicted using Prodigal. Closest annotations were determined after aligning all ORFs to the RefSeq protein database. TABLE 1 \| General assembly characteristics of each binned genome. The assembly length and GC content were calculated by Quast. Open reading frames were predicted using Prodigal. Closest annotations were determined after alignin all ORFs to the RefSeq protein database. Distinct Nodule Community Across Sampling Sites types are highly overlapping and less diverse than surrounding soils (Lundberg et al., 2012). The community selected by C. glauca was distinct from the soil and rhizosphere communities across a steep environmental gradient and was signiﬁcantly less diverse, which is like the pattern of the A. thaliana microbiome. The similar patterns of microbiome recruitment by C. glauca and A. thaliana suggests that phylogenetically distant plant groups tend to select for a particular endophytic community across environmental gradients. Multi-response permutation procedures analysis found that nodule samples formed a statistically signiﬁcant group compared to the rhizosphere and soil samples (Figure 5 and Supplementary Figures 4–6). Nodule samples are separated from the rhizosphere and soil samples by the ﬁrst principal coordinate axis, which explains the most taxonomic information in the samples. This result is also conﬁrmed with the jackknifed UPGMA tree built from the unweighted UniFrac distance matrix (Figure 4), which shows a distinct clade where all nodule samples clustered, despite location. Alpha diversity of the nodule samples was also signiﬁcantly lower than for the rhizosphere or soil samples (Supplementary Figure 1). These observations are similar to comparative analysis of the root microbiome of the model plant Arabidopsis thaliana, which has shown that endophytic communities from plants growing in chemically distinct soil Frontiers in Microbiology \| www.frontiersin.org Functions of Taxa in Nodules From metagenome analysis, seven predominant genomes were identiﬁed by binning (Figure 6) and were assigned to six diﬀerent genera: Frankia, Micromonospora, Bacillus (two isolates), Phyllobacterium, Paenibacillus, and Aﬁpia. Reads from the humid sampling site Tamra, contains the most reads mapping to the binned Frankia draft genome (60%), then December 2021 \| Volume 12 \| Article 749760 Frontiers in Microbiology \| www.frontiersin.org 11 Metagenomic Analysis of Actinorhizal Root Nodule Ghodhbane-Gtari et al. TABLE 2 \| Secondary metabolite biosynthetic gene clusters identiﬁed each genome bin by antiSMASH. Bin Genus Type Most similar known cluster Percent identity Frankia Terpene Sioxanthin 60% Type-Two PKS Frankiamicin 100% Lassopeptide- Type-One PKS Maklamicin 13% Terpene Hopene 38% Other Streptolydigin 7% NRPS Triostin 16% Type-One PKS Divergolide 13% Terpene Collismycin A 7% Type-Two PKS Medermycin 30% Ketide Synthase- Butyrolactone Abyssomicin 10% Other A47934 10% Type-Three Polyketide Synthase Feglymycin 52% Micromonospora T1PKS Maklamicin 19% NRPS-T1PKS Nostopeptolide 37% Other Diazepinomicin 75% Oligosaccharide- PKS-Terpene Brasilicardin A 54% Siderophore Desferrioxamine B 80% T2PKS Xantholipin 16% Terpene Nocathiacin 4% Lantipeptide Pentalenolactone 15% NRPS Friulimicin 12% T3PKS Alkyl-O- Dihydrogeranyl- Methoxyhydroquinone 71% Nrps-T1pks- Lantipeptide Bleomycin 12% Terpene Sioxanthin 80% Nrps-Lantipeptide- T1pks-Others Naphthyridinomycin 14% T1PKS Calicheamicin 13% Terpene- Bacteriocin Lymphostin 30% T1pks-Nrps Rifamycin 35% T1pks Leucanicidin 100% Paenibacillus Ectoine Ectoine 75% Nrps Bacillibactin 53% Trans-At Pks Bacillaene 21% Bacillus (1) Siderophore Desotamide 9% Nrps Koranimine 87% Bacillus (2) Terpene Carotenoid 33% Siderophore 16% Aﬁpia Terpene Malleobactin 11% Phyllobacterium Nrps Vicibactin 77% TABLE 2 \| Secondary metabolite biosynthetic gene clusters identiﬁed each genome bin by antiSMASH. surprisingly, the Frankia MAG closely matched the Frankia sp. strain BMG5.23 genome (Ghodhbane-Gtari et al., 2014). This strain was isolated from Tunisian soils and probably represents the local strain in this region. By the use of the antiSMASH program (Blin et al., 2017), secondary metabolite biosynthetic pathways were predicted from the draft genomes rebuilt from the metagenomic data set (Table 2). Not surprisingly, the Actinobacteria, Frankia and Micromonospora, genomes contained the most secondary metabolite biosynthetic clusters. Actinobacteria are well known for their large assembly of secondary metabolic pathways producing a wide array of natural products. p g y p The presence of non-Frankia bacteria within the nodules conﬁrms other observations (Guillén et al., 1993; Niner et al., 1996; Solans and Vobis, 2003; Valdes et al., 2005; Trujillo et al., 2006; Solans, 2007; Ghodhbane-Gtari et al., 2010, 2014, 2019; Solans et al., 2011). Functions of Taxa in Nodules In particular, the presence of Micromonospora is noteworthy having been identiﬁed as an endophyte of actinorhizal and leguminous plants (Trujillo et al., 2006, 2007; Solans et al., 2011) and considered a normal occupant of actinorhizal nodules (Carro et al., 2013). Two diﬀerent Bacillus genomes were identiﬁed with one being closely related to B. simplex and the other being closely related to B. aquimaris and B. vietnamensis. Bacillus simplex is known for its adaptations to arid environments (Sikorski and Nevo, 2005) and has been shown to acts as a helper-bacteria for the symbiosis between Pisum sativum and Rhizobium leguminosarum bv. viciae (Schwartz et al., 2013). Bacillus isolates are helper-bacteria for the Frankia- Casuarina symbiosis (Echbab et al., 2004). Paenibacillus genome revealed secondary metabolic clusters for siderophore production, ectoine, (an osmoprotectant), and bacillaene (an antibiotic). In a nursery trail, co-inoculation of C. equisetifolia with Paenibacillus polymyxa and Glomus geosporum results in the highest seedling quality (Muthukumar and Udaiyan, 2010), showing the potential beneﬁts of this nodule occupant. One hypothesis is that these non-Frankia Actinobacteria act as helper-bacteria in the formation of the actinorhizal symbiosis (Ghodhbane-Gtari et al., 2010, 2019; Solans et al., 2011; Ghodhbane-Gtari and Tisa, 2014). The absence of Nocardia within these nodule communities was surprising. Nocardia strains (BMG51109 and BMG111209) were isolated from Casuarina glauca in Tunisia (Ghodhbane-Gtari et al., 2010) and shown to have plant-growth promoting properties (Ghodhbane-Gtari et al., 2019). Co-infection studies showed that Nocardia casuarinae strain BMG51109 plays a role as a “helper bacteria” promoting an earlier onset of nodulation (Ghodhbane-Gtari et al., 2019). Besides generating metagenomes, the shotgun data set was functionally mined to identify potential plant-growth-promoting genes (PGPG) and to phylogenetically analyze these data (Figures 7–9) suggesting a wide berth of potential PGPG genetic capability for plant growth promotion. Besides this phylogenetic analysis, these coding regions were predicted to produce enzymes and metabolites of relevance to the beneﬁting plant health. All three metagenomes contained the 1-aminocyclopropane-1- carboxylate (ACC) deaminase gene. ACC deaminase plays an important role in the nodulation process of leguminous plants Sidi Bouzid (1.7%) and Medenine (1.6%). The less abundance of Frankia in semi-arid and arid sites can be related to the harsh conditions, dryness and poor composition of soils. Not Sidi Bouzid (1.7%) and Medenine (1.6%). The less abundance of Frankia in semi-arid and arid sites can be related to the harsh conditions, dryness and poor composition of soils. Functions of Taxa in Nodules Not December 2021 \| Volume 12 \| Article 749760 Frontiers in Microbiology \| www.frontiersin.org 12 Metagenomic Analysis of Actinorhizal Root Nodule Ghodhbane-Gtari et al. FIGURE 7 \| Cladograms of functional genes from Site 1 (Tamra), that were produced by the LCA Algorithm in MEGAN (Version 5). The sizes of colored circles represent that relative contribution of a taxa to the functional genes analyzed in a given cladogram. Numbers in Brackets represent the number of genes that were annotated to a given taxon for that set of genes. (A) Potential indole acetic acid biosynthetic genes. Total genes, 24. (B) Cellulose degrading genes. Total genes identiﬁed, 89. (C) Alkaline phosphatase genes. Total genes identiﬁed, 26. (D) ACC deaminase genes. Total genes Identiﬁed, 2. (E) Chitin degrading genes. Total genes identiﬁed, 14. (F) Pectinase genes. Total genes identiﬁed, 2. (G) Phytase genes. Total genes identiﬁed, 2. FIGURE 7 \| Cladograms of functional genes from Site 1 (Tamra), that were produced by the LCA Algorithm in MEGAN (Version 5). The sizes of colored circles represent that relative contribution of a taxa to the functional genes analyzed in a given cladogram. Numbers in Brackets represent the number of genes that were annotated to a given taxon for that set of genes. (A) Potential indole acetic acid biosynthetic genes. Total genes, 24. (B) Cellulose degrading genes. Total genes identiﬁed, 89. (C) Alkaline phosphatase genes. Total genes identiﬁed, 26. (D) ACC deaminase genes. Total genes Identiﬁed, 2. (E) Chitin degrading genes. Total genes identiﬁed, 14. (F) Pectinase genes. Total genes identiﬁed, 2. (G) Phytase genes. Total genes identiﬁed, 2. FIGURE 7 \| Cladograms of functional genes from Site 1 (Tamra), that were produced by the LCA Algorithm in MEGAN (Version 5). The sizes of colored circles represent that relative contribution of a taxa to the functional genes analyzed in a given cladogram. Numbers in Brackets represent the number of genes that were annotated to a given taxon for that set of genes. (A) Potential indole acetic acid biosynthetic genes. Total genes, 24. (B) Cellulose degrading genes. Total genes identiﬁed, 89. (C) Alkaline phosphatase genes. Total genes identiﬁed, 26. (D) ACC deaminase genes. Total genes Identiﬁed, 2. (E) Chitin degrading genes. Total genes identiﬁed, 14. (F) Pectinase genes. Total genes identiﬁed, 2. (G) Phytase genes. Total genes identiﬁed, 2. Functions of Taxa in Nodules (Nascimento et al., 2019) and has been shown to improve plant growth under arid (Bessadok et al., 2020) or saline regions (Orozco-Mosqueda et al., 2020). Other major functions involved in nutrient recycling, suppressing phytopathogens and mineralization of organic matter were also highly abundant within all three metagenomes. The enzymes, chitinase, phytase, cellulase, pectinase and alkaline phosphatase are well known to be involved in the preservation of the plant fertility and maintaining the healthy plant growth (Neeraja et al., 2010; Maksimov et al., 2011; Jain and Singh, 2017). These hydrolytic enzymes serve as an important defense strategy to defend against phytopathogenic fungi (Lynd et al., 2002). These enzymes may serve to help the Casuarina to survive from fungal pathogens. Genes for production of phytohormones were also present at all three sites in variable proportions. Genes for IAA and PAA December 2021 \| Volume 12 \| Article 749760 Frontiers in Microbiology \| www.frontiersin.org 13 Metagenomic Analysis of Actinorhizal Root Nodule Ghodhbane-Gtari et al. Ghodhbane Gtari et al. Metagenomic Analysis of Actinorhizal Root Nodu FIGURE 8 \| Cladograms of functional genes from Site 2 (Sidi Bouzid), that were produced by the LCA Algorithm in MEGAN (Version 5). The sizes of colored circles represent that relative contribution of a taxa to the functional genes analyzed in a given cladogram. Numbers in Brackets represent that number of genes that annotated to a given taxon for that set of genes. (A) Phytase genes. Total genes identiﬁed, 8. (B) Pectinase enzymes. Total genes identiﬁed, 5. (C) ACC deaminase genes. Total genes identiﬁed, 3. (D) Potential indole acetic acid biosynthesis genes. Total genes identiﬁed, 27. (E) Cellulose degrading enzymes. Total genes identiﬁed, 97. (F) Chitin degrading enzymes. Total genes identiﬁed, 25. (G) Potential phenyl acetic acid biosynthesis genes. Total genes identiﬁed, 3. (H) Alkaline phosphatase genes. Total genes identiﬁed, 15. production were signiﬁcantly enriched in the semi-arid and arid Sugawara et al., 2015). The increased number of IAA and PA FIGURE 8 \| Cladograms of functional genes from Site 2 (Sidi Bouzid), that were produced by the LCA Algorithm in MEGAN (Version 5). The sizes of colored circles represent that relative contribution of a taxa to the functional genes analyzed in a given cladogram. Numbers in Brackets represent that number of genes that annotated to a given taxon for that set of genes. (A) Phytase genes. Total genes identiﬁed, 8. (B) Pectinase enzymes. Total genes identiﬁed, 5. Functions of Taxa in Nodules (C) ACC deaminase genes. Total genes identiﬁed, 3. (D) Potential indole acetic acid biosynthesis genes. Total genes identiﬁed, 27. (E) Cellulose degrading enzymes. Total genes identiﬁed, 97. (F) Chitin degrading enzymes. Total genes identiﬁed, 25. (G) Potential phenyl acetic acid biosynthesis genes. Total genes identiﬁed, 3. (H) Alkaline phosphatase genes. Total genes identiﬁed, 15. Sugawara et al., 2015). The increased number of IAA and PAA genes suggest that these phytohormones may alter the plant physiology to respond to these arid conditions. production were signiﬁcantly enriched in the semi-arid and arid climates. IAA and PAA play a key role in many aspects of plant growth and cell elongation and division (Champion et al., 2015; December 2021 \| Volume 12 \| Article 749760 Frontiers in Microbiology \| www.frontiersin.org 14 Metagenomic Analysis of Actinorhizal Root Nodule Ghodhbane-Gtari et al. dhbane-Gtari et al. Metagenomic Analysis of Actinorhizal Root Nodul GURE 9 \| Cladograms of functional genes from Site 3 (Medenine), that were produced by the LCA Algorithm in MEGAN (Version 5). The sizes of colored circles present that relative contribution of a taxa to the functional genes analyzed in a given cladogram. Numbers in Brackets represent that actual number of genes that nnotated to a given taxon for that set of genes. (A) Cellulose degrading genes. Total genes identiﬁed, 108. (B) Alkaline phosphatase genes. Total genes identiﬁed, 9. (C) Potential indole acetic acid biosynthetic genes. Total genes identiﬁed, 30. (D) Potential phenyl acetic acid biosynthesis genes. Total genes identiﬁed, 5. ) ACC deaminase genes. Total genes identiﬁed, 6. (F) Chitin degrading genes. Total genes identiﬁed, 24. (G) Pectinase genes. Total genes identiﬁed, 8. H) Phytase genes. Total genes identiﬁed, 2. FIGURE 9 \| Cladograms of functional genes from Site 3 (Medenine), that were produced by the LCA Algorithm in MEGAN (Version 5). The sizes of colored circles represent that relative contribution of a taxa to the functional genes analyzed in a given cladogram. Numbers in Brackets represent that actual number of genes that annotated to a given taxon for that set of genes. (A) Cellulose degrading genes. Total genes identiﬁed, 108. (B) Alkaline phosphatase genes. Total genes identiﬁed, 19. (C) Potential indole acetic acid biosynthetic genes. Total genes identiﬁed, 30. (D) Potential phenyl acetic acid biosynthesis genes. Total genes identiﬁed, 5. (E) ACC deaminase genes. Total genes identiﬁed, 6. (F) Chitin degrading genes. Total genes identiﬁed, 24. (G) Pectinase genes. Total genes identiﬁed, 8. REFERENCES Caporaso, J. G., Lauber, C. L., Walters, W. A., Berg-Lyons, D., Huntley, J., Fierer, N., et al. (2012). Ultra-high-throughput microbial community analysis on the illumina HiSeq and MiSeq platforms. ISME J. 6, 1621–1624. doi: 10.1038/ismej. 2012.8 Albertsen, M., Hugenholtz, P., Skarshewski, A., Nielsen, K. L., Tyson, G. W., and Nielsen, P. H. (2013). Genome sequences of rare, uncultured bacteria obtained by diﬀerential coverage binning of multiple metagenomes. Nat. Biotechnol. 31, 533–538. doi: 10.1038/nbt.2579 Carro, L., Pujic, P., Trujillo, M. E., and Normand, P. (2013). Micromonospora is a normal occupant of actinorhizal nodules. J. Biosciences 38, 685–693. doi: 10.1007/s12038-013-9359-y Aronesty, E. (2011). ea-utils: “Command-Line Tools for Processing Biological Sequencing Data”. Available online at: http://code.google.com/p/ea-utils Chaia, E. E., Wall, L. G., and Huss-Danell, K. (2010). 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CT7.15 as a tool for improving Calicotome villosa nodulation and growth in Arid regions of Tunisia. FUNDING This material is based upon work supported by the New Hampshire Agricultural Experiment Station, through joint funding of the National Institute of Food and Agriculture, United States Department of Agriculture, and State of New Hampshire. This is Scientiﬁc Contribution Number 2913. This project (LT) was supported by the USDA National Institute of Food and Agriculture Hatch 1019869 (LT), College of Life Science and Agriculture at the University of New Hampshire, Durham, and Ministère de l’Enseignement Supérieur et de la Recherche Scientiﬁque, Tunisia (LR03ES03) (FG-G). This material is based upon work supported by the New Hampshire Agricultural Experiment Station, through joint funding of the National Institute of Food and Agriculture, United States Department of Agriculture, and State of New Hampshire. This is Scientiﬁc Contribution Number 2913. This project (LT) was supported by the USDA National Institute of Food and Agriculture Hatch 1019869 (LT), College of Life Science and Agriculture at the University of New Hampshire, Durham, and Ministère de l’Enseignement Supérieur et de la Recherche Scientiﬁque, Tunisia (LR03ES03) (FG-G). DATA AVAILABILITY STATEMENT The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi.nlm. nih.gov/bioproject/, PRJNA482626. CONCLUSION LT analyzed the data. FG-G, TD’A, MG, and LT wrote the manuscript. All authors approved the manuscript. LT analyzed the data. FG-G, TD’A, MG, and LT wrote the manuscript. All authors approved the manuscript. LT analyzed the data. FG-G, TD’A, MG, and LT wrote the manuscript. All authors approved the manuscript. The microbial community of the nodules appears to be shaped by the diﬀerent bioclimates found in Tunisia. Beside Frankia microsymbiont, the niche builder of root nodules, other bacteria may occur within. However, further work is required to understand the PGP mechanisms of the diﬀerent co-inhabitants of the nodule and more diverse samples from a wide range of actinorhizal plant nodules is needed to deepen this study. Functions of Taxa in Nodules (H) Phytase genes. Total genes identiﬁed, 2. December 2021 \| Volume 12 \| Article 749760 Frontiers in Microbiology \| www.frontiersin.org 15 Metagenomic Analysis of Actinorhizal Root Nodule Ghodhbane-Gtari et al. The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2021.749760/full#supplementary-material The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2021.749760/full#supplementary-material FG-G, MG, and LT conceived the study. FG-G and TD’A performed the research. 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https://openalex.org/W2903181364	https://hal.sorbonne-universite.fr/hal-01953038/document	English	null	Differentiation of haploid and diploid fertilities in Gracilaria chilensis affect ploidy ratio	BMC evolutionary biology	2,018	cc-by	9,237	To cite this version: Vasco M. N. C. S. Vieira, Aschwin H. Engelen, Oscar R. Huanel, Marie-Laure Guillemin. Differenti- ation of haploid and diploid fertilities in Gracilaria chilensis affect ploidy ratio. BMC Evolutionary Biology, 2018, 18, pp.183. 10.1186/s12862-018-1287-x. hal-01953038 Differentiation of haploid and diploid fertilities in Gracilaria chilensis affect ploidy ratio Vasco M. N. C. S. Vieira, Aschwin H. Engelen, Oscar R. Huanel, Marie-Laure Guillemin Differentiation of haploid and diploid fertilities in Gracilaria chilensis affect ploidy ratio Vasco M. N. C. S. Vieira, Aschwin H. Engelen, Oscar R. Huanel, Marie-Laure Guillemin Abstract Background: Algal isomorphic biphasic life cycles alternate between free-living diploid (tetrasporophytes) and haploid (dioicious gametophytes) phases and the hypotheses explaining their maintenance are still debated. Classic models state that conditional differentiation between phases is required for the evolutionary stability of biphasic life cycles while other authors proposed that the uneven ploidy abundances observed in the field are explained by their cytological differences in spore production. Results: We monitored the state and fate of individuals of the red seaweed Gracilaria chilensis periodically for 3 years in five intertidal pools from two sites with distinct conditions. We tested for differentiation in fecundity and spore survival among the gametophyte males and females (haploids) and the tetrasporophytes (diploids). We tested for the influence of fecundity and spore survival on the observed uneven ploidy abundances in recruits. The probability of a frond becoming fecund was size-dependent, highest for the haploid males and lowest for the haploid females, with the diploids displaying intermediate probabilities. Fecund diploids released more tetraspores than carpospores released by the haploid females. Spore survival depended on ploidy and on the local density of co-habiting adult fronds. An advantage of diploid over haploid germlings was observed at very low and very high adult fronds densities. Conclusions: Neither spore production nor spore survival determined the highly variable ploidy ratio within G. chilensis recruits. This result invalidates the hypothesis of natural cytological differences in spore production as the only driver of uneven field ploidy abundances in this species. Diploid spores (carpospores) survived better than haploid spores (tetraspores), especially in locations and time periods that were associated with the occurrence of strong biotic and abiotic stressors. We hypothesise that carpospore survival is higher due to support by their haploid female progenitors passing-on nutrients and chemical compounds improving survival under stressful conditions. Keywords: Gametophyte, Tetrasporophyte, Isomorphic biphasic life cycle, Phase ratio, Ploidy ratio, Recruitment (also known as biphasic life cycles) commonly found, for example, in many green, red and brown seaweeds [1]. In these life cycles, free-living haploid gametophyte and diploid sporophyte phases are morphologically distinct from one another (heteromorphic) to seemingly identical to each other (isomorphic). In the heteromorphic case, gametophytes and sporophytes generally display marked differences in size, morphology and physiology, and have been observed to occupy different spatio-temporal niches (see for review Thornber [4]). HAL Id: hal-01953038 https://hal.sorbonne-universite.fr/hal-01953038v1 Submitted on 12 Dec 2018 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Vieira et al. BMC Evolutionary Biology (2018) 18:183 https://doi.org/10.1186/s12862-018-1287-x Open Access Abstract Thus, it is not surprising that most developed hypotheses and models explaining the maintenance of haploid-diploid life cycles rely on the existence of some degree of niche differentiation between Vasco M. N. C. S. Vieira1* , Aschwin H. Engelen2, Oscar R. Huanel3,4 and Marie-Laure Guillemin3,5 sco M. N. C. S. Vieira1* , Aschwin H. Engelen2, Oscar R. Huanel3,4 and Marie-Laure Guillemin3,5 © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Introduction An alternation between haploid and diploid nuclear phases is a necessary consequence of eukaryotic sexual- ity. However, due to variation in the relative timing of meiosis and syngamy in life cycles, the duration of these two phases vary widely among organism [1]. Although the multicellular haploid generation went extinct in the life cycle of vascular plants roughly 400 million years ago [2, 3], it persists in the haploid-diploid life cycles * Correspondence: vasco.vieira@tecnico.ulisboa.pt 1MARETEC, Instituto Superior Técnico, Universidade Técnica de Lisboa, Av. Rovisco Pais, 1049-001 Lisbon, Portugal Full list of author information is available at the end of the article * Correspondence: vasco.vieira@tecnico.ulisboa.pt 1MARETEC, Instituto Superior Técnico, Universidade Técnica de Lisboa, Av. Rovisco Pais, 1049-001 Lisbon, Portugal Full list of author information is available at the end of the article Page 2 of 11 Page 2 of 11 Vieira et al. BMC Evolutionary Biology (2018) 18:183 the two phases [5–7]. Mirroring the classical Lotka–Vol- terra model of interspecific competition [8, 9], the coexist- ence of haploid and diploid individuals in a population requires only slight differentiation in adaptation to differ- ent environments (i.e., differences in temperature, light levels or grazing pressure [7]). isomorphic biphasic life cycles is sustained by the niche partitioning among ploidies, driven by the conditional dif- ferentiation of their individuals [7]. Many studies have re- ported uneven field abundances of haploids and diploids (i.e., commonly reported as the ratios of H:D or G:T, where H and G stands for the haploid gametophytes and D and T for the diploid tetrasporophytes). Furthermore, these un- even abundances often vary between species or even be- tween populations of the same species [11–13, 15, 26–30]. Such differences in the haploid-diploid ratio have been associated to ecological dissimilarities between phases in fecundity, recruitment, growth or mortality [11, 12, 15]). In this context, the evolutionary stability of the iso- morphic biphasic life cycle is complex to explain. Theoret- ically, two similar entities completely overlapping their niche cannot sustainably co-exist as one inevitably elimi- nates the other. However, the fact that phases in isomorphic biphasic life cycles have similar gross morphology does not necessarily mean that they are ecologically similar. Such ecological differentiation has been revealed in numerous cases during the last decades (e.g. [10–15]). Differences in chemical compounds have been shown to influence the fate of endophytic infection [16], palatability [17–20], tolerance to wave exposure [21] and desiccation [22]. So, these differ- ences can cause profound differentiation in mortality and fertility rates between isomorphic phases. Even for iso- morphic species for which no conspicuous biochemical dif- ferences have been revealed, ecological phase differences have been observed at both microscopic and macroscopic stages [13, 15, 23–25]. Therefore, as for heteromorphic life cycles, it has been argued that the evolutionary stability of y g y In red algae, the complex haploid-diploid life cycle is modified and includes a third phase: the carposporophyte (Fig. 1). In these organisms, the female gamete is retained on the thallus where fertilization occurs. The zygote under- goes successive mitotic divisions and develops into a multi- cellular carposporophyte, while remaining connected to the female thallus. Each carposporophyte can form thousands of carpospores that are, ultimately, released into the water. All photographs by Paula Valenzuela Page 3 of 11 Page 3 of 11 Vieira et al. BMC Evolutionary Biology (2018) 18:183 Vieira et al. BMC Evolutionary Biology (2018) 18:183 Vieira et al. BMC Evolutionary Biology (2018) 18:183 by the availability of male gametes or their lack of mobility. The production of tetraspores is considered to require only the growth of diploid tetrasporophytes, tetrasporangia de- velopment and tetraspore production by meiosis [33]. In contrast, carpospore production has to be preceded by the growth of both male and female haploid gametophytes, gametes production, fertilization, carposporophyte develop- ment, as well as carpospore production. Moreover, even though it has been hypothesized that algae invest few re- sources toward reproduction (i.e., no ancillary reproductive structures and all reproductive structures are photosyn- thetic [34]), the possibility of trade-offs in resources among survival, growth and reproduction have been postulated [15]. This is especially so for female gametophytes supply- ing nutrients for the development of cystocarps [35]. Be- cause of these particularities, different fecundity rates may reasonably be expected for haploid gametophytes and diploid sporophytes in red algae, and have indeed been reported in a large number of species (e.g., [10, 12–14, 17, 19, 20, 24, 36, 37]). These empirical results have been pro- posed to support the models where the observed uneven field abundances of haploids and diploids emerge as a nat- ural consequence of the differences in phase fertility with- out the necessity of any niche partitioning [12, 38, 39]. However, as is common in red algae, Gracilaria gracilis sheds both tetraspores and carpospores in abundance in nature [11]. Moreover, population persistence and growth were mostly driven by survival of the gametophyte and tetrasporophyte stages and it was postulated that differences in fertility could be ecologically irrelevant [11]. Indeed, the- oretical works support that conditional differentiation of the traits related to survival rates, and not fertility, was most effi- cient to partition niches between phases in haploid-diploid organisms [40–43]. Materials and methods Field surveys y The red macroalga Gracilaria chilensis is common along the Chilean shore. Individuals, fixed by a holdfast, inhabit the intertidal rocky bottom and may survive and re-grow new fronds after losing old ones [25]. Its complex life cycle alternates between isomorphic free living tetrasporophytes (diploids) and gametophytes (haploids) and is commonly named “isomorphic biphasic” or “haploid-diploid” (Fig. 1). Red algae life cycle also includes the grow of the carpos- porophyte after syngamy, a diploid structure that is some- time referred to as a third diploid phase and some authors refer to their life cycle as “triphasic”. However, since car- posporophytes are short-lived and dependent on the hap- loid female thallus for development [35], within the present work we refer to our model species life cycle as bi- phasic or haploid-diploid, as commonly done in previous studies [4, 7, 10–15, 18–20, 25, 39–43]. In G. chilensis, tet- rasporophyte adults produce haploid tetraspores after meiosis and release them to the environment. Settled tet- raspores, germinate and grow into adult haploid male or female gametophytes. The gametophyte males release gametes and fertilization occurs on female individuals. From the fertilized oogonia, a short-lived diploid epiphytic stage (the carposporophyte, Fig. 1d) develops on the fe- male gametophyte, producing thousands of diploid spores (i.e., carpospores, Fig. 1e) [35]. The carpospores are re- leased into the environment where they settle, germinate and grow into diploid tetrasporophyte adults. If the survival and fertility of both phases is equivalent, demographic models predict that the proportion of diploids present in a haploid-diploid population at the equilibrium should be 0.41 [12, 44]. Though most G. gracilis popula- tions studied showed diploid proportions close to 0.41 [11], this was not the case for the closely related species G. chi- lensis [45]. Indeed, in G. chilensis the proportion of diploids varies widely among populations – from clear excess of haploids to clear excess of diploids - with a tendency to show more populations with excess of diploids (7 of the 11 natural populations studied show a proportion of diploids > 0.41, [45]). Interestingly, in G. chilensis, the existence of a clear conditional differentiation of survival between phases was demonstrated in the field [15]. In any case, these hypotheses are not mutually exclusive and more complex models are emerging where the differences in fertility could explain the general trend in haploid-diploid ratio characterizing each species, while site specificity and spatial and seasonal variability can only result from conditional differentiation of the traits re- lated to survival [12 41] shown to survive more in stressful conditions (namely under very low or very high densities) than the haploid males and diploids and, when fertile, the survival of haploid females also surpassed the survival of the diploid tetraspor- ophytes [15]. In this case, differences in survival between phases did not seem to explain the generally high frequency of diploids observed in previous studies [45]. Differences in fertility or recruitment have not been thoughtfully tested yet in the field for this species, but previous experiments in controlled laboratory conditions have, though, given some insight in the existence of possible phase differences for spore attachment and survival and growth of germlings [25]. Here, using natural populations of G. chilensis moni- tored for 28 months, we test for field evidences of differ- ences in fertility and recruitment among haploid males, haploid females and diploid tetrasporophytes, and how these factors could influence natural haploid-diploid ratios. It has been postulated that this mitotic amplification of the zygote may have evolved to compensate for low natural fertilization success due to the lack of motile male gametes in this group [31]. However, Engel et al. [32] demonstrated that fertilization success in Gracilaria gracilis is not limited A B C D E Fig. 1 Isomorphic biphasic (haploid-diploid) life-cycle of Gracilaria chilensis. a Life cycle showing the free-living tetrasporophytes (diploids) and dioicious gametophytes (haploids, male and female) stages. Tetrasporophyte, when mature produce haploid tetraspores after meiosis and release them to the environment. Tetraspores settle and grow into adult male or female gametophytes. The gametophyte males release gametes that fertilize female gametophytes. From the fertilized oogonia, a short-lived diploid epiphytic stage (the carposporophyte) develops on the female thallus. Within each carposporophyte, the zygote undergoes successive mitoses allowing to produce many thousand of carpospores that are then released into the environment, where they settle and grow into diploid tetrasporopytes. b Mature male bearing gametocysts (i.e., ovate white spots). Male gametes are released in the water column. c Mature tetrasporophyte thallus with tetrasporocysts (i.e., structures where tetraspores are produced) visible. Deep red pigmented tetrasporocysts still contain tetraspores while round white spots correspond to empty tetrasporocysts. d Female thallus after fertilization bearing carposporophytes. e Detail of a carposporophyte liberating carpospores. All photographs by Paula Valenzuela B A A D D E Fig. 1 Isomorphic biphasic (haploid-diploid) life-cycle of Gracilaria chilensis. a Life cycle showing the free-living tetrasporophytes (diploids) and dioicious gametophytes (haploids, male and female) stages. Tetrasporophyte, when mature produce haploid tetraspores after meiosis and release them to the environment. Tetraspores settle and grow into adult male or female gametophytes. The gametophyte males release gametes that fertilize female gametophytes. From the fertilized oogonia, a short-lived diploid epiphytic stage (the carposporophyte) develops on the female thallus. Within each carposporophyte, the zygote undergoes successive mitoses allowing to produce many thousand of carpospores that are then released into the environment, where they settle and grow into diploid tetrasporopytes. b Mature male bearing gametocysts (i.e., ovate white spots). Male gametes are released in the water column. c Mature tetrasporophyte thallus with tetrasporocysts (i.e., structures where tetraspores are produced) visible. Deep red pigmented tetrasporocysts still contain tetraspores while round white spots correspond to empty tetrasporocysts. d Female thallus after fertilization bearing carposporophytes. e Detail of a carposporophyte liberating carpospores. Materials and methods Field surveys The volume (v, in cm3) of a cylinder of equal length and diameter was used as a proxy for ramet biomass since this estimate correlated with dry weight (r2 = 0.877; P < 0.0001; n = 281). Every individual absent after 4 months was re-checked after 8 months for confirmation and considered dead when absent in the re-check. Materials and methods Field surveys Haploid females were Demographic monitoring of individuals was performed in five intertidal rock-pools (‘Corral 1’, ‘Corral 2’, ‘Niebla 1’, ‘Nie- bla 2’ and ‘Niebla 3’) within two sites (Corral 39°52′27″S / 73°24′02″ W and Niebla 39°55′47″S / 73°23′57″W) along the margins of the Valdivia River estuary, from October 2009 to February 2011 at four-month intervals. In the Southern Hemisphere, the interval between the February Vieira et al. BMC Evolutionary Biology (2018) 18:183 Page 4 of 11 Page 4 of 11 the asymptotic maximum, bf is the displacement of the curve along the x-axis and cf is the increment rate (Fig. 2). The parameters Kf, bf and cf were estimated by vertical least squares regression. However, because the least squares have no closed form (i.e, analytical) solution for the Gompertz curve, it required numerical minimization by the Newton-Rahpson method or Levemberg-Marquardt algo- rithm. Its detailed application is presented in Additional file 1. A total of 18 Gompertz curves (i.e., three “sea- sons” × two sites (Niebla and Corral) × 3 stages (haploid females, haploid males and diploid tetrasporophytes)) were estimated describing the size-dependent fecundity (Fig. 2). Their goodness-of-fit was estimated from χ2 tests with the significance corresponding to the probability of the curves not fitting the observations. With 7 ≤i ≤9 size classes and j = 3 parameters needing estimation, there were i-j-1 degrees of freedom. These curves were com- pared among life cycle stages, at each site and season. Their pair-wise resemblances were estimated from χ2 tests of homogeneity with the significance corresponding to the probability of two curves matching. With 7 ≤i ≤9 size classes and j = 2 stages being compared, there were (i-1)(j-1) degrees of freedom. and June, June and October, and October and February cen- sus mostly corresponds to the autumn, winter and spring and summer, respectively. All individuals within each rock-pool were mapped relative to a pair of fixed points (see Engel et al. [11]). A small fragment of the thallus was col- lected from each individual for the identification of repro- ductive males (M), females (F) and tetrasporophytes (D for diploids) by their reproductive structures under a binocular microscope. Vegetative individuals, were identified using the sex-specific molecular markers developed in Guillemin et al. [25]. At each census, frond length and diameter were re- corded for each observed individual. Probability of becoming fecund (ρ) The observed probability of an individual to become fec- und (ρobs) was inferred for each site, season and size class by ρobs = nf/nt, where nf was the count of fecund individ- uals and nt the total count of individuals. Nine size classes were defined: ln(v) < −1.5, seven equally spaced classes within −1.5 < ln(v) < 5.5, and 5.5 < ln(v); where v was the frond volume in cm3. The ρobs were fitted to a Gompertz curve of the form ρest = Kf∙exp.(−bf∙exp.(−cfx)), where Kf is Spore production rates The number of spores produced per unit frond volume was estimated for all mature female gametophytes and diploid tetrasporophytes. For each mature female Fig. 2 Probability of an individual of Gracilaria chilensis to be fecund (ρ) across different frond size categories (ln(v)). Labels are (M) males, (F) females, and (D) diploids, (obs.) observed and (est.) estimated. Individual sizes are given by the frond volume (v) in cm3. Gompertz curves fitted by the Newton-Rahpson method and confirmed by Levenberg-Marquardt algorithm. The probabilities of M, F and D curves to be equal, as estimated by χ2 tests, were always below 0.0002 Fig. 2 Probability of an individual of Gracilaria chilensis to be fecund (ρ) across different frond size categories (ln(v)). Labels are (M) males, (F) females, and (D) diploids, (obs.) observed and (est.) estimated. Individual sizes are given by the frond volume (v) in cm3. Gompertz curves fitted by the Newton-Rahpson method and confirmed by Levenberg-Marquardt algorithm. The probabilities of M, F and D curves to be equal, as estimated by χ2 tests, were always below 0.0002 Vieira et al. BMC Evolutionary Biology (2018) 18:183 Vieira et al. BMC Evolutionary Biology (2018) 18:183 Page 5 of 11 Page 5 of 11 gametophyte, the number of cystocarps (i.e., the short-lived diploid epiphytic stage growing on haploid fe- male thallus) found on the 10-cm distal extremity of three branches was counted [11]. Four hundred cystocarps were sampled from 20 mature female gametophytes chosen ran- domly (i.e., 20 from each female) and these 400 cystocarps were placed together in 30 mL of filtered seawater in a Fal- con tube kept at 4 °C. Note that a small part of the female thallus was excised jointly with the cystocarps. The number of diploid carpospores produced per cystocarp was counted after 24 h under a binocular microscope (i.e., mean over 10 independent counting of seawater drops of 50 μL). In the same way, 80 cm of mature thallus were cut from 20 ran- dom diploid tetrasporophytes (i.e., 4 cm from each tetra- sporophyte) and these 80 thallus fragments were placed together in 30 mL of filtered seawater in a Falcon tube kept at 4 °C. The number of haploid tetraspores produced per centimeter of tetrasporophyte thallus was counted after 24 h under a binocular microscope (i.e., mean over 10 inde- pendent counting of seawater drops of 50 μL). Correlation between vital rates and field abundances Correlation between vital rates and field abundances We tested for the relation between the ploidy ratio of the abundances (i.e., the haploid-diploid ratio or H:D) and the ploidy ratio of the vital rates, the latter ratio requiring esti- mation according to the ploidy to which each vital rate is contributing. Thus, vital rates contributing to the haploids (production and survival of haploid tetraspores) must ap- pear in the numerator whereas vital rates contributing to the diploids (production and survival of diploid carpospores) must appear in the denominator (see [40–43]). Hence, the ratios used to compare these rates and relate them to the H:D were the ptet:pcarp, the stet:scarp and the RH:RD. The direct use of the haploid-diploid ratio when testing and comparing the effects of its driving factors is inad- equate because the addition of x haploid fronds to the nu- merator or of x diploid fronds to its denominator do not have symmetrical effects. Hence, the haploid-diploid ratio inevitably shows widely different sensitivities and elasticities to ecologically equivalent vital rates just because these con- tribute to different sides of the ratio. For this same reason the haploid-diploid ratio of a species cannot follow a nor- mal distribution (or any symmetrical distribution for that matter) particularly when there is change in phase domin- ance among locations or seasons. Since statistical tests using the haploid-diploid ratio as dependent (response) variable are inadequate, the haploid-diploid ratio was re- placed by the log(H:D) [40, 43, 47]. The point of such pro- cedure is exemplified in the following text. Consider the situation (H:D0) with the abundances balanced at 1:1, the alternative H:DA unbalanced at 10:1 and the alternative H:DB unbalanced at 1:10. The direct use of the haploid-dip- loid ratio metric falsely suggests that the alternative A rep- resents a change (A-0) of magnitude 9 whereas the alternative B represents a change (B-0) of magnitude 0.9. An honest comparison between the alternatives A and B re- quires the use of logarithms (e.g., [40, 43, 47]). In this Spore production rates Measures of the number of carpospores released per cystocarp and tet- raspores released per centimeter of tetrasporophyte thallus were performed independently for Corral and Niebla and implemented after each sampling except in October 2009. For each haploid female gametophyte, the number of spores produced (pcarp) was inferred by multiplying the average number of diploid carpospores released per cysto- carp by the average number of cystocarps per cm3 of thallus (assuming a branch diameter of 1 mm) and by the total vol- ume of the female individual. For each diploid tetrasporo- phyte, the number of spores produced (ptet) was inferred by multiplying the average number of haploid tetraspores re- leased by cm−3 of thallus (assuming a branch diameter of 1 mm) by the total volume of the individual. Furthermore, tetraspores could be male (Mtet) or female (Ftet). We as- sumed an even sex ratio and thus pMtet = pFtet = ptet/2. Iterative Reweighted Least Squares (IRLS) estimation method. The local frond density (Vp) was quantified by the sum of all frond volumes at each pool, yet, excluding the larger frond to avoid bias from exceptionally large fronds [15, 46]. The frond density was dependent on loca- tion and season. Since they were not numerically orthog- onal to the factor density and did not increase the explanatory power of the model, the effects of these fac- tors were not tested. Nevertheless, it was possible to com- pare between Corral and Niebla sites. The goodness-of-fit was estimated from χ2 tests with the significance corre- sponding to the probability of the curves not fitting the observations. The number of observations were i = 2 pools× 9 census at Corral, i = 3 pools× 9 census at Niebla and j = 3 parameters needed to be estimated, resulting in 14 = i-j-1 degrees of freedom for Corral and 23 = i-j-1 degrees of freedom for Niebla. Results and discussion Probability of fronds to become fecund The probability of fronds becoming fecund (ρ) was size-dependent, with larger fronds generally fecund (i.e., ρ ≈1), and well fitted by Gompertz curves (Fig. 2). The exceptions were the haploid female gametophytes and the diploid tetrasporophytes at Niebla during the spring-summer. Their decreasing ρ was better fitted by quadratic functions. All these curves fitted the observa- tions remarkably well (χ2 p < 0.007). Fecundity was highly seasonal: during the austral autumn and winter most fronds became fecund, even the small. On the other hand, the austral spring-summer was less favorable for becoming fecund. At Corral, many of the small and medium sized fronds - particularly of the haploid females and diploids - were not becoming fecund. At Niebla, even the large haploid females and diploids were not becoming fecund. The ρ was dependent on life cycle stage, with advantage for the haploid males. The χ2 tests showed that the Gompertz curves estimated for each stage were always different (χ2 p < 0.0002). However, during the autumn and winter, the differences among stages were mostly restricted to the smaller fronds and only during the spring-summer did they generalize to the full frond size range. This contrast among stages may represent a contrast between strategies for resource allocation in times of shortage or intense competition; with small and medium sized diploids and haploid females investing in survival, and haploid males invest- ing in fecundity. Furthermore, our companion study about G. chilensis survival [15] revealed a low survival of males when compared to females and diploids, especially for small individuals. In fact, the haploid female (F), hap- loid male (M) and diploid tetrasporophyte (D) differenti- ation of survival [15] was perfectly symmetric to their differentiation of becoming fecund (ρ). This mirroring pattern between probabilities of survival and becoming fecund support the existence in the field of a trade-off between survival and reproduction [48–50] that had already been reported for G. chilensis reared in the la- boratory [51]. Within the long list of theoretical works exploring potential differences between phases in haploid-diploid organisms, Lewis [52] proposed that, when resources are scarce, haploids might benefit from having half the cost of production and upkeep of DNA than diploids. Lewis theory has been further developed Spore survival rates The spore survival probabilities were estimated from eq. (1), where ‘spore’ holds for diploid carpospores (carp), hap- loid male tetraspores (Mtet) and haploid female tetraspores (Ftet). This estimation required the production of the re- spective spores (pspore) at time t and in each site (Corral or Niebla). We assumed that the spores produced at each site spread evenly among its respective pools. Therefore, each pool received a fraction of the spores produced that was equal to its relative area rApool = Apool/Asite, where A is area. The spores that survived to time t + 1 were detected as re- cruits of the respective stage (rstage) in the following census. sspore ¼ rstage rApool pspore ð1Þ ð1Þ The dependency of spore survival from the local frond density was fit to a second-degree polynomial using the Vieira et al. BMC Evolutionary Biology (2018) 18:183 Page 6 of 11 Page 6 of 11 into the “nutrient limitation hypothesis” [53] and this non-genetic explanation for the evolution of life cycles has been sustained by experimental studies showing that haploids grew faster than diploids when cultivated in nutrient-poor conditions [23]. Resources become scarce under intense competition or when the adaptations re- quired to overcome hydrodynamic stress, desiccation or herbivory are resource demanding. In all such cases the G. chilensis haploid females survived better than any other stage [15] while the haploid males had greater probability of becoming fecund than any other stage (this study). It is possible that resources saved in hap- loids by producing half the DNA are differently allocated depending on sex: to ensure a better survival in female and a higher fecundity in males. example log10(1:1) = 0, log10(10:1) = 1 and log10(1:10) = −1, revealing that the alternatives A and B represent changes of equal magnitude but opposite directions. The statistic log(H:D) may perfectly well follow a normal distribution without restrictions about which phase is dominant. Hence, in this work the haploid-diploid ratio of abundances and vital rates were always tested and plotted in logarithmic scales. The dependent variable ‘spore production’ was log-transformed to ascertain the homogeneity of variances within groups. The d.f.error = 40. Significant p-values shown in bold Fecundity Besides the differentiation of the probability of becoming fecund (ρ), a differentiation of fecundity itself (i.e., the production of spores per cm3 of thallus) was observed between haploid females and diploids. The 3-way per- mutation test with 1000 randomizations determined that the difference among stages, among sites and among seasons were all significant, as was the stage×season interaction (Table 1). Tetrasporophytes produced an average of 7939 tetraspores (haploid spores) per cm3 of frond whereas the female gametophytes produced an average of 1406 carpospores (diploid spores) per cm3. Average tetraspore production per individual was 6.97 times higher than carpospore production. This domin- ance of haploid spores production matches those previ- ously reported in haploid-diploid algae and was argued to be one of the probable causes of dominance of hap- loid gametophytes in natural stands [12, 38, 39]. During the austral spring-summer fewer carpospores were pro- duced than during the other seasons, resulting in a more Table 1 Three-way ANOVA on the spore production of Gracilaria chilensis across the stages haploid females, haploid males and diploids; the seasons autumn, winter and spring-summer; and the pools Corral 1, Corral 2, Niebla 1, Niebla 2 and Niebla 3 Factor effect d.f. F p Stage Fixed 1 37.957 < 0.001 Season Fixed 2 8.157 0.005 Site Fixed 4 3.103 0.039 Stage×season Fixed 2 4.902 0.007 Stage×site Fixed 4 1.714 0.154 Season×site Fixed 8 0.057 0.999 Stage×site×season Fixed 8 1.525 0.186 The dependent variable ‘spore production’ was log-transformed to ascertain the homogeneity of variances within groups. The d.f.error = 40. Significant p-values shown in bold Table 1 Three-way ANOVA on the spore production of Gracilaria chilensis across the stages haploid females, haploid males and diploids; the seasons autumn, winter and spring-summer; and the pools Corral 1, Corral 2, Niebla 1, Niebla 2 and Niebla 3 Vieira et al. BMC Evolutionary Biology (2018) 18:183 Page 7 of 11 Page 7 of 11 Page 7 of 11 tetraspore dominated spore production (Fig. 3). Overall, the summer was the season with the lowest G. chilensis fecundity: fronds were less likely to become fecund and the fecund fronds produced fewer spores. Relative to the factor ‘Site’, much more spores were produced at the Niebla pools than at the Corral pools (Fig. 3). Fecundity Since, the fronds at Corral dried on the flat bare rock during low tide, it is possible that the stress from desiccation at this site may demand considerable resource allocation to survival therefore enhancing trade-off between survival and fertility. maternal care may be at the origin of the diploid advan- tage. In fact, a generalized haploid advantage over the adult fronds of stressed intertidal red algal stands has been reported [11, 18, 21, 29, 44, 56–58] while a generalized diploid advantage was observed for sporelings [23, 25, 44], suggesting that maternal care by the haploid females pro- tecting their carpospore progeny in adverse environments could be a generalize tendency in red macroalgae. g y g Our results showed the negative effect of high frond densities on the survival of the spores and sporelings. The high density – low survival relation is well docu- mented in algal species as a consequence of intraspe- cific competition, leading to the famous self-thinning rule [59, 60]. This negative effect has been reported in G. chilensis [15] and has even been observed between sporelings in other seaweed species [61]. During our survey, at high frond densities, the increased mortality of settled spores was pronounced at the Niebla site for all spore types, while at the Corral site it was restricted to the haploid tetraspores (Fig. 4). As said above, even though both survey sites where located in the upper intertidal, they differ in terms of desiccation and UV stress levels during low tide. While, Niebla rock pools retain seawater during low tide protecting fronds from severe dehydration, the fronds at Corral dried on the flat bare rock. We propose that, at Corral, the positive effect of protection against desiccation and UV stress provided by the adult fronds override the negative effect of competition unless the stands were outstand- ingly crowded. Interestingly, we observed that when- ever spore survival depended on competition with adult fronds (i.e., at high frond density), the carposporelings (diploids) outperformed the tetrasporelings (haploids). This advantage of the diploid over the haploid sporel- ings contrasts with results showing the advantage of G. chilensis adult haploid female fronds over the diploid fronds under the same stress from competition [15]. These results could suggest the existence of maternal care by the haploid females. Fecundity Indeed, females, by provid- ing nutrients to their carpospores progeny, can lead to an advantage of diploid carpospores over haploid tetra- spores under scarcity of resources. The maternal care by the haploid females providing nutrients and thus benefiting the production, development and germin- ation of diploid carpospores has been reported in three other red algal species [35]. Spore survival A pattern of differentiation of spore survival among life cycle stages common to all algae, or even all Gracilariales, was im- possible to find so far. Studies have shown that advantages toward carpospores (diploids) or tetraspores (haploids) for settlement and germination varied depending on species, season or environmental conditions [13, 18, 23–25, 36, 37, 54, 55]. Since our census was done at four-month intervals, the estimation of spore survival included all the processes taking place during this time frame, namely their survival while drifting, settlement, germination and the survival of the sporelings. Despite the coarse temporal resolution of our data, the second degree polynomials were able to clearly identify the sporelings’ survival dependency on the local frond density (Fig. 4). Posterior simulations demonstrated that using these polynomials resulted in good fits to the ob- served haploid:diploid spore survival ratios (stet:scarp) (Fig. 4). This ratio was mostly beneficial to the diploids, particu- larly at the Corral site and under the lowest frond dens- ities (Fig. 4). In the following paragraphs we detail this spore survival dynamics and raise the hypothesis that Fig. 3 Tetra- and carpospore production rates of Gracilaria chilensis. Spores per cm3 of parental thallus observed during three seasons in five intertidal pools at sites (C) Corral and (N) Niebla. Contour lines represent log(ptet/pcarp). Because the study lasted almost 3 years, some seasons show 2 replicates while others show 3 replicates Our results also showed the negative effect of low frond densities on the survival of the spores and sporel- ings. Low frond densities have been shown to be stress- ful, exposing individuals to desiccation [8, 18, 22, 62, 63] or to hydrodynamic stress [8, 18, 21, 64], affecting the survival of intertidal algae (i.e., Allee effects). Our work showed that in Corral the diploid spores and sporelings survived better than the haploid spores when growing at Fig. 3 Tetra- and carpospore production rates of Gracilaria chilensis. Spores per cm3 of parental thallus observed during three seasons in five intertidal pools at sites (C) Corral and (N) Niebla. Contour lines represent log(ptet/pcarp). Because the study lasted almost 3 years, some seasons show 2 replicates while others show 3 replicates Fig. 3 Tetra- and carpospore production rates of Gracilaria chilensis. Spores per cm3 of parental thallus observed during three seasons in five intertidal pools at sites (C) Corral and (N) Niebla. Contour lines represent log(ptet/pcarp). Spore survival Because the study lasted almost 3 years, some seasons show 2 replicates while others show 3 replicates Vieira et al. BMC Evolutionary Biology (2018) 18:183 Page 8 of 11 Fig. 4 Spore survival rates of Gracilaria chilensis. Labels are (obs.) observed and (est.) estimated. Pool densities are given by their total frond volume (Vp) excluding the larger frond. Also shown are the non-linear coefficients of determination (R2) and Pearson’s χ2 goodness-of-fit test (p) Fig. 4 Spore survival rates of Gracilaria chilensis. Labels are (obs.) observed and (est.) estimated. Pool densities are given by their total frond volume (Vp) excluding the larger frond. Also shown are the non-linear coefficients of determination (R2) and Pearson’s χ2 goodness-of-fit test (p) low frond densities (Fig. 4). These differences in survival were observed during summer for the Corral algal stands (where G. chilensis dry on the flat bare rock dur- ing low tide). We propose that the advantage of diploid spores under these adverse conditions could also come from maternal care by the haploid female, who can pass-on to their diploid progeny the chemical com- pounds conferring resistance to UV and herbivory, that the sporelings are unable to produce and accumulate on their own. Supporting our hypothesis, the better survival of the G. chilensis adult haploid female fronds under desiccation and UV stress was also particularly evident at Corral during the summer [15] and has been linked to a possible accumulation of chemical compounds related to photoprotection [65, 66] and antioxidative enzymes [67]. A better ability of the diploid carpospores to with- stand UV radiation than the haploid tetraspores has been demonstrated in other red algae [68]. Consistently, haploid females in red algae have been reported to present higher concentrations of protective chemical compounds Fig. 5 Correlation between the ploidy ratios of Gracilaria chilensis spore vital rates at time t and the ploidy ratios of recruitment at time t + 1. Labels are (s) survival, (p) production, (R) recruitment, (ln) Neper’s logarithm, (carp) carpospores, (tet) tetraspores, (H) haploids, and (D) diploids. Grey areas represent incompatibility between the ploidy ratios of the spore vital rates and the ploidy ratios of the recruitment Fig. 5 Correlation between the ploidy ratios of Gracilaria chilensis spore vital rates at time t and the ploidy ratios of recruitment at time t + 1. Additional files within cystocarps than along the main thallus [20], a pat- tern that suggested the existence of a mechanism of strong protection of the cystocarps and diploid carpospores. Additional file 1: Gompertz. (DOCX 39 kb) Additional file 2: All.mat. (MAT 64 kb) Additional file 3: Weibull. (DOCX 78 kb) Additional file 1: Gompertz. (DOCX 39 kb) Additional file 2: All.mat. (MAT 64 kb) Additional file 3: Weibull. (DOCX 78 kb) Funding g This research was supported by CONICYT (Fondo Nacional de Desarrollo Científico y Tecnológico FONDECYT) under grant number 1090360 and 1170541. VV was funded by ERDF Funds of the Competitiveness Factors Operational Programme - COMPETE and national funds of the FCT - Foundation for Science and Technology under the project UID/EEA/50009/ 2013. AHE was supported by fellowships SFRH/BPD/63703/2009, SFRH/BPD/ 107878/2015 and UID/Multi/04326/2016 of the National Science Foundation FCT of Portugal. The funders took no part in the design of the study, in the collection, analysis, and interpretation of data, and in writing the manuscript. Availability of data and materials The dataset supporting the conclusions of this article is included within the Additional file 2. The non-linear regression methods are available in Additional file 1 and Additional file 3. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Abbreviations Recruitment is fundamental for the determination of the future population structure. However, recruitment itself is an intricate process resulting from the inter- action of spore production, survival, settlement, ger- mination and the development of the sporelings. While spore production has been proposed as the fundamen- tal driver of the ploidy ratio of abundances [12, 38, 39], others have argued that settlement and germination rates are more important factors [36, 37]. Concomi- tantly with the ranking of such rates, shifts in ploidy dominance are commonly found when moving from spore production to spore settlement and germination [13, 14, 36, 37]. The present study shows that in G. chi- lensis the production of tetraspores (haploids) is usually higher than of carpospores (diploids), whereas the sur- vival of the diploid carposporelings dominates over the survival of haploid tetrasporelings. However, the corre- lations between the ploidy ratio of recruitment and the ploidy ratios of spore production or of spore survival were very weak, and none of these processes dominated the recruitment of G. chilensis young fronds (Fig. 5). The importance of each of these two components in explaining the ploidy ratio of the recruits depended greatly on site and season. This finding rejects previous hypothesis (i.e, [12, 36–39]) proposing that the popula- tion structure (namely, its ploidy ratio) is determined by one specific aspect within fertility. On the contrary, our results suggest that an advantage of haploids or of diploids depends on a multitude of biological processes and their differential responses to the local conditions. ANOVA: Analysis of Variance; C: Corral sampling site; C1: Corral pool #1; C2: Corral pool #2; carp: Carpospores; D: Diploids; DNA: Deoxyribonucleic acid; F: Female haploids; Ftet: Female tetraspores; G: Gametophytes; G:T: Gametophyte-to- tetrasporophyte ratio; H: Haploid; H:D: Haploid-to-diploid ratio; IRLS: Iterative Reweighted Least Squares; M: Male haplois; Mtet: Male tetraspores; N: Niebla sampling site; N1: Niebla pool #1; N2: Niebla pool #2; N3: Niebla pool #3; ptet:pcarp: Ratio of tetraspore-to-carpospore production; RH:RD: Ratio of haploid-to- diploid recruits; RNA: Deoxyribonucleic acid; stet:scarp: Ratio of tetraspore-to- carpospore survival; T: Tetrasporophytes; tet: Tetraspores; UV: Ultra-violet radiation Spore survival Labels are (s) survival, (p) production, (R) recruitment, (ln) Neper’s logarithm, (carp) carpospores, (tet) tetraspores, (H) haploids, and (D) diploids. Grey areas represent incompatibility between the ploidy ratios of the spore vital rates and the ploidy ratios of the recruitment Vieira et al. BMC Evolutionary Biology (2018) 18:183 Page 9 of 11 Perspective on haploid-diploid life cycle maintenance C l b l f h d l f d Perspective on haploid-diploid life cycle maintenance Contrasting results about survival of the adult fronds of G. chilensis [15], with their fecundity and the survival of their spores (the present work) allow a more complete understanding of the species population dynamics. We propose that tetrasporophyte (diploid) fronds present a more r-oriented strategy. On the other hand, female gametophyte (haploid) fronds, seem to present a more K-oriented strategy; investing further resources in their survival, producing fewer spores, but investing in the survival of their carpospore (diploid) progeny through maternal care. The evolutionary stability of this life cycle suggests that, for this type of organisms in their habitat, complementary ploidy phases with different life strat- egies has more success than a monophasic alternative. Competing interests The authors declare that they have no competing interests. Competing interests The authors declare that they have no competing interests. Authors’ contributions VV developed the models and software, designed and performed the data analysis, interpreted the results and wrote the article. AE interpreted the results and reviewed the article. OH designed and performed the experiments, and reviewed the article. MLG designed and performed the experiments, interpreted the results and reviewed the article. All authors read and approved the final manuscript. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Consent for publication Not applicable. 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https://openalex.org/W4321603383	https://www.degruyter.com/document/doi/10.1515/opag-2022-0152/pdf	English	null	The fate of probiotic species applied in intensive grow-out ponds in rearing water and intestinal tracts of white shrimp, <i>Litopenaeus vannamei</i>	Open Agriculture	2,023	cc-by	8,003	Open Agriculture 2023; 8: 20220152 Abstract Odeyemi: Research Division, University of Tasmania, Launceston, Australia; HeTA Food Research Centre of Excellence, School of Chemical Engineering, University of Birmingham, Birmingham, UK Muhamad Ali: Laboratory of Microbiology and Biotechnology, Faculty of Animal Sciences, University of Mataram, Indonesia Awik P. D. Nurhayati: Department of Biology, Faculty of Science and Data Analytics, Institut Teknologi Sepuluh Nopember (ITS), Surabaya, Indonesia Open Access. © 2023 the author(s), published by De Gruyter. License. Muhamad Amin, Yoga Pramujisunu, Mirni Lamid, Yudi Cahyoko, Olumide A. Odeyemi, Muhamad Ali, Awik P. D. Nurhayati The fate of probiotic species applied in intensive grow-out ponds in rearing water and intestinal tracts of white shrimp, Litopenaeus vannamei https://doi.org/10.1515/opag-2022-0152 received October 2, 2022; accepted November 9, 2022 species was traced by collecting the rearing water and shrimp’s intestines on day 47 of culture to monitor their composition and abundance using high-throughput sequencing. This work is licensed under the Creative Commons Attribution 4.0 International Abstract Introduction ‒ Probiotics have been commonly prac- ticed in commercial shrimp farms to increase pond pro- duction. However, these possibilities were based on the results of in vitro studies or laboratory in vivo trials. While studies on probiotic applications in commercial-scale farms are still rarely investigated, this study addresses the fate of probiotic species in ponds and the intestinal tract of white shrimps reared in an intensive aquaculture system. Results ‒ None of the commercial probiotic species could be detected from both rearing water and shrimp intestinal tracts. These results suggest that the probiotic species had low viability and adaptability in the rearing pond as well as the shrimp intestines when applied on commercial-scale farms. These facts may explain the high variation in the yield among shrimp ponds in spite of having similar treatments. Conclusion ‒ Probiotic strains had low viability and adaptability in commercial farms. Thus, methods and strategies in probiotic application to commercial-scale shrimp farms should be evaluated and further developed to increase probiotic eﬃcacy. Material and methods ‒ Four commercial probiotic species (Lactobacillus plantarum, Lactobacillus fermentum, Bacillus subtilis, and Pseudomonas putida) were applied to the commercial shrimp ponds (@800 m2 area of high-den- sity polyethene ponds) in the morning at a dose of 5 ppm once every 2 days in the ﬁrst month, and once a week from second month onward. Then, the presence of the probiotic Keywords: food production, GI tract, microbiome, NGS, probiotics, water Corresponding author: Muhamad Amin, Department of Aquaculture, Faculty of Fisheries and Marine, Universitas Airlangga, Campus C Jl Mulyorejo Surabaya, East Java 60115, Indonesia, e-mail: muhamad.amin@fpk.unair.ac.id Yoga Pramujisunu: Aquaculture Study Program, Faculty of Fisheries and Marine, Universitas Airlangga, Surabaya, East Java 60115, Indonesia * Corresponding author: Muhamad Amin, Department of Aquaculture, Faculty of Fisheries and Marine, Universitas Airlangga, Campus C Jl Mulyorejo Surabaya, East Java 60115, Indonesia, e-mail: muhamad.amin@fpk.unair.ac.id Yoga Pramujisunu: Aquaculture Study Program, Faculty of Fisheries and Marine, Universitas Airlangga, Surabaya, East Java 60115, Indonesia Mirni Lamid: Faculty of Veterinary Science, Universitas Airlangga, Surabaya, East Java 60115, Indonesia Yudi Cahyoko: Department of Aquaculture, Faculty of Fisheries and Marine, Universitas Airlangga, Campus C Jl Mulyorejo Surabaya, East Java 60115, Indonesia Olumide A. hor(s), published by De Gruyter. This work is licensed under the Creative Commons Attribution 4.0 International  * Corresponding author: Muhamad Amin, Department of Aquaculture, Faculty of Fisheries and Marine, Universitas Airlangga, Campus C Jl Mulyorejo Surabaya, East Java 60115, Indonesia, e-mail: muhamad.amin@fpk.unair.ac.id Yoga Pramujisunu: Aquaculture Study Program, Faculty of Fisheries and Marine, Universitas Airlangga, Surabaya, East Java 60115, Indonesia Mirni Lamid: Faculty of Veterinary Science, Universitas Airlangga, Surabaya, East Java 60115, Indonesia Yudi Cahyoko: Department of Aquaculture, Faculty of Fisheries and Marine, Universitas Airlangga, Campus C Jl Mulyorejo Surabaya, East Java 60115, Indonesia Olumide A. Odeyemi: Research Division, University of Tasmania, Launceston, Australia; HeTA Food Research Centre of Excellence, School of Chemical Engineering, University of Birmingham, Birmingham, UK Muhamad Ali: Laboratory of Microbiology and Biotechnology, Faculty of Animal Sciences, University of Mataram, Indonesia Awik P. D. Nurhayati: Department of Biology, Faculty of Science and Data Analytics, Institut Teknologi Sepuluh Nopember (ITS), Surabaya, Indonesia Open Access. © 2023 the author(s), published by De Gruyter. License. Open Access. © 2023 the author(s), published by De Gruyter. This work is licensed under the Creative Commons Attribution 4.0 International License. 2 polyethylene ponds (@800 m2 area and a water depth of 120 cm). The pond consisted of three plots with an area of 800 m2 and a stocking population of 220,000 individuals. The commercial probiotics were Lactobacillus plantarum, L. fermentum, Bacillus subtilis, and Pseudomonas putida. The probiotic consortia was applied in the morning at a dose of 5 ppm once every 2 days. Siphoning of solid wastes (feces and uneaten feed) was carried out 1 day before the stocking of the fry and during the rearing period for as much as 2–5 days to adjust the age of the shrimp. Feeding of shrimps was done manually 1–5 times a day according to the shrimp sizes. waste in culture ponds including solid organic waste or soluble toxic chemicals such as ammonia (NH3) or nitrite (NO2) [5–7]. Despite the beneﬁts of the usage of probiotics in aquaculture, most of these studies were based on in vivo studies or in vivo laboratory trials and very small- scale rearing systems where environmental conditions were easily controlled. Some studies conﬁrmed that the results of in vitro and in vivo studies are frequently uncor- related. In a review by Toledo et al. [8], it was stated that many studies had inconsistent results concerning the eﬃ- cacy of probiotic treatments on shrimp survival and growth performance by in vitro and in vivo studies. These inconsistent results were due to the fact that environ- mental conditions in commercial shrimp ponds could vary, ﬂuctuate, and may be very diﬃcult to control. According to a study by Huerta-Rábago et al. [9], it was reported that commercial probiotics consisting of Bacillus spp., Lactobacillus spp., and Saccharomyces spp. intro- duced to a commercial shrimp farm could not be detected in rearing water due to competition with native micro- ﬂora in the rearing water, and diﬀerent environmental conditions. The probiotics addition also had a signiﬁcant eﬀect on the speciﬁc growth rate or survival rate of white shrimp. Salinity for instance in marine aquaculture is very critical for the survival of some bacteria which were iso- lated from the terrestrial organism [10]. Do these various limitations pose a question of whether probiotic strains can survive and signiﬁcantly contribute to the quality of rearing water, digestibility, or disease resistance as being reported by many in vitro or laboratory-scale studies? 2.2 Collection of water and shrimps’ intestinal samples Water sampling was carried out according to the protocol previously described by Gomes et al. [11] with a slight modiﬁcation. Water samples were collected from six ponds using a long pole sampling device and a 20 mL sterile plastic cup. The collected water was stored in a 50 mL falcon tube which was previously ﬁlled with 30 mL of absolute ethanol for DNA preservation. Samples were kept on ice until processed in the laboratory within the next 8 h. The shrimp intestine was sampled as previously described by Amin et al. [12]. A total of 30 healthy shrimps showing no symptoms of the disease were collected from 3 shrimp ponds (10 shrimps per pond) on day 47. The length and weight of shrimps were measured individually using a ruler and balance. Thereafter, each shrimp was washed with sterile distilled water, followed by 76% alcohol and rewashed with sterile distilled water to remove exogenous bacterial contamination. Then, the intestinal tract of each shrimp was dissected aseptically and placed into a sterile 1.5 mL microcentrifuge tube containing RNAlaterTM (R0901, SIGMA) and stored at −20°C until DNA extraction. To address this question, there is a need for a study that will trace the composition and abundance of com- mercial probiotic species applied in commercial shrimp farms (pond and the intestinal tract of white shrimps) in an intensive aquaculture system using a high-throughput sequencing. Ethics approval: The conducted research is not related to either human or animal use. 1 Introduction Probiotics have been considered an eco-friendly approach to increasing the yield of aquaculture production through several mechanisms including maintaining water quality, growth performance, or survival rate of aquatic organisms [1]. For example, studies have conﬁrmed that probiotic application has enabled us to signiﬁcantly reduce anti- biotic use in aquaculture industries and avoid the occur- rence of antibiotic resistance genes of microbes [2]. Some probiotics have been documented to produce digestive enzymes such as protease, amylase, lipase, alginate lyase, and cellulase which help animal hosts to digest ingested diets [3]. Probiotic strains were documented to produce antimicrobial compounds active against bacterial patho- gens [4]. Also, some probiotic species have the capacity to degrade and prevent the accumulation of aquaculture Muhamad Ali: Laboratory of Microbiology and Biotechnology, Faculty of Animal Sciences, University of Mataram, Indonesia Awik P. D. Nurhayati: Department of Biology, Faculty of Science and Data Analytics, Institut Teknologi Sepuluh Nopember (ITS), Surabaya, Indonesia 2  Muhamad Amin et al. The fate of probiotics applied in commercial shrimp ponds  3 Figure 1: KRONA visually displays the analysis result of taxonomic annotation. Circles from inside to outside stand for diﬀerent taxonomic ranks, and the area of the sector means a respective proportion of diﬀerent OTU annotation results. The abundance of Lactobacillus spp. Was detected from the rearing water of commercial shrimp ponds on the day of culture (DOC) 47. P1 is pond 1, P2 is pond 2, and P3 is pond 3. Figure 1: KRONA visually displays the analysis result of taxonomic annotation. Circles from inside to outside stand for diﬀerent taxonomic ranks, and the area of the sector means a respective proportion of diﬀerent OTU annotation results. The abundance of Lactobacillus spp. Was detected from the rearing water of commercial shrimp ponds on the day of culture (DOC) 47. P1 is pond 1, P2 is pond 2, and P3 is pond 3. 2.3 Extraction and ampliﬁcation of bacterial DNA A commercial shrimp farm located at the ordinate point, 113°01′14.7′′ E and 6°52′59.3′′ L were selected for the pre- sent study. White shrimp, Litopenaeus vannamei, was cultivated through an intensive system (275 indiv/m2 and applied with commercial probiotics) in high-density DNA from pond water and the intestinal tract of white shrimp was extracted using the DNeasy PowerSoil Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. In brief, falcon tubes were decontaminated with The fate of probiotics applied in commercial shrimp ponds  3 3.1 Proﬁles of probiotic species in grow-out ponds The results showed that the number of bacteria classiﬁed as Ordo Lactobacillales was quite abundance in the three ponds (Figure 1). A total of 4,704 bacterial sequences or 5% of total bacteria detected in pond 1 were assigned to Ordo Lactobacillales, of which 4,375 sequences (93%) were identiﬁed as genus Lactobacillus and belonged to 12 bacterial species Table 1. From pond 2, a total of 4,572 bacterial sequences (5% of the total identiﬁed bacteria in pond 2) were assigned to Ordo Lactobacillales, of which 3,795 sequences (83%) were classiﬁed as genus Lactoba- cillus and belonged to 12 bacterial species, Table 1. Both pond 1 and pond 2 appeared to be very similar in terms of Lactobacillales proportions (5%) and the number of Lactobacillus species (12 species). Five most dominant species in both ponds 1 and 2 were Lactobacillus aviarius Member of genus Bacillus was not found in ponds 2 and 3, but was found only in pond 1, Figure 2. A total of 441 bacterial sequences or 0.4% of the total detected bacterial sequences were assigned to Ordo Bacillales. Of which 395 sequences or 99% were classiﬁed as Bacillus sp. (OTU_160). Other NGS results showed that Pseudomonas spp. were detected only from two ponds with very low abun- dance (Figure 3). A total of 39 bacterial sequences or 0.04% of total bacterial sequences detected from rearing water of pond 1 were assigned to Ordo Pseudomonadales, but none of them belonged to Pseudomonas spp. While in pond 2, 35 bacterial sequences were assigned to Ordo Pseudomonadales, and only one sequence was identiﬁed as Pseudomonas azotoformans. The highest abundance sequences of Ordo Pseudomonadales were detected from pond 3 which were 6,325 bacterial sequences, of which 303 sequences belonged to the genus Pseudomonas and were assigned to 3 species which were Pseudomonas psychroto- lerans (213 sequences), Pseudomonas azotoformans (81 sequences), and Pseudomonas sp. (9 sequences) (Table 2). These results indicated that Pseudomonas putida which came from commercial probiotics had diﬃculties adapting and proliferating in the rearing water of shrimp ponds. Based on NGS results, the most abundant species was P. Psychro- tolerans (213 sequences) followed by Pseudomonas azotofor- mans (81 sequences) and Pseudomonas sp. with 9 sequences. 2.4 Sequencing and bioinformatic analysis The only diﬀerence between the 2 ponds was that Lactobacillus iners (OTU_228) in pond 2 was more abundant than in pond 1, 64 and 4 for ponds 2 and 1, respectively. While the other 11 species were higher in pond 1. From pond 3, a total of 2,986 sequences or 3% of the total identiﬁed bacteria in pond 3 were assigned to Ordo Lactobacillales, of which 65 sequences (2% of Lactobacillales) were identiﬁed as genus Lactobacillus and belonged to three bacterial species which are Lactobacillus sp. (2 sequences), L. salivarius (60 sequences), and L. ruminis (3 sequences). However, none of the Lactobacillus species identiﬁed in the three ponds showed to be the introduced probiotic species which were L. plantarum and L. fermentum. 2.4 Sequencing and bioinformatic analysis 76%ethanolandwashedwithsteriledistilledwater.Thereafter, all the falcon tubes were centrifuged (3,220 × g, 10 min, 6°C) for DNA precipitation and the supernatant was discarded [11]. The precipitated pellet was mixed with a buﬀer con- tained in the PowerBead Tube (Qiagen, Hilden, Germany). Other steps were carried out based on the manufacturer’s protocol [13]. The DNA ampliﬁcation process was carried out through several stages, namely, denaturation, annealing, and extension which are carried out in as many cycles as needed using a thermocycler. The stages started with prede- naturationatatemperatureof94°Cfor5 min,thenthesample was heated in the annealing stage at 56°C for 1 min, and con- tinued heating at the extension stage at 72°C for 1 min. For the extensionstage,thesamplewasheatedat72°Cfor7 minfor40 repetitions [14]. 76%ethanolandwashedwithsteriledistilledwater.Thereafter, all the falcon tubes were centrifuged (3,220 × g, 10 min, 6°C) for DNA precipitation and the supernatant was discarded [11]. The precipitated pellet was mixed with a buﬀer con- tained in the PowerBead Tube (Qiagen, Hilden, Germany). Other steps were carried out based on the manufacturer’s protocol [13]. The DNA ampliﬁcation process was carried out through several stages, namely, denaturation, annealing, and extension which are carried out in as many cycles as needed using a thermocycler. The stages started with prede- naturationatatemperatureof94°Cfor5 min,thenthesample was heated in the annealing stage at 56°C for 1 min, and con- tinued heating at the extension stage at 72°C for 1 min. For the extensionstage,thesamplewasheatedat72°Cfor7 minfor40 repetitions [14]. DNA samples with a concentration of 50 ng/µL were sent to Novogene Biological Information Technology Co. (Singapore) for sequencing and community analysis of microbiota in the digestive tract of white shrimps using Next Generation Sequencing (NGS, Illumina platform) based on 16S rRNA gene. Prior to sequencing, the hypervariable V3–V4 region of the 16S rRNA gene was ampliﬁed by polymerase chain reaction with primer pairs 341F (5′-CCTAYGGGRBGCAS- CAG-3′) and 806R (5′-GGACTACNNGGGTATCTAAT-3′) [14]. The results of the bacterial sequences that have been obtained were then analyzed using the UPARSE software. Sequences with a similarity of 97% were designated as the same operational taxonomic unit 4  Muhamad Amin et al. 4 (OTU). The taxonomic classiﬁcation of each OTU repre- sentative sequence was carried out using the MOTHUR program through the SILVA database with a conﬁdence level of 95%. followed by Lactobacillus sp. (OUT_39), Lactobacillus sp. (OUT_97), Lactobacillus sp. (OUT_157), and Lactobacillus salivarius (OUT_165). 3.1 Proﬁles of probiotic species in grow-out ponds Table 1: Bacterial species identiﬁed from rearing water of com- mercial shrimp ponds on the day of culture (DOC) 47 Table 1: Bacterial species identiﬁed from rearing water of com- mercial shrimp ponds on the day of culture (DOC) 47 Consensus lineage Sequence numbers OTU ID P1 P2 P3 1. Lactobacillus aviarius 1,938 1,584 — OTU_19 1. Lactobacillus sp. 1,116 914 2 OTU_39 1. Lactobacillus sp. 687 591 — OTU_97 1. Lactobacillus sp. 386 456 — OTU_157 1. Lactobacillus salivarius 112 95 60 OTU_165 1. Lactobacillus sp. 91 86 — OTU_226 1. Lactobacillus iners 4 64 — OTU_288 1. Lactobacillus ruminis 6 4 3 OTU_363 1. Lactobacillus saerimneri 15 9 — OTU_514 1. Lactobacillus agilis 13 4 — OTU_535 1. Lactobacillus mali 6 4 — OTU_548 1. Lactobacillus coleohominis 8 2 — OTU_590 P1, P2, and P3 are shrimp pond 1, shrimp pond 2, and shrimp pond 3. OUT is operational taxonomic unit. “–” means not detected. 3.2.1 Lactobacillus in shrimp intestines From the shrimp intestines collected in pond 1, a total of 172 bacterial sequences or 0.2% of the total identiﬁed The fate of probiotics applied in commercial shrimp ponds  5 The fate of probiotics applied in commercial shrimp ponds  5 igure 2: KRONA visually displays the analysis result of taxonomic annotation. Circles from inside to outside stand for diﬀerent taxonomic anks, and the area of the sector means a respective proportion of diﬀerent OTU annotation results. The abundance of Bacillus spp. Were etected from the rearing water of commercial shrimp ponds. P1, P2, and P3 are ponds 1, 2, and 3, respectively. The fate of probiotics applied in commercial shrimp ponds  5 Figure 2: KRONA visually displays the analysis result of taxonomic annotation. Circles from inside to outside stand for diﬀerent taxonomic ranks, and the area of the sector means a respective proportion of diﬀerent OTU annotation results. The abundance of Bacillus spp. Were detected from the rearing water of commercial shrimp ponds. P1, P2, and P3 are ponds 1, 2, and 3, respectively. Figure 2: KRONA visually displays the analysis result of taxonomic annotation. Circles from inside to outside stand for diﬀerent taxonomic ranks, and the area of the sector means a respective proportion of diﬀerent OTU annotation results. The abundance of Bacillus spp. Were detected from the rearing water of commercial shrimp ponds. P1, P2, and P3 are ponds 1, 2, and 3, respectively. genus Lactobacillus, 84 sequences (5% of Lactobacillales) belonged to Streptococcus, 11 sequences (0.7% of Lactobacillales) belonged to Enterococcus, and one sequence belonged to Weisella (Figure 4). 1,569 Lacto- bacillus were identiﬁed as 12 species and the 3 top most abundant species were Lactobacillus sp. (469 sequences), followed by L. pentosus (339 sequences), and L. reuteri (287 sequences). While the lowest abundance species were L. agilis and L. acidipiscis with a single sequence each (Table 3). bacteria were assigned to Ordo Lactobacillales. Of these sequences, 90 sequences (52% of Lactobacillales) belonged to genus Streptococcus, 33 sequences (19% of Lactobacillales) belonged to genus Enterococcus, 17 sequences (10% of Lactobacillales) belonged to genus Lactobacillus, 9% (16 OTUs) belonged to genus Weisella, 5% (9 sequences) belonged to genus Lactococcus, and 4% (7 sequences) belonged to genus Leuconostoc (Figure 4). The 17 sequences of genus Lactobacillus were identiﬁed as 3 species which were L. ruminis (12 sequences), L. aviaries (4 sequences), and Lactobacillus sp. 3.2.1 Lactobacillus in shrimp intestines (1 sequence) (Table 3). Furthermore, a total of 1,265 bacterial sequences were assigned to Ordo Lactobacillales from the shrimp intestines collected from pond 3. Of these sequences, 945 sequences (75% of Lactobacillales) were identiﬁed as genus Lactobacillus. Lower taxonomic annotation From the shrimp intestines collected in pond 2, a total of 1,669 bacterial sequences or 2% of the total iden- tiﬁed bacteria were assigned to Ordo Lactobacillales. 1,569 sequences (94% of Lactobacillales) belonged to Figure 3: KRONA visually displays the analysis result of taxonomic annotation. Circles from inside to outside stand for diﬀerent taxonomic ranks, and the area of the sector means a respective proportion of diﬀerent OTU annotation results. The abundance of Pseudomonas spp. was detected from the rearing water of commercial shrimp ponds. P1 is pond 1, P2 is pond 2, and P3 is pond 3. 6  Muhamad Amin et al. 6  Muhamad Amin et al. Figure 3: KRONA visually displays the analysis result of taxonomic annotation. Circles from inside to outside stand for diﬀerent taxonomic ranks, and the area of the sector means a respective proportion of diﬀerent OTU annotation results. The abundance of Pseudomonas spp. was detected from the rearing water of commercial shrimp ponds. P1 is pond 1, P2 is pond 2, and P3 is pond 3. Table 2: Bacterial species identiﬁed from rearing water of com- mercial shrimp ponds on DOC 47 Consensus Lineage Sequence numbers #OTU ID P1 P2 P3 Pseudomonas psychrotolerans — — 213 OTU_145 Pseudomonas azotoformans — 1 81 OTU_266 Pseudomonas sp. — — 9 OTU_600 P1, P2, and P3 are shrimp pond 1, shrimp pond 2, and shrimp pond 3. OTU is operational taxonomic unit. “–” means not detected. 3.2.2 Bacillus in shrimp intestines In addition, from the shrimp intestines collected in pond 3, 12 bacterial sequences or 0.01% of the total identiﬁed The fate of probiotics applied in commercial shrimp ponds  7 Figure 4: KRONA visually displays the analysis result of taxonomic annotation. Circles from inside to outside stand for diﬀerent taxonomic ranks, and the area of the sector means a respective proportion of diﬀerent OTU annotation results. Number and proportion of Lactobacillus spp. in the GI tract of white shrimps are depicted in the ﬁgure. Each ﬁgure represents sampling locations (P1 is pond 1, P2 is pond 2, and P3 is pond 3). Each ﬁgure consists of ten pooled shrimp intestines. The fate of probiotics applied in commercial shrimp ponds  7 Figure 4: KRONA visually displays the analysis result of taxonomic annotation. Circles from inside to outside stand for diﬀerent taxonomic ranks, and the area of the sector means a respective proportion of diﬀerent OTU annotation results. Number and proportion of Lactobacillus spp. in the GI tract of white shrimps are depicted in the ﬁgure. Each ﬁgure represents sampling locations (P1 is pond 1, P2 is pond 2, and P3 is pond 3). Each ﬁgure consists of ten pooled shrimp intestines. While in pond 2, 28 sequences or 0.03% of total bacterial sequences were assigned to Ordo Pseudomonadales. Seven sequences (25% of Pseudomonadales) belonged to genus Pseudomonas, 5 sequences of P. geniculata, and 2 sequences of Pseudomonas sp. Furthermore, 13 sequences were assigned to Ordo Psedomonadales but none belonged to Pseudomonas spp. in pond 3. bacterial sequences were assigned into Family Bacillaceae (Figure 5). Of these, 7 sequences (58%) were identiﬁed as B. thermoamylovorans, while the other 5 sequences (42% of Bacillaceae) were “unclassiﬁed.” 3.2.3 Pseudomonas in shrimp intestines Pseudomonas spp. also appeared to be in very low abun- dance in the intestinal tract of white shrimp reared in commercial ponds (Table 5 and Figure 6). 3.2.2 Bacillus in shrimp intestines Table 2: Bacterial species identiﬁed from rearing water of com- mercial shrimp ponds on DOC 47 From the shrimp intestines collected in pond 1, 48 sequences or 0.05% of the total identiﬁed bacteria were classiﬁed as Family Bacillaceae. Of the sequence, 18 sequences were identiﬁed as Bacillus badius, 24 sequences as Bacillus sp., and 6 sequences were identiﬁed as B. thermoamylovorans (Figure 5). While from the shrimp intestines collected in pond 2, 43 sequences or 0.05% of the total identiﬁed bacteria were assigned to Family Bacillaceae (Figure 5). Of these, 36 sequences (84% of Bacillaceae) belonged to genus Oceano- bacillus. Six sequences (14% of Bacillaceae) belonged to genus Bacillus, and were identiﬁed as four species which were B. thermoamylovorans (2 sequences), B. badius (2 sequences), Bacillus coagulans (1 sequence), and Bacillus sp. (1 sequence) (Table 4). From the shrimp intestines collected in pond 1, 48 sequences or 0.05% of the total identiﬁed bacteria were classiﬁed as Family Bacillaceae. Of the sequence, 18 sequences were identiﬁed as Bacillus badius, 24 sequences as Bacillus sp., and 6 sequences were identiﬁed as B. thermoamylovorans (Figure 5). While from the shrimp intestines collected in pond 2, 43 sequences or 0.05% of the total identiﬁed bacteria were assigned to Family Bacillaceae (Figure 5). Of these, 36 sequences (84% of Bacillaceae) belonged to genus Oceano- bacillus. Six sequences (14% of Bacillaceae) belonged to genus Bacillus, and were identiﬁed as four species which were B. thermoamylovorans (2 sequences), B. badius (2 sequences), Bacillus coagulans (1 sequence), and Bacillus sp. (1 sequence) (Table 4). P1, P2, and P3 are shrimp pond 1, shrimp pond 2, and shrimp pond 3. OTU is operational taxonomic unit. “–” means not detected. P1, P2, and P3 are shrimp pond 1, shrimp pond 2, and shrimp pond 3. OTU is operational taxonomic unit. “–” means not detected. indicated that the sequences were classiﬁed into 12 bacterial species. The 3 top most abundant species were Lactobacillus sp. (216 sequences), followed by L. pentosus (209 sequences) and L. reuteri (101 sequences) (Table 3). indicated that the sequences were classiﬁed into 12 bacterial species. The 3 top most abundant species were Lactobacillus sp. (216 sequences), followed by L. pentosus (209 sequences) and L. reuteri (101 sequences) (Table 3). 8  Muhamad Amin et al. 8 Table 3: Lactobacillus identiﬁed from the gastrointestinal (GI) tracts of white shrimps reared in commercial shrimp ponds Table 3: Lactobacillus identiﬁed from the gastrointestinal (GI) tracts of white shrimps reared in commercial shrimp ponds Consensus lineage P1 P2 P3 OTU ID Lactobacillus aviarius 4 60 685 OTU_29 Lactobacillus sp. — 97 — OTU_36 Lactobacillus pentosus — 339 — OTU_51 Lactobacillus reuteri — 287 132 OTU_87 Lactobacillus sp. 1 157 22 OTU_96 Lactobacillus futsaii — 79 2 OTU_172 Lactobacillus salivarius — 46 9 OTU_174 Lactobacillus sp. — — 84 OTU_187 Lactobacillus ruminis 12 20 18 OTU_216 Lactobacillus saerimneri — 10 — OTU_532 Lactobacillus agilis — 4 1 OTU_534 Lactobacillus acidipiscis — 5 — OTU_610 Lactobacillus sp. — 469 1 OTU_793 P1, P2, and P3 are shrimp pond 1, shrimp pond 2, and shrimp pond 3. OTU is operational taxonomic unit. “–” means not detected. water quality, improving growth rate, and enhancing disease resistance [1]. However, positive results of pro- biotic applications are mostly based on in vitro studies or small-scale in vivo trials in which all environmental con- ditions are easily managed and controlled. Meanwhile, the application of probiotics on large scales such as on commercial shrimp farms is still less investigated. Thus, questions such as does introduced probiotics could cope or compete with native bacteria and contribute to the culture of organisms in commercial farms remained to be answered. The present study traced and identiﬁed four commercial probiotic species (L. plantarum, L. fer- mentum, B. subtilis, and P. putida) which were applied in three commercial shrimp ponds. Lactobacillus and Bacillus are among the most frequently used microorganisms as probiotics in both terrestrial and aquatic cultured species [8]. Bacillus-based probiotics generally improved speciﬁc P1, P2, and P3 are shrimp pond 1, shrimp pond 2, and shrimp pond 3. OTU is operational taxonomic unit. “–” means not detected. Figure 5: KRONA visually displays the analysis result of taxonomic annotation. Circles from inside to outside stand for diﬀerent taxonomic ranks, and the area of the sector means the respective proportion of diﬀerent OTU annotation results. Each ﬁgure represents sampling locations (P1 is pond 1, P2 is pond 2, and P3 is pond 3). Each ﬁgure consists of ten pooled shrimp intestines. Figure 5: KRONA visually displays the analysis result of taxonomic annotation. 4 Discussion In pond 1, a total of 106 sequences or 0.1% of the total identiﬁed bacterial sequences were assigned into Ordo Psedomonadales, of which only 4 sequences (4% of Pseudomonadales) were identiﬁed as Pseduomonas sp. The application of probiotics has been considered the most eco-friendly method to boost aquaculture produc- tion through several mechanisms including maintaining Table 4: Bacterial species identiﬁed from the GI tracts of white shrimps reared in commercial shrimp ponds Consensus lineage P1 P2 P3 OTU ID Bacillus badius 18 2 — OTU_335 Bacillus sp. 24 1 — OTU_358 Bacillus thermoamylovorans 6 2 1 OTU_365 Bacillus coagulans — 1 2 OTU_473 P1, P2, and P3 are shrimp pond 1, shrimp pond 2, and shrimp pond 3. OTU is operational taxonomic unit. “–” means not detected. Table 4: Bacterial species identiﬁed from the GI tracts of white shrimps reared in commercial shrimp ponds Table 5: Genus Pseudomonas were identiﬁed from the GI tracts of white shrimps reared in commercial shrimp ponds Consensus lineage P1 P2 P3 OTU ID Pseudomonas geniculata — 5 — OTU_623 Pseudomonas sp. 4 2 — OTU_395 P1, P2, and P3 are shrimp pond 1, shrimp pond 2, and shrimp pond 3. OTU is operational taxonomic unit. “–” means not detected. P1, P2, and P3 are shrimp pond 1, shrimp pond 2, and shrimp pond 3. OTU is operational taxonomic unit. “–” means not detected. P1, P2, and P3 are shrimp pond 1, shrimp pond 2, and shrimp pond 3. OTU is operational taxonomic unit. “–” means not detected. The result of this study indicated that none of the four commercial probiotics was able to be detected in the shrimp ponds nor the intestinal tracts of white shrimps sampled on DOC 47. Each shrimp pond appeared to develop speciﬁc microbial communities in both rearing water and the shrimp intestines. Ponds 1 and 2, for instance, had 12 Lactobacillus species and the most dominant species was L. aviarius, but pond 3 had only two species of Lactobacillus and was dominated by L. salivarius. Similarly, the genus growth rate and feed conversion ratio through several mechanisms such as digestive enzyme secretion and pro- duction of many supplemental nutrients such as biotin, vitamin B12, fatty acids, essential amino acids, and other necessary growth factors [3]. Meanwhile, Lactobacillus- based probiotics have been reported to produce antimicro- bial compounds to suppress bacterial pathogens [4]. Figure 6: KRONA visually displays the analysis result of taxonomic annotation. Circles from inside to outside stand for diﬀerent taxonomic ranks, and the area of the sector means the respective proportion of diﬀerent OTU annotation results. The image depicts the number and proportion of Pseudomonas in the GI tract of white shrimps. Circles from inside to outside stand for diﬀerent taxonomic ranks and the area of the sector means the respective proportion of diﬀerent OTU annotation results Each ﬁgure represents sampling Figure 5: KRONA visually displays the analysis result of taxonomic annotation. Circles from inside to outside stand for diﬀerent taxonomic ranks, and the area of the sector means the respective proportion of diﬀerent OTU annotation results. Each ﬁgure represents sampling locations (P1 is pond 1, P2 is pond 2, and P3 is pond 3). Each ﬁgure consists of ten pooled shrimp intestines. The fate of probiotics applied in commercial shrimp ponds  9 The results of the present study showed that probiotic supplementation appeared not to change the structure of microbial composi- tions in the GITs of shrimps, indicated by no signiﬁcant diﬀerent in the top threbacterial phyla in both probiotic- treatment and the controls, which were Proteobacteria, Bacteroidetes and Planctomycetes. At the genus level, Rho- dopirellula, Ketogulonicigenium, Ruegeria, and Sulfurimonas were the most dominant phyla regardless of probiotic treat- ment. The bacterial diversity (phyla and genera) in probiotic treatments was also very similar to the control. microbial compositions in the rearing water of the shrimp ponds largely varied in both rearing water and the intestine of white shrimps even though all sampling ponds had similar treatments. The large variation in the microbial composition of water ponds as well as intestinal tracts of white shrimps has been previously reported in many studies [12,22]. The present study indicated that the application of probiotics in commercial outdoor farms where environmental conditions are diﬃcult to control seems not eﬀective yet. This hypothesis has been sup- ported by many studies which reported no probiotic eﬀect of the productions [9,17]. Thus, more studies and investigations are still required to apply probiotics on commercial outdoor farms. Since the introduced probiotics were not viable in the target sites, a question to be answered is “are these com- mercial probiotics able to contribute to the aquaculture species? According to Chauhan and Singh [16], probiotic viability is a very important factor in aquaculture species and serves as one of the prerequisites in screening pro- biotics for aquaculture. Less viable probiotics may not contribute well because the commercial probiotics are not viable in the target sites; thus, they may not contri- bute to shrimp farms. This might be the reason why stu- dies reported that the probiotic application does not have a signiﬁcant eﬀect on the production yields. A study by Huerta-Rábago et al. [9] reported that the addition of commercial probiotics did not aﬀect the dominant bac- teria in both phyla and genus levels in rearing ponds. Similarly, a study by Arias-Moscoso et al. [17] also reported that the addition of commercial organic and ammonia- oxidizing bacteria does not have any signiﬁcant eﬀect on water quality or waste degradation in shrimp farms cultured Acknowledging these issues, the probiotic applica- tion in commercial outdoor shrimp farms should be eval- uated. All these facts suggest that methods and strategies in applying probiotics in aquaculture species should still be carefully restudied in order to increase their eﬃcacy. Bacillus which developed in rearing water was diﬀerent from commercial bacillus. In addition, bacillus was only detected in pond 1, and none was detected in ponds 2 and 3. While three Pseudomonas were detected in pond 3 (P. psychrotolerans, P. azotoformans, and Pseudomonas sp.), only one species in pond 2 (P. azotoformans) and none were detected in pond 1. A similar result was reported by Huerta- Rábago et al. [9], where three commercial probiotics (Bacillus sp., Lactobacillus sp., and Saccharomyces sp.) introduced into white shrimp ponds at nursery stages could not be detected on DOC 7, 21, and 42. These results may suggest that the introduced probiotics were unable to cope with their new environments and failed to proliferate and grow in the target sites (the intestinal tracts of white shrimps or rearing water). There were several possibilities as to why the commercial bacteria were unable to survive based on previous studies. First, the probiotic species were isolated from signiﬁcantly diﬀerent environmental condi- tions and therefore had diﬃculty in adapting to the envir- onmental condition in the shrimp ponds or intestines of shrimps. A large loss of viability has been frequently attributed to the high acid and bile salt concentrations in the stomach and intestines [15]. Conditions of rearing water that are diﬀerent from conditions in culture including dissolved oxygen, pH, salinity, temperature, and nutrient sources will aﬀect the growth rate of probiotic bacteria and total cell yields [1]. Another possibility is that native bacteria out-compete the introduced probiotics for the same organic substrate such as carbon [1]. This result might explain the inconsistent results concerning the eﬃcacy of probiotic treatments on the survival and growth performance of white shrimps [8]. Other authors explained that probiotics may modify the balance of microbial communities in the target sites [18,19]. A study by Torpee et al. [20], for instance, reported that the introduction of probiotics suppresses opportu- nistic and/or pathogenic bacteria in the intestinal tract of white shrimp. Similarly, Vargas-Albores et al. [21] docu- mented that probiotic strains keep the microbial balance of beneﬁcial bacteria by suppressing the growth of vibrio. Then, what is the eﬀect of probiotics in the present study on the microbial composition in general? Each ﬁgure represents sampling locations (P1 is pond 1, P2 is pond 2, and P3 is pond 3). Each ﬁgure consists of 10 pooled shrimp intestines. Figure 6: KRONA visually displays the analysis result of taxonomic annotation. Circles from inside to outside stand for diﬀerent taxonomic ranks, and the area of the sector means the respective proportion of diﬀerent OTU annotation results. The image depicts the number and proportion of Pseudomonas in the GI tract of white shrimps. Each ﬁgure represents sampling locations (P1 is pond 1, P2 is pond 2, and P3 is pond 3). Each ﬁgure consists of 10 pooled shrimp intestines.  Muhamad Amin et al. 10 Bacillus which developed in rearing water was diﬀerent from commercial bacillus. In addition, bacillus was only detected in pond 1, and none was detected in ponds 2 and 3. While three Pseudomonas were detected in pond 3 (P. psychrotolerans, P. azotoformans, and Pseudomonas sp.), only one species in pond 2 (P. azotoformans) and none were detected in pond 1. A similar result was reported by Huerta- Rábago et al. [9], where three commercial probiotics (Bacillus sp., Lactobacillus sp., and Saccharomyces sp.) introduced into white shrimp ponds at nursery stages could not be detected on DOC 7, 21, and 42. These results may suggest that the introduced probiotics were unable to cope with their new environments and failed to proliferate and grow in the target sites (the intestinal tracts of white shrimps or rearing water). There were several possibilities as to why the commercial bacteria were unable to survive based on previous studies. First, the probiotic species were isolated from signiﬁcantly diﬀerent environmental condi- tions and therefore had diﬃculty in adapting to the envir- onmental condition in the shrimp ponds or intestines of shrimps. A large loss of viability has been frequently attributed to the high acid and bile salt concentrations in the stomach and intestines [15]. Conditions of rearing water that are diﬀerent from conditions in culture including dissolved oxygen, pH, salinity, temperature, and nutrient sources will aﬀect the growth rate of probiotic bacteria and total cell yields [1]. Another possibility is that native bacteria out-compete the introduced probiotics for the same organic substrate such as carbon [1]. This result might explain the inconsistent results concerning the eﬃcacy of probiotic treatments on the survival and growth performance of white shrimps [8]. with bioﬂoc technology. conditions or strategies on how probiotics are applied in commercial outdoor farms should be done in future in order to increase the probiotic eﬃcacy in white shrimp production. life stages of white shrimp have not yet been established therefore a perfect time to introduce, stir and manipulate its microbial species. In addition, shrimp at larval and early post-larval stages have less developed immune systems which may exclude the introduced probiotics species [25]. Furthermore, probiotic species may also determine the viability of probiotics in outdoor commer- cial farms. Some studies revealed that several commercial probiotics were isolated from terrestrial which have very diﬀerent environmental conditions such as salinity, nutrient availability, pH, or dissolved oxygen. These diﬀerences made such probiotic species diﬃcult to adapt, grow, and proliferate in aquatic environments. Thus, it is highly recommended to isolate and develop native/indigenous probiotic strains from surrounding environments. The native/indigenous probiotics may more easily adapt and contribute to aquaculture production. life stages of white shrimp have not yet been established therefore a perfect time to introduce, stir and manipulate its microbial species. In addition, shrimp at larval and early post-larval stages have less developed immune systems which may exclude the introduced probiotics species [25]. Furthermore, probiotic species may also determine the viability of probiotics in outdoor commer- cial farms. Some studies revealed that several commercial probiotics were isolated from terrestrial which have very diﬀerent environmental conditions such as salinity, nutrient availability, pH, or dissolved oxygen. These diﬀerences made such probiotic species diﬃcult to adapt, grow, and proliferate in aquatic environments. Thus, it is highly recommended to isolate and develop native/indigenous probiotic strains from surrounding environments. The native/indigenous probiotics may more easily adapt and contribute to aquaculture production. Acknowledgments: The authors thank colleges at the Department of Aquaculture, Faculty of Fisheries and Marine Universitas Airlangga for all support and tech- nical advice during the experiment. Funding information: This study was also ﬁnancially sup- ported by The Ministry of Education, Culture, Science and Technology, the Republic of Indonesia through Fundamental Research with Grant no. 672/UN3/2022. Author contributions: M.A1. – Conceptualization, metho- dology, software, validation, data collection, data analysis, writing originaldraft,fundingacquisition,andsubmission; Y.P. – Methodology, investigation, data collection, data analysis, draft writing, and editing; M.L. – Data collection, writing original draft, review, editing, and approved the ﬁnal manuscript; Y.C. – Data collection, draft writing, editing, data analysis, review, and editing; O.A.O. – Data collection, sequencing, data analysis, draft writing, editing, review, and approved the ﬁnal manuscript; M.A2. – Experimental design, data collection, sequencing, data analysis, review, editing, and approved the ﬁnal manu- script; A.P.W.N. – Conceptualization, data collection, data analysis, review, and approved the ﬁnal manuscript. The concept of indigenous probiotics has been docu- mented to be more eﬀective in enhancing aquaculture productions and viability is the keyword behind the suc- cess. In addition, based on the present study results, Lactobacillus appeared to be good candidates in general both in rearing water and intestinal tracts since its avail- ability seems better than both Bacillus and Pseudomonas. Our observation that probiotic treatment is less eﬀective in earthen containers could be related to the diﬃculty of exerting control over variables (probiotic access, tem- perature, dose, farm hygiene, etc.). Lactobacillus is also a member of lactic acid bacteria whose members have generally regarded as safe status for probiotics [26]. The other approach is synbiotic, which is the application of probiotics and prebiotics at the same time. Prebiotics is a nutrient which is required by probiotics to grow and pro- liferate in target sites [27]. Few studies have recently reported the application of synbiotics in white shrimps [28]. These approaches should be investigated more to increase the eﬀectiveness and eﬃcacy of probiotics in commercial outdoor farms of white shrimps. Conﬂict of interest: The authors state no conﬂict of interest. Data availability statement: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. More studies are still required in order to develop more eﬀective strategies, especially in the commercial outdoor system. Applying probiotics directly as practiced in the present study should be avoided. Some factors such as time and frequency of administration, probiotic species, administration (encapsulation) method [23], and the supplementation of prebiotics to support the nutrient requirements for probiotic species should be considered. In terms of introducing time, probiotics may exert a better eﬀect when introduced during early life [24]. Previous studies also explained stable gut microﬂora in the early The fate of probiotics applied in commercial shrimp ponds 11 5 Conclusion [7] Amin M, Agustono A, Ali M, Prayugo P, Hum NNMF. Apparent nutrient utilization and metabolic growth rate of Nile tilapia, Oreochromis niloticus, cultured in recirculating aquaculture and bioﬂoc systems. Open Agric. 2022;7(1):445–54. [8] Toledo A, Frizzo L, Signorini M, Bossier P, Arenal A. Impact of probiotics on growth performance and shrimp survival: A meta-analysis. Aquac. 2019;500:196–205. [21] Vargas-Albores F, Martínez-Córdova LR, Hernández- Mendoza A, Cicala F, Lago-Lestón A, Martínez-Porchas M. Therapeutic modulation of ﬁsh gut microbiota, a feasible strategy for aquaculture? Aquac. 2021;544:737050. [9] Huerta-Rábago JA, Martínez-Porchas M, Miranda-Baeza A, Nieves-Soto M, Rivas-Vega ME, Martínez-Córdova LR. Addition of commercial probiotic in a bioﬂoc shrimp farm of Litopenaeus vannamei during the nursery phase: eﬀect on bacterial diversity using massive sequencing 16S rRNA. Aquac. 2019;502:391–9. [22] Zhou L, Qu Y, Qin JG, Chen L, Han F, Li E. Deep insight into bacterial community characterization and relationship in the pond water, sediment and the gut of shrimp (Penaeus japo- nicus). Aquac. 2021;539:736658. [10] Yan M, Zhang X, Hu L, Huang X, Zhou Q, Zeng G, et al. Bacterial community dynamics during nursery rearing of paciﬁc white shrimp (Litopenaeus vannamei) revealed via high-throughput sequencing. Indian J Microbiol. 2020;60(2):214–21. [23] Pato U, Ayu DF, Riftyan E, Restuhadi F, Pawenang WT, Firdaus R, et al. Cellulose Microﬁber Encapsulated Probiotic: Viability, Acid and Bile Tolerance during Storage at Diﬀerent Temperature. Emerg sci J. 2022;6(1):106–17. [11] Gomes GB, Hutson KS, Domingos JA, Chung C, Hayward S, Miller TL, et al. Use of environmental DNA (eDNA) and water quality data to predict protozoan parasites outbreaks in ﬁsh farms. Aquac. 2017;479:467–73. [24] Wang R, Guo Z, Tang Y, Kuang J, Duan Y, Lin H, et al. Eﬀects on development and microbial community of shrimp Litopenaeus vannamei larvae with probiotics treatment. AMB Express. 2020;10(1):1–14. [12] Amin M, Kumala RRC, Mukti AT, Lamid M, Nindarwi DD. Metagenomic proﬁles of core and signature bacteria in the guts of white shrimp, Litopenaeus vannamei, with diﬀerent growth rates. Aquac. 2022;550:737849. [25] Kulkarni A, Krishnan S, Anand D, Kokkattunivarthil Uthaman S, Otta SK, Karunasagar I, et al. Immune responses and immu- noprotection in crustaceans with special reference to shrimp. Rev Aquac. 2021;13(1):431–59. [13] Turner CR, Uy KL, Everhart RC. Fish environmental DNA is more concentrated in aquatic sediments than surface water. Biol Conserv. 2015;183:93–102. [26] García-Cano I, Rocha-Mendoza D, Kosmerl E, Zhang L, Jiménez-Flores R. 5 Conclusion [1] Hlordzi V, Kuebutornye FK, Afriyie G, Abarike ED, Lu Y, Chi S, et al. The use of Bacillus species in maintenance of water quality in aquaculture: A review. Aquac Rep. 2020;18:100503. [1] Hlordzi V, Kuebutornye FK, Afriyie G, Abarike ED, Lu Y, Chi S, et al. The use of Bacillus species in maintenance of water quality in aquaculture: A review. Aquac Rep. 2020;18:100503. Four commercial probiotic species applied in the com- mercial grow-out shrimp ponds could not be detected from the rearing water or intestinal tracts of the white shrimps. These facts might answer why commercial ponds applying probiotics had high yield variations. The charac- teristics of probiotic species and environmental conditions in commercial outdoor farms may explain these results. Thus, more studies on selecting proper probiotic strains having good tolerance in a wide range of environmental [2] Pepi M, Focardi S. Antibiotic-resistant bacteria in aquaculture and climate change: A challenge for health in the mediterra- nean area. Int J Env Res Public Health. 2021;18(11):5723. [3] Amin M. Marine protease-producing bacterium and its poten- tial use as an abalone probiont. Aquac Rep. 2018;12:30–5. [4] Amin M, Adams MB, Burke CM, Bolch CJ. Isolation and screening of lactic acid bacteria associated with the gastro- intestinal tracts of abalone at various life stages for probiotic candidates. Aquac Rep. 2020;17:100378. 12  Muhamad Amin et al. 12 addition in a bioﬂoc shrimp farm during the nursery phase in zero water exchange. Aquac Rep. 2018;11:47–52. [5] Elsabagh M, Mohamed R, Moustafa EM, Hamza A, Farrag F, Decamp O, et al. Assessing the impact of Bacillus strains mixture probiotic on water quality, growth performance, blood proﬁle and intestinal morphology of Nile tilapia, Oreochromis niloticus. Aquacult Nutr. 2018;24(6):1613–22. [18] Swain S, Hauzoukim SKG, Das SK, Roy A. Application of pro- biotics in aquaculture. J Pharm Innov. 2021;10(7):146–9. [19] Butt UD, Lin N, Akhter N, Siddiqui T, Li S, Wu B. Overview of the latest developments in the role of probiotics, prebiotics and synbiotics in shrimp aquaculture. Fish Shellﬁsh Immunol. 2021;114:263–81. [6] Amin M, Agustono A, Prayugo P, Ali M, Hum NNMF. Comparison of total nutrient recovery in aquaponics and conventional aquaculture systems. Open Agric. 2021;6(1):682–8. [20] Torpee S, Kantachote D, Rattanachuay P, Chiayvareesajja S, Tantirungkij M. Dietary supplementation with probiotic Rhodobacter sphaeroides SS15 extract to control acute hepa- topancreatic necrosis disease (AHPND)-causing Vibrio para- haemolyticus in cultivated white shrimp. J Invertebr Pathol. 2021;186:107585. 5 Conclusion Technically relevant enzymes and proteins produced by LAB suitable for industrial and biological activity. Appl Microbiol Biotechnol. 2020;104(4):1401–22. [14] Gao S, Pan L, Huang F, Song M, Tian C, Zhang M. Metagenomic insights into the structure and function of intestinal micro- biota of the farmed Paciﬁc white shrimp (Litopenaeus van- namei). Aquac. 2019;499:109–18. [27] Wee W, Hamid NKA, Mat K, Khalif RIAR, Rusli ND, Rahman MM, et al. The eﬀects of mixed prebiotics in aquaculture: A review. Aquac Fish. 2022, in press. [15] Cook MT, Tzortzis G, Charalampopoulos D, Khutoryanskiy VV. Microencapsulation of probiotics for gastrointestinal delivery. J Control Rel. 2012;162(1):56–67. [28] Zhou L, Li H, Qin JG, Wang X, Chen L, Xu C, et al. Dietary prebiotic inulin beneﬁts on growth performance, antioxidant capacity, immune response and intestinal microbiota in Paciﬁc white shrimp (Litopenaeus vannamei) at low salinity. Aquaculture. 2020;518:734847. [16] Chauhan A, Singh R. Probiotics in aquaculture: a promising emerging alternative approach. Symbiosis. 2019;77(2):99–113. [17] Arias-Moscoso JL, Espinoza-Barrón LG, Miranda-Baeza A, Rivas-Vega ME, Nieves-Soto M. Eﬀect of commercial probiotics
https://openalex.org/W2913590592	https://www.frontiersin.org/articles/10.3389/fmars.2019.00005/pdf	English	null	Recurring Episodes of Thermal Stress Shift the Balance From a Dominant Host-Specialist to a Background Host-Generalist Zooxanthella in the Threatened Pillar Coral, Dendrogyra cylindrus	Frontiers in marine science	2,019	cc-by	12,385	ORIGINAL RESEARCH published: 23 January 2019 doi: 10.3389/fmars.2019.00005 Keywords: pillar coral, bleaching resistance, host-specialist zooxanthella, Dendrogyra cylindrus, Florida Reef Tract, Symbiodiniaceae Recurring Episodes of Thermal Stress Shift the Balance From a Dominant Host-Specialist to a Background Host-Generalist Zooxanthella in the Threatened Pillar Coral, Dendrogyra cylindrus Cynthia Lewis1,2, Karen Neely3,4 and Mauricio Rodriguez-Lanetty1* 1 Department of Biology, Florida International University, Miami, FL, United States, 2 Florida Keys Marine Lab, Long Key, FL, United States, 3 Florida Fish and Wildlife Conservation Commission, Marathon, FL, United States, 4 Department of Marine and Environmental Sciences, Nova Southeastern University, Fort Lauderdale, FL, United States 1 Department of Biology, Florida International University, Miami, FL, United States, 2 Florida Keys Marine Lab, Long Key, FL, United States, 3 Florida Fish and Wildlife Conservation Commission, Marathon, FL, United States, 4 Department of Marine and Environmental Sciences, Nova Southeastern University, Fort Lauderdale, FL, United States Citation: Lewis C, Neely K and Rodriguez-Lanetty M (2019) Recurring Episodes of Thermal Stress Shift the Balance From a Dominant Host-Specialist to a Background Host-Generalist Zooxanthella in the Threatened Pillar Coral, Dendrogyra cylindrus. Front. Mar. Sci. 6:5. doi: 10.3389/fmars.2019.00005 Most scleractinian corals form obligate symbioses with photosynthetic dinoﬂagellates (family Symbiodiniaceae), which provide differential tolerances to their host. Previously, research has focused on the inﬂuence of symbiont composition and the dynamic processes of symbiont repopulation during single episodes of hyperthermal events, followed by years of less-stressful conditions. In contrast, this study characterized for the ﬁrst time, the role of Symbiodiniaceae species changes in response to annually recurring hyperthermal events, a scenario soon expected to become the norm. Consecutive hyperthermal events during summer 2014 and 2015 along the Florida Reef Tract offered a unique opportunity to study bleaching susceptibility and recovery under recurrent annual hyperthermal scenarios. We utilized Illumina amplicon sequencing of the chloroplast 23S DNA region to assess with ﬁne resolution the Symbiodiniaceae diversity associated with pillar coral, Dendrogyra cylindrus. Our ﬁndings show diverse assemblages of Symbiodiniaceae species and that some cryptic members are not transient associates but persistent and ecologically relevant, especially during recurrent annual warming events. This was evidenced by changes in relative abundance from the typically dominant host-specialist endosymbiont, Breviolum dendrogyrum, to B. meandrinium a host-generalist species common to corals in the family Meandrinidae but occurs at background densities in most coral colonies of D. cylindrus. The rise in abundance of B. meandrinium associated strongly with bleaching resistance in the coral host during two consecutive hyperthermal events. In some cases, host-compatible background symbionts can rapidly increase in abundance during episodes of stress and may impart physiological resilience to rapid environmental change and thus, represents a potentially important ecological process by which symbiotic corals acclimatize to changing ocean conditions. Edited by: Oren Levy, Bar-Ilan University, Israel Reviewed by: Emma Mary Gibbin, École Polytechnique Fédérale de Lausanne, Switzerland Anthony William Larkum, University of Technology Sydney, Australia Correspondence: Mauricio Rodriguez-Lanetty m.rodriguez-lanetty@ﬁu.edu Correspondence: Mauricio Rodriguez-Lanetty m.rodriguez-lanetty@ﬁu.edu Specialty section: This article was submitted to Coral Reef Research, a section of the journal Frontiers in Marine Science Received: 18 August 2018 Accepted: 08 January 2019 Published: 23 January 2019 Specialty section: This article was submitted to Coral Reef Research, a section of the journal Frontiers in Marine Science Received: 18 August 2018 Accepted: 08 January 2019 Published: 23 January 2019 BACKGROUND Understanding the role of Symbiodiniaceae diversity in bleaching susceptibility and recovery, and the physiological constraints and advantages they confer on their coral hosts has become of increasing importance with escalating climate change (Cunning and Baker, 2012; Silverstein et al., 2012; Logan et al., 2014). Currently there are seven described genera and a similar number of other divergent lineages requiring generic names (LaJeunesse et al., 2018). Inter- and intra-generic diversity displays clear physiological diﬀerences (Rodriguez-Lanetty et al., 2004, 2006; Rodriguez-Lanetty, 2003; Rowan, 2004; Warner et al., 2006; Abrego et al., 2008; Sampayo et al., 2008; Fitt et al., 2009) and provides diﬀerential environmental tolerances and sensitivities to the symbiotic partnership (Jones and Berkelmans, 2010; LaJeunesse et al., 2014; Hume et al., 2015). Seminal work by Berkelmans and Van Oppen (2006) demonstrated experimentally that corals can acquire increased thermal tolerance as a direct result of changes in the symbiont genus dominating their tissues by shuﬄing existing types already present within the coral host. This study targeted the rare and iconic pillar coral, Dendrogyra cylindrus (Ehrenburg, 1834), a slow-growing columnar species typically found in low abundance throughout its Caribbean range (Figure 1). This species is currently categorized as ‘vulnerable’ under the International Union for Conservation of Nature (IUCN) Red List criteria (Aronson et al., 2008) and listed as ‘threatened’ under the US Endangered Species Act (NOAA Fisheries, 2014) due to its susceptibility to bleaching, disease, and habitat degradation. We focused on three representative sites distributed geographically along the Florida Reef Tract (Figure 2). For this study we used Illumina amplicon sequencing of the chloroplast 23S hyper-variable region (cp23S- HV) to characterize for the ﬁrst time with ﬁne resolution, the Symbiodiniaceae community assemblage associated with D. cylindrus colonies at three sites and their temporal dynamics through recurrent hyperthermal events over a 2-year period. We detected the presence of low abundance background Symbiodiniaceae genera and, at one location, observed changes in the symbiont population from the normal host-specialist species, Breviolum dendrogyrum, to a sibling species, B. meandrinium, common to corals across the family Meandrinidae. The dramatic increase in abundance of this species corresponded to enhanced colony recovery from bleaching and resistance to consecutive hyperthermal events. BACKGROUND tissues, interpreting the ecological and functional signiﬁcance of these requires caution (Lee et al., 2016). Still reef corals are often compatible with more than one symbiont species and diﬀerences in their physiological tolerances may shift population dynamics allowing for a symbiont at low abundances to proliferate within the coral animal thus changing the composition of the symbiont population to one that is better adapted to prevailing environmental conditions (Berkelmans and Van Oppen, 2006; LaJeunesse et al., 2009a; Baums et al., 2014; Grottoli et al., 2014; Parkinson and Baums, 2014). Coral reefs worldwide have experienced dramatic declines in recent decades due to natural and anthropogenic factors (Gardner et al., 2003; Van Hooidonk et al., 2016; Hughes et al., 2017). Global impacts of climate change, resulting in hyperthermal coral bleaching events (loss of symbiotic photosynthetic algae and/or chlorophyll), have become more frequent in recent decades and are projected to be annual events by 2050, or sooner on some reefs (Hoegh-Guldberg et al., 2007; Van Hooidonk et al., 2015, 2016). One of the strongest El Niño Southern Oscillation (ENSO) events on record occurred from May 2014 to June 2016 (Lian et al., 2017), causing staggering losses to coral reefs worldwide, including the Florida Reef Tract, due to consecutive bleaching events and subsequent disease outbreaks (Precht et al., 2016; Hughes et al., 2017). Studies conducted over the last two decades have been fundamental in gauging the response and the ability of corals to acclimatize to increased temperature extremes under scenarios in which isolated thermal anomalies have been followed by years of non-bleaching temperatures. However, the 2014–2016 ENSO phenomenon led to a recurrent thermal stress scenario, resulting in mass coral bleaching which detrimentally impacted coral reefs worldwide, including the Florida Reef Tract, during the summers of 2014 and 2015 (Manzello, 2015). These consecutive hyperthermal events, not experienced on Florida’s reefs since the 1997-1998 ENSO (Causey, 2001), oﬀered a unique opportunity to document spatial and temporal bleaching and post-bleaching recovery under conditions not frequently seen, but expected to become the norm in the near future based on current predictions (Hoegh-Guldberg et al., 2007; Altizer et al., 2013; Van Hooidonk et al., 2016). g Most scleractinian corals form obligate symbiotic relationships with photosynthetic dinoﬂagellates within the family Symbiodiniaceae. This partnership is critical for coral health and vital to enhanced calciﬁcation in reef-building corals (Muscatine and Porter, 1977; Mallela, 2013). Citation: Citation: Lewis C, Neely K and Rodriguez-Lanetty M (2019) Recurring Episodes of Thermal Stress Shift the Balance From a Dominant Host-Specialist to a Background Host-Generalist Zooxanthella in the Threatened Pillar Coral, Dendrogyra cylindrus. Front. Mar. Sci. 6:5. doi: 10.3389/fmars.2019.00005 January 2019 \| Volume 6 \| Article 5 1 Frontiers in Marine Science \| www.frontiersin.org Shift From Dominant Host-Specialist Zooxanthella Lewis et al. BACKGROUND Molecular approaches to Symbiodiniaceae diversity and community assemblages now allow us to further investigate the functional signiﬁcance of genetic diversity within genera, thus prompting further questions into the roles of the symbionts during environmental stressors (Sampayo et al., 2008; Bay et al., 2016). Using higher resolution DNA markers, it has become apparent that there is a very large number of species in this family (Rodriguez-Lanetty et al., 2001, 2004; Rodriguez-Lanetty, 2003; LaJeunesse, 2002; Sampayo et al., 2009; LaJeunesse and Thornhill, 2011; Thornhill et al., 2014; Wilkinson et al., 2015; Lewis et al., 2018). Additionally, advances in high-throughput amplicon sequencing technology have allowed ﬁne-scale exploration of the Symbiodiniaceae community composition by discovering more cryptic, previously undetected symbiont types occurring at abundances less than 0.01% (Arif et al., 2014; Green et al., 2014; Quigley et al., 2014; Thomas et al., 2014; Boulotte et al., 2016; Cunning et al., 2017). While various kinds of Symbiodiniaceae can be detected in trace amounts from host MATERIALS AND METHODS Field Assessments and Sampling Geographically stratiﬁed monitoring and ﬁeld sampling of 163 D. cylindrus at 29 sites across the Florida Reef Tract (FRT), Field Assessments and Sampling Total Genomic DNA Extraction Three of these sites (Pickles: Upper Keys, N = 107 colonies; Coﬃns: Middle Keys, N = 55 colonies; Marker 32: Lower Keys, N = 16 colonies; represented by stars in Figure 2) were selected spatially to represent each region and logistically due to the greater number of D. cylindrus colonies present at each site for replication of observations. These sites were targeted for more frequent sampling to more closely observe changes during bleaching and recovery (2014: September, October, November, and December; 2015: January, March, April, September, October, and November; and 2016: January and April). Sites ranged in depth from 4 to 8 m. Colonies at all sites were mapped and photographed to create ﬁeld identiﬁcation sheets for each colony to facilitate accurate repeat assessment and sampling. Pendant data loggers (Onset HOBO Inc., Bourne, MA, United States), secured to the base of colonies at Pickles, Coﬃns, and Marker 32 sites, recorded hourly temperatures between April 2014 and April 2016 (Figure 3). Temperature data was used to calculate mean daily, mean monthly, and maximum weekly sea temperatures at each site. Archived temperature data for 2004– 2013 at Molasses Reef C-MAN station MLRF1, located in the Upper Keys 4.2 km from Pickles site (NOAA National Data Buoy Total Genomic DNA Extraction DNA was extracted using modiﬁed DNeasy Plant Mini kit protocols (Quiagen Corporation, Valencia, CA, United States) (LaJeunesse, 2002). Brieﬂy, half of each sample ﬁlter was placed in 400 µl supplied lysing buﬀer and ground with a sterile pestle. Sterile acid-washed glass beads (425–600 µm; Sigma-Aldrich, Saint Louis, MO, United States) were added and shaken for 3–5 min to disrupt symbiont cell walls, followed by the addition of 20 µl proteinase K (Promega Corporation, Madison, WI, United States) and incubated at 56◦C for 1–2 h. Standard kit protocols were then followed for the remainder of the extraction. conducted every 4 months from April 2014 to April 2016, allowed for comparison before, during and after the occurrence of two consecutive hyperthermal bleaching events in 2014 and 2015. Three of these sites (Pickles: Upper Keys, N = 107 colonies; Coﬃns: Middle Keys, N = 55 colonies; Marker 32: Lower Keys, N = 16 colonies; represented by stars in Figure 2) were selected spatially to represent each region and logistically due to the greater number of D. cylindrus colonies present at each site for replication of observations. These sites were targeted for more frequent sampling to more closely observe changes during bleaching and recovery (2014: September, October, November, and December; 2015: January, March, April, September, October, and November; and 2016: January and April). Sites ranged in depth from 4 to 8 m. Colonies at all sites were mapped and photographed to create ﬁeld identiﬁcation sheets for each colony to facilitate accurate repeat assessment and sampling. Pendant data loggers (Onset HOBO Inc., Bourne, MA, United States), secured to the base of colonies at Pickles, Coﬃns, and Marker 32 sites, recorded hourly temperatures between April 2014 and April 2016 (Figure 3). Temperature data was used to calculate mean daily, mean monthly, and maximum weekly sea temperatures at each site. Archived temperature data for 2004– 2013 at Molasses Reef C-MAN station MLRF1, located in the Upper Keys 4.2 km from Pickles site (NOAA National Data Buoy conducted every 4 months from April 2014 to April 2016, allowed for comparison before, during and after the occurrence of two consecutive hyperthermal bleaching events in 2014 and 2015. Field Assessments and Sampling Geographically stratiﬁed monitoring and ﬁeld sampling of 163 D. cylindrus at 29 sites across the Florida Reef Tract (FRT), January 2019 \| Volume 6 \| Article 5 Frontiers in Marine Science \| www.frontiersin.org 2 Shift From Dominant Host-Specialist Zooxanthella Lewis et al. FIGURE 1 \| The iconic pillar coral, Dendrogyra cylindrus. This unique species occurs in historically low abundance throughout the Greater Caribbean. Its unique columnar structure provides important habitat complexity to the reef ecosystem where it does occur (Photo: C. Lewis). Center, 2016), was used as a proxy to calculate 10-year mean monthly and mean monthly maximum sea temperatures on the FRT (Supplementary Table 1B). Using the calculated mean monthly maximum temperatures at MRLF1, degree heating weeks (◦C-weeks) were calculated for each site for July, August, and September 2014 and 2015 (Liu et al., 2006) (Supplementary Table 1A). At each sampling time point, D. cylindrus colonies were assessed for live coral tissue (visual estimate of percent live, percent old mortality, percent recent mortality, i.e., bright white, recently exposed skeleton), and coral bleaching status. Colony color scores were assigned using the CoralWatch Coral Health colorimetric chart developed by Siebeck et al. (2006) and scaled from 1 (white) to 6 (heavily pigmented; Supplementary Figure 1), served as a proxy for symbiont density and chlorophyll a content. The CoralWatch Health Chart was not utilized as a color reference until September 2014, therefore, colony coloration scores for April 2014 were estimated after reviewing colony photos. Colonies were sampled at each time point using a low-impact syringe micro-sampling technique (Kemp et al., 2008) to minimize damage to the colonies from repeated sampling (sampling: Pickles n = 10 colonies, Coﬃns n = 10 colonies, Marker 32 n = 12 colonies). Brieﬂy, tissue from three to ﬁve polyps per colony was aspirated using a 30 cc syringe, transported back to shore on ice, then ﬁltered through a 13 mm Swinnex ﬁlter system (Millepore Corporation, Billerica, MA, United States), using a 3.0 µm ﬁlter disk (A/D glass ﬁber ﬁlter, Pall Corporation, Port Washington, NY, United States). Each ﬁlter disk was preserved in 95% molecular grade ethanol. FIGURE 1 \| The iconic pillar coral, Dendrogyra cylindrus. This unique species occurs in historically low abundance throughout the Greater Caribbean. Its unique columnar structure provides important habitat complexity to the reef ecosystem where it does occur (Photo: C. Lewis). Cp-23S-HV Parallel Amplicon Sequencing and Symbiodiniaceae Community Analysis Amplicon sequencing diversity assays of the Symbiodiniaceae communities was performed on Illumina MiSeq platform with 2 × 300 base pairs paired-end read capability, utilizing length variation in Domain V of large sub-unit rDNA chloroplast 23S hyper-variable region (cp-23S-HV, Santos et al., 2003; Supplementary Table 2) at the Molecular Research LP sequencing facility (MR DNA; Shallowater, TX, United States). Resulting raw sequence data (Read1 & Read2.fastq ﬁle format) were processed using MR DNA pipeline analysis. Brieﬂy, paired-end sequence reads (r1 and r2) were joined after quality control (Q25) trimming of the ends, barcoding was January 2019 \| Volume 6 \| Article 5 Frontiers in Marine Science \| www.frontiersin.org 3 Shift From Dominant Host-Specialist Zooxanthella Lewis et al. FIGURE 2 \| Study sites on the Florida Reef Tract where monitoring and sampling was conducted on the Pillar Coral Dendrogyra cylindrus. Geographically stratiﬁed tri-annual monitoring and sampling occurred at 28 sites April 2014 to April 2016 (yellow circles). Pickles (Upper Keys), Cofﬁns (Middle Keys), and Marker 32 (Lower Keys) sites (red stars) were monitored and sampled monthly during bleaching recovery in 2014 and 2015. ∗MLRF1 – location of Molasses Reef C-man buoy used to calculate 10-year mean monthly temperatures. FIGURE 2 \| Study sites on the Florida Reef Tract where monitoring and sampling was conducted on the Pillar Coral Dendrogyra cylindrus. Geographically stratiﬁed tri-annual monitoring and sampling occurred at 28 sites April 2014 to April 2016 (yellow circles). Pickles (Upper Keys), Cofﬁns (Middle Keys), and Marker 32 (Lower Keys) sites (red stars) were monitored and sampled monthly during bleaching recovery in 2014 and 2015. ∗MLRF1 – location of Molasses Reef C-man buoy used to calculate 10-year mean monthly temperatures. removed, and sequences with <150 base pair overlap were discarded. Remaining sequences were de-noised, OTUs were generated, and chimera sequences were removed. Clustering of OTUs was determined at 97% similarity (3% divergence) across all samples. Resulting OTUs were taxonomically classiﬁed using BLASTn against a selected database created from cp23S Symbiodiniaceae sequence data from NCBI1 to determine relative abundance of Symbiodiniaceae types in D. cylindrus. To conﬁrm identities of the most abundant OTUs, a BLAST-search of GenBank was performed2. Further identiﬁcation of the two most abundant B1 symbiont type OTUs was independently veriﬁed using Symbiodiniaceae microsatellite analysis (B7Sym15 primers, Supplementary Table 2) (Pettay and Lajeunesse, 2007). 1www.ncbi.nlm.nih.gov 2https://www.ncbi.nlm.nih.gov/genbank RESULTS Baseline Symbiodiniaceae Community Diversity in Dendrogyra cylindrus Before 2014 and 2015 Hyperthermal Events Baseline Symbiodiniaceae Community Diversity in Dendrogyra cylindrus Before 2014 and 2015 Hyperthermal Events Illumina amplicon sequencing of the cp23S-HV gene region showed a single OTU (or phylogenetic species) within the genus Breviolum (formerly clade B; (LaJeunesse et al., 2018) to be the dominant Symbiodiniaceae in D. cylindrus pre- bleaching (April 2014), ranging from 75 to 81% relative abundance across sites (Supplementary Table 3). Members within Symbiodinium, Cladocopium, and Durusdinium (formerly Cp-23S-HV Parallel Amplicon Sequencing and Symbiodiniaceae Community Analysis Brieﬂy, selected samples (dominant OTU ≥70% relative abundance) were PCR-ampliﬁed using the B7Sym15 primers and visualized on ABI 3100 Genetic Analyzer. Peaks were identiﬁed in each sample and compared to known Symbiodiniaceae samples in the LaJeunesse Lab (Lewis et al., 2018). April 2014 through April 2016. CoralWatch bleaching scores for colonies at the three sites were also analyzed, using repeated measures ANOVA with post hoc Tukey and Bonferroni correction (α = 0.05; Pickles n = 10, Coﬃns n = 10, Marker 32 n = 12), to compare bleaching and recovery diﬀerences between sites and between years. To determine the relationships between coral bleaching and changes in the Symbiodiniaceae community between sites, multivariate analysis of variance (MANOVA α = 0.05) was used to compare colony bleaching index (colony scores) to the relative abundance of OTUs generated by amplicon sequencing of the cp23S-HV region (Pickles n = 6, Coﬃns n = 6, Marker 32 n = 6). 1www.ncbi.nlm.nih.gov 2https://www.ncbi.nlm.nih.gov/genbank Statistical Analysis Illumina amplicon sequencing of the cp23S-HV gene region showed a single OTU (or phylogenetic species) within the genus Breviolum (formerly clade B; (LaJeunesse et al., 2018) to be the dominant Symbiodiniaceae in D. cylindrus pre- bleaching (April 2014), ranging from 75 to 81% relative abundance across sites (Supplementary Table 3). Members within Symbiodinium, Cladocopium, and Durusdinium (formerly Repeated measures analysis of variance (repeated measures ANOVA, α = 0.05) was used to compare water temperature proﬁles between the three sites (Pickles, Coﬃns, and Marker 32) through two consecutive bleaching and recovery periods January 2019 \| Volume 6 \| Article 5 Frontiers in Marine Science \| www.frontiersin.org 4 Shift From Dominant Host-Specialist Zooxanthella Lewis et al. FIGURE 3 \| Water temperatures at three sites in the Florida Keys recorded between April 2014 and April 2016. The Florida Reef Tract (FRT) in the Florida Keys is divided into distinct regions based on hydrologic inﬂuences, represented here by three sites: Pickles, Upper Keys; Cofﬁns, Middle Keys; and Marker 32, Lower Keys. Red dashed lines indicate the local bleaching threshold for the FRT (30.5◦C). Gaps in data are due to lost or damaged temperature loggers. (A) Mean monthly water temperature data. Dotted black line is the calculated 10-year mean monthly water temperature (error bars +1 SD) for Molasses Reef 2004-2013 (MLRF1, National Data Buoy Center). (B) Mean daily water temperature data. l d d l ( l ) k b d ll l FIGURE 3 \| Water temperatures at three sites in the Florida Keys recorded between April 2014 and April 2016. The Florida Reef Tract (FRT) in the Florida Keys is divided into distinct regions based on hydrologic inﬂuences, represented here by three sites: Pickles, Upper Keys; Cofﬁns, Middle Keys; and Marker 32, Lower Keys. Red dashed lines indicate the local bleaching threshold for the FRT (30.5◦C). Gaps in data are due to lost or damaged temperature loggers. (A) Mean monthly water temperature data. Dotted black line is the calculated 10-year mean monthly water temperature (error bars +1 SD) for Molasses Reef 2004-2013 (MLRF1, National Data Buoy Center). (B) Mean daily water temperature data. FIGURE 3 \| Water temperatures at three sites in the Florida Keys recorded between April 2014 and April 2016. Frontiers in Marine Science \| www.frontiersin.org Statistical Analysis The Florida Reef Tract (FRT) in the Florida Keys is divided into distinct regions based on hydrologic inﬂuences, represented here by three sites: Pickles, Upper Keys; Cofﬁns, Middle Keys; and Marker 32, Lower Keys. Red dashed lines indicate the local bleaching threshold for the FRT (30.5◦C). Gaps in data are due to lost or damaged temperature loggers. (A) Mean monthly water temperature data. Dotted black line is the calculated 10-year mean monthly water temperature (error bars +1 SD) for Molasses Reef 2004-2013 (MLRF1, National Data Buoy Center). (B) Mean daily water temperature data. in GeneBank. Using Symbiodiniaceae microsatellite analysis (B7Sym15 primers, Supplementary Table 2), the identity of these two sequences was veriﬁed as ITS2 types B1-4k and B1-3 (species novo: Breviolum dendrogyrum and B. meandrinium, respectively; LaJeunesse et al., 2018; Lewis et al., 2018). There was no diﬀerence in relative abundances of B. dendrogyrum or B. meandrinium compared between sites in April 2014 prior to bleaching (two- way ANOVA α = 0.05, p > 0.05). clades A, C, and D respectively; (LaJeunesse et al., 2018) were also detected in cryptic low abundance (<0.1%). Amplicon diversity analysis yielded 266 OTUs (0.97% similarity cut- oﬀ). All but fourteen OTUs were taxonomically classiﬁed as genus Breviolum (formerly clade B Symbiodinium). Moreover, the combined relative abundance of all 252 Breviolum OTUs represented > 99% of the Symbiodiniaceae community at all three representative sites along the Florida Reef Tract (Pickles, Coﬃns, and Marker 32). Two hundred thirty-two Breviolum OTUs were rare members of the Symbiodiniaceae community detected at <0.01% relative abundance. The two most abundant OTUs accounted for >80% of the symbiont community at all three sites. These two sequences were both identiﬁed as sub- genera Breviolum type cp-23S B184 (or ITS2 type B1) phylotypes Ninety-four OTUs belonging to the genus Symbiodinium (formerly clade A) were detected in cryptic low abundance at Pickles, Coﬃns, and Marker 32 in April 2014 (Supplementary Table 3). These OTUs were conﬁrmed by BLAST-search as belonging to Symbiodinium strains A2, A3, and A13 (strain A13, putatively S. necroappetens). Three cryptic Cladocopium OTUs January 2019 \| Volume 6 \| Article 5 Frontiers in Marine Science \| www.frontiersin.org 5 Shift From Dominant Host-Specialist Zooxanthella Lewis et al. (formerly clade C) were detected only at Pickles and Marker 32 sites. A single OTU, classiﬁed as Durusdinium (strain D1a, putatively D. trenchii), was only detected at Marker 32 in April 2014. Consecutive Hyperthermal Bleaching on the Florida Reef Tract in 2014 and 2015 the Florida Reef Tract in 2014 and 2015 Florida’s reefs exceeded 5◦C-weeks (degree heating weeks) during the summers of 2014 and 7◦C-weeks in 2015, based on NOAA’s Coral Reef Watch 50-km Satellite Monitoring (NOAA Coral Reef Watch, 2000), updated twice-weekly; NOAA OSPO, 2018) (Supplementary Table 1A). Data loggers at the three study sites recorded sea water temperatures which exceeded the FRT bleaching index of 30.5◦C (Manzello, 2015) (Figure 3), causing severe bleaching in most coral species across the FRT (personal observation and Precht et al., 2016). Maximum weekly temperatures in 2014 exceeded the FRT bleaching threshold (30.5◦C) 10 weeks at Coﬃns and 8 weeks at Marker 32 (Figure 3 and Supplementary Table 1B). Data loggers were lost at the Pickles site from April to September 2014 and thus meaningful temperature analysis could not be included. In 2015, maximum weekly temperatures exceeded the bleaching threshold 12 weeks at Pickles and 13 weeks at both Coﬃns and Marker 32. Using mean maximum monthly calculations from MRLF1, degree heating weeks at Coﬃns (Middle Keys) exceeded 7◦C-weeks in 2014 and 5◦C-weeks in 2015 (Supplementary Table 1B). Degree heating weeks at Marker 32 (Lower Keys) was nearly 3◦C-weeks in 2014 and 4◦C-weeks in 2015. Degree heating weeks at Pickles (Upper Keys) was 0◦C-weeks in 2015 (no data from 2014). Utilizing the FRT bleaching threshold (30.5◦C), calculated DHW was greater at the three sites (Supplementary Table 1B). The number of weeks maximum water temperatures exceeded the bleaching threshold were greater in 2015. Summary of water temperatures from June through October (i.e., warmest summer months) in 2014 and 2015 show diﬀerences in temperature characteristics between sites and between years (Figure 4; repeated measures ANOVA p < 0.04), except for July 2015 (p = 0.076). While all sites experienced temperatures exceeding the bleaching threshold, Coﬃns experienced the highest temperatures in August 2014, however, Marker 32 was warmer (maximum and median temperatures) than the other two sites in September 2014 (p < 0.01). During the second hyperthermal event in 2015, Coﬃns and Marker 32 sites experienced warmer temperatures than Pickles site (maximum and median temperatures; p < 0.01). During the second hyperthermal event in August-September 2015, site-speciﬁc diﬀerences in bleaching and recovery were observed. Colonies at Coﬃns and Marker 32 again bleached, although Coﬃns colonies were less severely bleached than September 2014, indicated by higher coloration scores (two- way ANOVA, p < 0.01; Figure 5 and Supplementary Table 4). Breviolum Species Switched Dominance Associated With Hyperthermal Events Associated With Hyperthermal Events The Symbiodiniaceae community in D. cylindrus was dynamic in response to hyperthermal bleaching and the subsequent recovery processes following two consecutive bleaching events. Relative abundance of the endosymbionts varied between three representative sites, especially the two species within Breviolum described above (Figure 6). At the Pickles site (Upper Keys) and associated with the August-September 2014 hyperthermal event, previously cryptic B. meandrinium (<10% relative abundance) became the dominant endosymbiont species in October 2014 and persisted through April 2015 while B. dendrogyrum declined in relative abundance through December 2014 to 20.60% but then slowly increased to 33.35% through April 2015 (Figure 6A and Supplementary Table 3A), but not to pre-bleaching abundance of 75.68% in April 2014. At the Coﬃns site (Middle Keys), B. dendrogyrum remained dominant through bleaching and recovery in 2014. Breviolum meandrinium remained at low Statistical Analysis In response to hyperthermal events in 2014 and 2015, patterns of bleaching and recovery diﬀered between sites as well as between years. During the ﬁrst bleaching event on the FRT (August–September 2014), all D. cylindrus colonies, including at Pickles, Coﬃns, and Marker 32 sites were severely bleached (Figure 5 and Supplementary Table 4). Colonies at Marker 32 were the most severely bleached, followed by Coﬃns colonies and ﬁnally Pickles (two-way ANOVA; p < 0.01). Pickles and Coﬃns colonies regained normal coloration by December 2014 and January 2015. From January to April 2015, colonies at these two sites paled (two-way ANOVA, p < 0.01). Marker 32 colonies recovered slowly, remaining pale through April 2015 (Figure 5 and Supplementary Table 4). Consecutive Hyperthermal Bleaching on the Florida Reef Tract in 2014 and 2015 However, unlike these two sites, Pickles colonies were more resistant to bleaching in 2015, with no signiﬁcant change in colony coloration from April to September 2015 (two-way ANOVA, p = 0.82). While there was no bleaching observed in these colonies during the hyperthermal event, there was an increase in colony coloration from September to October (p = 0.01) which remained constant through January 2016 (Figure 5 and Supplementary Table 4). Coﬃns colonies also recovered normal coloration by January 2016. Both Pickles and Coﬃns colonies again paled during the 2016 winter-spring transition (p = 0.01). Although many colonies at Marker 32 remined pale after September 2015, they appeared to recover slightly when compared with the previous year, indicated by near- normal coloration scores in November 2015 and January 2016 (mean scores: 3.46 ± 0.33 and 3.76 ± 0.33; two-way ANOVA, p < 0.01). Colonies at Marker 32 were considered pale again in April 2016 (mean score: 3.01 ± 1.13; one-way ANOVA p = 0.06; Figure 5 and Supplementary Table 4). Differential Bleaching Resistance and Resilience The CoralWatch Coral Health colorimetric chart developed by Siebeck et al. (2006) was used to determine the bleaching status of colonies. Healthy, non-thermally stressed colonies ranged in color between 3.5 and 4.5 on this chart (Supplementary Figure 1). Colonies with scores between 1.5 and 3.5 were considered pale, while colonies with scores < 1.5 were considered bleached. January 2019 \| Volume 6 \| Article 5 Frontiers in Marine Science \| www.frontiersin.org 6 Lewis et al. Shift From Dominant Host-Specialist Zooxanthella FIGURE 4 \| Summary of water temperature proﬁles at three sites on the Florida Reef Tract during summer 2014 and 2015. Five-number summary of temperature proﬁles at three sites (Pickles: Upper, Cofﬁns: Middle, Marker 32: Lower) along the Florida Reef Tract. Hourly water temperatures from pendant data loggers were used to create temperature proﬁles from June through October 2014 and 2015. Box plots indicate interquartile temperature ranges (Q2–Q3) and median temperatures. ‘Whiskers’ show maximum and minimum water temperature during this time period. Gray triangles indicate incomplete temperature proﬁles due to lost data loggers. FIGURE 4 \| Summary of water temperature proﬁles at three sites on the Florida Reef Tract during summer 2014 and 2015. Five-number summary of temperature proﬁles at three sites (Pickles: Upper, Cofﬁns: Middle, Marker 32: Lower) along the Florida Reef Tract. Hourly water temperatures from pendant data loggers were used to create temperature proﬁles from June through October 2014 and 2015. Box plots indicate interquartile temperature ranges (Q2–Q3) and median temperatures. ‘Whiskers’ show maximum and minimum water temperature during this time period. Gray triangles indicate incomplete temperature proﬁles due to lost data loggers. abundance but showed a slight but signiﬁcant increase from October to December 2014 (two-way ANOVA p = 0.01), reaching maximum 20.15% relative abundance in April 2015 (Figure 6B and Supplementary Table 3B). Similar to the Coﬃns site, B. dendrogyrum remained the dominant species at Marker 32 (Lower Keys; Figure 6C and Supplementary Table 3C). again dynamic and site-speciﬁc. At the Pickles site, ﬂuctuation in dominance was again detected in which B. dendrogyrum brieﬂy increased in abundance and re-established dominance by September 2015 (53.41%; two-way ANOVA, p = 0.02) however, this was short-lived. Relative abundance of B. meandrinium slowly declined through September 2015 to 30.28% but then quickly increased during the recovery months to persist as the dominant endosymbiont through April 2016 (51.55%). Differential Bleaching Resistance and Resilience In response to the second hyperthermal event in August- September 2015, the two most abundant Breviolum species were January 2019 \| Volume 6 \| Article 5 Frontiers in Marine Science \| www.frontiersin.org 7 Shift From Dominant Host-Specialist Zooxanthella Lewis et al. FIGURE 5 \| Mean bleaching index based on coloration scores of D. cylindrus colonies at three sites on the FRT between 2014 and 2016. The coloration scores were determined using the CoralWatch Coral Health Chart for colorimetric reference (version: Project Aware https://coralwatch.org; Siebeck et al., 2006). Coloration ranges for D. cylindrus based on triannual assessments of 168 colonies: healthy 3.5–4.0; pale: 2.0–3.0; bleached: 1.0–1.5. Dashed gray line represents trajectory of estimated colony scores from April 2014. Pickles (n = 12) Upper Keys; Cofﬁns (n = 12) Middle Keys; Marker 32 (n = 11) Lower Keys. Error bars ± 1 standard deviation. FIGURE 5 \| Mean bleaching index based on coloration scores of D. cylindrus colonies at three sites on the FRT between 2014 and 2016. The coloration scores were determined using the CoralWatch Coral Health Chart for colorimetric reference (version: Project Aware https://coralwatch.org; Siebeck et al., 2006). Coloration ranges for D. cylindrus based on triannual assessments of 168 colonies: healthy 3.5–4.0; pale: 2.0–3.0; bleached: 1.0–1.5. Dashed gray line represents trajectory of estimated colony scores from April 2014. Pickles (n = 12) Upper Keys; Cofﬁns (n = 12) Middle Keys; Marker 32 (n = 11) Lower Keys. Error bars ± 1 standard deviation. FIGURE 5 \| Mean bleaching index based on coloration scores of D. cylindrus colonies at three sites on the FRT between 2014 and 2016. The coloration scores were determined using the CoralWatch Coral Health Chart for colorimetric reference (version: Project Aware https://coralwatch.org; Siebeck et al., 2006). Coloration ranges for D. cylindrus based on triannual assessments of 168 colonies: healthy 3.5–4.0; pale: 2.0–3.0; bleached: 1.0–1.5. Dashed gray line represents trajectory of estimated colony scores from April 2014. Pickles (n = 12) Upper Keys; Cofﬁns (n = 12) Middle Keys; Marker 32 (n = 11) Lower Keys. Error bars ± 1 standard deviation. From September 2015 to April 2016, B. dendrogyrum slowly declined to 29.97% (Figure 6A and Supplementary Table 3A). At the Coﬃns site, B. dendrogyrum remained the dominant species throughout the second bleaching and recovery (2015) and returned to similar abundance observed in April 2014 baseline (Figure 6B and Supplementary Table 3B). Differential Bleaching Resistance and Resilience Breviolum meandrinium reached a maximum abundance of 20.39% in September 2015 at this site and declined to 0.12% in January 2016, remaining unchanged through April 2016 (two-way ANOVA, p < 0.01). And ﬁnally, at the Marker 32 site, relative abundance of B. dendrogyrum and B. meandrinium did not change from April to September 2015 or thereafter, and B. meandrinium remained cryptic (<0.1% abundance) through April 2016 (Figure 6C and Supplementary Table 3C). During the second hyperthermal event in August-September 2015, while B. dendrogyrum brieﬂy regained dominance by September, B. meandrinium once again became the dominant species, persisting through recovery until April 2016. This change was closely associated with the observation that colonies at Pickles did not bleach during the second hyperthermal event (Figure 5 and Supplementary Table 4), indicating acquired resistance to annual thermal stress (water temperatures exceeding 30.5◦C, Figures 3, 4). This resistance was strongly associated with a ﬂuctuation in Symbiodiniaceae species, speciﬁcally an increase and persistence in abundance of B. meandrinium as it became the dominant species in D. cylindrus colonies at the Pickles site (MANOVA p < 0.001). Colonies at the Coﬃns site (Middle Keys) also bleached severely in August-September 2014 (Figure 5 and Supplementary Table 4). As normal colony coloration returned in December 2014, cryptic B. meandrinium slightly increased in abundance, reaching 20.15% by April 2015 (Figure 6B and Supplementary Table 3B). Although B. meandrinium never became the dominant Breviolum species at Coﬃns, colonies did not bleach as severely the second year and recovered quickly to normal coloration by January 2016 (Figure 5 and Supplementary Table 4). This strongly suggests at least partial resistance to bleaching associated with an increased relative abundance in cryptic B. meandrinium (MANOVA p = 0.068). Colonies at Marker 32 (Lower Keys) bleached severely both in 2014 and 2015 (Figure 5 and Supplementary Table 4) while B. meandrinium remained at cryptic low levels throughout, reaching a maximum abundance of only 2.60% in September Bleaching Resistance and Resilience The magnitude of change in relative abundance of dominant Breviolum dendrogyrum and cryptic B. meandrinium during two consecutive hyperthermal bleaching events was site speciﬁc and closely associated with diﬀerential resistance and resilience to bleaching and recovery. Colonies at the Pickles site (Upper Keys) bleached severely during August-September 2014 and then recovered by December 2014 (Figure 5 and Supplementary Table 4). Concurrently, the relative abundance of dominant endosymbiont B. dendrogyrum declined while cryptic B. meandrinium increased to become the dominant symbiont. Relative abundance of these two species did not return to pre- bleaching levels (Figure 6A and Supplementary Table 3A). January 2019 \| Volume 6 \| Article 5 Frontiers in Marine Science \| www.frontiersin.org 8 Lewis et al. Shift From Dominant Host-Specialist Zooxanthella E 6 \| Relative abundance of Symbiodiniaceae symbionts from the genus Breviolum associated with D. cylindrus on the Florida Reef Tract between 2014 and The dynamics of Breviolum dendrogyrum (blue) and B. meandrinium (green) between three sites and between years, by percent relative abundance of um spp. OTUs. B. meandrinium increased in relative abundance during bleaching and recovery, becoming the dominant symbiont species at the Pickles site and 2015. (A) Pickles (n = 6) Upper Keys, (B) Cofﬁns (n = 6) Middle Keys, (C) Marker 32 (n = 6) Lower Keys. ‘All other Breviolum’ (blue-gray) represents 250 ombined. Asterisk (∗) indicates bleaching months. FIGURE 6 \| Relative abundance of Symbiodiniaceae symbionts from the genus Breviolum associated with D. cylindrus on the Florida Reef Tract between 2014 and 2016. The dynamics of Breviolum dendrogyrum (blue) and B. meandrinium (green) between three sites and between years, by percent relative abundance of Breviolum spp. OTUs. B. meandrinium increased in relative abundance during bleaching and recovery, becoming the dominant symbiont species at the Pickles site in 2014 and 2015. (A) Pickles (n = 6) Upper Keys, (B) Cofﬁns (n = 6) Middle Keys, (C) Marker 32 (n = 6) Lower Keys. ‘All other Breviolum’ (blue-gray) represents 250 OTUs combined. Asterisk (∗) indicates bleaching months. January 2019 \| Volume 6 \| Article 5 Frontiers in Marine Science \| www.frontiersin.org 9 Lewis et al. Shift From Dominant Host-Specialist Zooxanthella 2014 (Figure 6C and Supplementary Table 3C; MANOVA, p = 0.981). 2014 (Figure 6C and Supplementary Table 3C; MANOVA, p = 0.981). associated with greater bleaching resistance during consecutive hyperthermal events. associated with greater bleaching resistance during consecutive hyperthermal events. Abundance were only detected from April to October 2014 and then not after at the Pickles site (Supplementary Table 3). Cladocopium spp. were detected at Coﬃns after the ﬁrst bleaching in September 2014 and persisted intermittently during recovery until November 2015. At Marker 32, Cladocopium spp. were detected at cryptic levels in the April 2014 baseline sampling (<0.001%) and persisted through April 2016 (0.21% abundance). Cladocopium spp. were only detected from April to October 2014 and then not after at the Pickles site (Supplementary Table 3). Cladocopium spp. were detected at Coﬃns after the ﬁrst bleaching in September 2014 and persisted intermittently during recovery until November 2015. At Marker 32, Cladocopium spp. were detected at cryptic levels in the April 2014 baseline sampling (<0.001%) and persisted through April 2016 (0.21% abundance). A single OTU, classiﬁed as Durusdinium sp. and identiﬁed by BLAST-search as type D1a (putatively D. trenchii, formerly S. trenchii), was ﬁrst detected in cryptic low abundance at Pickles and Coﬃns sites in October 2014 after the 2014 bleaching event and then intermittently through the second bleaching and recovery period until January 2016 (Supplementary Table 3). At Marker 32, Durusdinium sp. was detected pre-bleaching in April 2014 and then intermittently throughout both bleaching and recovery periods through April 2016. A single OTU, classiﬁed as Durusdinium sp. and identiﬁed by BLAST-search as type D1a (putatively D. trenchii, formerly S. trenchii), was ﬁrst detected in cryptic low abundance at Pickles and Coﬃns sites in October 2014 after the 2014 bleaching event and then intermittently through the second bleaching and recovery period until January 2016 (Supplementary Table 3). At Marker 32, Durusdinium sp. was detected pre-bleaching in April 2014 and then intermittently throughout both bleaching and recovery periods through April 2016. Abundance Although Breviolum (formerly clade B) remained the dominant genus in D. cylindrus through two consecutive bleaching and recovery events, cryptic Symbiodinium, Cladocopium, and Durusdinium species (formerly clades A, C, and D, respectively) were also detected and often persisted through recovery (Supplementary Table 3). Symbiodinium spp. were detected in cryptic low abundance (<0.01–5.38%) from April 2014 through December 2014 and then not thereafter at Pickles site (Upper Keys). At the Coﬃns site (Middle Keys), Symbiodinium spp. were detected intermittently through both bleaching and recovery events. At Marker 32 (Lower Keys), Symbiodinium spp. were detected in April 2014 and persisted at cryptic low abundance though April 2016 (≤0.2%). By revealing a Symbiodiniaceae community represented by 266 OTUs, with the majority in very low relative abundances (<0.1%), our ﬁndings indicate that past studies have considerably underestimated the actual diversity of endosymbionts associated with D. cylindrus, and likely many other coral species, although at least some of these rare and cryptic OTUs may in fact be sequencing artifacts (Arif et al., 2014). Most symbiont types detected in our study (>99%) belonged to the genus Breviolum spp. (formerly clade B). Three other genera, Symbiodinium, Cladocopium, and Durusdinium (formerly clades A, C, and D, respectively) were detected at extremely low levels (0.001– 5.38%) in D. cylindrus. Discovery of this tremendous sequence diversity within the genus Breviolum associated with D. cylindrus opens new questions regarding whether these symbiont types are the reﬂection of population variability within species or are indeed independent evolutionary lineages (i.e., species). Symbiont assemblages across sites and within individual D. cylindrus colonies were dominated by a single Breviolum species of endosymbiont. Using single copy microsatellite markers (see Materials and Methods), it was conﬁrmed that this symbiont corresponded to the recently described Breviolum dendrogyrum (Lewis et al., 2018). Furthermore, low concentrations of Breviolum meandrinium, which was also identiﬁed and conﬁrmed with the use of diagnostic microsatellites, increased in relative abundance as a function of environmental stress. Breviolum meandrinium is common to corals in the family Meandrinidae from shallow habitats (1-10 m) across the Greater Caribbean. Dendrogyra cylindrus is one notable exception of the family in that it harbors a unique host-specialist, which appears adapted to associating only with this host. These ﬁndings highlight how a host-generalist, B. meandrinium, normally rare in D. cylindrus, can proliferate in colonies subjected to severe thermal stress. Cladocopium spp. Frontiers in Marine Science \| www.frontiersin.org DISCUSSION This diﬀerence across sites seems not linked to diﬀerent temperature proﬁles during the two hyperthermal events, since all sites experienced similar exposure to elevated temperatures above the 30.5◦C bleaching threshold for the FRT (Figures 3, 4). While we cannot explain why the shift of Symbiodiniaceae assemblages did not occur across all sites, it is important to note that baseline abundance of the cryptic B. meandrinium prior to the ﬁrst hyperthermal event in April 2014 was higher in Pickles colonies than the other two sites (Supplementary Table 3). Perhaps some critical minimum abundance may be required for a rare symbiont to out-compete dominant symbionts when the opportunity arises, such as during bleaching and recovery. However, the relative abundance of B. meandrinium in Coﬃns colonies was higher (20.15%) prior to the second hyperthermal event and still we did not see a shift in symbiont assemblages during the second bleaching and recovery event at this site. This suggests the existence of other site-speciﬁc factors inﬂuencing the dynamics and competition processes of symbiont repopulation after environmental stressors. Alternatively, genotypic diﬀerences in the coral animal may contribute to the observed diﬀerential symbiont ﬂexibility, but their role is thus far unknown. Recent work on genetic diversity in Florida’s D. cylindrus population indicates that each of these three sites is represented by unique coral genotypes (Chan et al., 2018). All colonies at the Pickles site comprised one genotype, indicating a high level of clonality at this local. The same pattern of clonality was also detected for colonies at the Coﬃns site. The Marker 32 site had the most genetic diversity, composed of ﬁve diﬀerent coral animal genotypes, and yet was the population most aﬀected by these hyperthermal events. Continued work on the role of genetic diversity in the coral host and its symbiont requires further investigation. p g The Adaptive Bleaching Hypothesis posits that when corals bleach, they expel less thermally tolerant endosymbionts and then acquire new, more favorable endosymbionts, allowing them to acclimatize and adapt to environmental stressors (Buddemeier and Fautin, 1993; Baker et al., 2004; Buddemeier et al., 2004). DISCUSSION Research over the last two decades has documented the inﬂuence of the symbiont composition and the dynamic processes of symbiont repopulation during the coral response to single episodes of hyperthermal stress events, followed by years of normal environmental conditions (Edmunds et al., 2014; Kemp et al., 2014). Our study characterized changes in symbiont species within some colonies of Dendrogyra cylindrus in response to a consecutive hyperthermal event. This study contributes to further understanding how coral-algal mutualisms may respond through shifts in partnerships under long-predicted environmental scenarios (annual bleaching) (Hoegh-Guldberg, 1999; Grottoli et al., 2014), which coral reefs worldwide are now experiencing (Hughes et al., 2017). Our ﬁndings show unexpectedly diverse assemblages of Symbiodiniaceae associated with Dendrogyra cylindrus, and that one low abundant host-compatible species was not a transient associate but persistent and ecologically relevant symbionts that plays a role during thermal stress. Furthermore, site-speciﬁc shifts in Symbiodiniaceae species dominance were It has been suggested that most cryptic Symbiodiniaceae are transitory and likely provide minimal ecological signiﬁcance for their coral hosts (Lee et al., 2016). However, recent studies have challenged this view and suggested that rare symbionts tend to be non-random clusters of coral host-symbiont communities and may provide environmental resilience for the coral holobiont (Ziegler et al., 2015, 2017). In agreement with this, our data show a clear association between a site-speciﬁc increase and persistence in relative abundance of the low abundance background B. meandrinium and the overall stability of the host– symbiont community during subsequent hyperthermal stress. At the Pickles site in the Upper Keys region of the Florida January 2019 \| Volume 6 \| Article 5 Frontiers in Marine Science \| www.frontiersin.org 10 Shift From Dominant Host-Specialist Zooxanthella Lewis et al. Reef Tract, B. meandrinium increased in relative abundance during the ﬁrst hyperthermal bleaching event in 2014 and rapidly switched to become the dominant symbiont during the ﬁrst 7 months of recovery (Figure 6A and Supplementary Table 3). This change in the relative abundances of two species persisted for at least 11 months under normal conditions, but by September 2015, B. dendrogyrum displaced B. meandrinium as the dominant symbiont. DISCUSSION This reversal of endosymbiont assemblages is consistent with other studies that have monitored changes before and after non-recurrent hyperthermal events, where changes in symbionts during bleaching episodes reverted to the original state after several months or even years (Thornhill et al., 2006; Sampayo et al., 2008; LaJeunesse et al., 2009b; Grottoli et al., 2014). Nevertheless, the reversion to the normal symbiont in D. cylindrus was short-lived as B. meandrinium again become the dominant symbiont species among colonies at the Pickles site in response to the second hyperthermal event in August–September 2015. These host-symbiont combinations persisted and, at the time of the last sampling for this study in April 2016, B. meandrinium remained the dominant species (Figure 6A and Supplemental Table 3A). Importantly, corals at this site did not lose color during this second event (Figure 5); the relative abundance of B. meandrinium was 3–4 times higher during the onset of the 2015 hyperthermal event in comparison to pre-bleaching levels in April 2014. Under scenarios of annual hyperthermal bleaching events, impacted coral communities may not have suﬃcient time to fully recover their stable host-symbiont pairings. pairings may be longer-lived and maintain higher relative proportions within the Symbiodiniaceae community as the period of less-stressful environmental conditions between disturbances becomes shorter (Grottoli et al., 2014). Such was the case at the Pickles site where relative abundance of B. meandrinium remained near 50% and, while trace amounts of Durusdinium persisted through April 2016, Symbiodinium sp. and Cladocopium sp. disappeared altogether (Supplementary Table 3A). Our data did not show a shift in dominance at the other two sites (Figures 6B,C) and, unlike Pickles colonies, they bleached again during the second hyperthermal event in August-September 2015 (Figure 5). However, at the Coﬃns site (Middle Keys), we detected an increase in relative abundance of B. meandrinium to 20.39% during the second hyperthermal event (August–September 2015; Figure 6B and Supplementary Table 3B), which was associated with only paling in most colonies at this site (Figure 5 and Supplementary Table 4). While B. meandrinium did not become the dominant Symbiodiniaceae species, perhaps some threshold abundance may also impart at least partial bleaching resistance for the coral animal (Bay et al., 2016), as was observed at the Coﬃns site. The Lower Keys site (Marker 32) showed minimal ﬂuctuation in the endosymbiont community but also bleached more severely and recovered more slowly after both bleaching events. Frontiers in Marine Science \| www.frontiersin.org CONCLUSION While it is encouraging to substantiate that coral symbioses respond ecologically and thus “acclimatize” to a changing climate through shifts in the dominant symbiont partner, it may not be enough for their long-term survival in the Anthropocene. As annual thermal bleaching is predicted to become more prevalent on many reefs in the coming decades, the collateral damage to biological, physiological, and immunological functions of the coral holobiont (Bellantuono et al., 2012; Pinzón et al., 2015) may negate their innate ability to acclimatize. It is imperative that we address the issues of environmental stressors in the hopes that at least some reef ecosystems will be able to acclimate and survive in a changing climate. Even this glimmer of hope for survival may prove to be ‘too little, too late’ as alarming coral losses due to escalating disease outbreaks in warming oceans may overcome these slow-growing monarchs of the reef more quickly than they can adapt. NOMENCLATURE • cp23S-HV primers - utilize length variation in Domain V of large sub-unit rDNA chloroplast 23S hyper-variable gene region g • ENSO - El Niño/ La Niña Southern Oscillation is characterized by oscillating changes from expected sea surface temperatures in the eastern and central equatorial Paciﬁc Ocean (El Niño – warm phase, and La Niña – cool phase) • FRT – Florida Reef Tract • Illumina Mi-Seq platform – high resolution parallel amplicon sequencing diversity assay • Symbiodiniaceae Family (LaJeunesse et al., 2018) – photosynthetic endosymbiotic dinoﬂagellates One of three cryptic Symbiodinium OTUs detected in D. cylindrus and identiﬁed putatively as Symbiodinium necroappetens (strain A13), is considered an opportunist, emerging transiently to associate with thermally stressed or diseased corals (LaJeunesse et al., 2015). Our data show that Symbiodinium spp. were part of the cryptic community in D. cylindrus even prior to the 2014–2015 thermal events (April 2014; Supplementary Table 3). At sites showing an increased abundance of B. meandrinium in response to annual thermal events (Pickles and Coﬃns), Symbiodinium and Cladocopium genera were not detected, and may have been displaced as B. meandrinium approached 20% relative abundance. Alternatively, the occurrence and persistence of cryptic symbionts in D. cylindrus and other Caribbean corals ◦Symbiodinium spp. (living together, whirling) – formerly Clade A ◦Breviolum spp. (short and small) – formerly Clade B ◦Cladocopium spp. (branch and plenty) - formerly Clade C ◦Durusdinium spp. (tough and whirling) – formerly Clade D Persistent Cryptic Communities as a Source of More Thermally Tolerant Strains for Acclimatization and Adaptation Our data support that under certain circumstances, a low abundant symbiont may emerge during thermal bleaching and/or recovery to enhance acclimatization of the coral host (Kemp et al., 2014). Durusdinium sp., known to be opportunistic in certain Caribbean corals exposed to stress (LaJeunesse et al., 2009b), remained at trace levels (<0.03%) during the 2014 and 2015 bleaching and recovery events on the FRT. Due to the high resolution of this study, this is the ﬁrst reporting of Durusdinium trenchii (formerly Symbiodinium trenchii) in Dendrogyra cylindrus, suggesting an expansion of this invasive, more thermally tolerant species into another Caribbean coral host (LaJeunesse et al., 2010, 2014; Wham et al., 2011). Durusdinium trenchii is a stress-tolerant species within the genus Durusdinium, commonly found in the Indo-Paciﬁc and known to impart thermal tolerance and bleaching resistance to its coral host (Pettay and Lajeunesse, 2009). This species is considered invasive in the Greater Caribbean and has been increasingly found in corals inhabiting marginal habitats or under high environmental stress, particularly after bleaching events (Pettay and Lajeunesse, 2009). DISCUSSION Berkelmans and Van Oppen (2006) demonstrated that thermal stress may induce changes in the dominant symbionts among experimental corals, thus providing thermal tolerance and decreased mortality to the coral animal, supporting the hypothesis that the presence of a thermally tolerant endosymbiont, even in low abundance, may impart an ecological advantage to their coral host (LaJeunesse et al., 2009b). Sampayo et al. (2009) demonstrated that shifts in dominance between closely related species in the genus Cladocopium can also impart diﬀerential thermal tolerance and, ultimately, diﬀerential colony survival. However, unusual host-symbiont partnerships that emerge during bleaching events revert to the original state after normal environmental conditions return (Stat et al., 2009). It appears that during a scenario of hyperthermal stress events, followed by multiple years of less-stressful environmental conditions, new host-symbiont combinations that appear following the bleaching event are short-lived and the Symbiodiniaceae community eventually reverts back to its original state (Berkelmans and Van Oppen, 2006; Sampayo et al., 2008). This is likely as normal host-compatible symbionts are more eﬀective at growing inside their hosts under non-stressful conditions (Jones and Berkelmans, 2010). However, under sustained disturbance events, such as annually recurring hyperthermal events, new thermally tolerant partner January 2019 \| Volume 6 \| Article 5 Frontiers in Marine Science \| www.frontiersin.org 11 Shift From Dominant Host-Specialist Zooxanthella Lewis et al. It has been proposed that colonies exposed to moderate thermal stress are better conditioned to dealing with episodes of severe thermal stress (Oliver and Palumbi, 2011; Ainsworth et al., 2016). Thermal proﬁles showing a pre- bleaching spike in water temperatures, followed by a recovery period of cooler temperatures, prior to a hyperthermal event, reduced the severity of bleaching. Temperature proﬁles at our three study sites show a similar sub-bleaching spike in water temperatures in June 2014 and May 2015 (Figure 3B), however, severe bleaching occurred in August–September both years. This may be due to the recovery period being too short or the ensuing hyperthermal stress was too severe (i.e., temperatures exceeding the bleaching threshold for too many weeks, Supplementary Table 1), exceeding the capacity of the thermally primed corals. As predicted by Ainsworth et al. (2016), climate change leading to annual bleaching and excessive thermal stress may indeed disable this protective thermal priming scenario. DISCUSSION may also be an indicator of long-term physiological stress due to deteriorating environmental factors, e.g., water quality (Boyer et al., 1999; Boyer and Jones, 2002; Wagner et al., 2010) and pulsed thermal events (i.e., previous Florida Reef-wide bleaching events in 1987, 1990, 1997, 1998, 2005, as well as other localized bleaching episodes (Manzello et al., 2007). REFERENCES evidence for symbiont switching in reef-building corals. ISME J. 10, 2693–2701. doi: 10.1038/ismej.2016.54 evidence for symbiont switching in reef-building corals. ISME J. 10, 2693–2701. doi: 10.1038/ismej.2016.54 Abrego, D., Ulstrup, K. E., Willis, B. L., and van Oppen, M. J. (2008). Species– speciﬁc interactions between algal endosymbionts and coral hosts deﬁne their bleaching response to heat and light stress. Proc. R. Soc. B Biol. Sci. 275, 2273–2282. doi: 10.1098/rspb.2008.0180 Boyer, J. N., Fourqurean, J. W., and Jones, R. D. (1999). 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(2008). Dendrogyra cylindrius. Cambridge: The IUCN Red List of Threatened Species 2008. Chan, A., Lewis, C. L., Neely, K. L., and Baums, I. B. (2018). FUNDING The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmars. 2019.00005/full#supplementary-material This research was supported by US National Science Foundation Rapid Response Research (NSF RAPID) grant DATA AVAILABILITY Water temperature data at Pickles, Coﬃns, and Marker 32 sites: NSF RAPID project 637743 https://www.bco-dmo.org/dataset/ 699988. 10-year mean monthly water temperatures: National Frontiers in Marine Science \| www.frontiersin.org January 2019 \| Volume 6 \| Article 5 12 Shift From Dominant Host-Specialist Zooxanthella Lewis et al. Data Buoy Center, Molasses Reef C-MAN station MLRF1 data: https://www.ncbi.nlm.nih.gov/sra. Cp23S-Illumina Sequencing: all fastq ﬁles deposited in NCBI Sequence Read Archive – BioProject #PRJNA513329. #1503483 (to MR-L and Laurie L. Richardson) and conducted under Florida Keys National Marine Sanctuary permits FKNMS-2014-004-A1 and FKNMS-2013-085-A1. Data Buoy Center, Molasses Reef C-MAN station MLRF1 data: https://www.ncbi.nlm.nih.gov/sra. Cp23S-Illumina Sequencing: all fastq ﬁles deposited in NCBI Sequence Read Archive – BioProject #PRJNA513329. Data Buoy Center, Molasses Reef C-MAN station MLRF1 data: https://www.ncbi.nlm.nih.gov/sra. Cp23S-Illumina Sequencing: all fastq ﬁles deposited in NCBI Sequence Read Archive – BioProject #PRJNA513329. AUTHOR CONTRIBUTIONS We thank the Florida Keys Marine Laboratory, Long Key, FL, United States and Florida Fish and Wildlife Conservation/Fish and Wildlife Research Institute, Marathon, FL, United States for logistical support, and scientiﬁc divers from KML, FWRI, and FIU for assistance with ﬁeldwork. We also thank A. Lewis and T. LaJeunesse (Pennsylvania State University) for Symbiodiniaceae species conﬁrmation. This is contribution number 363 from the Tropical Biology Program of Florida International University. CL conceived experimental design and sampling strategy, site selection, sampling methodologies for ﬁeldwork, sample processing and data analysis, manuscript preparation including ﬁgures and tables, and reviewed manuscript drafts. KN contributed site selection, ﬁeld assessments and dive support, assisted with tables and ﬁgures, and reviewed manuscript drafts. CL conceived experimental design and sampling strategy, site selection, sampling methodologies for ﬁeldwork, sample processing and data analysis, manuscript preparation including ﬁgures and tables, and reviewed manuscript drafts. KN contributed site selection, ﬁeld assessments and dive support, assisted with tables and ﬁgures, and reviewed manuscript drafts. 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Coral Reefs 28, 709–713. doi: 10.1007/ s00338-009-0509-5 Conﬂict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Thomas, L., Kendrick, G., Kennington, W., Richards, Z., and Stat, M. (2014). Exploring Symbiodinium diversity and host speciﬁcity in Acropora corals from geographical extremes of Western Australia with 454 amplicon pyrosequencing. Mol. Ecol. 23, 3113–3126. doi: 10.1111/mec. 12801 Copyright © 2019 Lewis, Neely and Rodriguez-Lanetty. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. January 2019 \| Volume 6 \| Article 5 REFERENCES No use, distribution or reproduction is permitted which does not comply with these terms. Copyright © 2019 Lewis, Neely and Rodriguez-Lanetty. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Thornhill, D. J., LaJeunesse, T. C., Kemp, D. W., Fitt, W. K., and Schmidt, G. W. (2006). Multi-year, seasonal genotypic surveys of coral-algal symbioses reveal prevalent stability or post-bleaching reversion. Mar. Biol. 148, 711–722. doi: 10.1007/s00227-005-0114-2 January 2019 \| Volume 6 \| Article 5 Frontiers in Marine Science \| www.frontiersin.org 15
https://openalex.org/W1979729993	https://zenodo.org/records/576674/files/ZK_article_2209.pdf	English	null	First report and morphological, molecular characterization of Xiphinema chambersi Thorne, 1939 (Nematoda, Longidoridae) in Canada	ZooKeys	2,010	cc-by	3,507	Keywords Xiphinema, dagger nematode, diagnostic, Canada, rDNA sequencing First report and morphological, molecular characterization of Xiphinema chambersi Thorne, 1939 (Nematoda, Longidoridae) in Canada Qing Yu1, Ahmed Badiss1, Zhidong Zhang2, Weimin Ye3 1 Environmental Health Program/Invertebrate Biodiversity, Agriculture and Agri-Food Canada, Ottawa, ON Canada 2 PhD student from the Inner Mongolia Agriculture University, Hohhot, Inner Mongolia, China 3 Nematode Assay Section, Agronomic Division, North Carolina Department of Agriculture & Consumer Services; Raleigh, NC 27607, USA Corresponding author: Qing Yu (Qing.Yu@agr.gc.ca) g \| y \| p 5 J \| J Citation: Yu Q et al. (2010) First report and morphological, molecular characterization of Xiphinema chambersi Th orne, 1939 (Nematoda, Longidoridae) in Canada. ZooKeys 49: 13–22. doi: 10.3897/zookeys.49.473 Abstract A Xiphinema species, new to Canada was recovered from rhizosphere of oak trees in Ontario, Canada. Th e identity was confi rmed with morphological and molecular methods as X. chambersi Th orne, 1939. Female bodies are 2.1–2.4 mm long; odontostyle lengths are 110–118 μm; tail 110–177 μm long, arcu- ate, elongate-conoid, with hyline region 22 - 43 μm long. Vagina directed about 30 degrees posteriorly. Reproductive system is monodelphic with ovary refl exed anteriorly, vulva opening at 23–26% of the body. Males were not found. Th e 18S and ITS1 sequences of this population had 3–4 bp diff erences (99% identity) and 30 bp diff erences (97% identity) from two Arkansas populations respectively. Th e nematode population had three juvenile stages. Some variations of the morphometrics were observed comparing with the other populations. Th is is the fi rst report of X. chambersi in Canada. First report and m ZooKeys 49: 13–22 (2010) doi: 10.3897/zookeys.49.473 www.pensoftonline.net/zookeys First report and m ZooKeys 49: 13–22 (2010) doi: 10.3897/zookeys.49.473 www.pensoftonline.net/zookeys First report and m ZooKeys 49: 13–22 (2010) doi: 10.3897/zookeys.49.473 www.pensoftonline.net/zookeys molecular characteriza RESEARCH ARTICLE Copyright Qing Yu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Introduction Xiphinema chambersi Th orne, 1939 was described from specimens from Virginia, USA. A more complete description of this species was added by Cohn and Sher (1972) based on the lectotypes. In USA, X. chambersi has been reported in 16 states and is widely Qing Yu et al. / ZooKeys 49: 13–22 (2010) 14 distributed (Cohn and Sher 1972, Robbins and Brown 1991, Ye 2002, Lamberti et al. 2002). It was also reported in Japan (Shishida 1983) and Korea (Cho et al. 1992). Males were only reported in Arkansas (Ye 2002). Th e Japan (Shishida 1983) and Flori- da (Lamberti et al. 2002) populations have been reported having 3 juvenile stages, the other reported populations have not been specifi ed on the number of juvenile stages. Although Th orne (1939) described and illustrated a male, no males were seen on the syntype slides. Only 2 males were found in Arkansas (Ye 2002). Th is species has been reported to cause damage on sweet gum (Liquidambar styracifl ua) in Georgia (Ruehle 1971) and strawberry (Perry 1958). During a survey of nematodes of grasslands in Ontario, a population of Xiphinema was discovered. It was identifi ed as X. chambersi. Th e objective of this paper is to report and characterize X. chambersi in Ontario, Can- ada with morphological and molecular methods. Materials and methods Nematodes. Soil samples were collected with a soil probe from the rhizosphere of oak trees (Quercus rubra L.) at the Turkey Point Provincial Park of Ontario (42°42.460'N, 80°20.375'W), Canada in 2009. Nematodes were extracted with the modifi ed pan method (Townshend 1963), fi xed in TAF, processed to glycerine and mounted on slides (Hooper 1970) for compound microscopic studies. A portion of the nematodes were frozen at -20°C in water for molecular studies. Microscopic observation. Specimens were examined using Leica DM5500 B compound microscope using diff erential interference contrast and pictures were taken with Leica DFC 420 digital camera. Th e observed characters of the adults were com- pared with those of the specimens described by Th orne (1939) and the description by Cohn and Sher (1972). Measurements were made using a Leica micro application system on the images, and dimensions are expressed in a formula suggested by de Man (1880). Drawings were aided using a drawing tube.ii SEM. Nematodes were fi rst fi xed in TAF (7% formalin, 2% triethanolamine, 91% distilled water), and then transferred to Seinhorst solution 1 (1% glycerol; 4% for- malin: 95% distilled water) in a foam capsule. Th e nematodes then were processed through a serial alcohol dehydration by placing in the foam capsule in succession into 40, 60, 70, 90, and 100% ethanol each for 24 h. the foam capsule with the nematodes was placed into a Polarum E3100 Jumbo II CPD, where critical point drying of the nematodes was accomplished utilizing carbon dioxide. Th e nematodes were then glued on pins using the wood glue as adhesive. Th e pins with nematodes were then placed in an Emitech K550X Sputter Coater and coated for 1 min with gold-palladium. Nema- todes were observed using a Philips XL 30 Scanning Electron Microscope. SEM. Nematodes were fi rst fi xed in TAF (7% formalin, 2% triethanolamine, 91% distilled water), and then transferred to Seinhorst solution 1 (1% glycerol; 4% for- malin: 95% distilled water) in a foam capsule. Th e nematodes then were processed through a serial alcohol dehydration by placing in the foam capsule in succession into 40, 60, 70, 90, and 100% ethanol each for 24 h. the foam capsule with the nematodes was placed into a Polarum E3100 Jumbo II CPD, where critical point drying of the nematodes was accomplished utilizing carbon dioxide. Results and discussion Th e specimens are deposited in the Canadian National Collection of Nematode. Mor- phometrics data of the females and 3 stages of juveniles are in Table 1. Materials and methods Th e nematodes were then glued on pins using the wood glue as adhesive. Th e pins with nematodes were then placed in an Emitech K550X Sputter Coater and coated for 1 min with gold-palladium. Nema- todes were observed using a Philips XL 30 Scanning Electron Microscope. Molecular study. Two populations of X. chambersi from Arkansas were included for the molecular comparisons. Both ITS and 18S genes of the Ontario population and Arkansas populations were PCR amplifi ed and sequenced. Primers were the same as described in Ye et al. (2004) and Neilson et al. (2004). Th e 25 μl PCR contained First report and morphological, molecular characterization of Xiphinema chambersi... 15 12.5 μl 2X GoTaq DNA polymerase mix (Promega Corporation, Madison, WI 53711, USA), 1 μl each of 0.4-μM forward and reverse primers and 1 μl of DNA template. Th e thermal cycling program was as follows: denaturation at 95°C for 6 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s and extension at 72°C for 1 min. A fi nal extension was performed at 72°C for 10 min. PCR prod- ucts were cleaned by Montage™ PCR Centrifugal Filter Devices (Billerica, MA, USA). PCR primers were used for direct sequencing by dideoxynucleotide chain termination using an ABI PRISM BigDye terminator cycle sequencing ready reaction kit (Applied Biosystems) in an Applied Biosystems 377 automated sequencer (Applied Biosystems) by Eurofi ns MWG Operon (Huntsville, AL35805, USA). Th e sequence was depos- ited in Genbank. Th e sequence was compared with other nematode species stored at the GenBank using the BLAST homology search program. Th e closest sequences were selected in the phylogenetic analysis. DNA sequences were aligned by Clustal W (http://workbench.sdsc.edu, Bioinformatics and Computational Biology group, Dept. Bioengineering, UC San Diego, CA). Maximal-parsmony (MP) analysis (Saitou and Nei 1987) was conducted on both ITS and 18S. Sites with missing data or gaps were treated as missing characters for the analysis. Th e robustness of the MP tree was tested using the bootstrap method (Felsenstein 1985) based on 2,000 replicates. Morphological characterization Th e main characters of X. chambersi from Ontario, Canada match well with the lecto- type described by Cohn and Sher (1972) and Arkansas populations (Ye 2002) regard- ing to the body shape, size, the tail shape, hyline tail length, the odontostyle length, and the female gonad. It has only 3 juvenile stages, and no male was found.f Female. Body is C- to L- shaped when relaxed, tapering off gradually at both ends, more so posterior. Lip region is slightly setoff from the rest of body by a faint depression. Amphid aperture is about four-fi fth of the width of lip region. Reproductive system is monodelphic, opistodelphic with ovary refl exed. Vagina directed about 30 degrees angle posteriorly, lips thick. Intestine is transparent, pre-rectum is visible, and rectum is 45 μm long. Tail is 132 μm long, arcuate, elongate-conoid with 3 pairs of caudal pores, terminating in a cylindroid nonprotoplasmic tip with hyline region of 37.4 μm long. Most of the morphometirc measurements of the females agree with those described by Cohn and Sher (1972) and Ye (2002) in the USA and Japan, some morphometrics variations have been observed: Th e averaged body length of the female of the population in Ontario, Canada is 2.2 ± 0.1 (2.1–2.4) mm is smaller than that of the populations 16 16 Qing Yu et al. / ZooKeys 49: 13–22 (2010) Figure 1. Drawings of female of Xiphinema chambersi from Ontario, Canada, A entire body B tail region C gonad D pharynx. A B D C A 100 μm 20 μm 25 μm B C, D A B D C A 100 μm 20 μm 25 μm B C, D D Figure 1. Drawings of female of Xiphinema chambersi from Ontario, Canada, A entire body B tail region C gonad D pharynx. A B D C 100 μm Figure 2. Juveniles and female of Xiphinema chambersi from Ontario, Canada, A fi rst stage juvenile B second stage juvenile C third stage juvenile D female. A B D C 100 μm C D B Figure 2. Juveniles and female of Xiphinema chambersi from Ontario, Canada, A fi rst stage juvenile B second stage juvenile C third stage juvenile D female. B from Iowa, USA, the Florida population averaged 2.5±0.15 (2.4–2.7) μm (Lamberti et al. Morphological characterization 2002) the Arkansas population averaged 2.5 (2.1–2.8) μm (Ye 2002) and the Iowa population 2.4 (2.2–2.5) μm (Cohn and Sher 1972); but is larger than that of the popu- lation in Japan averaged 1.9 (1.8–2.0) μm (Shishida 1983). Th e odontostyle lengths of females from these populations are similar. Th e hyline tail length of the Ontario popula- tion averaged 37.4 ± 6.1 (22.0–43.4) μm is much longer than that of the population from Iowa, USA, the Florida population averaged 2.5±0.15 (2.4–2.7) μm (Lamberti et al. 2002) the Arkansas population averaged 2.5 (2.1–2.8) μm (Ye 2002) and the Iowa population 2.4 (2.2–2.5) μm (Cohn and Sher 1972); but is larger than that of the popu- lation in Japan averaged 1.9 (1.8–2.0) μm (Shishida 1983). Th e odontostyle lengths of females from these populations are similar. Th e hyline tail length of the Ontario popula- tion averaged 37.4 ± 6.1 (22.0–43.4) μm is much longer than that of the population First report and morphological, molecular characterization of Xiphinema chambersi... 17 Figure 3. Comparison of tails of Xiphinema chambersi from Ontario, Canada, A fi rst stage juvenile B second stage juvenile C third stage juvenile D female. A B D C 20 μm D B Figure 3. Comparison of tails of Xiphinema chambersi from Ontario, Canada, A fi rst stage juvenile B second stage juvenile C third stage juvenile D female. Figure 3. Comparison of tails of Xiphinema chambersi from Ontario, Canada, A fi rst stage juvenile B second stage juvenile C third stage juvenile D female. Figure 3. Comparison of tails of Xiphinema chambersi from Ontario, Canada, A fi rst stage juvenile B second stage juvenile C third stage juvenile D female. Figure 4. Comparison of pharyngeal regions of diff erent stage of juveniles and female of Xiphinema chambersi from Ontario, Canada, A fi rst stage juvenile B second stage juvenile C third stage juvenile D female. A B D C 50 μm A, B, C, D A B D C 50 μm A, B, C, D D A B Figure 4. Comparison of pharyngeal regions of diff erent stage of juveniles and female of Xiphinema chambersi from Ontario, Canada, A fi rst stage juvenile B second stage juvenile C third stage juvenile D female. Qing Yu et al. / ZooKeys 49: 13–22 (2010) 18 Figure 5. Morphological characterization Micrographs of Xiphinema chambersi from Ontario, Canada, A SEM image of lip region of female B anus region C head D vulva E tail. A C D E B 20 μm 20 μm 20 μm 20 μm B 20 μm E 20 μm E Figure 5. Micrographs of Xiphinema chambersi from Ontario, Canada, A SEM image of lip region of female B anus region C head D vulva E tail. 0 0 20 40 60 80 100 Style length (μm) Body length (μm) 120 140 500 J1 J2 J3 Female Odontostyle Replacement 1000 1500 2000 2500 3000 Figure 6. Scatter diagram plotting body, odontostyle, and its replacement of individual juveniles and females of X. chambersi from Ontario, Canada. 0 0 20 40 60 80 100 Style length (μm) Body length (μm) 120 140 500 J1 J2 J3 Female Odontostyle Replacement 1000 1500 2000 2500 3000 Figure 6. Scatter diagram plotting body, odontostyle, and its replacement of individual juveniles and females of X. chambersi from Ontario, Canada. First report and morphological, molecular characterization of Xiphinema chambersi... 19 Character J1 J2 J3 Female n 5 2 3 9 L (mm) 0.9±0.1 1.2±0.1 1.7±0.2 2.22±0.1 (0.8–1.0) (1.2–1.2) (1.5–1.8) (2.1–2.4) Total stylet 103±2.7 122.1±0.6 152.3±3 180.5±3.3 (98.5–105.8) (121.5–122.6) (148.3–155.8) (173.0–185.1) Odontostyle 64.1±1.2 72.9±0.1 94.9±1.5 114.7±1.9 (62.4–66.1) (72.8–73.0) (92.8–96.0) (110.5–118.1) Odontophore 38.9±2.6 49.1±0.5 57.4±1.8 65.9±2.4 (35.1–43.1) (48.7–49.6) (55.5–59.9) (62.5–70.2) Odontostyle replacement 72.5±2.2 93.4±0.4 115.3±14.3 - 70.1–75.4 92.99–93.9 113.9–117.3 - a 40.4±3.8 48.1±1.6 50.5±2 51.9±2.5 (32.7–43.3) (46.5–49.8) (47.7–52.3) (47.7–55.7) b 3.7±0.3 3.8±0.2 4.4±0.2 5.8±0.3 (3.2–4.1) (3.7–4.0) (4.2–4.7) (5.4–6.2) c 11.4±0.4 12±0.1 14.2±1.1 17±1.9 (10.7–11.9) (11.9–12.1) (12.6–15.2) (12.5–19.7) c’ 5.7±0.5 5.8±0.3 5.3±0.3 5.3±0.7 (5.1–6.6) (5.5–6.1) (5.1–5.7) (4.4–7.0) V - - - 24.4±0.9 - - - (23.1–26.4) Lip region width 7.9±0.2 8.8±0.1 9.9±0.1 10.9±0.6 (7.6–8.1) (8.7–8.9) (9.7–10.0) (10.0–12.0) Lip region height 2.8±0.2 4.3±0 4.2±0.1 5.3±0.8 (2.6–3.0) (4.3–4.3) (4.1–4.4) (4.5–7.2) Guiding ring from anterior end 56.7±1.8 72.9±0.6 88.1±0.2 110±3.3 (53.2–57.9) (72.4–73.6) (87.8–88.3) (105.4–115.0) Guiding sheath 19.9±6.1 10.3±0.3 6.3±1.9 10.2±3.7 (8.6–25.2) (10.1–10.6) (4.4–8.9) (4.4–15.7) Flanges width 8.1±0.4 8.6±0.3 10.3±0.4 10.1±0.6 (7.4–8.4) (8.3–8.8) (9.8–10.8) (9.2–11.0) Body width at midbody 23.1±3.2 25.8±1.1 33±1.9 42.9±2.4 (20.1–28.9) (24.7–26.8) (31.1–35.7) (39.9–47.8) Pharyngeal bulb length 63.8±1.8 74.1±0.9 89.3±0.9 95.9±5.4 (61.4–65.9) (73.2–75.1) (88.5–90.2) (86.4–103.0) Pharyngeal bulb width 12.3±0.9 12.9±1 15.6±1.5 17.2±1.4 (11.0–13.2) (12.0–14.0) (14.2–17.7) (15.7–20.0) Pharynx 252.3±6.8 323.2±12.8 376.7±14.7 384.9±13.5 (245.6–264.2) (310.4–336.0) (356.9–392.2) (359.4–403.0) G1 - - - 16.5±1.7 - - - (13 9–21 0) Table 1. Morphometric data of X. chambersi from Canada Table 1. Morphological characterization Morphometric data of X. chambersi from Canada Qing Yu et al. / ZooKeys 49: 13–22 (2010) 20 Note: unless specifi ed in the table, the unit for the measurements is μm. Character J1 J2 J3 Female n 5 2 3 9 G1% - - - 0.7±0.1 - - - (0.6–0.9) G2 - - - 195.7±37.2 - - - (154.6–289.2) G2% - - - 8.8±1.8 - - - (7.2–13.3) Prerectum 111.1±19.9 163.4±27.5 184.3±20.2 278.7±67 (72.3–128.4) (135.9–190.8) (155.8–199.0) (215.0–413.7) Rectum 14.7±0.9 25.5±0.9 35.4±2 45.2±5.8 (13.4–15.8) (24.5–26.5) (33.4–37.4) (37.5–56.9) Tail length 81±2.8 102.9±1.6 117.6±2.3 132.1±17.6 (76.3–84.6) (101.2–104.5) (115.1–120.7) (110.2–177.3) Body width at anus 14.4±1.3 17.8±1.3 22.1±1.2 24.9±1.6 (12.4–16.1) (16.6–19.1) (20.5–23.1) (23.4–28.9) Hyaline tail tip 11.9±0.7 18.4±2.2 28.4±1.8 37.4±6.1 (11.0–13.1) (16.2–20.5) (26.0–30.2) (22.0–43.4) H% 14.7±0.6 17.9±2.4 24.1±1.1 28.5±4.8 (13.9–15.5) (15.5–20.3) (22.6–25.0) (19.9–34.4) Note: unless specifi ed in the table, the unit for the measurements is μm. Note: unless specifi ed in the table, the unit for the measurements is μm. from Florida averaged 20.4 ±2.1 (17.6–23) μm, and Arkansas’s averaged 29.7 (15.0– 35.0), is shorter than that of the population from Japan averaged 43.7 (38–47) μm.hh Juvenile. Th e presence of three juvenile stages was determined. Th e body of the fi rst stage juvenile ventrally curved slightly when heat relaxed, the anterior end of the replacement odontostyle almost adjacent to the posterior end of the functional odon- tostyle, the tail has one pair of caudal pores. Th e body of the second stage juvenile is curved slightly more than the fi rst stage juvenile, the anterior end of the replacement odontostyle is behind the posterior end of the functional odontostyle. Th e tail has 2 pairs of caudal pores. Th e body of the third stage juvenile is ventrally curved slightly more than the second stage juvenile, especially in the tail region, the anterior end of the replacement odontostyle is more posterior to the end of the functional odontostyle. Th e tail has three pairs of caudal pores. Fig. 6 revealed X. chambersi from Canada only has 3 juvenile stages. Male: not found Molecular characterization DNA Sequence: 2610 bp ribosome DNA segment consisting of the near-full-length 18S ribosomal RNA gene, the internal transcribed spacer 1 and partial 5.8S ribosomal First report and morphological, molecular characterization of Xiphinema chambersi... 21 RNA gene was PCR amplifi ed and sequenced for Ontario population of X. cham- bersi (genBank accession number HM138503). Th e 18S sequence of this population had only 3–4 bp diff erences (99% identity) from two Arkansas populations sequenced (AY283174 for population Xiph-41 and HM191718 for population Xiph-61). ITS1 region between Ontario population and Arkansas population Xiph-61 had 30 bp dif- ferences (97% identity, 1029 total characters) and 4 gaps. Phylogenetic trees based on the 18S and ITS of rDNA agreed with the results from previous studies (Ye et al. 2004, Neilson et al., 2004) (trees not shown). Both trees revealed the closest relationships be- ing in the same highly supported monophyletic clade for the Ontario population and the Arkansas populations of X. chambersi. Due to the short branch length diff erence in phylogenetic trees and lack of suffi cient morphological diff erences among X. chambersi populations studied, the DNA sequence diff erences on 18S and ITS were considered as intraspecies variation when many Xiphinema species and populations were examined in the phylogenetic trees. Acknowledgements Th e authors thank the Ontario Parks for the sampling permits and the Ontario Tall- grass Association for assistance the soil samplings. Perry VG (1958) Parasitism of two species of dagger nematodes (Xiphinema americanum and X. chambersi) to strawberry. Phytopathology 48: 420–423. References Cohn E, Sher SA (1972) A contribution to the taxonomy of the Genus Xiphinema Cobb, 1913. Journal of Nematology 4: 36–65. Cho YE, Choi YS, Cho MR (1992) Nematodes associated with forest trees in Korea. III. A new species of Xiphinemella Loos, 1950 and four unrecorded species of Xiphinema Cobb, 1913. Korean Journal of Applied Entomology 31: 416–426. Hasagawa M, Kishino H, Yano TA (1985) Dating of the human ape splitting by a molecular clock of mitochondrial-DNA. Journal of Molecular Evolution 22: 160–174. Hooper DJ (1970) Handling, fi xing, staining and mounting nematodes. In: Southey JF (d) Laboratory methods for work with plant and soil nematodes. London, 59–80 Lamberti F, Luca FDe, Molinari S, Duncan LW, Agostineli A, Coiro ML, Dunn D, Radicci V (2002) Xiphinema chambersi and Xiphinema naturale sp. n., two monodelphic Longidorids (Nematoda, Dorylaimida) from Florida. Nematologia Mediterranea 25: 55–61. Neilson R, Ye W, Oliveira CMG, Hübschen J, Robbins RT, Brown DJF, Szalanski AL (2004) Phylogenetic relationships of selected species of Longidoridae (Nematoda: Longidoridae) from North America inferred from 18S rDNA gene sequence data. Helminthologia 41: 209–215. Perry VG (1958) Parasitism of two species of dagger nematodes (Xiphinema americanum and X. chambersi) to strawberry. Phytopathology 48: 420–423. Qing Yu et al. / ZooKeys 49: 13–22 (2010) 22 Robbins RT, Brown DJF (1991) Comments on the taxonomy, occurrence and distribution of Longidoridae (Nematoda) in North America. Nematologica 37: 395–419. Robbins RT, Brown DJF (1991) Comments on the taxonomy, occurrence and distribution of Longidoridae (Nematoda) in North America. Nematologica 37: 395–419. Ruehle JL (1968) Plant-parasitic nematodes associated with southern hardwood and coniferous forest trees. Plant Disease Report 52: 837–839. Saitou N, Nei M (1987) Th e neighbor-joining method, a new method for reconstructing phy- logenetic trees. Molecular Biology Evolution. 4: 406–425. Shishida Y (1983) Studies on nematodes parasitic on woody plants 2. Genus Xiphinema Cobb, 1913. Japanese Journal of Nematology 12: 1–14.h Th orne G (1939) A monograph of the nematodes of the super-family Dorylaimoidea. Capita Zoologica 8: 1–261. Townshend JL (1963) A modifi cation and evaluation of the apparatus for the Oostenbrink direct cottonwool fi lter extraction method. Nematologica 9: 106–110. Ye W (2002) Morphological and molecular taxonomy of Longidorus and Xiphinema (Nemato- da: Longidoridae) occurring in Arkansas, USA. PhD dissertation, University of Arkansas, 380 pp. References Ye W, Szalanski AL, Robbins RT (2004) Phylogenetic relationships and genetic variation in Longidorus and Xiphinema species (Nematoda: Longidoridae) using ITS1 sequences of nu- clear ribosomal DNA. Journal of Nematology 36: 14–19.
https://openalex.org/W3111582363	https://www.duo.uio.no/bitstream/10852/81798/1/12898_2020_Article_338.pdf	English	null	Hydrology influences breeding time in the white-throated dipper	BMC ecology	2,020	cc-by	10,895	Nilsson et al. BMC Ecol (2020) 20:70 https://doi.org/10.1186/s12898-020-00338-y Nilsson et al. BMC Ecol (2020) 20:70 https://doi.org/10.1186/s12898-020-00338-y BMC Ecology © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativeco mmons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract Background: Earlier breeding is one of the strongest responses to global change in birds and is a key factor deter‑ mining reproductive success. In most studies of climate effects, the focus has been on large-scale environmental indices or temperature averaged over large geographical areas, neglecting that animals are affected by the local con‑ ditions in their home ranges. In riverine ecosystems, climate change is altering the flow regime, in addition to changes resulting from the increasing demand for renewable and clean hydropower. Together with increasing temperatures, this can lead to shifts in the time window available for successful breeding of birds associated with the riverine habitat. Here, we investigated specifically how the environmental conditions at the territory level influence timing of breeding in a passerine bird with an aquatic lifestyle, the white-throated dipper Cinclus cinclus. We relate daily river discharge and other important hydrological parameters, to a long-term dataset of breeding phenology (1978–2015) in a natural river system. Results: Dippers bred earlier when winter river discharge and groundwater levels in the weeks prior to breed‑ ing were high, and when there was little snow in the catchment area. Breeding was also earlier at lower altitudes, although the effect dramatically declined over the period. This suggests that territories at higher altitudes had more open water in winter later in the study period, which permitted early breeding also here. Unexpectedly, the largest effect inducing earlier breeding time was territory river discharge during the winter months and not immediately prior to breeding. The territory river discharge also increased during the study period. Conclusions: The observed earlier breeding can thus be interpreted as a response to climate change. Measur‑ ing environmental variation at the scale of the territory thus provides detailed information about the interactions between organisms and the abiotic environment. Keywords: Breeding phenology, Climate change, Environmental heterogeneity, Hydropower, Long-term study, Passerine bird, River discharge, Snow, Spatial scale, Stream global change and is a key factor determining reproduc- tive success [7, 37, 54]. Most studies of climatic effects have focused on large-scale estimates of environmental variation, such as the North Atlantic Oscillation (NAO) or temperature averaged over large geographical areas, neglecting the fact that organisms are affected by the local conditions in their home ranges [34]. Even on the scale of a population, an individual might use a very lim- ited proportion of the population space; thus, popula- tion-specific measurements might not accurately reflect Hydrology influences breeding time in the white‑throated dipper Anna L. K. Nilsson1,2* , Thomas Skaugen3, Trond Reitan1,3, Jan Henning L’Abée‑Lund3, Marlène Gamelon4, Kurt Jerstad5, Ole Wiggo Røstad6, Tore Slagsvold1, Nils C. Stenseth1, L. Asbjørn Vøllestad1 and Bjørn Walseng7 © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativeco mmons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Background Spring phenology, the annual timing of recurring life- history events in spring, is well-known to have advanced (become earlier) in response to climate change [42]. Ear- lier breeding in birds is one of the strongest responses to Correspondence: anna.nilsson@nina.no 2 Norwegian Institute for Nature Research, Thormøhlens Gate 55, 5006 Bergen, Norway Full list of author information is available at the end of the article Correspondence: anna.nilsson@nina.no 2 Norwegian Institute for Nature Research, Thormøhlens Gate 55, 5006 Bergen, Norway Full list of author information is available at the end of the article © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativeco mmons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Nilsson et al. BMC Ecol (2020) 20:70 Page 2 of 11 Page 2 of 11 the exposure of an individual to the current environmen- tal conditions [11, 24]. In fact, organisms experience the local weather and its direct effects on the availability of food and shelter. This determines whether resources are to be accumulated or if stored resources are depleted, and determines the allocation of resources to growth, survival and reproduction. In addition to the local weather condi- tions, the environmental challenges faced by organisms may also be influenced by other aspects of the local area they inhabit. The local area is often defended against con- specifics and is then termed a territory. Territories are heterogeneous in geography and topography, which can have implications for the availability of breeding sites and foraging opportunities; the availability of food resources might vary at small spatial scales [24]. The robustness to environmental variation such as droughts and floods might furthermore vary between territories. Such heter- ogeneity can lead to differences in territory quality [46] determining variation in occupancy between years [13]. Environmental variation measured at the scale of the ter- ritory might thus affect individual life history decisions, such as the timing of breeding. temperature is important for initiating breeding, but spring temperature has so far only been measured at the regional level [37]. The documented effect of spring temperature most likely reflects an extrapolation of local microclimatic conditions on the scale of the territory, such as local river discharge dynamics and snow condi- tions. Thus, in this study, we investigate how environmen- tal variation at the scale of the territory, such as the local river discharge (defined as the volume of water flowing past a cross-section of a river per unit time), hydrological dynamics, territory and individual characteristics, influ- ence the timing of breeding. In this study system, Skau- gen et al. [48] developed a model allowing simulations of daily values of a number of biologically important hydro- logical variables at each dipper territory in our study system during 1978–2015. We here relate these unique hydrological data to our long-term dipper breeding data to test the hypothesis that local environmental variation, particularly runoff, at the scale of the territory influences timing of breeding. Furthermore, our study uses a novel method, the biologically-based “trigger date”, to deal with the statistically unsound problem of circularity in causal- ity. This is often encountered in phenology analyses when using sliding window-approaches to determine periods of importance, which then are included in the statistical analyses (using response to find appropriate period and then using period to predict response). Temperature and precipitation have increased rapidly during the last 100 years in northern Europe [22]. This has naturally led to changes in snow conditions and the extent of ice cover during winter [47] and potentially changed the timing of snow melt, and ultimately the tim- ing of spring floods. Similarly, this has changed water discharge in rivers and streams, and consequently the occurrence and size of floods and droughts [29]. Further climate change is predicted to lead to more alterations in precipitation, discharge, evaporation, and shifts in drought and flood patterns [33], which will have impor- tant implications for species closely associated with the riverine habitat [43]. In addition, freshwater ecosystems are under pressure in the pursuit of clean and renew- able energy resources, where hydropower is an attractive alternative to fossil fuels. Hydropower development has major impacts on ecosystem functioning as the construc- tion of dams and reservoirs interrupts the natural flow regime, which is the main driver of all ecological pro- cesses in rivers and streams [39]. General analysesh The median hatching date of the dipper was the 8th of May (hatching-day-of-year = 128, mean = 129.6, stand- ard deviation, SD = 13.2 days). Hatching date advanced (earlier) 10.2 days during the study period (1978–2015) as shown in a mixed-effects linear regression model with only year as a linear predictor and year as the random effect (random effect, year SD = 7.7, residual SD = 10.9; fixed effect year: b = − 0.27, t = − 2.2, P = 0.035; Fig. 1). Male dippers arrived at the breeding grounds 2.5 days before females (mean = 2.43, SD = 18.65; t = 6.2, df = 2278, P < 0.0001). Trigger dates varied from 1st of February (day-of-year = 31) to 28th of April (day- of-year = 119; median = 43 (13th of Feb), mean = 54.9 (24th of Feb)), and the annual variation in trigger dates among territories was on average 38 days (SD = 25.3, range 0–87). In random factor decomposition, we found a standard deviation of 10.6 days between the territo- ries, while the within-territory standard deviation was 23.0 days. This means that the within-territory variation explained 82.6% of the variation in trigger date. During the study period, there was a positive temporal trend in the relative winter discharge (trigger period 17 (Dec– Feb; see Table S1, Additional file 1); mixed-effects linear In birds, most studies of breeding phenology have focused on species that depend on terrestrial food resources [7, 14, 55], but see [10]. Here, we report the results from a long-term study of a passerine bird that collects most of its resources year-round under water, namely the white-throated dipper Cinclus cinclus (here- after dipper). This aquatic life style confers some special challenges in the northern range of the dippers’ distri- butions, because winter survival and onset of breeding are dependent on the presence of ice-free (open) water [18, 35, 37], and thus on hydrological dynamics. Spring Nilsson et al. BMC Ecol (2020) 20:70 Page 3 of 11 model: random effect: year SD = 0.53, residual SD = 0.28, fixed effect year: b=0 02 t=2 1 P=0 04) Fig. 1 The advancement in breeding time in the white-throated dipper population in Lyngdalselva. The advancement in hatching date (hatching day-of-year, solid line) in the white-throated dipper population in Lyngdalselva, in Norway. General analysesh Estimated fixed effects and variance decomposition of fixed and random effects in a linear mixed effects model explaining the variation in hatching date in the white-throated dipper, where “spring” and “winter” is applied to enhance understanding of the terms SCA (Snow Covered Area), Qnorm (relative river discharge) and NAO (North Atlantic Oscillation). Numbers in brackets in the variable names denote the trigger period, mean and sd denote mean and standard deviation of the variable in the specified trigger period General analysesh Grey points denote observed annual hatching days and black points the observed annual average hatching date in 1978–2015 Table 1 Estimate fixed effects and variance decomposition explaining the variation in breeding time Estimated fixed effects and variance decomposition of fixed and random effects in a linear mixed effects model explaining the variation in hatching date in the white-throated dipper, where “spring” and “winter” is applied to enhance understanding of the terms SCA (Snow Covered Area), Qnorm (relative river discharge) and NAO (North Atlantic Oscillation). Numbers in brackets in the variable names denote the trigger period, mean and sd denote mean and standard deviation of the variable in the specified trigger period Variables Estimate Std. Error Variance decomposition Intercept 136 3.15 – Spring SCA sd [6] 7.0 2.14 0.4 Spring Groundwater mean [14] − 0.067 0.02 1.1 Winter Qnorm mean [17] − 6.6 0.95 10.3 Spring NAO [26] − 0.78 0.24 1.2 M novely 2.2 0.43 0.8 beta polygyny indicator 6.7 0.73 2.2 Age f − 5.7 0.67 – Age f2 0.57 0.08 2.8 Altitude 0.055 0.004 – Year − 0.10 0.09 – Altitude × Year − 0.00082 0.00 29.2 Std. Deviation Correlation – Year 8.4 – Qspec sd [23] \|Year 0.080 − 0.85 20.5 Territory id 2.5 4.3 F id × Territory id 3.1 6.7 Residual 5.5 – Table 1 Estimate fixed effects and variance decomposition explaining the variation in breeding time Fig. 1 The advancement in breeding time in the white-throated dipper population in Lyngdalselva. The advancement in hatching date (hatching day-of-year, solid line) in the white-throated dipper population in Lyngdalselva, in Norway. Grey points denote observed annual hatching days and black points the observed annual average hatching date in 1978–2015 Fig. 1 The advancement in breeding time in the white-throated dipper population in Lyngdalselva. The advancement in hatching date (hatching day-of-year, solid line) in the white-throated dipper population in Lyngdalselva, in Norway. Grey points denote observed annual hatching days and black points the observed annual average hatching date in 1978–2015 model: random effect: year SD = 0.53, residual SD = 0.28, fixed effect year: b = 0.02, t = 2.1, P = 0.04). model: random effect: year SD = 0.53, residual SD = 0.28, fixed effect year: b = 0.02, t = 2.1, P = 0.04). Model selectionh The most supported model to account for the variation in hatching date in the dipper population contained the environmental variables normalized mean winter river discharge (Winter Qnorm mean 17), spring NAO (Spring NAO 26), spring ground water levels (Spring Ground- water mean 14), spring variation in snow cover (Spring SCA sd 6), and the temporal trend in altitude, where the effect of altitude on timing of breeding changed during the study period (Table 1). The best model also included the individual variables female age (the quadratic effect, Age f2), male polygyny status (beta polygyny indicator) and whether the territory occupied by the male was new to him or not (M novelty; Table 1). Among the random effects, the interaction between female (F id) and terri- tory ID accounted for 6.7% of the variation in hatching date, while territory ID itself accounted for 4.3% and the variation in specific river discharge in combination with year accounted for 20.5% (Qspec sd 23, random slope; Table 1) of the variation in hatching date (Table 1). Hatch- ing date advanced (was earlier) with increasing normal- ized mean winter river discharge (Winter Qnorm mean 17), spring NAO (spring NAO 26), and spring ground- water levels (Spring Groundwater mean 14; Fig. 2). There was a quadratic effect for female age (Age f2), with delayed hatching date for females younger than four years old (fifth calender year; Fig. 3) and for females older than that. On the other hand, hatching date was delayed by increasing variation in spring snow cover (Spring SCA sd 6) at the lowest part of each territory’s catchment (Fig. 2), male territory novelty (M novelty) and polygyny (beta polygyny indicator), and altitude (Fig. 4). During the study period, the delay in hatching date decreased from 5.5 to 2.5 days per 100 m increase in altitude (Fig. 4), indicating that the difference in hatching date at high and low altitudes diminished over the study period. The random slope variation in specific river discharge during the previous spring and summer (Qspec sd 23) could not be explained by annual variation or any other predictor variable, although within the interaction, year explained 91% of the variation. Model selectionh a Mean relative river discharge at each territory during the preceding Dec–Feb (Winter Qnorm mean [17]), b the North Atlantic Oscillation (NAO) 10–21 days after the trigger date at each territory (Spring NAO [26], c the minimum ground water levels for the last 20 days before the trigger date (Spring groundwater mean [14]), and d the maximum Snow Covered Area (SCA) the last 30 days before the trigger date (Spring SCA sd (06)), where 0 is no snow cover and 1 is full snow cover. Solid lines denote the effect and vertical dotted lines denote the 5 and 95% quantiles of the raw data. The trigger date is defined as the first date when the daily air temperature exceeded 0 °C for five consecutive days Fig. 3 The effect of age on timing of breeding. The effect of age (calendar year) on timing of breeding (hatching day-of-year) shown as a quadratic line (for all other variables set at their average values) in the white-throated dipper population in Lyngdalselva 1978–2015. Grey dots correspond to observed hatching dates per age, with ages jittered in order to increase visibility of the full dataset Fig. 4 The effect of altitude on the timing of breeding. The effect of altitude (all other variables set at their average values) on the timing of breeding (hatching day-of-year) in 1978 (solid line), 1987 (stapled line), 1996 (dotted), 2005 (stapled and dotted), 2015 (long staples) in the white-throated dipper population in Lyngdalselva 1978–2015 Fig. 2 The influence of the hydrological and climatic predictor variables on the timing of breeding. The influence of the hydrological and climatic predictor variables on hatching date (hatching day-of-year) in the white-throated dipper population in Lyngdalselva 1978–2015. a Mean relative river discharge at each territory during the preceding Dec–Feb (Winter Qnorm mean [17]), b the North Atlantic Oscillation (NAO) 10–21 days after the trigger date at each territory (Spring NAO [26], c the minimum ground water levels for the last 20 days before the trigger date (Spring groundwater mean [14]), and d the maximum Snow Covered Area (SCA) the last 30 days before the trigger date (Spring SCA sd (06)), where 0 is no snow cover and 1 is full snow cover. Solid lines denote the effect and vertical dotted lines denote the 5 and 95% quantiles of the raw data. Model selectionh The effect of altitude (all other variables set at their average values) on the timing of breeding (hatching day-of-year) in 1978 (solid line), 1987 (stapled line), 1996 (dotted), 2005 (stapled and dotted), 2015 (long staples) in the white-throated dipper population in Lyngdalselva 1978–2015 Fig. 3 The effect of age on timing of breeding. The effect of age (calendar year) on timing of breeding (hatching day-of-year) shown as a quadratic line (for all other variables set at their average values) in the white-throated dipper population in Lyngdalselva 1978–2015. Grey dots correspond to observed hatching dates per age, with ages jittered in order to increase visibility of the full dataset Fig. 3 The effect of age on timing of breeding. The effect of ag Model selectionh The random slope in specific river discharge nevertheless had a significant effect on the timing of breeding (see Figure S3, Additional file 2), and this means that the effect of river discharge the previ- ous spring and summer could be negative in 1 year and positive in another. The 95% confidence interval for the model prediction was ± 10.7 days (SD = 5.5). old (fifth calender year; Fig. 3) and for females older than that. On the other hand, hatching date was delayed by increasing variation in spring snow cover (Spring SCA sd 6) at the lowest part of each territory’s catchment (Fig. 2), male territory novelty (M novelty) and polygyny (beta polygyny indicator), and altitude (Fig. 4). During the study period, the delay in hatching date decreased from 5.5 to 2.5 days per 100 m increase in altitude (Fig. 4), indicating that the difference in hatching date at high and low altitudes diminished over the study period. The random slope variation in specific river discharge during the previous spring and summer (Qspec sd 23) could not be explained by annual variation or any other predictor variable, although within the interaction, year explained 91% of the variation. The random slope in specific river discharge nevertheless had a significant effect on the timing of breeding (see Figure S3, Additional file 2), and this means that the effect of river discharge the previ- ous spring and summer could be negative in 1 year and positive in another. The 95% confidence interval for the model prediction was ± 10.7 days (SD = 5.5). Nilsson et al. BMC Ecol (2020) 20:70 Page 4 of 11 Discussion Timing of breeding advanced 10 days during the study period 1978–2015. Earlier breeding in birds is one of the Fig. 2 The influence of the hydrological and climatic predictor variables on the timing of breeding. The influence of the hydrological and climatic predictor variables on hatching date (hatching day-of-year) in the white-throated dipper population in Lyngdalselva 1978–2015. Model selectionh The trigger date is defined as the first date when the daily air temperature exceeded 0 °C for five consecutive days Fig. 2 The influence of the hydrological and climatic predictor variables on the timing of breeding. The influence of the hydrological and climatic predictor variables on hatching date (hatching day-of-year) in the white-throated dipper population in Lyngdalselva 1978–2015. a Mean relative river discharge at each territory during the preceding Dec–Feb (Winter Qnorm mean [17]), b the North Atlantic Oscillation (NAO) 10–21 days after the trigger date at each territory (Spring NAO [26], c the minimum ground water levels for the last 20 days before the trigger date (Spring groundwater mean [14]), and d the maximum Snow Covered Area (SCA) the last 30 days before the trigger date (Spring SCA sd (06)), where 0 is no snow cover and 1 is full snow cover. Solid lines denote the effect and vertical dotted lines denote the 5 and 95% quantiles of the raw data. The trigger date is defined as the first date when the daily air temperature exceeded 0 °C for five consecutive days Discussion Timing of breeding advanced 10 days during the study period 1978–2015. Earlier breeding in birds is one of the Fig. 3 The effect of age on timing of breeding. The effect of age (calendar year) on timing of breeding (hatching day-of-year) shown as a quadratic line (for all other variables set at their average values) in the white-throated dipper population in Lyngdalselva 1978–2015. Grey dots correspond to observed hatching dates per age, with ages jittered in order to increase visibility of the full dataset Fig. 4 The effect of altitude on the timing of breeding. The effect of altitude (all other variables set at their average values) on the timing of breeding (hatching day-of-year) in 1978 (solid line), 1987 (stapled line), 1996 (dotted), 2005 (stapled and dotted), 2015 (long staples) in the white-throated dipper population in Lyngdalselva 1978–2015 Fig. 4 The effect of altitude on the timing of breeding. The effect of altitude (all other variables set at their average values) on the timing of breeding (hatching day-of-year) in 1978 (solid line), 1987 (stapled line), 1996 (dotted), 2005 (stapled and dotted), 2015 (long staples) in the white-throated dipper population in Lyngdalselva 1978–2015 Fig. 4 The effect of altitude on the timing of breeding. Discussion [24], where the timing of oak leaf emergence in the territory accurately predicted breeding phenology. Evidently, there is immense environmental heterogeneity within single populations, and large-scale climate indi- ces or population-level measurements might not reliably capture the level of environmental variation the individu- als are exposed to [11, 24].h An unexpected effect was the variation in response to discharge variation during the previous spring and sum- mer between years (the random slope). A possible expla- nation might be that discharge variation might have a very different effect in a year with moderate compared to high mean discharge. However, it may also be that this effect is correlated with an unmeasured climatic driver that vary from year to year.i Dippers breeding at high altitudes were delayed, a find- ing also reported in other bird species [23, 28, 44, 56]. Altitude was the parameter that contributed the single most to explain the variation in the timing of breeding. As there is a steep altitudinal gradient in this population and temperature drops with 0.65 °C by 100 m increase in altitude, altitude was probably indicating when in spring a territory would become available to prospec- tive breeders. Surprisingly, the altitudinal effect on the timing of breeding was decreasing over the study period, from 6 days to merely 1 day delay in hatching per 100 m increase in altitude. It might be that after mild winters, more territories are accessible prior to the breeding sea- son also at higher elevations, rending the altitudinal gra- dient less influential. Because of global warming, winters have generally become warmer and this might have led to a more synchronous breeding across altitudes in our study population. The environmental conditions prevailing on the terri- tories around the trigger date, but also the winter condi- tions, affected breeding time. Territory river discharge was particularly influential in determining the breeding time in the dipper, as suggested by D’Amico et al. [12]. Surprisingly, it was not the river discharge immediate before breeding, around the trigger date, but during the winter months prior to the breeding season that had the greatest effect on the breeding time. As the food sources exploited by the dippers are hidden under water, the water levels, velocity and turbidity could influence dip- per foraging success during winter [15], which might have implications for when the individual bird is physi- ologically capable of starting to breed. Discussion Timing of breeding advanced 10 days during the study period 1978–2015. Earlier breeding in birds is one of the Nilsson et al. BMC Ecol (2020) 20:70 Page 5 of 11 conditions, because this species is negatively affected by the ice conditions in winter [35]. In the 3–4 weeks prior to the trigger date at each territory, increasing minimum ground water levels and decreasing maximum snow- covered area (SCA) advanced breeding time. This criti- cal time period precedes territory establishment and nest building. The minimum groundwater levels may reflect some aspects of the discharge at a vital time, while the snow conditions may indicate the timing of spring floods. Flooding might be particularly detrimental to the dipper, because the nest is positioned immediately above fast- flowing water and may be flushed away by the current if water levels increase. Flooding may also reduce water visibility and hamper foraging success [8]. Judging from the relative importance of the investigated variables, we conclude that the long-term (months) hydrologi- cal conditions specific to a particular territory prior to the breeding season seem surprisingly important com- pared to the weather conditions prevailing shortly before breeding. most reported responses to climate change [14, 38, 42]. Dunn and Winkler [14] argued that variation in popu- lation density and habitat quality might influence the interpretation of phenology responses. However, this is unlikely to have been the case in our study. Although the population density of the dipper is predicted to increase in the future because of warmer winters [18], currently there are strong fluctuations but no obvious long-term trend [35]. Previous work in this study system [19, 37] also indicates that high population density results in earlier breeding, which is opposite to the prediction by Dunn and Winkler [14]. The advancement in breeding time reported here may thus be interpreted as a response to climate change. Environmental variation measured at the territory had a significant influence on the timing of breeding. The level of environmental heterogeneity between territories was immense, as reflected by the variation in the trig- ger date (the first date when temperature was above 0 °C for five consecutive days), which varied by more than a month on average and in some years almost by 3 months. Thus, individuals even within close range of each other, in this case within 70 km, experienced very different environmental conditions. This supports the findings by Hinks et al. Study species and study systemh The dipper is common across the Palearctic mountain- ous regions and it breeds in close proximity to rapidly flowing parts of rivers and streams. Territories are estab- lished in early spring and both sexes build the nest and feed the young, except in instances of polygyny where the male contribution is lower. The clutch of 3–6 eggs are incubated by the female for a period of 17 days and the young remain in the nest for approximately 20 days before fledging [19, 37, 52]. The study population is par- tially migratory, where part of the population undertakes migration to southern Sweden, Denmark, and northern Germany and Poland [4].h We also found that breeding time depended on female age, with four-year olds breeding earlier than both younger and older females. Reproductive performance and associated traits such as the timing of breeding usu- ally improve with age and then level off [16]. However, age-specific reproductive output is more commonly reported than age-specific differences in timing of breed- ing (but see [5, 20, 30, 32, 53, 59]. Supposedly, the same mechanisms governing age-specific reproductive out- put, such as selective disappearance of poor breeders and individual improvement with increasing age and experi- ence [20, 32, 41], might also explain the observed pattern in breeding phenology. y The study system is situated in river Lyngdalselva and its tributaries in southernmost Norway (58° 08′–58° 40′ N, 6° 56′–7° 20′ E). The system is subject to a strong alti- tudinal gradient, reaching almost from the river mouth in Lyngdalsfjorden to 60 km inland and 700 m above sea level. The population has been monitored accord- ing to a standardised methodology since 1978 [35]. The population size has fluctuated between 20 and 117 pairs (defined as the number of breeding females). Almost all (94%) breeding birds have been individually identi- fied. Birds are caught in mist nets at first encounter and ringed with a metal ring and an individual colour code comprising two plastic rings. Individual colour codes enable later recognition of individual identity without having to recapture the bird. Arrival date was registered as first encounter of each bird each year, by ring reading or capture at first encounter. In some instances, arrival date was not registered until well into the breeding cycle. The breeding outcome of almost all nests is known and nearly all young are ringed. Study species and study systemh For more details on the study system, see Nilsson et al. [35]. Dippers prefer nest sites where the opening of the nest is situated over fast-flow- ing water [52]. Because nest sites are limited and spatially segregated, there is a limited number of individual terri- tories containing one or more nest sites, namely 158, as recorded within the whole river system during the entire study period. Polygyny is well documented in the dipper, but the frequency varies both between populations and years [17, 31, 58]. We found that breeding time did not differ between females mated to monogamous males and pri- mary females mated to polygynous males, but that the onset of breeding of females that settled with an already mated male was delayed. In this species, late breeding reduces reproductive success [37] which might be a sig- nificant cost of polygyny. Discussion In general, pre- cipitation in southern Norway has increased (1961–2009; [47] and, similarly, the mean winter temperature in the study area has increased at least by 3 °C (1978–2008; [35], meaning that less precipitation falls as snow. During the study period, there was a tendency towards increased river discharge, which might contribute to the temporal trend in breeding phenology. Most importantly, high dis- charge indicates open water [26, 40] and thus favourable We found a significant interaction between territory and female identity that affected the timing of breed- ing, i.e., certain combinations of individuals and terri- tories were associated with earlier breeding and others with later breeding. The ability to take advantage of the resources in a given territory may differ between indi- viduals and be reflected by the timing of breeding. In Nilsson et al. BMC Ecol (2020) 20:70 Page 6 of 11 Page 6 of 11 some species, immigrants and locally recruited birds may differ both in laying time and prey choice, and this may reflect genetic differences, differences in quality, or the early learning environment [50]. Interestingly, males that established themselves in unknown territories as well as first-time breeding males, bred on average 2 days later than males that established themselves in familiar territo- ries where they had previous breeding experience. Thus, not only female experience but also male familiarity with a territory may influence the time of breeding. Female site familiarity has previously been shown to be impor- tant [45], but it has only recently been shown that male experience influence breeding time [57]. Foraging and prey choice may however improve equally with experi- ence in the two sexes [50]. Depending on their local expe- riences, males might differ in eagerness and contribution to nest building, thus affecting the time for initiation of breeding. This finding highlights the importance of male territory familiarity and thus proving that breeding time is not solely a female trait. winter period as well as in early spring. This also applies to rivers and streams outside the breeding areas, because the dipper is a short-distance partial migrant. This study is by nature an explorative approach, where the results provide the first step in seeking out biologically relevant hydrological and other environmental drivers of the tim- ing of breeding. Further work is required to understand how discharge is affecting riverine birds in the long-term, particularly in the immediate proximity to hydropower developments. Conclusions In conclusion, we have shown how hydrological dynam- ics and other prominent local characteristics of the envi- ronment at the territory scale influence the timing of breeding in the dipper. Particularly, the river discharge and the altitudinal effect were of great importance to this riverine specialist bird. From a management perspective, it should be emphasized that the dipper, often used as an indicator species, is particularly sensitive to changes in river discharge and the snow conditions during the entire Nilsson et al. BMC Ecol (2020) 20:70 Page 7 of 11 The timing of breeding is estimated as the hatching date of the first clutch of a female in a year (see [36]. In short, hatching date is based on the growth trajectory of the largest young in the brood, i.e. the individual that is closest to the maximum physiological growth rate. This implies that the phenology of nests that did not produce young, or produced young that were not measured, could not be estimated by this method. All-together, a total of 1184 breeding events with estimated breeding time dur- ing the study period are analysed here. A total of 1271 breeding events with no estimated breeding time were excluded, and another 209 breeding events with breed- ing time were excluded due to inaccuracies in the ringing data or absence of hydrology data. In the study area, territory river discharge is of vastly different magnitudes. For example, the last dipper terri- tory in the main river before the outlet into the fjord has a very large catchment area and thus discharge compared to the territories located in small brooks in the moun- tains (mean discharge varied between territories, from 0.008 m3/s to 32 m3/s). To allow comparison among ter- ritories, river discharge was standardized with two com- peting methods. First, river discharge was standardized as specific discharge, which is discharge per catchment area, measured in l/s/km2, at each breeding territory (Qspec; Table 2). Second, it was standardized as the rela- tive river discharge (defined as discharge divided by the territory mean discharge for the study period; Qnorm; Table 2). Because access to open water is of vital importance for the dippers’ foraging and thus breeding opportunities in seasonal environments, we defined an annual trigger date based on the daily temperature at each individual terri- tory (Additional file 1). Conclusions Hence, the trigger date was used for solving the problem of which environmental variable at which time period was of importance for the timing of breeding, with a biological basis. The hydrological simu- lations produced daily measurements, for 38 full years, of many potentially important hydrological and other environmental variables for timing of breeding in the dipper. The common procedure in dealing with tempo- ral data is to use a sliding window approach [2], which is far from ideal when the period length is unknown and when it likewise is unknown whether the period relates to a date or a biological trigger. By using the response variable, which in studies of phenology is temporal, to find the time period of the exploratory variable with the best fit, the final analysis predicting the response with the best-fit exploratory variable is using the future to predict the past, and thus not a statistically sound procedure. Instead, we solve the problem by allowing the main anal- ysis of the response to find the time period that actually explains the response. The statistics is expanded upon in the Statistics section, but we simply let a multivari- ate framework select the time period of the explanatory variable(s) that explains the variation in the response. Time periods had a biological basis, built on the “trigger date”; thus, at each territory, the annual trigger date was defined as the first date when the daily air temperature had exceeded 0 °C for five consecutive days (Additional file 1). Such a duration of time will speed up the melting of snow and ice. We then defined a number of different time periods, defined as trigger periods, with different starting dates and of various duration related to the trig- ger date (see Table S1, Additional file 1). This was done in order to catch weather variation at and around the critical moments when egg laying first becomes feasible Hydrology data d l l d Hydrological data are normally restricted to gauged sites, which often are inconveniently located with respect to the biological study systems of interest. In Lyngdalselva, only two sites are gauged. However, the rainfall-runoff model (the Distance Distribution Dynamics (DDD) model, [48]) has produced predictions of river discharge in ungauged basins in the Lygne basin at each unique dipper breeding territory (except for 13 out of 158 terri- tories whose catchments were too small). The model for Lyngdalselva is extremely good when comparing model predictions and gauged sites; the Kling Gupta Efficiency criterion (KGE; [21, 27]) for both gauged sites in Lyng- dalselva is 0.94. Thiemig et al. [51] regard values KGE 0.5–0.75 as intermediate, and 0.75–0.9 as good; a KGE of 0.94 for Lyngdalselva is thus extraordinarily good. Sev- eral of the model parameters of DDD are estimated from digitized maps of terrain and river network. In addition, regressed relations between model parameters and catch- ment characteristics, like fractions of forest and bare rock, wetland, and so on are determined by calibrating the model against observed runoff from several gauged catchments in Norway. Daily river discharge data in the Lygne basin have therefore been estimated for the whole study period 1978–2015. The data input for the rainfall- runoff-model, precipitation (P; Table 1) and temperature (T; Table 2), stems from the interpolated meteorological grid, and contains also environmental variables specific to each territory. In addition, snow covered area (SCA; Table 2), snow water equivalent (SWE; Table 2; [49] and groundwater levels (Groundwater; Table 2) are predicted by the DDD model. All are variables that may prove to be significant biological predictors of the timing of breeding in the dipper, reflecting microclimatic or other important environmental variation at each territory (for a full list, see Table 2; for further details on the hydrological vari- ables, see [48]. Nilsson et al. Hydrology data d l l d BMC Ecol (2020) 20:70 Page 8 of 11 Table 2 Predictor variables and their explanation used in analysing variation in breeding time Variable type Predictor Explanation Territory–indicating microclimate Altitude Altitude of most frequented nest site at the territory Distancea Distance from the river outlet to the most frequented nest site at the territory Qmeana Absolute mean annual discharge Catchment–indicating microclimate Area Size of catchment area (m2) Altitude median Median elevation of catchment (m) Altitude diff Altitudinal range of catchment (m) Bog fraction Fraction of bogs in catchment, which contribute to flow when saturated Bog max Maximum of distance distribution for bogs (m) Bog mean Mean of distances (m) between grid-points in the catchment classified as bogs to the nearest river reach, indicating the flow dynamics from saturated bogs Bog fractionb Areal fraction of grid cells with zero distance to the river network for bogs S mean Mean of distances (m) between grid-points in the catchment classified as soils to the nearest river reach, indicating the flow dynamics from soils Soil fraction Areal fraction of grid cells with zero distance to the river network for soils L mean Mean of the distances (m) between points in the river network, a measure of river network complexity L sd Standard deviation of distances between points in the river network to the outlet, a measure of river network complexity L max Maximum of distance distribution of the river network, a measure of river network complexity Evotranspiration Degree-day factor for evotranspiration (mm/day/ °C) Trigger Trigger day Trigger day index for first five-day period above 0 °C Qspec* Specific discharge (l/s/km2, discharge per area unit) Qnorm* Normalized discharge (discharge/annual mean discharge) T* Temperature (°C) P* Precipitation (mm) Groundwater* Dynamic groundwater storage (mm). Hydrology data d l l d The part of groundwater storage that fluctuates with runoff indicating hydrological drivers SCA* Fraction of the lowest 10% of the catchment that is snow covered SWE* Snow water equivalent at the lowest 10% of the catchment (mm) Ql10* Indicating whether discharge exceeded 10 l/s or not (indicating extreme drought) in period Ql100* Indicating whether discharge exceeded 100 l/s or not (indicating drought) in period Global climate NAOb Daily North Atlantic Oscillation Index NAOcurr North Atlantic Oscillation Index current year NAOprev North Atlantic Oscillation Index previous year Individual Age fa Female age indicating individual experience and possible age restrictions Age ma Male age indicating individual experience and possible age restrictions Polyandry Polyandry status: monogamous or polyandrous brood Polygyny all Polygyny status: monogamous, alpha, beta, gamma, successive alpha, and successive beta brood Polyandry history 0/1 indicator for whether the female has been polyandrous, 1 = yes Polygyny history 0/1 indicator for whether the male has been polygynous, 1 = yes polygyny indicator 0/1 indicator for whether the male was detectably polygynous for this brood, 1 = yes beta polygyny indicator 0/1 indicator for whether the male was detectably polygynous and this wasn’t the alpha brood, 1 = yes polyandry indicator 0/1 indicator for whether the female was detectably polyandrous for this brood, 1 = yes male first 0/1 indicator for whether this was the first hatching for this male, 1 = yes female first 0/1 indicator for whether this was the first hatching for this female, 1 = yes M novelty Male territory novelty, 1 = first time for this male in this territory F novelty Female territory novelty, 1 = first time for this female in this territory Other lintime Temporal time trend, indicating possible global climatic change Other Page 9 of 11 Nilsson et al. Hydrology data d l l d In addi- tion, we introduced two indicator variables for drought, using the original discharge threshold at 0.01 m3/s and 0.1 m3/s. Lastly, we included the daily North Atlantic Oscillation index (NAO) to account for fluctuations in the global climate [25], that might not have been cap- tured by the variation in the hydrological simulations. (Additional file 1). In addition, some of our possible peri- ods are statically defined, using fixed start and end dates. For each trigger period in each year and territory, we estimated the mean, maximum, minimum and standard deviation. For river discharge, this was calculated for spe- cific discharge as well as for relative discharge. In addi- tion, we introduced two indicator variables for drought, using the original discharge threshold at 0.01 m3/s and 0.1 m3/s. Lastly, we included the daily North Atlantic Oscillation index (NAO) to account for fluctuations in the global climate [25], that might not have been cap- tured by the variation in the hydrological simulations. Hydrology data d l l d BMC Ecol (2020) 20:70 Table 2 (continued) Variable type Predictor Explanation Random effects Yeara Annual variation not explained by climatic variables M id Male identity (ring number) indicating individual variation F id Female identity (ring number) indicating individual variation Territory id Territory identity indicating spatial variation not explained by territory and catchment variables A list of all predictor variables and their biological explanation used in analysing the variation in the timing of breeding in the white-throated dipper population in Lyngdalselva 1978–2015 Mean, min, max and sd were estimated for all trigger periods a The quadratic effect was also included b From the National Weather service, Climate Prediction Center, http://www.cpc.ncep.noaa.gov/products/precip/CWlink/pna/nao.shtml qf rom the National Weather service, Climate Prediction Center, http://www.cpc.ncep.noaa.gov/products/precip/CWlink/pna/nao.shtml interest for hatching date, namely the trigger date and the winter river discharge, by investigating the univari- ate regression of each of these variables in turn. We then modelled hatching date with multiple effects using linear mixed-effects models to account for random effects, such as male and female ID, territory ID and year. All analyses were conducted in the programming environment R (ver- sion 3.2.5) with add-on packages ‘lme4’ for linear mixed- effects models [6]. Possible predictors included the hydrological variables and other climatic variables such as precipitation, temperature and NAO using the trigger periods (Table 2). Location-specific variables from the rainfall-runoff model were also added as possible predic- tors, as well as altitude and random location intercepts. We investigated temporal trends in breeding time both by using numeric year as a possible linear predictor and by using categorical year as a random intercept or a ran- dom slope for climatic variables. Last, we included the individual characteristics of the breeding birds, namely male and female age, territory novelty (M and F novelty; Table 2) and polygamy status (Polyandry, Polygyny all, etc.; Table 2). For a full list of predictor variables used in modelling the timing of hatching, see Table 2. A dis- cussion of model uncertainty can be found in Additional file 3. (Additional file 1). In addition, some of our possible peri- ods are statically defined, using fixed start and end dates. For each trigger period in each year and territory, we estimated the mean, maximum, minimum and standard deviation. For river discharge, this was calculated for spe- cific discharge as well as for relative discharge. Abbreviations NAO: North Atlantic Oscillation; SD: Standard Deviation; Qnorm: Normalized river discharge, which is discharge divided by territory mean discharge for the study period; Qspec: Specific river discharge, discharge per catchment area l/s/km2; Groundwater: Ground water levels; SCA: Snow Covered Area; Age f: Female age; Age m: Male age; Age f2: Quadratic female age; beta polygyny indicator: Male polygyny; F novelty: Whether a territory was new to a female; M novelty: Whether a territory was new to a male; F id: Female identity; M id: Male identity; territory ID: Territory identity; trigger date: The first date when temperature was above 0 degrees Celsius for five consecutive days; trigger periods: Time periods with different starting dates and of various duration related to the trigger date: in addition to some periods with fixed start and end date; DDD: Distance Distribution Dynamics model; KGE: Kling Gupta Effi‑ ciency criterion; P: Precipitation; T: Temperature; SWE: Snow water equivalent; BIC: Bayesian Information Criterion. Ethics approval and consent to participate The field work was conducted with respect for the animals’ well-being and adheres to the Guidelines for the Use of Animals in Research [3]. It com‑ plies with the laws and regulations for animals used in research in Norway (FOR-2003-03-18-349, LOV-2009-06-19-100, LOV-1981-05-29-38§26). Ethical approval is not required for bird ringing (FOR-2003-03-18-349§§21-24), but Ringing licenses were issued and approved by the Norwegian Bird Ringing Centre on behalf of the Norwegian Environment Agency. p g The authors declare that they have no competing interests. Additional file 1. Trigger date and periods. The defined trigger periods, relating trigger date or absolute dates, used on the runoff and catchment predictor variables when modelling timing of breeding in the white- throated dipper in Lyngdalselva 1978–2015 g We are grateful to all the people involved in the many years of field work. 6. Bates DM, Maeschler M, Bolker B. Package ‚lme4‘.2018. https://cran.r-proje ct.org/web/packages/lme4/lme4.pdf. Accessed 3 May 2018. 6. Bates DM, Maeschler M, Bolker B. Package ‚lme4‘.2018. https://cran.r-proje ct.org/web/packages/lme4/lme4.pdf. Accessed 3 May 2018. Supplementary information ac /10 1186/ 12898 020 00338 Supplementary information accompanies this paper at https://doi. org/10.1186/s12898-020-00338-y. Availability of data and materials such decompositions. In the case where many quite similar variables were included (such as many differ- ent discharge contributions), we used the variance con- tributions for the set of such variables, which for fixed effects meant the variance of the sum of coefficients multiplied by the variables. This should at least alleviate the problem of variance decomposition due to covaria- tion in the explanatory variables. In addition, we com- pared the variation in hatching date in the first years of the study period (1978–1996) with the last years (1997–2015) using the F-test. The datasets analysed during the current study are not publicly available, due to it being a unique long-term collection of individual data on a breeding dip‑ per population, which has cost an enormous effort in the field 1973–present, but are available from the corresponding author on reasonable request. The complete dipper data are stored at the Norwegian University of Life Sciences. Author details 1 Centre for Ecological and Evolutionary Synthesis (CEES), Department of Biosciences, University of Oslo, P. O. Box 1066, Blindern, 0316 Oslo, Norway. 2 Norwegian Institute for Nature Research, Thormøhlens Gate 55, 5006 Bergen, Norway. 3 Norwegian Water Resource and Energy Directorate, P. O. Box 5091, Majorstua, 0301 Oslo, Norway. 4 Centre for Biodiversity Dynamics, Department of Biology, Norwegian University of Science and Technology, 7491 Trondheim, Norway. 5 Jerstad Viltforvaltning, Aurebekksveien 61, 4516 Mandal, Norway. 6 Faculty of Environmental Sciences and Natural Resource Management, Norwegian University of Life Sciences, P.O. Box 5003, NMBU, 1432 Ås, Norway. 7 Norwegian Institute for Nature Research, Gaustadallén 21, 0349 Oslo, Norway. Additional file 2. Variation in specific discharge affects timing of breed‑ ing. The effect of the standard variation in specific discharge during the period Apr–Aug the preceeding year on the timing of breeding (hatching day-of-year), where each year is denoted in a different colour, in the white- throated dipper in Lyngdalselva 1978–2015 Additional file 3. Model uncertainty. Alternative models with the defined trigger periods, relating trigger date or absolute dates, used on the runoff and catchment predictor variables when modelling timing of breeding in the white-throated dipper in Lyngdalselva 1978–2015 Received: 9 September 2019 Accepted: 25 November 2020 Authors’ contributions g p g p y 7. Both C, Bouwhuis S, Lessells CM, Visser ME. Climate change and popula‑ tion declines in a long-distance migratory bird. Nature. 2006;441:81–3.l 7. Both C, Bouwhuis S, Lessells CM, Visser ME. Climate change and popula‑ tion declines in a long-distance migratory bird. Nature. 2006;441:81–3. ALKN and JHLL conceived the idea. KJ collected the field data. OWR organised the dipper data collection. ThS produced the hydrology data. TR analysed the data. ALKN and TR wrote the manuscript with significant input from MG, ThS, JHLL, LAV, ToS, OWR, BW, KJ and NCS. ALKN, TR, LAV, and MG contributed substantially to the revised version. All authors contributed to the article. All authors read and approved the final manuscript. 8. Chiu M-C, Kuo M-H, Hong S-Y, Sun Y-H. Impact of extreme flooding on the annual survival of a riparian predator, the brown dipper Cinclus pal- lasii. Ibis. 2013;155:377–83. 8. Chiu M-C, Kuo M-H, Hong S-Y, Sun Y-H. Impact of extreme flooding on the annual survival of a riparian predator, the brown dipper Cinclus pal- lasii. Ibis. 2013;155:377–83. 9. Claeskens G, Hjort NL. Model selection and model averaging. Cambridge: Cambridge University Press; 2008. 9. Claeskens G, Hjort NL. Model selection and model averaging. Cambridge: Cambridge University Press; 2008. 10. Clark RG, Pöysä H, Runko P, Paasivaara A. Spring phenology and timing of breeding in short-distance migrant birds: phenotypic responses and offspring recruitment patterns in common goldeneyes. J Avian Biol. 2014;45:457–65. 10. Clark RG, Pöysä H, Runko P, Paasivaara A. Spring phenology and timing of breeding in short-distance migrant birds: phenotypic responses and offspring recruitment patterns in common goldeneyes. J Avian Biol. 2014;45:457–65. Referencesii 1. Afifi AA, Clark V. Computer-aided multivariate analysis. 3rd ed. London: Chapman & Hall; 2007. 2. Ahola M, Laaksonen T, Sippola K, Eeva T, Raino K, Lehikoinen E. Variation in climate warming along the migration route uncouples arrival and breed‑ ing dates. Glob Change Biol. 2004;10:1610–7. 2. Ahola M, Laaksonen T, Sippola K, Eeva T, Raino K, Lehikoinen E. Variation in climate warming along the migration route uncouples arrival and breed‑ ing dates. Glob Change Biol. 2004;10:1610–7. 3. [Anon]. Guidelines for the treatment of animals in behavioural research and teaching. Anim. Behav. 2006;71:245–53. 4. Bakken V, Runde O, Tjørve E. Norsk ringmerkningsatlas. Stavanger: Stavan‑ ger Museum; 2006. 5. Balbontin J, Hermosell IG, Marzal A, Reviriego M, de Lope F, Møller AP. 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https://openalex.org/W1978434336	https://europepmc.org/articles/pmc2917372?pdf=render	English	null	Localized Fetomaternal Hyperglycemia: Spatial and Kinetic Definition by Positron Emission Tomography	PloS one	2,010	cc-by	8,628	Abstract Background: Complex but common maternal diseases such as diabetes and obesity contribute to adverse fetal outcomes. Understanding of the mechanisms involved is hampered by difficulty in isolating individual elements of complex maternal states in vivo. We approached this problem in the context of maternal diabetes and sought an approach to expose the developing fetus in vivo to isolated hyperglycemia in the pregnant rat. Methodology and Principal Findings: We hypothesized that glucose infused into the arterial supply of one uterine horn would more highly expose fetuses in the ipsilateral versus contralateral uterine horn. To test this, the glucose tracer [18F]fluorodeoxyglucose (FDG) was infused via the left uterine artery. Regional glucose uptake into maternal tissues and fetuses was quantified using positron emission tomography (PET). Upon infusion, FDG accumulation began in the left-sided placentae, subsequently spreading to the fetuses. Over two hours after completion of the infusion, FDG accumulation was significantly greater in left compared to right uterine horn fetuses, favoring the left by 1.960.1 and 2.860.3 fold under fasted and hyperinsulinemic conditions (p,10211 n = 32-35 and p,10212 n = 27–45) respectively. By contrast, centrally administered [3H]-2-deoxyglucose accumulated equally between the fetuses of the two uterine horns. Induction of significant hyperglycemia (103 mg/dL) localized to the left uterine artery was sustained for at least 48 hours while maternal euglycemia was maintained. Conclusions and Significance: This approach exposes selected fetuses to localized hyperglycemia in vivo, minimizing exposure of the mother and thus secondary effects. Additionally, a set of less exposed internal control fetuses are maintained for comparison, allowing direct study of the in vivo fetal effects of isolated hyperglycemia. Broadly, this approach can be extended to study a variety of maternal-sided perturbations suspected to directly affect fetal health. Editor: Gian Paolo Fadini, University of Padova Medical School, Italy Received April 26, 2010; Accepted July 14, 2010; Published August 6, 2010 pyright: 2010 Yao et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, whic e, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the American Diabetes Association’s Amaranth Diabetes Fund 1-08-RA-142 (to A.W.N.), National Institutes of Health (NIH) R01-DK081548 (to A.W.N.), NIH P30 CA086862 (cancer center support grant for the University of Iowa Small Animal Imaging Core), and the Burlington Care for Kids fund. Abstract The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: andrew-norris@uiowa.edu Jianrong Yao1, Chunlin Wang1, Susan A. Walsh2, Shanming Hu1, Alexander B. Sawatzke1, Diana Dang1, Jeffrey L. Segar1, Laura L. B. Ponto2,3, John J. Sunderland2,3, Andrew W. Norris1* 1 Department of Pediatrics, University of Iowa Carver College of Medicine, Iowa City, Iowa, United States of America, 2 Small Animal Imaging Core, University of Iowa Carver College of Medicine, Iowa City, Iowa, United States of America, 3 Department of Radiology, University of Iowa Carver College of Medicine, Iowa City, Iowa, United States of America PLoS ONE \| www.plosone.org August 2010 \| Volume 5 \| Issue 8 \| e12027 Introduction [9], inflammatory agents [10], and reactive oxygen species [11]. It often is difficult to induce isolated single maternal perturbations related to the above complex phenomena and in vivo model approaches which dissect specific maternal-sided contributors to fetal pathology are needed. The fetus normally is maintained in a protected environment, sheltered by maternal and placental homeostasis and detoxifica- tion mechanisms. When these protections fail or are breached, significant health consequences to the fetus can occur, inducing malformations [1], neonatal maladies [2], and/or disease susceptibilities that may not manifest for decades [3]. We have approached this problem in the context of fetal diabetes exposure, which recapitulates many of the above characteristics. Diabetes exposure during early fetal development leads to birth defect risk [7] whereas exposure later in gestation leads to neonatal maladies [2] as well as risk of insulin resistance and type 2 diabetes later in life [12], [13]. The Freinkel hypothesis of fuel-mediated teratogenesis posits that the excess exposure to metabolic fuels increased by maternal diabetes leads to the adverse fetal and offspring outcomes [9]. Studies using embryo culture [14], [15] and epidemiologic correlation [16] have lent credence to this hypothesis, as have fuel infusions sufficient to induce systemic maternal elevations [17] though the latter are complicated by metabolic In simple cases, fetal consequences are induced by direct action of a single inciting agent on the fetus or non-maternal extra-fetal tissues [4]. By contrast, in complex cases, the maternal state consists of compound perturbations, such that it is often unclear which specific perturbations of the fetal environment are responsible for the adverse fetal outcomes [5], [6]. Examples include diabetes mellitus [7] and obesity [8]. In these complex phenomena, potential mediators of fetal damage may include alterations in numerous circulating micro- and macro- nutrients 1 August 2010 \| Volume 5 \| Issue 8 \| e12027 Local Fetal Hyperglycemia to define the temporal and spatial disposition of locally infused glucose. to define the temporal and spatial disposition of locally infused glucose. spillover [18] such as insulin resistance due to lipid infusion or hyperlipidemia due to glucose infusion [19]. The overall conclusion from these studies is that although hyperglycemia is likely the primary fuel that incites fetal pathology [20] additional fuels contribute as well, including ketones, fatty acids, triglycerides, and branched chain amino acids [21]-[23]. Uterine artery catheter Blood flow to each uterine horn is primarily provided by its uterine artery, reaching flow rates of 1-2 mL/min near the end of gestation [24], [26]. An infusion catheter was advanced from the left femoral artery such that the tip lay just upstream of the left uterine artery takeoff (Fig. 1a). Competing blood flow was reduced by either simply ligating the iliac artery just downstream of the uterine artery takeoff (surgical approach ‘‘A’’) or by, more extensively, also tying off two nearby competing arteries (approach ‘‘B’’) (Fig. 1b). Catheter placement was performed on gestational day #18 and rats were studied by PET scan on gestational day #20 (Fig. 1c). Maternal weight increased pre-surgery to pre-scan from 32364 to 33064g (p,0.05, n = 11). To further address this need, it is hypothesized that glucose infused into the arterial supply of the uterus would highly expose fetuses to hyperglycemia while minimizing maternal exposure and secondary effects. We took advantage of the bicornate nature of the rat uterus, wherein embryos develop in either the left or the right horns of the uterus. This is of great experimental utility, since the two horns of the uterus have distinct blood supplies [24]. Because circulating glucose is rapidly cleared from plasma [25], the amount of infused glucose reaching the central circulation of the mother and thus the contralateral fetuses should be significantly less than the amount reaching the fetuses on the side of the infusion. Here, we validate this approach, testing for differential uterine horn hyperglycemic exposure using positron emission tomography (PET) imaging and glucose metabolic tracers Introduction However, direct determi- nation of the effects of individual fuels in vivo on fetal development and offspring health has been difficult to achieve. Disposition of infused glucose tracer An 8.961.1 min bolus infusion of FDG was initiated and the animals were scanned for the ensuing 120 minutes (Fig. 1d). Serum 18F concentration peaked at 20 minutes, showing good correlation between samples collected from the tail or right femoral artery (Fig. S1a,b). Serum glucose levels remained euglycemic (13262 mg/dL, n = 226) and unchanged throughout the scan (Fig. S1c,d). The 18F content of tissues, fetuses and placentae were determined after euthanasia at 16163 in. As expected, brain and heart showed high degrees of FDG accumulation, while liver and skeletal muscle experienced less (Fig. 2a). Like heart and brain, right-sided fetuses and placentae were exposed to FDG only after it entered the systemic circulation. The right-sided fetuses showed intermediate avidity for FDG, accumulat- ing FDG levels nearer that of brain than of muscle, whereas right- sided placentae showed avidity for FDG that rivaled that of brain. Importantly, left-sided fetuses and placentae accumulated significant- ly more FDG than their right-sided counterparts (Fig. 2a). The uterine horn positions closest to the cervix showed the greatest differential uptake (Fig. 2b,c), though the left-greater-than-right pattern was present at nearly all positions. On average, each left-sided fetus accumulated 2.360.1% of the total administered dose whereas each right-sided fetus accumulated only 1.560.1% (p,10-8 for left versus right, n =76-79). (Fig. 2a). Like heart and brain, right-sided fetuses and placentae were exposed to FDG only after it entered the systemic circulation. The right-sided fetuses showed intermediate avidity for FDG, accumulat- ing FDG levels nearer that of brain than of muscle, whereas right- sided placentae showed avidity for FDG that rivaled that of brain. Importantly, left-sided fetuses and placentae accumulated significant- ly more FDG than their right-sided counterparts (Fig. 2a). The uterine horn positions closest to the cervix showed the greatest differential uptake (Fig. 2b,c), though the left-greater-than-right pattern was present at nearly all positions. On average, each left-sided fetus accumulated 2.360.1% of the total administered dose whereas each right-sided fetus accumulated only 1.560.1% (p,10-8 for left versus right, n =76-79). euglycemic (13262 mg/dL, n 226) and unchanged throughout the scan (Fig. S1c,d). The 18F content of tissues, fetuses and placentae were determined after euthanasia at 16163 in. As expected, brain and heart showed high degrees of FDG accumulation, while liver and skeletal muscle experienced less differential uptake (Fig. 2b,c), though the left greater than right pattern was present at nearly all positions. Disposition of infused glucose tracer We hypothesized that glucose infused via the left uterine artery catheter would have a greater chance of being taken up by the Figure 1. A model for selective fetal exposure. (a) The vast majority of blood flow to each uterine horn is supplied by each horn’s uterine artery [24], which derives from the iliac artery. Therefore, infusate from a catheter appropriately placed in the left iliac artery should experience first pass exposure to fetuses in the left, but not right, uterine horn. The fetuses in the right uterine horn thus serve as internal controls. (b) In surgical approach ‘‘A’’, ligatures (green) were tied around the left internal iliac artery just inferior to the uterine artery take off and around the femoral artery. Surgical approach ‘‘B’’ included additional ties (blue) around the left superior gluteal and hypogastric trunk arteries. (c) Experimental time-line. (d) Dynamic PET of the abdomen was initiated upon infusion of FDG, followed by a whole-body scan (WB) at 90-120 minutes, transmission and emission contamination scans (TE) at 120-160 minutes, and tissue collection after euthanasia at 160 minutes. doi:10.1371/journal.pone.0012027.g001 Figure 1. A model for selective fetal exposure. (a) The vast majority of blood flow to each uterine horn is supplied by each horn’s uterine artery [24], which derives from the iliac artery. Therefore, infusate from a catheter appropriately placed in the left iliac artery should experience first pass exposure to fetuses in the left, but not right, uterine horn. The fetuses in the right uterine horn thus serve as internal controls. (b) In surgical approach ‘‘A’’, ligatures (green) were tied around the left internal iliac artery just inferior to the uterine artery take off and around the femoral artery. Surgical approach ‘‘B’’ included additional ties (blue) around the left superior gluteal and hypogastric trunk arteries. (c) Experimental time-line. (d) Dynamic PET of the abdomen was initiated upon infusion of FDG, followed by a whole-body scan (WB) at 90-120 minutes, transmission and emission contamination scans (TE) at 120-160 minutes, and tissue collection after euthanasia at 160 minutes. doi:10.1371/journal.pone.0012027.g001 August 2010 \| Volume 5 \| Issue 8 \| e12027 PLoS ONE \| www.plosone.org 2 Local Fetal Hyperglycemia fetuses in the left uterine horn as compared to those in the right uterine horn. To test this directly, we used the glucose tracer [18F]fluorodeoxyglucose (FDG) and positron emission tomography (PET). osition of centrally administered glucose tracer As a control for the unilaterally infused FDG, we centrally administered [3H]-2-deoxyglucose (2DG), a similar glucose tracer, via subcutaneous injection just prior to initiation of the PET scans. The resultant 3H serum activity exhibited a similar profile to that of 18F (Fig. S1e,f). As with FDG, brain and heart showed greater avidity for 2DG than did liver and skeletal muscle (Fig. 2d). Fetuses and placentae also showed great avidity for 2DG. However, in contrast to 18F, the accumulation of 3H was symmetric between the fetuses and placentae from the two Disposition of infused glucose tracer On average, each left-sided fetus accumulated 2.360.1% of the total administered dose whereas each right-sided fetus accumulated only 1.560.1% (p,10-8 for left versus right, n =76-79). Figure 2. Final accumulation of glucose tracers. (a) Per gram accumulation of 18F in various tissues at 160 minutes after FDG infusion. (* p,1025 for left versus right, n = 76–79). Per fetus (b) and per placenta (c) accumulation of 18F, based on uterine horn position. The fetus/placenta closed to the cervix is counted as position 1. * p,0.05 left versus right. (d) Clearance of 3H into various tissues as measured after completion of the scan (n.s. not significant for left versus right). Per fetus (e) and per placenta (f) accumulation of 3H, based on uterine horn position. doi:10.1371/journal.pone.0012027.g002 Figure 2. Final accumulation of glucose tracers. (a) Per gram accumulation of 18F in various tissues at 160 minutes after FDG infusion. (* p,1025 for left versus right, n = 76–79). Per fetus (b) and per placenta (c) accumulation of 18F, based on uterine horn position. The fetus/placenta closed to the cervix is counted as position 1. * p,0.05 left versus right. (d) Clearance of 3H into various tissues as measured after completion of the scan (n.s. not significant for left versus right). Per fetus (e) and per placenta (f) accumulation of 3H, based on uterine horn position. doi:10.1371/journal.pone.0012027.g002 PLoS ONE \| www.plosone.org August 2010 \| Volume 5 \| Issue 8 \| e12027 3 Local Fetal Hyperglycemia uterine horns (Fig. 2d). This was true across all uterine horn positions (Fig. 2e,f). uterine horns (Fig. 2d). This was true across all uterine horn positions (Fig. 2e,f). Dynamic PET imaging Dynamic PET images were acquired for the first 90 minutes after initiating FDG infusion, with the field of view encompassing the abdomen. By several minutes, the infusion catheter was clearly visible (Fig. 3a). Early frames showed accumulation of FDG in discoid structures superior to the catheter tip, the location and size of the accumulation being consistent with the placentae of the left was symmetric between the fetuses and placentae from the two of the accumulation being consistent with the placentae of the left Figure 3. Dynamic PET imaging. (a) Representative dynamic PET images, shown as maximum intensity projections. Dynamic scans were centered on the abdomen. Minutes (‘‘m’’) after initiation of infusion are shown on each image. (b) SUV evolution for several anatomic structures. (c) SUV evolution in the left and right uterine horns. doi:10.1371/journal.pone.0012027.g003 Figure 3. Dynamic PET imaging. (a) Representative dynamic PET images, shown as maximum intensity projections. Dynamic scans were centered on the abdomen. Minutes (‘‘m’’) after initiation of infusion are shown on each image. (b) SUV evolution for several anatomic structures. (c) SUV evolution in the left and right uterine horns. doi:10.1371/journal.pone.0012027.g003 Figure 3. Dynamic PET imaging. (a) Representative dynamic PET images, shown as maximum intensity projections. Dynamic scans were centered on the abdomen. Minutes (‘‘m’’) after initiation of infusion are shown on each image. (b) SUV evolution for several anatomic structures. (c) SUV evolution in the left and right uterine horns. doi:10.1371/journal.pone.0012027.g003 PLoS ONE \| www.plosone.org August 2010 \| Volume 5 \| Issue 8 \| e12027 4 Local Fetal Hyperglycemia Figure 4. PET whole-body imaging. (a) Representative maximum intensity projection whole-body image. Color-intensity gradations are indicated from least to greatest. (b) Correlation between standardized uptake values (SUV) for volumes of interest as measured from the final whole body image (SUVscan) and for the corresponding tissues as determined by gamma well counter measurement, dose administered, and weight of the rat and the tissue (SUVwell) (r = 0.82, n = 66, p,10-16). Abbreviations: RU – right uterine horn; LU – left uterine horn. doi:10.1371/journal.pone.0012027.g004 uterine horn. Over the ensuing frames, the entire left uterine horn regions became progressively more uniformly populated with radioactivity, representing movement of FDG into fetal tissues as well. As expected, the kidneys became symmetrically visible and then faded as FDG accumulated in the urinary bladder (Video S1). Whole-body PET imaging y g g A whole-body PET image was acquired at 90-120 minutes after FDG infusion (Fig. 4a). High accumulations of FDG were evident in glucose-avid tissues including the brain, harderian glands and variably the heart and thoracic aspect of the catheter. As expected, FDG concentrations were high within the bladder. The uterine horns also were dominant structures of FDG accumulation, with higher concentrations in the left uterine horn. There was good correlation between SUVs determined either from the whole-body scan for selected volumes of interest (VOIs) or from direct well- counter-based assay of the 18F content of the corresponding tissues collected after the scan (Fig. 4b), confirming the above qualitative image-based impressions. Despite the overall excellent correlation between in vivo imaging and ex vivo radioactivity concentration determinations (Fig. 4b), minor discrepant trends were apparent. In particular, PET-based SUV tended to underestimate uterine and heart SUV. This likely is due to inactive uterine structures (e.g. amniotic fluid) included in the image-based VOI analyses but not in the radioactivity concentration determinations of extracted tissue such as the placentae and fetuses which were blotted dry prior to weighing and counting. The same may apply to the heart, where the image-based VOI analyses would include the blood in the ventricles that would not be included in the weights and well counter-based assays. In contrast, PET-based SUV of brain tended to be higher than well-counter SUV, perhaps due to bias in drawing brain regions on ‘‘hotter’’ portions of the images. Dynamic PET imaging These visualized phenomena were recapitulated in mean standardized uptake values (SUVs) measured for these regions of interest (Fig. 3b), reinforcing the qualitative impressions gleaned from visualization of the dynamic images. The infusion catheter SUV peaked early and intensely, fading significantly by 20 minutes. Renal SUV peaked at about 20 minutes, whereas right uterus SUV reached a near plateau by 90 minutes, with far lower SUV in skeletal muscle. Importantly, the left uterine horn accumulated more FDG than the right uterine horn (Fig. 3c), primarily due to enhanced FDG accumulation during the catheter infusion phase, consistent with greater first pass exposure to glucose tracer. PLoS ONE \| www.plosone.org Uterine artery catheter drainage Some scans exhibited a lack of differential FDG accumulation between the left and right uterine horn structures. We noted that the scans lacking differential uptake typically also demonstrated a predominance of early FDG distribution into non-uterine, caudal structures such as the tail or left hindquarter (Fig. S3). We reasoned that this was likely due to competing drainage of infusate into other nearby arterial trunks arising from the iliac artery. By dissection, we identified the hypogastric trunk and superior gluteal arteries as potential diverting arterial drainages (Fig. 1b). We thus tested the hypothesis that surgical approach ‘‘B’’, in which we additionally tied off these two arterial trunks, would result in a more differential FDG accumulation between the two uterine horns. There was left-dominant FDG accumulation in both surgical approaches, however surgical approach ‘‘B’’ as compared to approach ‘‘A’’ resulted in greater left-sided selectivity calculated either per mother (Fig. 5a) or per fetus (left/right = 1.960.1 versus 1.360.1, n = 27-30,38 for ‘‘B’’ and ‘‘A’’ respectively). By contrast, the ratio of 2DG accumulation between the left and right horns was essentially 1.0 in both surgical approaches. We further tested the early FDG infusate drainage pattern for association with the eventual left-versus-right uterine FDG accumulation. We divided the scans into those that showed strong or weak left-versus-right differential FDG accumulation on the 90 minute PET image. For each animal, we defined two volumes of interest, one cranial and one caudal to the infusion catheter tip, representing flow into the left uterine region or into caudal structures of the rump, tail and hindquarter. The animals with eventual strong left-versus-right differential accumulation exhibit- ed increasing cranially-directed FDG peaking at 5 minutes, indicative of immediate flow of the catheter infusate into the left uterine structures (Fig. 5b). By contrast, among animals exhibiting eventual weak left-versus-right differential accumulation, the pattern was reversed, with a caudally-directed inverted peak at 5 minutes, indicative of direct infusate flow not towards the uterine horn but rather into inferior structures. Thus, in a portion of our scans, the infusate failed to experience first-pass exposure to the left uterine horn; a situation improved by surgical approach ‘‘B’’. To further enhance the degree of left-versus-right uterine horn FDG exposure, these scans (Fig. 6) were performed under hyperinsulinemic conditions during euglycemic clamp. Optimizing differential exposure p g p To explore the above considerations and optimize differential exposure, we performed an additional series of PET scans in which we infused 15 mm fluorescent microspheres (FMS) via the left uterine artery catheter at the conclusion of the scan. The FMS are unable to traverse the arterial to venous circulation, and thus are used to quantitate blood flow to tissues including placenta [29]. As expected, far greater FMS accumulated in placentae of the left uterine horn than in structures not drained by the left uterine artery including heart, liver and brain (Fig. 6a). However, the first two right-sided fetuses also received a large portion of FMS on average, though the terminal two right-sided fetuses did not. Interestingly, the ratio of left-versus-right FMS was extremely discrepant from scan to scan, with most scans showing a strong left-sided selectivity but a few scans showed a right-sided selectivity paradoxically (Fig. 6b). The change in selectivity was due primarily to a reduction in left-sided (500680-fold, p,0.0005, n = 8-19) and less so to an increase in right-sided (863-fold, p,0.005, n = 8-15) FMS accumulation. The reasons for this alteration in flow pattern were not readily discerned on dissection. When these right-sided- FMS-flow scans were excluded, the remaining animals showed a very high degree of left-sided flow selectivity (Fig. 6c). As hypothesized, the right-sided-FMS-flow scans exhibited a caudal- ly-directed pattern of FDG at 5 minutes as compared to the strongly cranially-directed FDG appearance in the left-sided-FMS- flow scans (Fig. 6d). Thus, a portion of catheterizations were unsuccessful in directing infusate flow to the left uterus. Importantly, across all scans, the chances of a selective infusion, as defined as a fetal left-versus-right FDG selectivity ratio .1.6, were far greater with surgical approach ‘‘B’’ (10 of 11) than ‘‘A’’ (2 of 6). Fetal response to catheter p Ligation of the uterine artery leads to intrauterine growth retardation [27]. Gestational days 18-20 are a time of tremendous growth; for example, one rat strain experiences a nearly tripling of fetal weight during this interval [28]. It is thus conceivable that our uterine artery catheter approach might impede blood flow to the uterus thus impairing fetal growth. Arguing against this possibility, there was no difference in fetal weight between the left and right uterine horns at 3.5260.06 versus 3.5460.06 g (p = 0.87, n = 76- 79). There was a modest increase in placental size in the left compared to right uterine horns at 0.5060.01 versus 0.4660.01 g (p,0.005, n = 76-79). Comparing by uterine position, no differ- ences between left and right fetuses were apparent, whereas the slightly increased placental size on the left was observed in several positions (Fig. S2). Fetuses and placentae which developed further from the cervix tended to be smaller than those more proximal to the cervix, a trend which was observed both on the right and left sides. No fetal deaths or resorptions were observed among these scans and there was no difference in the number of fetuses between Figure 4. PET whole-body imaging. (a) Representative maximum intensity projection whole-body image. Color-intensity gradations are indicated from least to greatest. (b) Correlation between standardized uptake values (SUV) for volumes of interest as measured from the final whole body image (SUVscan) and for the corresponding tissues as determined by gamma well counter measurement, dose administered, and weight of the rat and the tissue (SUVwell) (r = 0.82, n = 66, p,10-16). Abbreviations: RU – right uterine horn; LU – left uterine horn. doi:10.1371/journal.pone.0012027.g004 PLoS ONE \| www.plosone.org PLoS ONE \| August 2010 \| Volume 5 \| Issue 8 \| e12027 PLoS ONE \| www.plosone.org 5 Local Fetal Hyperglycemia the left and right uterine horns (left 6.960.6 fetuses, right 7.260.4 fetuses, p = ns, N = 11). Uterine artery catheter drainage (g) Final accumulation of FDG in left and right fetuses and placenta in scans showing left- or right- dominant FMS accumulation. (p,10-4, for left versus right 18F, n = 19–25; ns p = not significant, n = 8–20). doi:10.1371/journal.pone.0012027.g006 Local Fetal Hyperglycemia Figure 6. Impact of infusate flow on differential glucose exposure. (a) Accumulation of fluorescent microspheres (FMS) in placenta by laterality and position (1 is closest to cervix; z is closest to ovary) and tissues (L, liver; H, heart; B, brain). The number of FMS normalized to weight and total number, such that a tissue receiving an average number per gram would have a relative drainage of 1. (b) Relative accumulation of FMS between left and right, dividing the average number per left placenta by the average number per right placenta, among six individual scans. (c) Accumulation of FMS in placenta and tissues only among scans showing left-dominant FMS accumulation. (d) The ratio of FDG concentration cranial versus caudal to the catheter tip for scans that exhibited a left- or right-dominant FMS pattern (p,0.05, n = 2–4). (e-f) SUV in left uterine (LU) versus right uterine (RU) horns for (e) left-dominant or (f) right-dominant FMS patterns. (g) Final accumulation of FDG in left and right fetuses and placenta in scans showing left- or right- dominant FMS accumulation. (p,10-4, for left versus right 18F, n = 19–25; ns p = not significant, n = 8–20). doi:10.1371/journal.pone.0012027.g006 takeoff [30]. Because several nearby arteries can divert a portion of the infusate to structures other than the uterus, there are competing interests between ligating arteries that divert flow and minimizing surgical dissection. In our study, ligating two nearby arteries improved uterine horn selectivity with a minimum of additional dissection. Although additional ties could be placed, we believe our approach ‘‘B’’ represents a reasonable balance the left and right uterine horns (Fig. 6g, right-flow group). By selecting left-sided-FMS-flow animals only under hyperinsulinemic conditions, there was a 3.7-fold increased accumulation of FDG in the left- versus right- uterine horn fetuses (p,10-4, n = 19-25) (Fig. 6g, left-flow group; this data is shown by uterine horn position in Fig. S4). Chronic glucose infusions. We assessed the ability of this approach to maintain differential glucose exposure between the left uterine artery and systemic maternal circulation. A double lumen catheter was placed on gestational day #18 using surgical approach ‘‘B’’. Uterine artery catheter drainage Glucose was infused for 48 hours via the upstream port at 4 mg/min and blood sampled via the downstream port. This produced sustained and severe localized hyperglycemia at the left uterine artery takeoff, while normal glycemia was maintained in the maternal systemic circulation (Fig. 7). Figure 7. Chronic glucose infusion. Glucose was infused on gestational days #18-20 at 4 mg/min via the left artery catheter. Serum was sampled downstream of the infusion catheter tip (LU, black squares) and from the maternal tail tip (mat, open circles). p,0.05. doi:10.1371/journal.pone.0012027.g007 Uterine artery catheter drainage We reasoned that, since insulin action shortens the plasma half-life of circulating glucose, this would reduce the relative amount of left uterine artery infused FDG reaching the right uterine horn. Indeed, uterine horn SUVs were much stronger on the left than on the right among the animals with left-sided-FMS-flow (Fig. 6e), a pattern not seen in right-sided-FMS-flow animals despite hyper- insulinemia (Fig. 6f). Furthermore, the right-sided-FMS-flow animals showed a lack of differential FDG accumulation between Figure 5. Impact of early FDG pattern on differential glucose exposure. (a) Impact of surgical approach on differential 18F (black bars) but not 3H (white bars) accumulation (* p,0.05 for difference between approaches, n = 5–6). (b) The ratio of 18F cranial versus caudal to the catheter tip is plotted over time for scans that exhibited a final left/right uterine horn 18F ratio of .1.6 (SEL) or ,1.6 (NON). (p,0.01, #p,0.001, n = 5–6). doi:10.1371/journal.pone.0012027.g005 Figure 5. Impact of early FDG pattern on differential glucose exposure. (a) Impact of surgical approach on differential 18F (black bars) but not 3H (white bars) accumulation ( p,0.05 for difference between approaches, n = 5–6). (b) The ratio of 18F cranial versus caudal to the catheter tip is plotted over time for scans that exhibited a final left/right uterine horn 18F ratio of .1.6 (SEL) or ,1.6 (NON). (p,0.01, #p,0.001, n = 5–6). doi:10.1371/journal.pone.0012027.g005 August 2010 \| Volume 5 \| Issue 8 \| e12027 PLoS ONE \| www.plosone.org 6 Local Fetal Hyperglycemia Figure 6. Impact of infusate flow on differential glucose exposure. (a) Accumulation of fluorescent microspheres (FMS) in placenta by laterality and position (1 is closest to cervix; z is closest to ovary) and tissues (L, liver; H, heart; B, brain). The number of FMS normalized to weight and total number, such that a tissue receiving an average number per gram would have a relative drainage of 1. (b) Relative accumulation of FMS between left and right, dividing the average number per left placenta by the average number per right placenta, among six individual scans. (c) Accumulation of FMS in placenta and tissues only among scans showing left-dominant FMS accumulation. (d) The ratio of FDG concentration cranial versus caudal to the catheter tip for scans that exhibited a left- or right-dominant FMS pattern (p,0.05, n = 2–4). (e-f) SUV in left uterine (LU) versus right uterine (RU) horns for (e) left-dominant or (f) right-dominant FMS patterns. PLoS ONE \| www.plosone.org Discussion Here, we quantify an approach to expose the fetus in vivo to isolated hyperglycemia by infusion of glucose into one of the unilateral uterine arteries. As hypothesized, the ipsilateral fetuses were exposed to significantly more hyperglycemia than contralat- eral fetuses, with substantive first pass clearance of glucose into the ipsilateral placentae and fetuses. To best take advantage of this approach, ideally all of the catheter infusate would be directed into uterine artery flow. Although this could be achieved by direct catheterization of the distal uterine artery, this would likely impede uterine blood flow. We used a surgically simpler approach, similar to that reported in studies of maternal-fetal metabolite [30] and toxin [31] transfer, placing the catheter tip in the iliac artery near the uterine artery Figure 7. Chronic glucose infusion. Glucose was infused on gestational days #18-20 at 4 mg/min via the left artery catheter. Serum was sampled downstream of the infusion catheter tip (LU, black squares) and from the maternal tail tip (mat, open circles). p,0.05. doi:10.1371/journal.pone.0012027.g007 August 2010 \| Volume 5 \| Issue 8 \| e12027 7 Local Fetal Hyperglycemia between selectivity and surgical intervention. In some animals, catheter flow to the left uterine horn was unexpectedly negligible as assessed by early images from PET scanning, corroborated by fluorescent microspheres (FMS). Importantly, this result illustrates that FMS can be used as a means to assess catheter drainage without the need for PET. FMS could be used to select those animals which exhibit the desired flow pattern, thus removing from consideration infusions that otherwise would dilute the effect under study. On the other hand, strictly standardizing by FMS flow could reduce the within-experiment variation in unintended ways, reducing the degree to which results could be generalized [32]. It is important to note that selection by flow pattern was not required to obtain statistically significant degrees of differential uterine horn exposure even when averaging across a small number of animals. rapidly cleared diabetes-related metabolites such as fatty acids [40], triglycerides [41], and ketones [42]. Thus, this model is ideally suited to test components of the Freinkel hypothesis in vivo. Additionally, this method is likely applicable to other categories of agents that affect mother and fetus, such as various toxins, pharmaceuticals, and reactive oxygen species. Discussion We conclude that this model approach creates isolated hyperglycemic fetal exposure while maintaining relatively unexposed control fetuses in the opposing uterine horn and that this approach is likely well-suited to study of other agents that may impact on fetal health. Uterine artery catheter On gestational day 18, a vascular catheter draining into the left- sided uterine artery was placed as follows. Anesthesia was induced and maintained with inhalational isoflurane mixed with oxygen. The femoral triangle was opened, and a stretched, sterilized PE10 catheter (Instech Laboratories Inc., Plymouth Meeting, PA) was inserted and secured 1.65 cm retrograde into the femoral artery, thus placing the tip several mm superior to the uterine artery takeoff from the common iliac. The left inferior peritoneal space was then opened and a silk ligature tied around the iliac artery inferior to the uterine artery takeoff, typically just inferior to the hypogastric trunk artery. In selected surgeries, the superior gluteal and hypogastric trunk arteries were identified [43] and tied with silk ligature at their origin. A right sided femoral artery catheter was likewise placed except without intraperitoneal procedure. Catheters were tunneled subcutaneously to a mid-scapular exit and connected to a dual channel infusion swivel (Instech) allowing the rat freedom of movement. Postoperative analgesia included bupivacaine and buprenorphine. Saline with 5 U/mL heparin was infused at 10 ml/min to maintain each catheter’s patency. This approach has several limitations. As implied above, this approach does not recapitulate the full effects of diabetes during pregnancy, but rather is limited to the effects of hyperglycemia local to the fetus. However, this limitation is of value to help identify the individual metabolic fuels that contribute to fetal risk. It is important to know which maternal metabolic fuels elevated in diabetes to target in order to reduce fetal pathology [38], especially given that maternal-sided interventions might have advantages over fetal-sided interventions [39]. Additionally, the present work was limited to late gestation, during which diabetes adversely affects fetal growth, neonatal health [2], and programming of adult disease [12], [13] but does not cause teratogenesis [7]. To adapt this technique to the study of diabetes-related birth defects, the catheter would need to be placed earlier in gestation to allow hyperglycemic infusion during fetal organogenesis. Animals All procedures were performed within the regulations of the Animal Welfare Act and the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and were approved by the Institutional Animal Care and Use Committee of the University of Iowa. Timed, pregnant Hsd:Sprague Dawley SD rats (Harlan Laboratories, Inc.) were allowed one week to acclimate. Gestational day 0 was defined as the day of initial vaginal plug detection. The approach of directly providing excess macronutrients in vivo to fetuses in one horn of the uterus has several advantages over systemic induction of hyperglycemia [33] or diabetes. Importantly, the induced hyperglycemia is localized. For example, infusion of 9 and 15 mg/min glucose induces mild and marked systemic hyperglycemia in pregnant rats [34], whereas we find that infusion of 4 mg/min glucose into the uterine artery at term raises the local glucose concentration markedly but has little effect on maternal systemic glucose levels. Another advantage is that the contralateral fetuses serve as an ideal internal control. Fetuses from both sides are exposed to essentially the same maternal milieu, with the exception that the left-sided fetuses are additionally exposed to the localized hyperglycemia. This approach can be thought of as an in vivo compliment to in vitro embryo culture, which has been very helpful in determining the effects of metabolic fuel excesses on fetal development but does not perfectly recapitulate the in vivo situation [33], [35], [36]. In fact, the idea of providing excess macronu- trients in vivo to fetuses in one horn of the uterus is not new, having been explored via transamniotic feeding [37] for example. One additional advantage of directly infusing glucose into one uterine artery is that the timing of hyperglycemia exposure can be finely controlled temporally. For example, the fetal effects of brief repeated episodes of hyperglycemia, such as occur during pregnancy complicated by glucose variability in unstable type 1 diabetes, could be tested. PLoS ONE \| www.plosone.org PET imaging On gestational day 20, rats were anesthetized with 1.5% isoflurane after overnight fast (13.660.9 h). Each animal was maintained on a heated bed in dorsal recumbency, blood glucose measured, a bladder catheter and rectal temperature probe placed, and positioned in a MOSAIC microPET scanner (Philips Medical Systems, Milpitas, CA, USA) with the abdomen completely within the field of view as confirmed by transmission scan using 137Cs. FDG was delivered in its standard buffer, containing a trivial amount of glucose (,,6 mg/dL, formed as by-product of the FDG synthesis), used for human clinical infusions. At time zero, a dose of 3362 MBq 18FDG in approximately 175 mL was infused at 20 mL/min via the uterine artery catheter. Dynamic PET imaging (120 transaxial slices, 1 mm slice thickness) and blood sampling commenced at the start of the FDG infusion (t = 0). At 90-120 minutes, a whole-body image of the rat from lower extremities to head was acquired (230 transaxial slices, 1 mm slice thickness) by movement of the scanner bed through the gantry. The rat and its fetuses were then euthanized with 150 mg/kg intravenous pentobarbital and the Our immediate plans with this model are to study the effects of localized hyperglycemia on placental blood flow, fetal growth, and islet development late in gestation. We also note that this approach could be applied to other agents beyond glucose. A general requirement for utility in this approach is that the agent tested needs to be cleared rapidly from the circulation, otherwise significant amounts will reach the contralateral horn. The half- life of glucose in the circulation of the rat is on the order of 10-40 minutes [25]. This technique may be well suited to study other August 2010 \| Volume 5 \| Issue 8 \| e12027 August 2010 \| Volume 5 \| Issue 8 \| e12027 8 Local Fetal Hyperglycemia fetuses and placentae collected by Caesarean section. The native positioning of the left and right-sided fetuses was photodocumen- ted prior to removal. Plasma samples and excised tissues were weighed and the 18F content determined by assay in a previously calibrated gamma NaI well-counter with decay correction to the start of the radiotracer infusion. infused via the upstream catheter tip at a rate of 4 mg/min and 20 ml/min. Euglycemic hyperinsulinemic clamp during PET Euglycemic hyperinsulinemic clamp during PET After induction of anesthesia, insulin was infused via the right femoral vein at 20 mU/kg/min. Glucose was infused via the same catheter to maintain blood glucose at 90 mg/dL. FDG infusion and PET imaging were initiated upon achievement of steady state glucose infusion, at 9863 min after the initiation of insulin infusion. The clamp was maintained during the PET imaging, with the glucose infusion rate averaging 2660.4 mg/kg/min and the blood glucose averaging 9061.7 mg/dL. PET imaging The second lumen tip, downstream by 2.5 mm, was used for blood sampling and kept patent by 0.9% saline with 5 units/mL heparin infused at 10 mL/min. Simultaneous mater- nal blood was obtained by tail-nick. Supporting Information Figure S1 Plasma sampling during PET. (a) Average serum 18F concentration as sampled from the tail or right femoral artery, showing correlation (b) from samples collected from both sites simultaneously (r = 0.80, p,10-23, N = 104). (c) Serum glucose as measured from the tail or right femoral artery, showing correlation (d) from samples collected from both sites simultaneously (r = 0.83, p,10-24, N = 92). (e) Average serum 3H concentration as sampled from the tail or right femoral artery, showing correlation (f) from samples collected from both sites simultaneously (r = 0.87, p,10-28, N = 95). F d t: d i:10 1371/j l 0012027 001 (0 44 MB TIF) Acknowledgments We thank PET technologists Dean A. Clermont CNMT, Christine A. Mundt CNMT, John C. W. Richmond CNMT, and Julie A. Riggert MA CNMT, and thank Gordon L. Watkins PhD and Katherine R. Thede- Reynolds M.S. for radiolabeled FDG, and thank Art A. Spector MD, Fred S. Lamb MD PhD and Stacia L. Koppenhafer for advice (all acknowledged are from the University of Iowa). We thank PET technologists Dean A. Clermont CNMT, Christine A. Mundt CNMT, John C. W. Richmond CNMT, and Julie A. Riggert MA CNMT, and thank Gordon L. Watkins PhD and Katherine R. Thede- Reynolds M.S. for radiolabeled FDG, and thank Art A. Spector MD, Fred S. Lamb MD PhD and Stacia L. Koppenhafer for advice (all acknowledged are from the University of Iowa). Quantitative image analysis Error bars represent standard error of the means. Comparisons between two independent samples were performed using two- tailed homoscedastic Student’s t-tests. Paired samples were compared using two-tailed repeated measures t-tests. g y Transaxial slices were reconstructed using a 3D Row Action Maximum Likelihood Algorithm (Philips Medical Systems) on projection data corrected for random coincidences, scatter, and dead time. All images were attenuation- and decay- corrected. PET images were scaled in standardized uptake values (SUV) by normalizing to the administered FDG dose and the weight of the animal. Image analysis was performed using PMOD 3.0 (PMOD Technologies Ltd., Zurich, Switzerland). VOIs were defined manually on transverse sections while viewing SUV data displayed in four dimensions (transverse, sagittal, and coronal planes and time). The anatomic regions representing the left and right uterine horns were defined while comparing PET images to the respective photodocuments. Central 2-deoxyglucose tracing In selected animals, 80 mCi of 2-[1,2-3H(N)]-deoxy-D-glucose (2DG) (New England Nuclear, PerkinElmer, Waltham, MA) was injected subcutaneously in the axilla immediately before initiating 18FDG infusion. Tritium content was determined in tissue and plasma samples after 18F decay. Tissues were dissolved in 4N KOH overnight, the supernatant neutralized, and 3H content determined by liquid scintillation counting. Glucose concentra- tions were measured on whole-blood or plasma with a One Touch Ultra meter (LifeScan Inc., Milpitas, CA). Found at: doi:10.1371/journal.pone.0012027.s001 (0.44 MB TIF) Figure S2 Fetal and placental weights by uterine horn position. Fetal (a) and placental (b) weights are shown by uterine horn position. Position 1 is closest to the uterine cervix, with further positions closer to the ovarian end of the uterine horn. Found at: doi:10.1371/journal.pone.0012027.s002 (0.13 MB TIF) Figure S3 Impact of early FDG pattern on differential glucose exposure. Dynamic imaging, displayed as maximum intensity projections, shows early FDG disposition caudal relative to the catheter tip, in a scan that ultimately did not exhibit differential glucose accumulation between uterine horns. Minutes (‘‘m’’) after initiation of infusion are listed on each image. Found at: doi:10.1371/journal.pone.0012027.s003 (1.45 MB TIF) Fluorescent microspheres At the conclusion of selected PET scans, 16105 fluorescent microspheres (FMS) (polystyrene, 15 mm, # F-8844, Invitrogen Corp., Carlsbad, CA) were infused at 20 mL/min via the uterine artery catheter. After 18F decay, the FMS content of selected tissues was determined as follows. Each tissue was dissolved in 2.5 mL/g-tissue 4N KOH:2% Tween 80 overnight, then FMS collected by filtration through a 5 mm pore size polycarbonate membrane. After rinsing with 2% Tween 80, FMS were dissolved from the membrane in 1 ml 2-ethoxyethyl acetate. Fluorescence was quantitated in polypropylene multiwell plates using excitatio- n:emission frequencies of 492:520 nm and compared to a standardized curve. Figure S4 Fetal and placental 18F by uterine horn position. Position 1 is closest to the uterine cervix, with further positions closer to the ovarian end of the uterine horn. Per fetus (a) and per placenta (b) accumulation of 18F. p,0.05 for left versus right. Found at: doi:10.1371/journal.pone.0012027.s004 (0.07 MB TIF) Video S1 Video of representative dynamic PET. Serial dynamic images collected during time 0-90 minutes, shown as maximum intensity projections. Found at: doi:10.1371/journal.pone.0012027.s005 (1.33 MB GIF) PLoS ONE \| www.plosone.org References 1. Finnell RH, Waes JG, Eudy JD, Rosenquist TH (2002) Molecular basis of environmentally induced birth defects. Annu Rev Pharmacol Toxicol 42: 181–208. 22. 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Barton RN (1976) Effect of ischaemic limb injury on the rates of metabolism of ketone bodies in starved rats.. Biochem J 156: 233–238. 21. Moore DC, Stanisstreet M, Clarke CA (1989) Morphological and physiological effects of beta-hydroxybutyrate on rat embryos grown in vitro at different stages. Teratology 40: 237–251. ketone bodies in starved rats.. Biochem J 156: 233–23 43. Green E (1935) Anatomy of the Rat. References Trans Am Philos Soc 2 PLoS ONE \| www.plosone.org August 2010 \| Volume 5 \| Issue 8 \| e12027 10
https://openalex.org/W2531310868	http://journal.teflin.org/index.php/journal/article/download/335/271	English	null	THE INFLUENCE OF STUDENTS’ L1 AND SPOKEN ENGLISH IN ENGLISH WRITING: A CORPUS-BASED RESEARCH	Teflin Journal	2,016	cc-by-sa	7,803	THE INFLUENCE OF STUDENTS’ L1 AND SPOKEN ENGLISH IN ENGLISH WRITING: A CORPUS-BASED RESEARCH Prihantoro (prihantoro2001@yahoo.com) Universitas Diponegoro Jl. Prof. Soedharto Tembalang, Semarang, Jawa Tengah Abstract: Academic writing requires both style and grammatical correct- ness; however, efforts in improving the quality of English academic writing by non-native students have been focused on grammar. Structures observed in this study were grammatically correct, but considered unnatural in aca- demic writing genre. This research involves a group of non-native English speaking students who were assigned to submit two different kinds of writ- ing to an online repository: a research paper abstract and a free writing arti- cle. A survey to understand the sources of English exposure is also conduct- ed. The objectives of this study are to describe unnatural sequences/Multi Words Units (MWUs) used by the students and to identify the motives of us- ing such sequences. The tools for corpus processing used are Unitex and Antconc. Corpus of Contemporary American English and British National Corpus are also used as reference corpora for English while the SEAlang In- donesian Corpus is used to validate the influence of first language (L1). The analysis of these sequences with comparison to reference corpora indicated the influence of spoken English and students’ L1 (Indonesian). This corre- sponds to the results of the survey that most of the students are exposed to English mostly via spoken, and non-academic sources (songs, movies, social media, etc). Keywords: corpus, recurrent patterns, lexical bundle, L1, L2, Academic Writing Keywords: corpus, recurrent patterns, lexical bundle, L1, L2, Academic Writing Keywords: corpus, recurrent patterns, lexical bundle, L1, L2, Academic Writing DOI: http://dx.doi.org/10.15639/teflinjournal.v27i1/217-245 DOI: http://dx.doi.org/10.15639/teflinjournal.v27i1/217-245 DOI: http://dx.doi.org/10.15639/teflinjournal.v27i1/217-245 Students pursuing higher education are always required to write academic works such as essays or papers. This can be a challenging task, given that writ- 217 218 TEFLIN Journal, Volume 27, Number 2, July 2016 ten language needs to be distinctive from spoken language (see Biber (2006a); Biber (2006b); and Swales (1990)). For that reason, even a native speaker of a language may find academic writing demanding. As for writing academically in a foreign language, the problem is definitely more complicated. Consider some evaluative expressions that are commonly present in everyday spoken English, but rarely used in academic texts as shown by example (1): (1) It’s fantastic. That is a fabulous work. I love this method Authors of academic writing are most likely to resort to more objective evaluation structures such as: This research is of a crucial importance. The work is significant, or The method is commonly preferred. As for students whose L1 is not English, the evaluation of the writing is mostly on grammar; which is how the students can express their ideas logically through grammatically correct sentences. However, errors still happen, particularly for beginner students. One of the reasons for these errors to take place is L1 influence (interference or negative transfer) as evidentially shown by Sawalmeh (2013), Isaac (2008), Figueredo (2006) and Arabski (2006). In countries where English is not widely used, the curriculum of English departments normally begin with some basic competence skills, including writing/composition. This writing/composition classes are usually the primary requirements for academic writing. At this point, teachers start to introduce the stylistics of academic writing papers. The general purpose of this research is to collect data on the academic writing performance of non-native speakers of English, whose L1 is Indonesian. Specifically, it seeks confirmation whether unnatural sequences used by the students are under the influence of L1 or spoken English (or both). The processing is completely carried out by corpus processing softwares and the analysis of this data is cross referenced with two reference corpora to confirm the validity of the author’s evaluation. This research also attempts to validate whether performance, as expressed in the writing, corresponds straight to the sources that the students have been exposed to. Some following sub-sections here also review related studies about interference specifically related to Indonesian as L1. The relevancies of these studies to the present research and in what respect it differs will be explained here. Methodology section describes in detail how this study was carried out. It also describes the tools and corpora used in this study. The core of this study is Prihantoro, Influence of L1 and Spoken English on English Writing 219 presented in the finding and discussion section. While the finding section focuses more on the quantitative analysis, the discussion section explores the data with more qualitative approach. The conclusion section correlates the corresponding qualitative to quantitative natures of the data as well as proposes recommendation for further studies. Written and Spoken Language: Corpus Validation The distinction of spoken and written languages is necessary especially in the field of genre analysis and text production, as described by Halliday (1989) and Hasan (1986). They focused on providing concepts and baselines that characterize the difference between spoken and written language. More re- cent works, such as Biber (2006), Szmrecsanyi & Hinrichs (2008), applied the- se concepts to more controlled variables, which are college students. The results of research in spoken and written language is also documented in standard dictionares. The documentation of written language in specific domain is usually shown by <written> special annotation to the related entries. Consid- er the annotation for entry <exclaim>, as shown by figure 1. Figure 1: Annotation for Entry <exclaim> Figure 3: Frequency Chart of <it is commonly believed> in COCA Figure 3: Frequency Chart of <it is commonly believed> in COCA Prihantoro, Influence of L1 and Spoken English on English Writing 221 How corpus data can be used to validate judgment is relevant to my re- search as the dynamic corpus is always updated to record the actual language use. Besides amplifying the analysis, corpus data is also useful to show the in- fluence of L1. There are some patterns observed in L2 writing that correspond to L1 structure. This early hypothesis can be validated with help of reference corpora. Figure 1: Annotation for Entry <exclaim> Figure 1 is taken from Longman Dictionary of Contemporary English, where the entry <exclaim> is marked (written) as it is used more frequently in written language as compared to spoken language. However, since features of language may change from time to time, we need to make sure that the diction- ary is recent or we can validate this using dynamic corpora (corpora that are regularly updated). Focus on the retrieval of <exclaim> in Corpus of Contem- porary American English (COCA) and its genre distinction are shown in Figure 2. The result indicates that <exclaim> is used widely in fiction and other text types (note that in COCA, besides spoken section, all data are taken from writ- ten texts). The lowest frequency is on spoken section. Thus, it validates the claim that <exclaim> is widely used in written language. 220 TEFLIN Journal, Volume 27, Number 2, July 2016 Besides the choice of word, the distinction can also be shown by sequence or pattern preference. The term sequence or pattern refers to a string of words, which is considered pre-fabricated expressions. These expressions are syntacti- cally composed, but used paradigmatically. A variety of terms have been pro- posed. Hyland (2008) and Allen (2009) refer to the sequence as lexical bundle, as also used in Allen (2009) and Chen and Baker (2010). While in the field of computational linguistics, the term lexical chain or multiword units (MWUs) is also used (see Paumier, 2008). Figure 2: Lowest Frequency of <exclaim> in Spoken Section of Corpus Data Figure 2: Lowest Frequency of <exclaim> in Spoken Section of Corpus Data It is important to understand what makes MWUs essential in the field of language learning. Rather than concatenating words by words, using pre- fabricated sequence will reduce the risk of making mistakes. In turn, this will improve the quality of students’ academic writing. Besides, this will also help characterize the texts to a specific domain, as the identification of a text to a specific genre may derive from lexical bundles (Hyland, 2008). Meanwhile, Figure 3 below introduces us to the expression <it is commonly believed> which according to COCA has zero occurrences in spoken section, but the fre- quency is very high in academic section. Studies on Interference Some of the research in academic writing, especially where the subjects of research are students, whose L1 is not English, is often focused on error analy- sis. The research usually describes and categorizes students’ errors. The catego- ries where errors take place are prioritized target of improvement for teaching. Error analysis patterns may further be compared to the linguistic features of students’ L1 to validate whether the error is transferred from L1. When writing in English, for example, it is quite common for Indonesian students to drop ar- ticles since this linguistic feature is not present in Indonesian (Wijaya, 2012). In the languages where such markers are absent, similar problems tend to occur (Bautista & Gonzalez, 2008). Some other academic research focuses not on grammar, but on the style and word choice of the writing. It tries to measure how appropriate the style and the word choice of the writing to the genre where the language is used. One of such research studies the preference of pronoun ‘I’ that is getting more and more common these days in academic writing (Harwood, 2005). Some of the works dedicated to the interference of Indonesian in English were conducted by Fauziati (2003), Roni (2006), Yembise (2011) and Moehkardi (2012). While Fauziati’s (2003) respondents were middle schoolers, my respondents in this research are all college students. Roni’s (2006) respondents were college-level students, but they were not from English majors. In my research, the students were all from English department and they were all senior undergraduate students who received minimum B- (grade) on Academic Writing. Moehkardi (2012) describes some lexical bundles in English and lists some possible L1 transfers patterns. Unlike Mohkardi (2012) whose research did not take account of authentic data, my research is fully data driven, and all the data are processed by using a corpus processing software. None of the previous research employed any corpus processing software and none of them used reference corpus as well. The validation with corpora 222 TEFLIN Journal, Volume 27, Number 2, July 2016 reference, not to disregard the introspective competence of the researchers, is of a crucial importance as the variables of corpora are overt, and most importantly, the data in the corpora are authentic, updated and evidential. Reasons for interference to occur may vary, but one classical factor, as also mentioned in the previous studies, is the L1 of the students (Husein & Mohammad, 2012). METHOD The respondents of this study are a group of Undergraduate English De- partment students of the 6th semester from Universitas Diponegoro, Indonesia. I accessed the on-line academic information system and screened the students with the following variables: 1) students who have passed academic writing class, 2) students with at least a final grade of B-. Of around 120 students, 117 passed the first screening, and 79 passed the second one. Of this number, 69 students volunteered. Of the 69 students, 65 responses were collected; two stu- dents failed to submit the tasks due to failure in establishing a stable Internet connection while the other two dropped the tasks for unknown reasons. There are two types of responses; the first one is response to questionnaires, and the second one is response to writing tasks. Studies on Interference Another factor that may contribute to interference is the spoken language. Šimčikaitė (2012) discovers that students use some English spoken discourse markers in writing like I mean, anyway, and by the way. The interference of spoken language is well documented by Krauthamer (1999). He refers to the interference as SLIP (Spoken Language Interference Patterns). The present study is aimed at testing whether these two factors contribute to students’ writing. Cross reference is conducted by using COCA (Davies, 2008), BNC (Aston & Burnard, 1998) and SEAlang Indonesian Corpus (Scannel, 2010). This research is also digital data driven, which means that all data are digi- tally processed. The corpus methodology in this research follows the works of Pang (2010), Allen (2009), Hyland (2008) and Yoon (2008), where the recur- rent lexico-grammatical patterns were retrieved by corpus processing software and analyzed both quantitatively and qualitatively. Figure 4: Screenshot of the On-Line Questionnaire The second crucial task is the task to write two articles. The first one is to write a research paper abstract, and the second one is free writing. Questionnaires and Writing Tasks The questionnaire is crucial as it describes the students’ exposures to English via different sources. It will be used further in data processing to con- Prihantoro, Influence of L1 and Spoken English on English Writing 223 firm whether the exposure corresponds to their academic writing skills or not. In the survey, students can score the source of exposure to English from the lowest (1) to the highest (5), as is shown in Figure 4 below. Figure 4: Screenshot of the On-Line Questionnaire Corpora and Corpus Processing Software Results of the survey are saved in a spreadsheet file, while the articles are saved in a raw text file allowing Unitex and AntConc, the corpus processing software used in this research, to further process the texts collection as a cor- pus. Word Frequency computation and concordance extraction is performed to obtain necessary information that will be presented in the findings section. Analysis to the findings is performed with reference to COCA and SEAlang Indonesian corpus. While Unitex is used to analyze the texts used in the re- search, COCA provides an online reference to standard corpora. The analysis is fully presented in the discussion section. Prior to the writing of this paper, the result of the analysis was presented to the students. Interview session was also held to understand the motivation of why such unnatural sequences were se- lected by the students. As for COCA, the presentation may include more than concordance lines (see Figure 5). Figure 5 shows a chart generated by COCA to show frequency of occur- rence on the basis of corpus section (left side) and historical trend (right side). The highest frequency corresponds to the other section and it always reaches the top ceiling in the section as shown by academic section on the left side. The historical trend itself is grouped in a four-year term. As a companion to the concordance, this chart takes a very significant role. Actual concordance lines 224 TEFLIN Journal, Volume 27, Number 2, July 2016 correspond directly to the frequency. Presenting all concordance lines can be wordy; therefore, I decided to randomly choose any concordance lines to be shown and explained. These selected concordance lines that are presented in the findings and discussion section are representative to the aims of this study. Figure 5: Chart Showing Sections, Historical Progress and Frequency in COCA Token Frequency Table 2. Token Frequency Extraction Type Frequency Type Frequency Type Frequency The 481 in 188 English 110 And 219 study 177 Foreign 99 Be 207 language 151 students 97 but 111 need 90 Table 2. Token Frequency Extraction Token frequency extraction is useful in determining topics and keywords. The result shown in Table 2 suggests a focus on English as Foreign language in the collection of abstracts. It is an interesting notion since the term ‘foreign’ is used instead of ‘second language’. Table 1. Data Segmentation Data Segmentation The survey also showed that students were exposed more to spoken than written English. I categorized direct conversations, lectures, songs and movies as spoken English, while the rest (journals, theses, assignments, social media, comic books, magazines, assignments and newspapers) as written English. Even though the variables of written English outnumbered spoken English, the average of spoken English (69%) is higher than that of written English (31%). Token Frequency Findings The survey showed that the students’ largest exposure to English included movies, songs and social media (>4). It is important to notice that lecture and assignment are only one level below (3-4). This was expected, as the students are English department students and the classes and assignments are conducted in English. The level of exposure to textbook is 3, which is equal to direct con- versations, comic books and newspapers. Theses, dissertations and journals, which are actually significant reference for academic writing are down on level 0-1 Figure 6: Student Exposure to English 0 1 2 3 4 5 movies magazine or newspaper lecture social media journals assigment Figure 6: Student Exposure to English Prihantoro, Influence of L1 and Spoken English on English Writing 225 Ironically, students were least exposed to academic sources such as jour- nals, theses and dissertations. Students also claimed that the highest exposure came from social media like Twitter, Facebook, Path, etc. A round score (0) was obtained for corpus as a source of exposure. Overall responses indicated that students were more exposed to non-academic, and spoken languages as compared to academic and written language. This is shown in Table 1 below. Table 1. Data Segmentation Data Segmentation Written Spoken Academic Non-Academic 31% 69% 37% 63% Table 1. Data Segmentation Data Segmentation Written Spoken Academic Non-Academic 31% 69% 37% 63% Lemma Distribution and 3-Gram While word frequency is useful in determining the topics or keywords, some other means are required to retrieve sequences/MWUs. With this in mind, I decided to first observe lemma distribution. The processing indicates that 226 TEFLIN Journal, Volume 27, Number 2, July 2016 226 there are three lemmas, content words, which occurred in almost each of the students’ writing (both abstracts and free-writing) as shown in Table 3. Table 3. The Distribution of <suggest>, <help>, and <goal> Suggest Help Goal Present 94% 97% 92% Absent 6% 3% 8% Table 3. The Distribution of <suggest>, <help>, and <goal> Suggest Help Goal Present 94% 97% 92% Absent 6% 3% 8% The identification of sequences used on the left and right context is per- formed by Local Grammar Graphs (LGGs). LGGs is one of the machine- readable grammars in Unitex used to retrieve words or sequences (Paumier, 2008). Figure 7: LGGs to Extract 3-Gram Sequences Figure 7: LGGs to Extract 3-Gram Sequences The LGGs, as shown in Figure 7, extracted three tokens on the left-right context of target lemma <suggest; help; goal>. This process gives reason to name it 3-Gram. When necessary, the retrieval of N-gram (where N may refer to any number) is possible. The angle brackets are required to indicate all word forms of a lemma. As an illustration, the use of angle brackets in <go> retrieve all word forms conceived by corpus-processing software lexical resource which include go, went, gone, goes and going. LGGs in Figure 7 retrieved 3-gram sequences as shown in Figure 8. For <suggest>, and <help>, the recurrent sequence patterns are on the right context. The nouns that the verb <suggest> specifies are almost all human nouns, and some are represented by pronouns. After the nouns, <to><Vinf> are used. The same specification also applied for <help>; only, there are two pat- terns after the nouns, which is to use <to><Vinf>, or just <Vinf>. As for <goal>, it is interesting to observe the left context, especially the verbs that col- Prihantoro, Influence of L1 and Spoken English on English Writing 227 ligate with <goal>. These patterns are explored more with concordance in the discussion section. Figure 8: The 3-Grams for Left-Right Context <suggest;help;goal> in Abstracts Collection ligate with <goal>. These patterns are explored more with concordance in the discussion section. Figure 8: The 3-Grams for Left-Right Context <suggest;help;goal> in Abstracts Collection Students’ Feedback As stated previously, the findings of the study had been presented to the students where they were asked to provide some reasons as to why the suggest- ed structures were not used. Most of the students said that they were not aware of the presence of such structures. As for those who were aware, the reasons for not choosing the structures varied. See Table 4. Table 4. Awareness of Suggested Structures Positive (28%) Negative (72%) 6% Strong belief in the source 10% Strong belief in frequency 12% Strong belief in teacher’s instruction Table 4. Awareness of Suggested Structures There are three main reasons why they had the confidence in using the structures, even though they know that the suggested structures exist. The faith derived from consulting, specifically, existing final projects as the source of their writing (6%). The second one is because of the degree of frequency of the overall exposure. The third one is the strong belief in previous teacher’s in- structions/ descriptions, which are mostly spoken and acquired during the lec- ture. 228 TEFLIN Journal, Volume 27, Number 2, July 2016 Discussion In this section, I discuss the frequent Multi Word Expressions (MWUs) obtained from the 3-gram extraction of students writing corpus. The MWUs are as follows: MWU 1 Suggest that you do - Suggest you to do MWU 2 Help you to do - Help you do MWU 3 Score goal - make goal - print goal Should the results of the questionnaires be parallel to the findings, it is possible that domain shift from spoken to written may take place. It is also pos- sible that to some extent, learners replicate the L1 pattern, in this case Indone- sian, to English as the foreign language. The description is validated by corpora analysis. In doing so, I consulted two well-known English corpora from two different dialects: British English (BNC) and American English (COCA). To investigate language transfer possibility, the SEAlang Indonesian Corpus is al- so consulted. Analytical information from each corpus that corresponds to the MWUs will be presented to show the gap between authentic language and lan- guage used by learners of English as a second language. MWU 1: Corpus Data In this section, I will show how students use the verb <suggest>. The presence of this verb in my corpus is quite significant as students usually use this verb in the end of their writing to give recommendation. The retrieval was aimed at all verb forms of <suggest>: suggest, suggests, suggested, suggesting. Among these four forms, ‘suggesting’ was not found in the retrieval. As for the patterns, there were two verb patterns in use. The first one (---1) was <sug- gest><PRO><to><V>, and the second one (---2) is <sug- gest><that><PRO><Vinf>. Figure 9 below shows Concordance 1 that is gen- erated by Unitex. Prihantoro, Influence of L1 and Spoken English on English Writing 229 Figure 9: Concordance 1: The Retrieval of <suggest> in Abstracts Collec- tion Figure 9: Concordance 1: The Retrieval of <suggest> in Abstracts Collec- tion Figure 9: Concordance 1: The Retrieval of <suggest> in Abstracts Collec- tion Concordance lines are presented on the left, and pattern variation is shown on the right. Although some other patterns are observed, but these two patterns were quite similar with respect to the nouns that they specify. The nouns that each pattern specifies is human noun, and some of them are replaced by pro- noun. Other patterns occur, but the two patterns dominated the use. In the re- search abstracts, <suggest>Pers.PRO><to><Vinf> is observed to occur more than 75%. The same, even higher, domination was also observed in free writ- ing: Figure 10: Concordance 2: The Retrieval of <suggest> in Free Writing Collection Figure 10: Concordance 2: The Retrieval of <suggest> in Free Writing Collection 230 TEFLIN Journal, Volume 27, Number 2, July 2016 MWU 1: Corpus Validations Instead of determining the correctness of particular structures, I navigated the corpus to discover how the structures are present in actual use. The result of the first pattern retrieval in COCA is shown in Figure 11. Figure 11: Concordance of <suggest><PRO><to><V> Figure 11 shows that the first pattern is in actual use, but very low in fre- quency. The figure indicates that they are used only one or twice. At this point, I then refined the retrieval by focusing the pronouns to personal pronouns (you, him, them, us and etc.): Figure 12: Concordance of <suggest><Pers.PRO><to><V> MWU 1: Corpus Validations Instead of determining the correctness of particular structures, I navigated the corpus to discover how the structures are present in actual use. The result of the first pattern retrieval in COCA is shown in Figure 11. Figure 11: Concordance of <suggest><PRO><to><V> Figure 11: Concordance of <suggest><PRO><to><V> Figure 11: Concordance of <suggest><PRO><to><V> Figure 11 shows that the first pattern is in actual use, but very low in fre- quency. The figure indicates that they are used only one or twice. At this point, I then refined the retrieval by focusing the pronouns to personal pronouns (you, him, them, us and etc.): Figure 12: Concordance of <suggest><Pers.PRO><to><V> Figure 12: Concordance of <suggest><Pers.PRO><to><V> Prihantoro, Influence of L1 and Spoken English on English Writing 231 231 The result of the retrieval indicated by Figure 12 showed that the first pat- tern is most frequently used in spoken English, and the frequency is very low (5). Further, let us consider how the second pattern <suggest><that>, is used: Figure 13: The Source Text of the Concordance <sug- gest><that><PRO><Vinf> Figure 13: The Source Text of the Concordance <sug- gest><that><PRO><Vinf> The frequency of this sequence is quite high in the corpus. The highest frequency of the concordance <suggest><that> is observed in the academic English section (1737). This indicates that the second pattern is widely used in written academic English, even though both are used. In addition, it is neces- sary to check the occurrence in another corpus. British National Corpus (BNC) is another corpus of English that are com- posed by different text types. In this corpus, the result of the retrieval also showed the same tendency. The pattern <suggest that> displayed 169 hits in academic section, while only 69 hits in spoken section. The retrieval of <sug- gest><PRO> in academic section resulted in zero hit, while it only had one hit in spoken section and one hit in magazine section. 232 TEFLIN Journal, Volume 27, Number 2, July 2016 Figure 14: <suggest that> VS <suggest><pro> in BNC Figure 14: <suggest that> VS <suggest><pro> in BNC MWU 1: Analysis Negative transfer may take place not only from first to second language, but also across domains, for instance from spoken to written language. Krauthamer (1999) refers to the interference patterns of spoken language as SLIP (Spoken Language Interference Pattern). It ranges from vocabulary to style. One of the means to help determine whether the writing task is influ- enced by spoken language is by evaluating its lexical density. For this purpose, I used AntConc vocabulary profiler (Anthony, 2006) to measure the density of academic words. The list of academic words (which contains the token and type) is obtained from Coxhead (2000). The list was then improved by Davies (2008) with the help of COCA. The profiling here excludes function words like conjunctions and prepositions. Table 5. Comparison of Vocabulary Profile: Scientific Writing and Free Writing Vocabulary Profile (Token of academic word list) Number Scientific Writing Tasks Free Writing More than 60% 4 0 Less than 60% 61 65 Table 5. Comparison of Vocabulary Profile: Scientific Writing and Free Writing Vocabulary Profile (Token of academic word list) Number Scientific Writing Tasks Free Writing More than 60% 4 0 Less than 60% 61 65 Table 5. Comparison of Vocabulary Profile: Scientific Writing and Free Prihantoro, Influence of L1 and Spoken English on English Writing 233 Kwary (2013) asserts that when the token percentage of the vocabulary profile is more than 60%, it is statistically significant. Table 5 shows the evalu- ation of scientific and free writing profile. It shows that the number of scien- tific writing tasks where the profile receives evaluation more than 60% is only 4, while the number of writing tasks that receives less than 60% is 61. One of the reasons is the use of words that are not included in the academic wordlist such as: well, anyway, I mean. This is in line with Šimčikaitė (2012) when identifying spoken language vocabularies in the writing task. The presence of these words reduces the academic vocabulary densities. Krauthamer’s (1999) SLIPs also documented the influence of spoken language stylistics. Nuruzi, Farahani, and Farahani (2012), in studying the stylistics of academic writing, suggests that students use nominalization feature in academic writing. MWU 2: Corpus Data The previous sub-section has described how two structures differ under the influence of the text type (spoken and academic). Both structures are cor- rect, but one is more frequently used in the written academic section (another is used more in spoken section). p ) There are some lemmas in which the word forms are more frequently used in spoken language but less in written language, or vice versa. As for the verb <help>, the structure without ‘to’ infinitive is less dominant than its counterpart in student’s writing corpus. Figure 15: Concordance 3: The Retrieval of <help> in Abstracts Figure 15: Concordance 3: The Retrieval of <help> in Abstracts Figure 15: Concordance 3: The Retrieval of <help> in Abstracts 234 TEFLIN Journal, Volume 27, Number 2, July 2016 Figure 16: Concordance 4: The Retrieval of <help> in Magazine/News Ar- ticle Figure 16: Concordance 4: The Retrieval of <help> in Magazine/News Ar- ticle Figure 16: Concordance 4: The Retrieval of <help> in Magazine/News Ar- ticle Figure 16: Concordance 4: The Retrieval of <help> in Magazine/News Ar- ticle Both concordances indicate two structures <help><to><Vinf> and <help><N><Vinf>. The first one is used more frequently in both abstracts and free writing article. But the second pattern is used in abstracts writing only. Whether there is a preference of one genre over the other still requires a corpus validation. MWU 2: Corpus Validation Moving on from MWU 1, this section presents BNC and COCA vali- dation. Consider Figure 17, which presents the concordance of <help><N><Vinf> by COCA: Figure 17: The Concordance of <help><N><Vinf> Moving on from MWU 1, this section presents BNC and COCA vali- dation. Consider Figure 17, which presents the concordance of <help><N><Vinf> by COCA: Figure 17: The Concordance of <help><N><Vinf> Prihantoro, Influence of L1 and Spoken English on English Writing 235 Prihantoro, Influence of L1 and Spoken English on English Writing 235 Figure 17 suggests that first, there is a steady rise from 1990 to 2012 on how the structure <help><N><Vinf> is used. Second, both structures are pre- sent in actual use. Third, this structure is most frequently used in academic set- ting (see the highest frequency shown in the academic section (4547)). Further, a validation is required to observe whether the same trend applies to the second pattern <help><N><to><Vinf>. The result of COCA retrieval of this pattern is shown in the following figure: Figure 18: The Concordance for <help><N><to><Vinf> Figure 18 suggests that the structure is present in actual language use. Se- cond, the structure is used in the academic setting, but is lower in frequency (672). Third, unlike <help><N><Vinf> that undergoes a steady rise since 1990, the use of <help><N><to><Vinf> seems to be consistently used over the past 22 years. However, the frequency of the first pattern (as compared to the se- cond one) is high. Hence, this <help><N><Vinf> sequence seems to be more preferable in academic writing. Further, the following Figure 19 shows the comparison of the two structures in BNC. Figure 18: The Concordance for <help><N><to><Vinf> Figure 18 suggests that the structure is present in actual language use. Se- cond, the structure is used in the academic setting, but is lower in frequency (672). Third, unlike <help><N><Vinf> that undergoes a steady rise since 1990, the use of <help><N><to><Vinf> seems to be consistently used over the past 22 years. However, the frequency of the first pattern (as compared to the se- cond one) is high. Hence, this <help><N><Vinf> sequence seems to be more preferable in academic writing. Further, the following Figure 19 shows the comparison of the two structures in BNC. 236 TEFLIN Journal, Volume 27, Number 2, July 2016 Figure 19: Help you to do VS Help you do in BNC In BNC we can see that the two structures are in use both in academic and spoken sections. The occurrence in spoken and academic section is almost equal for the first structure, while for the second structure, the occurrence is relatively lower in academic section than in spoken section. The second struc- ture differs in a way that there is a striking difference between non-academic (489) and academic writing (250). Figure 19: Help you to do VS Help you do in BNC Figure 19: Help you to do VS Help you do in BNC In BNC we can see that the two structures are in use both in academic and spoken sections. The occurrence in spoken and academic section is almost equal for the first structure, while for the second structure, the occurrence is relatively lower in academic section than in spoken section. The second struc- ture differs in a way that there is a striking difference between non-academic (489) and academic writing (250). Prihantoro, Influence of L1 and Spoken English on English Writing 237 Therefore, we can safely assume that domain shift is relevant case-per- case. Studies in terms of the difference of spoken/written English, such as Biber (2006), and Carter & McCarthy (2006), or academic/non-academic Eng- lish, like Allen (2009) and Annelie & Erman (2012), cannot be generalized as there are a number of lexis or structures applied in more than one domains. Even when we notice Coxhead’s (2000) academic wordlist, which was later re- fined by Davies (2014) in COCA, some words are treated equally in spo- ken/written domains, such as negative (S2/W2). COCA data however, provides an interesting historical finding that the use of the structure with ‘to’ steadily increase over years (see Figure 17), while the use of the structure without ‘to’ is constant despite remaining higher in frequency. MWU 2: Analysis Similarities are observed from corpus validation. First, corpora investiga- tion to both BNC or COCA suggests that both structures (with or without ‘to’ infinitive’ are present in actual use (see Figure 17-19). However, COCA and BNC differ in terms of domination in academic domain. A closer observation can help us understand how COCA data suggests that the use of the structure without to infinitive is more frequent (see Figure 17 and 18). BNC data, how- ever, is interesting as the frequency of the structure with ‘to’ infinitive is al- most equal in academic (226) and non-academic (296) domains. Significant difference, however, is observed for structure without ‘to’ (489-250). That can be seen in Figure 16. Prihantoro, Influence of L1 and Spoken English on English Writing 237 MWU 3: Corpus Data L1 seems to interfere when the vocabulary is similar to L2, not always on the lexis but it can also apply on the grammar. This happens to <goal>, in which its Indonesian equivalence is gol. Negative transfer from students’ L1 affect certain lemmas with similar surface forms as their Indonesian translation. Clear-cut distinction of the lemma <goal> is observed between the two tasks. In the abstracts collection, the lemma is used completely in the sense of goal as an aim or objective. This is because the semantic of <goal> in Indonesian (tujuan) is not of the same equivalence. This might be the reason why students preferred to use purpose and intention instead of goal to express the same con- cept. Table 6. Expressions for Aim and Score Sense Lemma (frequency) Scientific Writing Free Writing Aim purpose (51), aim (19) purpose (11), intention (7) score - goal (39) Another sense of <goal> may also refer to points scored by team players in a sport game. In this case, the equivalence of <goal> in Indonesian, goal, is similar in form. This is the sense that is also present and dominating in the free writing. It is interesting to observe some verbs that collocate with <goal> as it suggests the interference of L1. Number --1,2,3,4, refers to the left context <make, create, score, print> respectfully: 238 TEFLIN Journal, Volume 27, Number 2, July 2016 Figure 20: Concordance 5: The Retrieval of <goal> in Free Writing Figure 20: Concordance 5: The Retrieval of <goal> in Free Writing Figure 20: Concordance 5: The Retrieval of <goal> in Free Writing I listed four verbs that collocate with <goal>. The first one, and the highest in frequency is <make> (line 1, 7, 8 and 10), <create> (line 2), <score> line 6, and <print> (line 9). One pattern in line 7 is not valid as it is grammatically wrong (goal is used as a verb). MWU 3: Corpus Validation The pattern <V><goal> is interesting to research as students have the ten- dency to choose verbs under the influence of Indonesian collocation patterns. The results of pattern matching of <V><gol> from Indonesian corpus indicated the same result. There are four verbs in Indonesian that collocate with <gol>, which are: <mencetak>, <membuat>, <bikin> and <menciptakan>. In this re- spect, <membuat> and <bikin> are similar; these verbs can be translated literal- ly as the followings: ‘to print’, ‘to make’, ‘to make (informal), and ‘to create’, respectively. See Figure 21. p y g The first part of Figure 21 from SEAlang Indonesian corpus (Scannel, 2010) has shown that there are three verbs that collocate with <gol> in Indone- sian. The literal translation of the verb <score> in pattern 3 is not observed in this corpus. The three verbs that have been mentioned previously were often preferred by the students, but the strings resulted by the collocation patterns are odd; they are merely the concatenation of the literal translation of those verbs. In this case, the verb <score> is the perfect collocate to <score>. This hypothe- sis can be validated by retrieval on COCA as the standard corpus of English. See Figure 22. Prihantoro, Influence of L1 and Spoken English on English Writing 239 Prihantoro, Influence of L1 and Spoken English on English Writing 239 Figure 21: Comparison of verbs specifying <gol> in Indonesian Corpus Figure 22: Concordance < V><goal> Figure 22: Concordance < V><goal> 240 TEFLIN Journal, Volume 27, Number 2, July 2016 Figure 20 describes verbs that do not necessarily relate to <goal> in the senses of points earned. The top six verbs (set and setting are two tokens of the same verb) do not seem to be the verbs that suffice the sport-definition of goal. However, this corresponds to the frequency. Figure 23 explains that <goal>, as ‘aim/purpose’, is frequently used more in the genre of ‘academic’ as compared to others. Figure 23: The Distribution Chart of <V><goal> In order to understand what verbs collocate with <goal> in the sense of scores, I refined the search to magazine section (Figure 24) specifically to the sport page (Figure 25). MWU 3: Corpus Validation Figure 24: <V><goal> in Magazine Corpus Figure 23: The Distribution Chart of <V><goal> Figure 23: The Distribution Chart of <V><goal> In order to understand what verbs collocate with <goal> in the sense of scores, I refined the search to magazine section (Figure 24) specifically to the sport page (Figure 25). In order to understand what verbs collocate with <goal> in the sense of scores, I refined the search to magazine section (Figure 24) specifically to the sport page (Figure 25). Figure 24: <V><goal> in Magazine Corpus Figure 24: <V><goal> in Magazine Corpus Prihantoro, Influence of L1 and Spoken English on English Writing 241 Figure 25: <V><goal> in Sport Section of Magazine Corpus Figure 25: <V><goal> in Sport Section of Magazine Corpus This refined search seems to generate positive result. As we specify close- ly, we begin to understand that the pattern <score><goal> is the most frequent pattern, which refers to the sense that we are looking for under sport domain within magazine section. MWU 3: Analysis Corpus data and corpus validation verify my proposition that the prefer- ence of other verbs such as <creates, make> is under the interference of stu- dents’ L1, in this case Indonesian. It is also parallel to the findings of Husein and Mohammad (2012), Annelie and Erman (2012), and Arabski (2006) that concerned the negative impact of L1 to L2. Although this negative transfer is common, to some extent it is dangerous when the choice of structures and vocabulary are considered peculiar. For peculiar use of the lemmas <create, make>, the readers perhaps can still deduce the meaning contextually. Domain shift is a common phenomena of metaphor study (Deignan, 2006). However, using <print> as a collocate to <goal> in terms of sport does not make any sense in English as <print> is a creation of textual image. It is true that some domain shift in metaphors might be shared across languages (Deignan & Potter, 2004), but cetak <print> and gol <goal> is a common metaphor in Indonesian but not in English. The shift might confuse native speakers of English. CONCLUSIONS AND SUGGESTIONS This research concludes that 1) students involved in this research were more exposed to spoken English rather than written English; 2) degree of expo- sure, as well as the students’ L1, has a great influence on their preference over 242 TEFLIN Journal, Volume 27, Number 2, July 2016 certain structures; and 3) although the degree of exposure varies, teacher’s in- struction is still prioritized. Considering the importance of teachers’ instruction in this research, I rec- ommend that teachers, especially in colleague-level academic writing class, in- struct the students to consult different sources proportionally, and use the refer- ence appropriately in accordance to the aim of their writing. Students cannot just rely completely on the teachers, or undergraduate final projects. I also strongly suggest that various corpora be used in the classroom in order to show authentic evidence of how language is used academically. Teachers can navi- gate the corpora together with the students in the class. The students are also encouraged to share the result of corpus exploration with teachers and other students. At this stage, teachers can always facilitate them in improving corpus exploration techniques, correcting false conclusions, or suggesting that they use a more specific/general corpus to explore. Biber, D. (2006a). Stance in spoken and written university registers. 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https://openalex.org/W4233218205	https://zenodo.org/records/1549121/files/article.pdf	German	null	Berichtigung.	Zeitschrift für die alttestamentliche Wissenschaft	1,910	public-domain	601	Kittel, Ben Chajjim und Eb. Nestle. g g g g g Unter diesen Umständen liegt aller Grund vor, auch für den Penta- teuch die oben gebrauchte Bezeichnung „GlNSBURG-NESTLE ?" in dem oben näher beschriebenen Sinn beizubehalten, daß NESTLE, auch wo er etwa nicht an der Arbeit beteiligt ist (so hier), doch durch seine warme Empfehlung des Werkes (vgl. noch oben S. 233 Anm. 2) für seine Be- schaffenheit mitverantwortlich ist. Berichtigung. In mein als Beiheft XVIII zu dieser Zeitschrift erschienenes Buch Die j ü d i s c h e n Baptismen bitte "ich nachstehende Berichtigungen eintragen zu wollen: S. 44 Z. Jl von unten lies: de oratione. S. 47 Z. 7 von oben 1 , lies: H3tf6. S. 51 Z. I von unten: 3 ff., lies: 8 ff. S. 119 Z. 8f. von oben: nachdem . .. achtgegeben hat, lies: indem.. : achtgibt. _ "_" W. BRANDT. Kittel, Ben Chajjim und Eb. Nestle. Kittel, Ben Chajjim und Eb. Nestle. 239 dazu die Apostrophen in ZAW 1908, S. 149! Am Schlüsse dankt N. ausdrücklich nochmal für die ,,schönste Bibel mit einem Apparat, der in einer Richtung nicht seinesgleichen hat" g g Kennt unser schneidiger Kritiker ihre Schwächen nicht? Der obige Satz zeigt, daß in dem Mann mit der Keule — es ist die Keule der Wahrheit — doch auch ein heimlicher Diplomat steckt. Tatsächlich weiß N. wohl, daß GlNSBURGs Verheißungen in betreff der alten Versionen nichtig sind; weiß auch, daß ihre Zitation unwissenschaftlich ist — wehe mir, wenn ich einmal <S statt <5 E drucken ließ oder eine LA aus SWETE statt dem Kodex selbst entnahm, vgl. Sept. Studien V; Theol. Lit. Bl. 1909 Nr. 22 —; kann wenigstens wissen, daß HSS nicht mit dem Scheffel gemessen, sondern mit der Wage gewogen werden sollten; aber der Mantel der christlichen Liebe ist von herzerfreuender Weite. Tatsächlich konnte er ferner schon durch meine Prolegomena wissen, daß auch der Text des neuen GlNSBURG keine wirkliche Kollation von B darstellte; desgleichen ahnt er wohl, daß es auch mit dem Apparat gar nicht sauber sei. Aber wie ausgewechselt gegen seine sonstigen Gepflogenheiten sagt er (Exp. T. 3i5a unten = Qu. Rec. 43 Mitte), er wolle Vokale und Akzente beiseitelassen (!) und nennt ein einziges Beispiel eines Versehens aus dem Konsonantentext. Ja er entschuldigt sich auch da noch ausdrücklich, daß er sich um solche Kleinigkeiten kümmere (/ do not even care about these minutiae). Und das bei einer massoretischen Bibel, die sich rühmt, alle Vokale und Akzente aufs genaueste wiederzugeben und angesichts der Tat- sache, daß bei BHK in ZAW 1910, S. 153 selbst die geringfügigsten Ab- weichungen von 23, ja selbst solche, die in den Prolegomena begründet und sogar Druckfehler, die schon in den Corrigenda namhaft gemacht waren, zur Begründung^des Vonvurfs der Mietlingsarbeit herangezogen werden! Unter diesen Umständen liegt aller Grund vor, auch für den Penta- teuch die oben gebrauchte Bezeichnung „GlNSBURG-NESTLE ?" in dem oben näher beschriebenen Sinn beizubehalten, daß NESTLE, auch wo er etwa nicht an der Arbeit beteiligt ist (so hier), doch durch seine warme Empfehlung des Werkes (vgl. noch oben S. 233 Anm. 2) für seine Be- schaffenheit mitverantwortlich ist. Berichtigung. In mein als Beiheft XVIII zu dieser Zeitschrift erschienenes Buch Die j ü d i s c h e n Baptismen bitte "ich nachstehende Berichtigungen eintragen zu wollen: S. 44 Z. Jl von unten lies: de oratione. S. 47 Z. 7 von oben 1 , lies: H3tf6. S. 51 Z. I von unten: 3 ff., lies: 8 ff. S. 119 Z. 8f. von oben: nachdem . .. achtgegeben hat, lies: indem.. : achtgibt. _ "_" W. BRANDT. g g W. BRANDT.
https://openalex.org/W4206266207	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0219614&type=printable	English	null	Correction: Hypofractionated radiation therapy and temozolomide in patients with glioblastoma and poor prognostic factors. A prospective, single-institution experience	PloS one	2,019	cc-by	262	Correction: Hypofractionated radiation therapy and temozolomide in patients with glioblastoma and poor prognostic factors. A prospective, single-institution experience Paola Anna Jablonska, Ricardo Diez-Valle, Jaime Ga´llego Pe´rez-Larraya, Marta Moreno- Jime´nez, Miguel A´ ngel Idoate, Leire Arbea, Sonia Tejada, Maria Reyes Garcia de Eulate, Luis Ramos, Javier Arbizu, Pablo Domı´nguez, Jose´ Javier Aristu There is an error in the XML of the article. As a result, the third author’s name is listed in the citation incorrectly. The correct citation is: Jablonska PA, Diez-Valle R, Ga´llego Pe´rez-Larraya J, Moreno-Jime´nez M, Idoate MA´, Arbea L, et al. (2019) Hypofractionated radiation therapy and temozolomide in patients with glioblastoma and poor prognostic factors. A prospective, single-institution experience. PLoS ONE 14(6): e0217881. https://doi.org/10.1371/journal. pone.0217881 OPEN ACCESS Citation: Jablonska PA, Diez-Valle R, Ga´llego Pe´rez-Larraya J, Moreno-Jime´nez M, Idoate MA´, Arbea L, et al. (2019) Correction: Hypofractionated radiation therapy and temozolomide in patients with glioblastoma and poor prognostic factors. A prospective, single-institution experience. PLoS ONE 14(7): e0219614. https://doi.org/10.1371/ journal.pone.0219614 1. Jablonska PA, Diez-Valle R, Pe´rez-Larraya JG, Moreno-Jime´nez M, Idoate MA´ , Arbea L, et al. (2019) Hypofractionated radiation therapy and temozolomide in patients with glioblastoma and poor prognostic factors. A prospective, single-institution experience. PLoS ONE 14(6): e0217881. https://doi.org/10. 1371/journal.pone.0217881 PMID: 31170245 Reference a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Published: July 8, 2019 Copyright: © 2019 Jablonska et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 1 / 1 PLOS ONE \| https://doi.org/10.1371/journal.pone.0219614 July 8, 2019
https://openalex.org/W2769540542	https://www.mdpi.com/2073-4352/7/12/359/pdf?version=1512385293	English	null	Structurally Complex Frank–Kasper Phases and Quasicrystal Approximants: Electronic Origin of Stability	Crystals	2,017	cc-by	6,280	Valentina F. Degtyareva * and Natalia S. Afonikova Valentina F. Degtyareva * and Natalia S. Afonikova Valentina F. Degtyareva * and Natalia S. Afonikova Institute of Solid State Physics, Russian Academy of Sciences, Chernogolovka 142432, Russia; natasha@issp.ac.ru * Correspondence: degtyar@issp.ac.ru Academic Editor: Dmitry A. Shulyatev Received: 11 November 2017; Accepted: 1 December 2017; Published: 4 December 2017 Valentina F. Degtyareva and Natalia S. Afonikova Institute of Solid State Physics, Russian Academy of Sciences, Chernogolovka 142432, Russia; natasha@issp.ac.ru * Correspondence: degtyar@issp.ac.ru Academic Editor: Dmitry A. Shulyatev Received: 11 November 2017; Accepted: 1 December 2017; Published: 4 December 2017 Institute of Solid State Physics, Russian Academy of Sciences, Chernogolovka 142432, Russia; natasha@issp.a * Correspondence: degtyar@issp.ac.ru Academic Editor: Dmitry A. Shulyatev Received: 11 November 2017; Accepted: 1 December 2017; Published: 4 December 2017 Abstract: Metal crystals with tetrahedral packing are known as Frank–Kasper phases, with large unit cells with the number of atoms numbering from hundreds to thousands. The main factors of the formation and stability of these phases are the atomic size ratio and the number of valence electrons per atom. The significance of the electronic energy contribution is analyzed within the Fermi sphere–Brillouin zone interaction model for several typical examples: Cu4Cd3, Mg2Al3 with over a thousand atoms per cell, and for icosahedral quasicrystal approximants with 146–168 atoms per cell. Our analysis shows that to minimize the crystal energy, it is important that the Fermi sphere (FS) is in contact with the Brillouin zones that are related to the strong diffraction peaks: the zones either inscribe the FS or are circumscribed by the FS creating contact at edges or vertices. Keywords: Frank-Kasper phases; icosahedral quasicrystal approximants; Hume-Rothery electron concentration rule crystals crystals crystals crystals www.mdpi.com/journal/crystals 2. Theoretical Background and Method of Analysis Formation of binary compounds at a certain alloy composition is deﬁned by some important factors characterizing the alloy constituents, such as the difference in atomic sizes, electronegativity etc. Beyond these factors, formation of metallic structures is deﬁned by effects of the Fermi sphere–Brillouin zone (FS–BZ) interaction. The Hume-Rothery mechanism has been identiﬁed to play a role in the stability of structurally-complex alloy phases, quasicrystals, and their approximants [2,13–16]. Formation of the complex structures of elemental metals under pressure can also be related to the Hume-Rothery mechanism [17–19]. Phase diagrams of binary alloys, such as Au-Cd represent several phases with complex structures that follow the Hume-Rothery rule of stability at certain electron concentrations [20]. The band structure contribution to the crystal structure energy can be estimated by analyzing conﬁgurations of Brillouin-Jones zone planes within the nearly free-electron model. A special program, BRIZ, has been developed [21] to construct FS–BZ conﬁgurations and to estimate some parameters, such as the Fermi sphere radius (kF), values of reciprocal wave vectors of BZ planes (qhkl), and volumes of BZ and FS. The BZ planes are selected to match the condition qhkl ≈2kF, which has a signiﬁcant structure factor. In this case an energy gap is opened on the BZ plane leading to the lowering of the electron band energy. The ratio of 1 2qhkl to kF is usually less than 1, and equal to ~0.95; it is called a “truncation” factor. In the FS–BZ presentations by the BRIZ program the BZ planes cross the FS whereas, in the real system, the Fermi sphere is deformed and accommodated inside the BZ due to an increase of the electron density and to a decrease in the electron energy near the BZ plane. The crystal structure of a phase chosen for the analysis by the BRIZ program is characterized by the lattice parameters and the number of atoms in the unit cell, which deﬁne the average atomic volume (Vat). The valence electron concentration (z) is the average number of valence electrons per atom that gives the value of the Fermi sphere radius kF = (3π2z/Vat)1/3. Further structure characterization parameters are the number of BZ planes that are in contact with the FS, the degree of the “truncation” factor, and the value of BZ ﬁlling by electronic states, deﬁned as a ratio of the volumes of FS and BZ. Crystals 2017, 7, 359 Crystals 2017, 7, 359 Classical Hume-Rothery phases with fcc→bcc→complex γ-brass→hcp are deﬁned by the number of valence electrons per atom, such as 1.35→1.5→1.62→1.75 [10]. In the case of quasicrystal approximants, these values are usually higher. Diffraction patterns of these compounds consist of several strong diffraction peaks that should be taken into account when considering the stability of their crystal structures. Classical Hume-Rothery phases with fcc→bcc→complex γ-brass→hcp are deﬁned by the number of valence electrons per atom, such as 1.35→1.5→1.62→1.75 [10]. In the case of quasicrystal approximants, these values are usually higher. Diffraction patterns of these compounds consist of several strong diffraction peaks that should be taken into account when considering the stability of their crystal structures. In the present paper the model of the Fermi-sphere–Brillouin zone (FS–BZ) interaction is applied to complex structures, considering the FS inscribed into the BZ, as well as the FS enveloped inner zones contacting the edges or vertices. Both cases of FS–BZ conﬁguration should affect the band-structure energy and decrease the crystal energy. 1. Introduction Common metallic structures are based on high-symmetry atomic cells, such as face-centered cubic (fcc), close-packed hexagonal (hcp), and body-centered cubic (bcc), that have a coordination number of either 12 or 8+6. These structures are found in elements in their metallic phases; they are also formed in binary alloys and compounds if constituent elements have small differences in atomic size and electronegativity. A special family of intermetallic alloys is based on tetrahedrally-packed phases, called Frank-Kasper phases, where tetrahedra form polyhedra with coordination numbers of 12 (icosahedron), 14, 15, and 16 [1]. Frank-Kasper polyhedra are basic structural units for many types of metallic alloys, including very complex structures with the number of atoms in the unit cell up to hundreds or thousands, as well as quasicrystals. Structures and properties of complex metallic alloys are considered in several books and review papers [2–7]. Crystal structures of very complex intermetallics are built with successive shells of atoms, including polyhedra, like icosahedrons, dodecahedrons, rhombic triacontahedrons, and truncated icosahedrons (soccer ball or “fullercage” [8]). These atomic arrangements are called “Russian doll” (“matryoshka”) clusters [9]. Similar atomic packings exist in icosahedral quasicrystals, known as Mackay-type, Bergman-type, and Tsai-type. These clusters build the structures of quasicrystal approximants with the periodic arrangements in the bcc cell (1/1 type) or more complex periodic structures. Quasicrystal and approximant phases exist at certain alloy compositions usually deﬁned by valence electron concentrations and represent a kind of Hume-Rothery phases [2–5]. The crystal energy is lowered by contact of the Fermi level to the Brillouin zone planes formed by strong diffraction peaks. Historically, Hume-Rothery phases were considered ﬁrst in the Cu-Zn alloys and related binary systems of Cu-group elements with the neighboring elements of higher valences [10–12]. Crystals 2017, 7, 359; doi:10.3390/cryst7120359 www.mdpi.com/journal/crystals 2 of 10 3. Results and Discussion In this work, two groups of complex intermetallic phases are selected: with very large unit cells containing above a thousand atoms, and quasicrystal 1/1 approximants of the Bergman-type and Tsai-type. For our consideration we selected compounds with s and sp valence electron elements (non-transition elements) to allow us to calculate deﬁnitely the valence electron concentration. Structural data for compounds are given in Table 1. X-ray diffraction patterns and constructed Brillouin-Jones zones for these structures are presented in Figures 1–3. Crystal structure descriptions are given following the Pearson notation [30]. Table 1. Structure parameters of several metallic compounds with giant unit cells and quasicrystal approximants. Fermi sphere radius kF, ratios of kF to the distances of the Brillouin zone planes 1 2qhkl, and the ﬁlling degree of the Brillouin zones by electron states VFS /VBZ are calculated by the program BRIZ [21]. Phase Cu4Cd3 Mg28Al45 CaCd6 Al30Mg40Zn30 Al5CuLi3 Au15Cd23Zn11 Pearson symbol cF1124 cF1168 cI168 cI162 cI160 cI146 Structural data Space group F43m Fd3m Im3 Im3 Im3 Im3 Lattice parameters (Å) a = 25.871 a = 28.24 a = 15.680 a = 14.355 a =13.891 a = 13.843 Vat. (Å3) 15.67 19.28 22.95 18.26 16.75 18.17 References [31] [32,33] [34,35] [36,37] [38,39] [14] FS–BZ data from the BRIZ program z (number of valence electrons per atom) 1.43 2.62 2 2.3 2.18 1.69 kF (Å−1) 1.401 1.590 1.372 1.551 1.568 1.403 Total number BZ planes 84 96 96 84 96 42 hkl: kF/( 1 2qhkl) (880):1.020 (955):1.008 (10.44):1.004 (11.33):0.979 (14.20):1.011 (10.86) (10.10.2):1.001 (631):1.010 (543):0.968 (701) (550) (543):1.002 (701) (550) (631):1.022 (543):0.988 (710) (503):1.060 (600):1.030 (532):1.003 VFS / VBZ 0.955 0.966 0.950 0.945 0.936 1.00 3.1. Giant Unit Cell Compounds Cu4Cd3 and Mg2Al3 3.1. Giant Unit Cell Compounds Cu4Cd3 and Mg2Al3 Complex compounds with more than 1000 atoms in the unit cell were found in Cu4Cd3 and in Mg2Al3 by Samson [31,32]. Interestingly, the Cu4Cd3 compound differs considerably from isoelectronic phases in neighboring systems of Cu-Zn and Au-Cd that have, in this composition, either bcc or ordered CsCl-type structures [20]. The decisive factor for this difference is that the atomic size ratio does not exceed 10% for Cu/Zn or Au/Cd, but is ∼20% for Cu/Cd. Therefore, in Cu4Cd3, instead of the simple bcc or CsCl structures, a tetrahedrally-packed complex compound is formed. The diffraction pattern of Cu4Cd3-cF1124 consists of diffraction peaks with relatively strong intensity grouped near the 2kF position (Figure 1a). Crystals 2017, 7, 359 Crystals 2017, 7, 359 1/1 approximants. The electrostatic energy of ion-ion interactions is usually deﬁned by sums in the real and the reciprocal space providing quick convergence [26–28]. In this relation, the conﬁguration of BZs formed by strong reﬂections and accommodated inside the FS is responsible for the contribution to the Ewald energy and usually prefers high-symmetry polyhedra. The oC16 structure observed in some metals and binary alloys under pressure should be mentioned as an example [29]. 1/1 approximants. The electrostatic energy of ion-ion interactions is usually deﬁned by sums in the real and the reciprocal space providing quick convergence [26–28]. In this relation, the conﬁguration of BZs formed by strong reﬂections and accommodated inside the FS is responsible for the contribution to the Ewald energy and usually prefers high-symmetry polyhedra. The oC16 structure observed in some metals and binary alloys under pressure should be mentioned as an example [29]. 2. Theoretical Background and Method of Analysis Presentations of the FS–BZ conﬁgurations are given with the orthogonal axes with the following directions in the common view: a* is looking forward, b* to the right and c* upward. The FS–BZ interaction for relatively simple structures, such as Cu-Zn phases, is usually described as a contact of the FS to the BZ planes resulting in the decrease of the electronic energy by the formation of the energy gap. In the case of complex structures, diffraction patterns consist of several strong reﬂections at wave vectors well below 2kF, forming BZs that are inscribed by the FS with contacts at the edges or vertices. These cases are constructed with the BRIZ program by proper choice of presentations: “sphere inside polyhedron” or “polyhedron inside sphere”. Structural stabilization due to the electronic band contribution where FS is enveloping the BZ was discussed for phase stability in In and Sn alloys [22–24] and for fcc-based phases in simple metals [25]. Signiﬁcant arguments for the stability of tetrahedrally-packed structures are related to electrostatic (Ewald or Madelung) contributions to the crystal energy because of the high packing density of atoms in Frank-Kasper polyhedra. These polyhedra are forming interpenetrating building blocks that are packed in the long-range structure with a high symmetry, as, for example, in bcc in the case of 3 of 10 3. Results and Discussion Below the diffraction pattern, FS–BZ constructions are shown for separate peaks and for the ﬁnal BZ, including 84 planes in close contact to the FS (lower-right). This consideration conﬁrms the inﬂuential role of the Hume-Rothery mechanism for the stability of a complex compound along with an atomic size factor. A similar approach that considers Brillouin zones in the discussion of tetrahedrally-packed structures is given in [14]. It is necessary to note that, for the Cu4Cd3 compound, the same group of diffraction peaks were selected for the BZ construction (see [14], Figure 40), however, the BZ form is slightly different from the construction made with the BRIZ program more accurate, as given in Figure 1a (lower-right). 4 of 10 Crystals 2017, 7, 359 intensity construc t th F mechanism for the stability of a complex compound along with an atomic size factor. (a) (b) Figure 1. Calculated diffraction patterns for complex intermetallic phases (upper panel) and corresponding Brillouin-Jones zones with the Fermi spheres (lower panel). The position of 2kF for a given valence electron number per atom z and the hkl indices of the planes used for the BZ construction are indicated on the diffraction patterns. (a) Cu4Cd3-cF1124; and (b) Mg28Al45-cF1168. Structural data and FS–BZ evaluation data are given in Table 1. A similar approach that considers Brillouin zones in the discussion of tetrahedrally-packed t t i i i [14] It i t t th t f th C Cd d th f Figure 1. Calculated diffraction patterns for complex intermetallic phases (upper panel) and corresponding Brillouin-Jones zones with the Fermi spheres (lower panel). The position of 2kF for a given valence electron number per atom z and the hkl indices of the planes used for the BZ construction are indicated on the diffraction patterns. (a) Cu4Cd3-cF1124; and (b) Mg28Al45-cF1168. Structural data and FS–BZ evaluation data are given in Table 1. (a) (b) (b) Figure 1. Calculated diffraction patterns for complex intermetallic phases (upper panel) and corresponding Brillouin-Jones zones with the Fermi spheres (lower panel). The position of 2kF for a given valence electron number per atom z and the hkl indices of the planes used for the BZ construction are indicated on the diffraction patterns. (a) Cu4Cd3-cF1124; and (b) Mg28Al45-cF1168. Structural data and FS–BZ evaluation data are given in Table 1. A similar approach that considers Brillouin zones in the discussion of tetrahedrally-packed Figure 1. 3. Results and Discussion Calculated diffraction patterns for complex intermetallic phases (upper panel) and corresponding Brillouin-Jones zones with the Fermi spheres (lower panel). The position of 2kF for a given valence electron number per atom z and the hkl indices of the planes used for the BZ construction are indicated on the diffraction patterns. (a) Cu4Cd3-cF1124; and (b) Mg28Al45-cF1168. Structural data and FS–BZ evaluation data are given in Table 1. g y p g p diffraction peaks were selected for the BZ construction (see [14], Figure 40), however, the BZ form is The complex compound Mg2Al3 has a similar structure, with 1168 atoms in the unit cell, as was deﬁned by Samson [32] and re-determined recently [33]. Structural data for this phase are given in Table 1 designated as Mg28Al45 following the Pauling ﬁle data [40]. The diffraction pattern for this phase (Figure 1b) is similar to that for Cu4Cd3 (Figure 1a) with the same group of strong reﬂections. However, for Mg28Al45 the position of 2kF for z = 2.62 falls well above the position of strong reﬂections. This case was discussed by Dubois with the suggestion that, for this compound, “the Fermi sphere overlaps the Jones zone” (see [7], Figure 4). Overlapping Brillouin-Jones zones by the FS for strong reﬂections are shown in Figure 1b (lower-left, middle). Near the position of 2kF there is a group of reﬂections forming BZ planes in contact with the inscribed FS (lower-right). 3.2.1. Approximants cI168 and cI162 Diffraction patterns for QCs-approximants CaCd6-cI168 and Al30Mg40Zn30-cI162 and FS–BZ conﬁgurations are shown in Figure 2. Both phases have similar groups of strong reﬂections, whereas the 2kF positions are different and are close to the diffraction peak (6 3 1) for z = 2 and to the diffraction peaks (5 4 3), (7 1 0), (5 5 0), for z = 2.3 (Figure 2a,b, respectively). Strong reﬂections at lower wave vector values form highly-symmetrical polyhedra that are enveloped by the FS, creating a contact at the edges of the polyhedron with planes (5 0 3) or vertices of the polyhedron with planes (5 3 2), (6 0 0). Due to space group symmetry Im3 there is a difference in the intensity for reﬂections within the same (hkl) set. For example, the structure CaCd6-cI168 results in an intensity ratio of ~6 for reﬂections (5 0 3) and (5 3 0). Assuming that only the (5 0 3) reﬂections contribute to the constructions of the BZ, the resulting polyhedron is of the pentagonal dodecahedron type (Figure 2a, lower-left). The BZ polyhedron for (5 3 2) and (6 0 0) reﬂections represents a rhombic triacontahedron (Figure 2a, lower-middle). Both kinds of polyhedra are inscribed into the FS and with contacts at the vertices. Thus, the polyhedra in reciprocal space are related to polyhedra in real space formed by atom clusters. It should be noted that the BZ that is in contact with the inscribed FS for CaCd6 consists mainly of (6 3 1) and (3 6 1) with nearly the same intensity (Figure 2a, lower-right). For the approximant Al30Mg40Zn30–cI162 the next diffraction peak with indices (5 4 3), (7 1 0), and (5 5 0) forms the BZ planes in essential contact to the FS, as shown in Figure 2b (lower-right). There are 84 facets in the BZ contacting the FS at the same distance. The ﬁlling of the BZ by electron states is 0.945 (see Table 1) which satisﬁes the Hume-Rothery rule well. The same BZ-FS conﬁguration for the Al30Mg40Zn30–approximant was considered in Figure 1 [37] with discussion of matching 2kF to the wave vectors of reﬂections (5 4 3), (7 1 0), and (5 5 0). 3.2. Quasicrystal Approximants Interesting groups of tetrahedrally-packed phases represent quasicrystal approximants of the 1/1-type and other types. Some of these phases have been known long before the discovery of quasicrystals: for example, Mg32(Zn,Al)49 and CaCd6 [34,36], and later they were assigned as approximants of icosahedral quasicrystals (QCs). Diffraction patterns of QCs and their approximants have strong reﬂections at nearly the same positions and both groups of phases exist at very close alloy 5 of 10 Crystals 2017, 7, 359 compositions that can be estimated as valence electron concentrations (z). It is commonly assumed that QCs and their approximants are stabilized by the Hume-Rothery mechanism [2–5] and exist in regions of z equal to ∼1.8, 2, and ∼2.2–2.3. For those z values, representative QCs-approximants are considered with the structural data listed in Table 1. Al30Mg40Zn30-cI162 and Al5CuL b l i h B 3.2.2. Approximants cI160 and cI146 belonging to the Bergman-type approximants. Diffraction patterns for both phases are similar, as can be seen from Figures 2b and 3a. The only difference is in the 2kF position: for Al5CuLi3 it is close to the reflection (6 3 1), while for Al30Mg40Zn30 it is close to reflections (5 4 3), (7 1 0), and (5 5 0), as shown in Figures 2b and 3a (lower-right), respectively. It should be noted that, because of space group Im3, the intensity of the (6 1 3) reflection is ~100 times less than that of (6 3 1) and the only (6 3 1) set is participating in the BZ construction. For Al5CuLi3, the BZ filling by electron state is 0.936, matching the Hume-Rothery criteria. In addition to this FS–BZ plane contact, it is also necessary to consider the FS overlap with the inner zones, such as the (5 0 3)-dodecahedron and rhombic Al30Mg40Zn30-cI162 and Al5CuLi3-cI160 have much similarity in their crystal structures, both belonging to the Bergman-type approximants. Diffraction patterns for both phases are similar, as can be seen from Figures 2b and 3a. The only difference is in the 2kF position: for Al5CuLi3 it is close to the reﬂection (6 3 1), while for Al30Mg40Zn30 it is close to reﬂections (5 4 3), (7 1 0), and (5 5 0), as shown in Figures 2b and 3a (lower-right), respectively. It should be noted that, because of space group Im3, the intensity of the (6 1 3) reﬂection is ~100 times less than that of (6 3 1) and the only (6 3 1) set is participating in the BZ construction. For Al5CuLi3, the BZ ﬁlling by electron state is 0.936, matching the Hume-Rothery criteria. In addition to this FS–BZ plane contact, it is also necessary to consider the FS overlap with the inner zones, such as the (5 0 3)-dodecahedron and rhombic triacontahedron formed by (5 3 2) and (6 0 0) planes, which should give a signiﬁcant contribution to the reduction of the electronic energy. Our next example of approximants—the phase Au15Cd23Zn11-cI146 was recently found [14] with the valence electron number z = 1.694. For this z value, the 2kF position is close to (5 3 2) and slightly overlaps the (5 0 3) and (6 0 0) reﬂections, as shown in Figure 3b. The resulting BZ (Figure 3b, lower-right) is completely ﬁlled by electron states. 3.2.1. Approximants cI168 and cI162 In addition to this, it is also necessary to consider the BZs constructed with the reﬂections (5 0 3) and (5 3 2)/(6 0 0) that are enveloped by the FS, as shown in Figure 2b (lower-left and middle). Crystals 2017, 7, 359 6 of 10 (a) Figure 2. Cont. (a) Figure 2. Cont. 6 of 10 Crystals 2017, 7, 359 (a) (b) Figure 2. Calculated diffraction patterns for quasicrystal approximants (upper panel) and corresponding Brillouin-Jones zones with the Fermi spheres (lower panel). The position of 2kF for a given valence electron number per atom z and the hkl indices of the planes used for the BZ construction are indicated on the diffraction patterns. (a) CaCd6-cI168; and (b) Al30Mg40Zn30-cI162. Structural data and FS–BZ evaluation data are given in Table 1. 3.2.2. Approximants cI160 and cI146 Figure 2. Calculated diffraction patterns for quasicrystal approximants (upper panel) and corresponding Brillouin-Jones zones with the Fermi spheres (lower panel). The position of 2kF for a given valence electron number per atom z and the hkl indices of the planes used for the BZ construction are indicated on the diffraction patterns. (a) CaCd6-cI168; and (b) Al30Mg40Zn30-cI162. Structural data and FS–BZ evaluation data are given in Table 1. ( ) (b) (b) Figure 2. Calculated diffraction patterns for quasicrystal approximants (upper panel) and corresponding Brillouin-Jones zones with the Fermi spheres (lower panel). The position of 2kF for a given valence electron number per atom z and the hkl indices of the planes used for the BZ construction are indicated on the diffraction patterns. (a) CaCd6-cI168; and (b) Al30Mg40Zn30-cI162. Structural data and FS–BZ evaluation data are given in Table 1. 3 2 2 Approximants cI160 and cI146 Figure 2. Calculated diffraction patterns for quasicrystal approximants (upper panel) and corresponding Brillouin-Jones zones with the Fermi spheres (lower panel). The position of 2kF for a given valence electron number per atom z and the hkl indices of the planes used for the BZ construction are indicated on the diffraction patterns. (a) CaCd6-cI168; and (b) Al30Mg40Zn30-cI162. Structural data and FS–BZ evaluation data are given in Table 1. Al30Mg40Zn30-cI162 and Al5CuL b l i h B 3.2.2. Approximants cI160 and cI146 It should be noted that BZ construction for this structure was reported (see [14], Figure 34), however, the BRIZ program gives a more accurate construction. Interestingly, for this phase with z ≈1.7 there are no other diffraction peaks with comparably high intensity, in contrast to the phases with higher z values. 7 of 10 Crystals 2017, 7, 359 g structure construct p y g y, p g (a) (b) Figure 3. Calculated diffraction patterns for quasicrystal approximants (upper panel) and corresponding Brillouin-Jones zones with the Fermi spheres (lower panel). The position of 2kF for a given valence electron number per atom z and the hkl indices of the planes used for the BZ construction are indicated on the diffraction patterns. (a) Al5CuLi3-cI160; and (b) Au15Cd23Zn11-cI146. Structural data and FS–BZ evaluation data are given in Table 1. 3.3. Similar Structural Features of Intermetallics with Giant Unit Cells and Approximants Figure 3. Calculated diffraction patterns for quasicrystal approximants (upper panel) and corresponding Brillouin-Jones zones with the Fermi spheres (lower panel). The position of 2kF for a given valence electron number per atom z and the hkl indices of the planes used for the BZ construction are indicated on the diffraction patterns. (a) Al5CuLi3-cI160; and (b) Au15Cd23Zn11-cI146. Structural data and FS–BZ evaluation data are given in Table 1. (b) d diff i f i l i ( (b) Figure 3. Calculated diffraction patterns for quasicrystal approximants (upper panel) and corresponding Brillouin-Jones zones with the Fermi spheres (lower panel). The position of 2kF for a given valence electron number per atom z and the hkl indices of the planes used for the BZ construction are indicated on the diffraction patterns. (a) Al5CuLi3-cI160; and (b) Au15Cd23Zn11-cI146. Structural data and FS–BZ evaluation data are given in Table 1. 3.3. Similar Structural Features of Intermetallics with Giant Unit Cells and Approximants Figure 3. Calculated diffraction patterns for quasicrystal approximants (upper panel) and corresponding Brillouin-Jones zones with the Fermi spheres (lower panel). The position of 2kF for a given valence electron number per atom z and the hkl indices of the planes used for the BZ construction are indicated on the diffraction patterns. (a) Al5CuLi3-cI160; and (b) Au15Cd23Zn11-cI146. Structural data and FS–BZ evaluation data are given in Table 1. 3.3. Similar Structural Features of Intermetallics with Giant Unit Cells and Approximants 3.3. Similar Structural Features of Intermetallics with Giant Unit Cells and Approximants 3.3. Similar Structural Features of Intermetallics with Giant Unit Cells and Approximants The complex intermetallic phases discussed in the present paper have similar local atomic arrangement based on tetrahedrally-packed Frank-Kasper polyhedra. Relations of atomic configurations organized in different structural types lead to relations in the form of Brillouin-Jones zones. Examples of such similarity provide the BZ constructions for Mg28Al45-cI1168 and Al30Mg40Zn30-cI162 shown in Figures 1b and 2b (lower-right). Diffraction plain sets for BZ construction contacting the FS are as follows: Mg28Al45 (10 8 6) (14 2 0) (10 10 2) Al30Mg40Zn30 (5 4 3) (7 1 0) (5 5 0) Mg28Al45 (10 8 6) (14 2 0) (10 10 2) Al30Mg40Zn30 (5 4 3) (7 1 0) (5 5 0) From the comparison of these sets, it can be concluded that the former cell is related to the latter cell as a 2 ×2 × 2 lattice parameter. In this case the number of atoms would be 162 × 8 = 1296, which can be reduced to 1168 by vacancies. It should be noted that, for Mg28Al45, a reﬂection (10 10 0) expected from this transformation also exists, but has very weak intensity, and for the construction of the BZ, the next reﬂection (10 10 2) was taken. Formation of a complex supercell by multiplication of 8 of 10 Crystals 2017, 7, 359 lattice parameters was demonstrated by a classical example of γ-brass phase Cu5Zn8. The structure cI52 is formed from bcc with the 2 × 2 × 2 increase of the lattice parameter and an induction of vacancies with some atomic movement that results in an appearance of additional peaks close to the FS, as was discussed by Jones [12]. Interestingly, the forms of the Brillouin zones with faces (8 8 0) and (11 3 3) for Cu4Cd3 and Mg28Al45 are similar (Figure 1) and related to the BZ for γ-brass with faces (3 3 0) and (4 1 1), which indicates the structural relationship between all these compounds. Structural presentations of complex intermetallics as multi-fold supercells of basic metallic close-packed lattices were considered in [25,41]. Structural relations of alloy phases that are built with similar short-range clusters are visible by comparison of BZs related to strong diffraction peaks. 3.3. Similar Structural Features of Intermetallics with Giant Unit Cells and Approximants For Al5CuLi3-cI160 the outer BZ with faces (5 0 3), (6 0 0), and (5 3 2), shown in Figure 3b (lower-right), represents a polyhedron that looks like the Brillouin-Jones zone of quasicrystals i-AlMgCu and i-AlLiCu constructed from the ﬁrst strong peaks [6]. It should be noted, that BZs with this type of polyhedron with faces (5 0 3), (6 0 0), and (5 3 2) exist for all approximants discussed where the only difference is in the location either outside or inside the FS. In both cases the FS–BZ interaction is signiﬁcant for the crystal structure energy. 4. Conclusions Structurally-complex alloy phases with tetrahedrally-packed polyhedra of Frank-Kasper type are discussed with reference to the free-electron model of Brillouin zone-Fermi sphere interactions. Electron energy reduction originates through the contact of the FS to the BZ planes, as usually assumed for classical Hume-Rothery phases with relatively simple structures. On their diffraction patterns, complex structures display several peaks of strong intensity that produce Brillouin zone polyhedra enveloped by the FS in contact with some edges or vertices. These FS–BZ conﬁgurations should be taken into account for the estimation of electron energy as a convincing reason for stability of such complex intermetallic structures. Complex tetrahedrally-packed intermetallic phases consist of successive atomic building blocks arranged as symmetrical polyhedra. The Brillouin-zone constructions for these structures reveal successive BZ polyhedra with nearly the same form as the atomic clusters in the real space. These effects should be regarded by the theoretical considerations. Acknowledgments: The authors gratefully acknowledge Dr. Olga Degtyareva for valuable discussions and comments. 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https://openalex.org/W4286486466	https://www.aanda.org/articles/aa/pdf/2022/10/aa43862-22.pdf	English	null	Orbital and dynamical analysis of the system around HR 8799	Astronomy & astrophysics	2,022	cc-by	25,120	n Access article, published by EDP Sciences, under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. This article is published in open access under the Subscribe-to-Open model. Subscribe to A&A to support open access publication. ABSTRACT From the photometric measurements obtained in different wavelengths, we estimated the masses of the planets following the evolutionary models. Results. We obtained updated parameters for the orbits with the assumption of coplanarity, relatively small eccentricities, and periods very close to the 2:1 resonance. We also refined the dynamical mass of each planet and the parallax of the system (24.49 ± 0.07 mas), which overlap with the recent Gaia eDR3/DR3 estimate. Hydrodynamical simulations suggest that inward migration of the planets caused by the interaction with the disk might be responsible for the planets being locked in resonance. We also conducted detailed N-body simulations indicating possible positions of a putative fifth planet with a mass below the present detection limits of ≃3 MJup. Key words. planets and satellites: dynamical evolution and stability – planet-disk interactions – stars: individual: HR8799 – instrumentation: adaptive optics – astrometry – techniques: image processing Hinz et al. 2010; Zuckerman et al. 2011; Bell et al. 2015), γ Dor-type variable star (Gray & Kaye 1999) with λ Boo-like abun- dance patterns. The mass of the star is 1.47+0.12 −0.17 M⊙(Sepulveda & Bowler 2022) and its distance is 40.88 ± 0.08 pc from Gaia measurements (Gaia Collaboration 2020). ABSTRACT Being the first multi-planetary system discovered with the direct imaging technique, it has been observed extensively since 1998. This wide baseline of astrometric measurements, counting over 50 observations in 20 years, permits a detailed orbital and dynamical analysis of the system. Aims. To explore the orbital parameters of the planets, their dynamical history, and the planet-to-disk interaction, we made follow-up observations of the system during the VLT/SPHERE guaranteed time observation program. We obtained 21 observations, most of them in favorable conditions. In addition, we observed HR 8799 with the instrument LUCI at the Large Binocular Telescope (LBT). Methods. All the observations were reduced with state-of-the-art algorithms implemented to apply the spectral and angular differential imaging method. We re-reduced the SPHERE data obtained during the commissioning of the instrument and in three open-time programs to have homogeneous astrometry. The precise position of the four planets with respect to the host star was calculated by exploiting the fake negative companions method. We obtained an astrometric precision of the order of 6 mas in the worst case and 1 mas in the best case. To improve the orbital fitting, we also took into account all of the astrometric data available in the literature. From the photometric measurements obtained in different wavelengths, we estimated the masses of the planets following the evolutionary models. g y p y y y Aims. To explore the orbital parameters of the planets, their dynamical history, and the planet-to-disk interaction, we made follow-up observations of the system during the VLT/SPHERE guaranteed time observation program. We obtained 21 observations, most of them in favorable conditions. In addition, we observed HR 8799 with the instrument LUCI at the Large Binocular Telescope (LBT). Methods. All the observations were reduced with state-of-the-art algorithms implemented to apply the spectral and angular differential imaging method. We re-reduced the SPHERE data obtained during the commissioning of the instrument and in three open-time programs to have homogeneous astrometry. The precise position of the four planets with respect to the host star was calculated by exploiting the fake negative companions method. We obtained an astrometric precision of the order of 6 mas in the worst case and 1 mas in the best case. To improve the orbital fitting, we also took into account all of the astrometric data available in the literature. Orbital and dynamical analysis of the system around HR 8799 New astrometric epochs from VLT/SPHERE and LBT/LUCI A Z l 1 2 3 K G ´d i ki4 C L i5 6 D M 6 P N i 1 S D id 6 R G 6 F M i7 (Affiliations can be found after the references) (Affiliations can be found after the references) Received 25 April 2022 / Accepted 15 June 2022 Received 25 April 2022 / Accepted 15 June 2022 ABSTRACT Context. HR 8799 is a young planetary system composed of four planets and a double debris belt. Being the first multi-planetary system discovered with the direct imaging technique, it has been observed extensively since 1998. This wide baseline of astrometric measurements, counting over 50 observations in 20 years, permits a detailed orbital and dynamical analysis of the system. Aims. To explore the orbital parameters of the planets, their dynamical history, and the planet-to-disk interaction, we made follow-up observations of the system during the VLT/SPHERE guaranteed time observation program. We obtained 21 observations, most of them in favorable conditions. In addition, we observed HR 8799 with the instrument LUCI at the Large Binocular Telescope (LBT). Methods. All the observations were reduced with state-of-the-art algorithms implemented to apply the spectral and angular differential imaging method. We re-reduced the SPHERE data obtained during the commissioning of the instrument and in three open-time programs to have homogeneous astrometry. The precise position of the four planets with respect to the host star was calculated by l i i h f k i i h d W b i d i i i f h d f 6 i h d 1 Context. HR 8799 is a young planetary system composed of four planets and a double debris belt. Being the first multi-planetary system discovered with the direct imaging technique, it has been observed extensively since 1998. This wide baseline of astrometric measurements, counting over 50 observations in 20 years, permits a detailed orbital and dynamical analysis of the system. Aims. To explore the orbital parameters of the planets, their dynamical history, and the planet-to-disk interaction, we made follow-up observations of the system during the VLT/SPHERE guaranteed time observation program. We obtained 21 observations, most of them in favorable conditions. In addition, we observed HR 8799 with the instrument LUCI at the Large Binocular Telescope (LBT). Context. HR 8799 is a young planetary system composed of four planets and a double debris belt. Being the first multi-planetary system discovered with the direct imaging technique, it has been observed extensively since 1998. This wide baseline of astrometric measurements, counting over 50 observations in 20 years, permits a detailed orbital and dynamical analysis of the system. Context. HR 8799 is a young planetary system composed of four planets and a double debris belt. A133, page 1 of 25 Open Access article, published by EDP Sciences, under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. This article is published in open access under the Subscribe-to-Open model. Subscribe to A&A to support open access publication. Astronomy & Astrophysics Astronomy & Astrophysics A&A 666, A133 (2022) https://doi.org/10.1051/0004-6361/202243862 © A. Zurlo et al. 2022 Orbital and dynamical analysis of the system around HR 8799 New astrometric epochs from VLT/SPHERE and LBT/LUCI A. Zurlo1,2,3 , K. Go´zdziewski4, C. Lazzoni5,6, D. Mesa6, P. Nogueira1, S. Desidera6, R. Gratton6, F. Marzari7, M. Langlois8,3, E. Pinna9, G. Chauvin10, P. Delorme10, J. H. Girard11, J. Hagelberg12, Th. Henning13, M. Janson14, E. Rickman15, P. Kervella16, H. Avenhaus13, T. Bhowmik1, B. Biller17, A. Boccaletti16, M. Bonaglia9, M. Bonavita18,6 M. Bonnefoy10, F. Cantalloube3, A. Cheetham12, R. Claudi6, V. D’Orazi6, M. Feldt13, R. Galicher16, E. Ghose9, A.-M. Lagrange10, H. le Coroller3, R. Ligi20, M. Kasper21, A.-L. Maire10, F. Medard10, M. Meyer19, S. Peretti12, C. Perrot16, A. T. Puglisi9, F. Rossi9, B. Rothberg22,23, T. Schmidt16, E. Sissa6, A. Vigan3, and Z. Wahhaj24 1. Introduction 2016; Wang et al. 2018; GRAVITY Collaboration 2019; Go´zdziewski & Migaszewski 2020). From the age of the system and the luminosity of the planets, evolutionary hot-start models derive masses of around 5–7 MJup (Marois et al. 2010; Currie et al. 2011; Sudol & Haghighipour 2012). These masses are compatible with the dynamical studies because small masses help the orbits to remain stable (e.g., Wang et al. 2018). On the other hand, Brandt et al. (2021) found a dynamical mass for the innermost planet HR 8799 e of 9.6+1.9 −1.8 MJup assuming that planets c, d, and e share the same mass within ∼20%. This result is 2 MJup higher than the prediction from the evolutionary models. UT date Filter ∆FoV (◦) S/NMin S/NMax 2014-07-12 DB_H23 17 9 44 2014-08-13 (a) BB_J 23 0 36 2014-12-04 (b) BB_H 9 14 24 2014-12-05 (b) BB_H 8 11 25 2014-12-06 (b) BB_H 8 15 25 2014-12-08 (a,b) BB_H 8 6 25 2015-07-03 DB_K12 18 12 75 2015-07-29 (a,b) DB_J23 82 4 51 2015-07-30 (b) DB_K12 81 21 123 2015-09-27 DB_K12 24 10 71 2016-11-17 DB_H23 17 15 52 2017-06-14 DB_H23 19 14 67 2017-10-07 (a) BB_H 76 0 0 2017-10-11 BB_H 74 45 90 2017-10-12 BB_H 78 24 100 2018-06-18 DB_H23 34 20 78 2018-08-17 BB_H 73 29 57 2018-08-18 BB_H 73 42 106 2019-10-31 DB_K12 24 22 62 2019-11-01 DB_K12 54 38 114 2021-08-20 DB_H23 20 21 59 Notes. (a)Discarded for bad weather. (b)IRDIS alone. UT date Filter ∆FoV (◦) S/NMin S/NMax p y In this paper, we present all the astrometric measurements obtained during the whole guaranteed time observation (GTO) of VLT/SPHERE for HR 8799 bcde. Ad hoc observations were designed for the orbital follow-up of the four planets; HR 8799 was observed 21 times in total with SPHERE, and only four observations were rejected based on quality criteria. To comple- ment the SPHERE measurement, we obtained one astrometric epoch with the instrument LUCI, installed at the LBT. This instrument observed HR 8799 as part of the commissioning phase of the new adaptive optics (AO) system. With a total of 18 epochs and the addition of all the astrometric measure- ments available in the literature (69 astrometric epochs in total), we performed the dynamical analysis of the system and the planet-to-disk interaction. Notes. (a)Discarded for bad weather. (b)IRDIS alone. The outline of the paper is as follows: in Sect. 1. Introduction Among the exoplanets discovered with the high-contrast imag- ing technique (e.g., Chauvin et al. 2004; Lagrange et al. 2010; Rameau et al. 2013; Keppler et al. 2018; Bohn et al. 2021), the system around HR 8799 is undoubtedly one of the most interesting. This is mainly because HR 8799 hosts a greater num- ber of planets detected with high-contrast imaging than any other system, only three systems that host two planets were detected: PDS 70 (Keppler et al. 2018; Haffert et al. 2019), TYC 8998-760-1 (Bohn et al. 2020), and β Pic (Lagrange et al. 2010; Nowak et al. 2020). HR 8799 is a perfect laboratory with which to study dynamical interaction in young planetary sys- tems, with its four planets (HR 8799 bcde; Marois et al. 2008, 2010) and a double debris belt (see, e.g., Su et al. 2009; Hughes et al. 2011; Matthews et al. 2014; Booth et al. 2016; Faramaz et al. 2021). The host star HR 8799 is a young (∼42 Myr, This system is an optimal target for high-contrast imaging observations, as its four planets are easily detectable with state- of-the-art imagers; their contrasts are about 2–8 × 10−6; and the separation of the closest planet HR 8799 e is ∼390 mas, which is further than the inner working angle of most current instru- ments designed for direct imaging. For these reasons, the system has been observed dozens of times starting from 1998, with dif- ferent instruments, wavelengths, and configurations. Thanks to this rich pool of archival data, HR 8799 can be studied in detail from an astrometric point of view, being the only multi-planetary system for which there are tens of astrometric data points on a baseline of more than 20 yr. These measurements began with A&A 666, A133 (2022) Table 1. Summary of the observations of HR 8799 with IRDIS and IFS during SHINE. Table 1. Summary of the observations of HR 8799 with IRDIS and IFS during SHINE. uncertainties of ∼20 mas, but have since reached unprecedented precision of below a milliarcsecond (0.1–0.2 mas) with optical interferometry (GRAVITY Collaboration 2019). Works regarding the analysis of the dynamical interaction of the four planets have been presented; most of these agree on the near coplanarity of the planets, and that they are locked in a 1:2:4:8 resonance, (see, e.g., Fabrycky & Murray-Clay 2010; Esposito et al. 2013; Konopacky et al. 2016; Zurlo et al. 1. Introduction 2, we present SPHERE and LUCI observations; in Sect. 3, we describe the reduction methods applied and the astrometric results that we obtained. In Sect. 4, we present the astrometric fitting for the four planets of HR 8799 and in Sect. 5 a possible interpretation of the history of the system. We also explored the possibility of the presence of a fifth planet, studying the planets–disk interaction (Sect. 6). We provide our conclusions in Sect. 7. the IWA is ∼0.′′12. For a detailed description of the observing sequence, we refer the reader to Zurlo et al. (2014, 2016). In gen- eral, the working sequence includes an image of the off-axis star point spread function (PSF) for the flux calibration, a long coro- nagraphic sequence with the satellite spots (Langlois et al. 2013) mode, a second image of the stellar PSF, and the sky images. The waffle mode was used on purpose to assure maximum astro- metric precision. The long waffle sequence was taken in pupil stabilized mode in order to apply the angular differential imag- ing (ADI; Marois et al. 2006) method. While IRDIS has a field of view of ∼11 × 11′′, IFS is smaller (1.7′′ × 1.7′′), and only the two interior planets are visible. The SHINE observations are sum- marized in Table 1. For almost all the epochs, the observation was with IRDIS and IFS working in parallel. On a few occa- sions, IRDIS was used alone; these observations are marked in the table. We discarded data for time periods where the condi- tions did not permit a clear detection of the planets or there was a very poor signal-to-noise ratio (S/N). In particular, we rejected data from 2014 August 13 ( only IFS data were considered), 2014 December 08, 2015 July 29, and 2017 October 07. All the other observations are taken into account in this analysis. 2.1. VLT/SPHERE HR 8799 was observed several times during the SpHere INfrared survey for Exoplanets (SHINE, papers I, II, III; Desidera et al. 2021; Vigan et al. 2021; Langlois et al. 2021) during the GTO of VLT/SPHERE. The instrument SPHERE (Beuzit et al. 2019) is a planet finder equipped with an extreme AO system (SAXO; Fusco et al. 2006; Petit et al. 2014) to characterize substel- lar companions with high-contrast imaging. The near-infrared (NIR) arm includes the IR dual-band imager and spectrograph (IRDIS; Dohlen et al. 2008) and an integral field spectrograph (IFS; Claudi et al. 2008). During the observations, these two sub- systems observed the target in parallel. HR 8799 was periodically observed for astrometric monitoring, the setup of the observa- tions included three different filter pairs: IRDIS in H2H3 bands (λH2 = 1.593 µm, λH3 = 1.667 µm), in BB_H (λH = 1.625 µm), and K1K2 bands (λK1 = 2.102 µm, λK2 = 2.255 µm). The coro- nagraph used for the shortest wavelengths was an apodized Lyot with a mask diameter of 185 mas and an inner working angle (IWA) of 0.′′09 (see Boccaletti et al. 2008), while for K1K2 band 2.2. Large Binocular Telescope/SOUL-LUCI HR 8799 was observed with LBT/SOUL-LUCI1 during its com- missioning on night 2020 September 29. LUCI1 (Seifert et al. 2010) is a NIR spectro-imager that can work in a diffraction- limited regime with a sampling of 15 mas pix−1. The AO A133, page 2 of 25 A133, page 2 of 25 A. Zurlo et al.: Dynamical analysis of HR 8799 from SPHERE and LUCI astrometry. A. Zurlo et al.: Dynamical analysis of HR 8799 from SPHERE and LUCI astrometry. Table 2. List of astrometric points from IRDIS observations. 2.2. Large Binocular Telescope/SOUL-LUCI We observed this target in pupil stabilization mode with two narrow-band filters: FeII (λFeII = 1.646 µm; ∆λ = 0.018 µm) and H2 (λH2 = 2.124 µm; ∆λ = 0.023 µm). The observations with the two filters were alternated during the whole sequence. 2.2. Large Binocular Telescope/SOUL-LUCI List of astrometric points from IRDIS observations. 2015 July 29, 2015 July 30, 2017 October 07, 2017 October 11, and 2017 October 12 (two epochs were excluded for poor qual- ity), and 2018 August, 17–18. Finally, we re-processed the IRDIS data presented by Wahhaj et al. (2021) and taken on October 31, 2019, and November 1, 2019. In the DC, a “fake negative planets” (e.g., Zurlo et al. 2014, and references therein) algo- rithm is implemented to calculate the precise position of each planet with respect to the central star. A summary of all the SPHERE astrometric points, both from the SHINE survey and published observations presented in Zurlo et al. (2016), Apai et al. (2016), Biller et al. (2021), and Wahhaj et al. (2021) is listed in Tables 2 (IRDIS) and 3 (IFS). We refer the reader to these latter publications for further information about the observ- ing conditions. The results of the updated astrometry are also listed in Table A.1, together with all the other data from different instruments presented in the literature. correction is provided by SOUL (Pinna et al. 2019), the upgrade of the FLAO system (Esposito et al. 2010). During this obser- vation, the SOUL system was correcting 500 modes with a rate of 1.7 kHz. No coronagraphic masks are available on LUCI, and therefore to avoid any saturation, HR 8799 was observed adopt- ing a sub-windowing of 256 × 256 pixels (corresponding to a field of view (FoV) of 3.84 × 3.84′′). In this way, we were able to set a minimum exposure time of 0.34 s. We observed this target in pupil stabilization mode with two narrow-band filters: FeII (λFeII = 1.646 µm; ∆λ = 0.018 µm) and H2 (λH2 = 2.124 µm; ∆λ = 0.023 µm). The observations with the two filters were alternated during the whole sequence. correction is provided by SOUL (Pinna et al. 2019), the upgrade of the FLAO system (Esposito et al. 2010). During this obser- vation, the SOUL system was correcting 500 modes with a rate of 1.7 kHz. No coronagraphic masks are available on LUCI, and therefore to avoid any saturation, HR 8799 was observed adopt- ing a sub-windowing of 256 × 256 pixels (corresponding to a field of view (FoV) of 3.84 × 3.84′′). In this way, we were able to set a minimum exposure time of 0.34 s. 2.2. Large Binocular Telescope/SOUL-LUCI Date Planet b Planet c Planet d Planet e ∆RA ; ∆Dec (mas) ∆RA ; ∆Dec (mas) ∆RA ; ∆Dec (mas) ∆RA ; ∆Dec (mas) 2014-07-12 1570 ± 3; 704 ± 3 −521 ± 3; 790 ± 9 −391 ± 2; −530 ± 2 −387 ± 2; −10 ± 3 2014-12-04 1574 ± 3; 701 ± 2 −514 ± 3; 798 ± 4 −399 ± 4; −525 ± 4 −389 ± 8; 11 ± 4 2014-12-05 1574 ± 4; 701 ± 3 −512 ± 3; 798 ± 4 −400 ± 4; −523 ± 4 −390 ± 7; 12 ± 4 2014-12-06 1573 ± 3; 701 ± 3 −512 ± 3; 797 ± 4 −403 ± 4; −524 ± 4 −383 ± 8; 11 ± 4 2015-07-03 1579 ± 1; 694 ± 1 −498 ± 1; 806 ± 1 −417 ± 1; −517 ± 1 −383 ± 9; 33 ± 5 2015-07-30 1580 ± 5; 689 ± 3 −495 ± 2; 806 ± 2 −419 ± 2; −516 ± 1 −386 ± 1; 36 ± 1 2015-09-27 1580 ± 1; 688 ± 1 −494 ± 1; 811 ± 1 –426 ± 1; −512 ± 1 −382 ± 9; 50 ± 5 2016-11-17 1589 ± 2; 666 ± 1 −464 ± 2; 824 ± 2 –454 ± 2; −490 ± 2 −378 ± 4; 90 ± 2 2017-06-14 1591 ± 1; 653 ± 1 −449 ± 1; 835 ± 1 −472 ± 2; −482 ± 2 −373 ± 3; 118 ± 2 2017-10-11 1595 ± 1; 647 ± 1 −441 ± 1; 839 ± 1 −480 ± 1; −477 ± 1 −369 ± 1; 128 ± 1 2017-10-12 1595 ± 1; 647 ± 1 −441 ± 1; 839 ± 1 −480 ± 1; −477 ± 1 −371 ± 2; 128 ± 2 2018-06-18 1601 ± 1; 635 ± 1 −424 ± 1; 848 ± 1 −497 ± 2; −463 ± 2 −358 ± 2; 156 ± 2 2018-08-17 1601 ± 2; 632 ± 3 −421 ± 1; 850 ± 1 −502 ± 1; −461 ± 1 −357 ± 1; 162 ± 2 2018-08-18 1600 ± 1; 632 ± 1 −421 ± 1; 851 ± 1 −502 ± 2; −458 ± 1 −358 ± 2; 163 ± 1 2019-10-31 1606 ± 2; 615 ± 2 −392 ± 2; 875 ± 2 −532 ± 3; −425 ± 2 −338 ± 2; 215 ± 2 2019-11-01 1611 ± 1; 611 ± 1 −388 ± 2; 870 ± 2 −530 ± 2; −430 ± 1 −335 ± 1; 210 ± 1 2021-08-20 1626 ± 1; 578 ± 2 −339 ± 2; 890 ± 2 −563 ± 2; −391 ± 2 −287 ± 4; 272 ± 2 Table 2. 3. Data reduction 3.1. SPHERE p From the IRDIS photometry, we also estimated the mass of each planet using evolutionary models. In particular, we applied evolutionary models from Baraffe et al. (2003, 2015) to the IRDIS filters photometry. The age of the system used in this estimation is 42 Myr and the parallax is 24.46 ± 0.05 mas, as in Gaia EDR3. The error reported is the standard deviation between different epochs taken with the same filter. Results are listed in Table 4. The reduction of the IRDIS data was carried out entirely with the Data Center (DC; Delorme et al. 2017) which uses the stan- dard SPHERE pipeline SpeCal (Galicher et al. 2018). For the astrometric calibration, we used a true north orientation of – 1.75 deg and a pixel scale value of 12.25 mas as reported in Maire et al. (2016). The reduction algorithm used by the DC for HR 8799 is the T-LOCI (Marois et al. 2014). To provide a homogeneous reduction of all the IRDIS data, we processed the observations presented in Zurlo et al. (2016) again, which were previously reduced with custom routines, which included seven epochs in 2014 (we excluded the sequence of 2014 August 13 for poor quality). The four epochs taken for variability monitor- ing – once per night (2014 December 4, 2014 December 5, 2014 December 6, 2014 December 8) – and presented in Apai et al. (2016) were reprocessed with the DC. As in Apai et al. (2016), we excluded the sequence of December 8 for poor conditions. In the same way, we used the DC to reduce the data from the vari- ability monitoring of Biller et al. (2021), with observation dates: Table 3. List of astrometric points from IFS observations. Table 3. List of astrometric points from IFS observations. Table 3. List of astrometric points from IFS observations. Table 3. List of astrometric points from IFS observations. Date Planet d Planet e ∆RA ; ∆Dec (mas) ∆RA ; ∆Dec (mas) 2014-07-12 −400 ± 4; −512 ± 4 −389 ± 1; −22 ± 2 2014-08-13 −396 ± 1; −524 ± 1 −389 ± 1; −17 ± 2 2015-07-03 −424 ± 4; −509 ± 3 −391 ± 1; 33 ± 2 2015-09-27 −420 ± 4; −513 ± 4 −392 ± 1; 39 ± 3 2016-11-17 −464 ± 1; −486 ± 2 −382 ± 2; 94 ± 6 2017-06-14 −473 ± 2; −476 ± 2 −377 ± 1; 115 ± 3 2017-10-11 −480 ± 2; −478 ± 2 −371 ± 1; 129 ± 2 2017-10-12 −492 ± 5; −463 ± 6 −370 ± 2; 135 ± 3 2018-06-18 −495 ± 1; −460 ± 2 −360 ± 2; 162 ± 2 2018-08-17 −509 ± 2; −452 ± 3 −361 ± 2; 166 ± 1 2018-08-18 −503 ± 1; −456 ± 2 −359 ± 1; 167 ± 2 2019-10-31 −527 ± 3; −432 ± 3 −337 ± 2; 210 ± 3 2019-11-01 −528 ± 1; −435 ± 2 −337 ± 2; 208 ± 1 2021-08-20 −569 ± 1; −390 ± 2 −292 ± 2; 276 ± 2 in the literature. Recently, Go´zdziewski & Migaszewski (2020) (GM2020 hereafter) reported an exact-resonance configuration (or “periodic orbits model” (PO) hereafter). This model is designed to explain the astrometric measurements through con- straints on geometry, planetary masses, and astrometric parallax of the system. Direct determination of planetary masses is crucial for calibrating astrophysical cooling models and for deter- mining the origin and long-term orbital evolution of the entire system, including its debris disk components. Here, we consider a less stringent condition on the stability of the system, assuming that it may be close to exact resonance but not necessarily fully periodic. This assumption may be nat- ural in the sense that migration may result in a system that is not exactly in a configuration of PO (e.g. Ramos et al. 2017). We want to determine whether such near-resonance models can yield statistically different or perhaps even better best-fitting solutions than those that appear in the PO scenario. 4.2. Mass and parallax priors We conducted MCMC sampling from the posterior defined through the general merit function in Eq. (1) and astrophysical and geometrical priors. The most important astrophysical priors are the masses of the star and the planets, and the geometrical prior is the parallax. Table 3. List of astrometric points from IFS observations. We define the fol- lowing merit functions as in Go´zdziewski & Migaszewski (2018, 2020): ln L(x) = −1 2χ2(x) −1 2 Nobs X i=1 h ln θ2 i,α + ln θ2 i,δ i −Nobs ln(2π), χ2(x) = Nobs X i=1  [αi −α(ti, x)]2 θ2 i,α + [δi −δ(ti, x)]2 θ2 i,δ , (1) (1) was switched off to allow the rotation of the FOV and be able to use the ADI method. Before performing the high-contrast imaging method, we obtained the position of the stellar PSF for each frame using the FIND procedure as implemented for IDL. Exploiting these positions, we then precisely registered the whole dataset, positioning the stellar PSFs at the center of each frame. The ADI was then implemented by exploiting the princi- pal component analysis (PCA; Soummer et al. 2012) technique. For the reduction, we tested a different number of principal com- ponents, but found that the best solution was to use ten of them. We were able to recover all the known companions with S/N ranging between 7 and 10. where (αi, δi) are the right ascension (RA) and declination (Dec) measurements at time ti; α(ti, x), δ(ti, x) are for their ephemeris (model) values at the same moments as implied by the adopted x parameters; and θ2 i,α and θ2 i,δ are the nominal measurement uncer- tainties in RA and Dec scaled in quadrature with the so-called error floor, θ2 i,α = (σ2 i,α + σ2 α,δ) and θ2 i,δ = (σ2 i,δ + σ2 α,δ), for each datum, respectively. Also, if Nobs is the number of observations then N = 2Nobs, because RA and Dec are measured in a single detection. The error floor can be introduced for unmodeled mea- surement uncertainties, such that the resulting best-fitting model should yield the reduced χ2 ν ≃1. However, in this work, as we rely on MCMC sampling and the error floor introduces little qualitative variability into the solutions, we omit this parameter, thereby also simplifying the astrometric model. We obtained the precise position of each planet, introducing fake negative companions and changing its position to minimize the standard deviation in a small region around the planet itself. The results of this procedure are listed in Table 5. 3.2. SOUL+LUCI The data were taken with a randomized offset of the position of the PSF on the detector. This was used to create a back- ground obtained by calculating the median of all the frames. This background was then subtracted from each science frame. The final dataset was composed of 60 and 61 files for the FeII and H2 observations, respectively. In both cases, each file was com- posed of 112 frames for a total exposure time of 2284.80 s and 2322.88 s, respectively. During the observations, the derotator A133, page 3 of 25 A133, page 3 of 25 A&A 666, A133 (2022) 4.1. Dynamical constraints on the astrometry Filter Planet e (MJup) Planet d (MJup) Planet c (MJup) Planet b (MJup) BB_H 8.9 ± 1.1 8.4 ± 0.9 8.5 ± 0.9 6.4 ± 0.6 D_H2 7.0 ± 0.3 6.6 ± 0.3 6.7 ± 0.3 4.6 ± 0.3 D_H3 8.5 ± 0.3 8.1 ± 0.2 8.3 ± 0.3 6.8 ± 0.3 D_K1 9.6 ± 0.3 9.3 ± 0.3 9.3 ± 0.2 6.7 ± 0.3 D_K2 10.5 ± 0.1 10.5 ± 0.1 10.4 ± 0.1 9.0 ± 0.3 Notes. The age of the system is assumed to be 42 Myr. The error is the standard deviation between different observations with the same filter. Table 5. List of astrometric points from the LBT/LUCI observation. Date Planet b Planet c Planet d Planet e ∆RA; ∆Dec (mas) ∆RA; ∆Dec (mas) ∆RA; ∆Dec (mas) ∆RA; ∆Dec (mas) 2020-09-29 1620 ± 1; 591 ± 3 −364 ± 2; 883 ± 1 −551 ± 1; −415 ± 4 −315 ± 3 ; 242 ± 5 Table 4. Values for the mass of each planet calculated using the evolutionary models in the different IRDIS filters. Filter Planet e (MJup) Planet d (MJup) Planet c (MJup) Planet b (MJup) m is assumed to be 42 Myr. The error is the standard deviation between different observations with the same filter. Table 5. List of astrometric points from the LBT/LUCI observation. Date Planet b Planet c Planet d Planet e ∆RA; ∆Dec (mas) ∆RA; ∆Dec (mas) ∆RA; ∆Dec (mas) ∆RA; ∆Dec (mas) 2020-09-29 1620 ± 1; 591 ± 3 −364 ± 2; 883 ± 1 −551 ± 1; −415 ± 4 −315 ± 3 ; 242 ± 5 significantly shifted from Π ≃24.22±0.08 mas in the Gaia DR2 and 24.8 ± 0.7 mas in Gaia DR1 catalog, respectively. The par- allax determinations in the Gaia catalogs appear subtly biased, depending on the luminosity and spectral type (Lindegren et al. 2021). However, compared to the uncertainties of the dynam- ical estimates here, the predicted parallax correction for Gaia eDR3/DR3 of <0.1 mas (a few tens of µas) would be insignifi- cant. Indeed, the parallax correction predicted by Lindegren et al. (2021) yields 24.50±0.05 mas (Kervella et al. 2022). This appar- ently very small shift of the order of 1σ uncertainty may still be meaningful when compared to the dynamical estimates, as is found below. 4.3. The exact Laplace resonance revisited It is common in the literature to assume smaller planet masses, that is, of ≃7 MJup, given the results of dynamical N-body simulations. In particular, Wang et al. (2018) report difficulty in finding dynamically stable solutions for planet masses larger than ≃7 MJup. Simultaneously, Go´zdziewski & Migaszewski (2018, 2020) found rigorously stable systems locked in the Laplace resonance for higher mass ranges as well that is, of, ≃8–9 MJup. Given systematic observationally constrained shifts to higher masses, as predicted by the exact Laplace MMR model, we decided to apply the mass priors in the ≃9 MJup range. Brandt et al. (2021) estimated the mass of HR 8799e as 9.6+1.9 −1.8 MJup based on the secular variation of the proper motion of the parent star in Gaia and HIPPARCOS catalogs. Moreover, in a very recent work, Kervella et al. (2022) similarly determined the dynamical mass of 12 ± 3.5 MJup for HR 8799e. This latter is less precise than in the prior work of Brandt et al. (2021), because Kervella et al. (2022) did not account for the actual orbital geometry. These high mass ranges for HR 8799e are consistent with the larger mass priors. Therefore, in some experiments we also used the mass priors Nm with the innermost mass changed to the value computed in Brandt et al. (2021).i In the first step, we verified whether or not the resonant model in GM2020 based on measurements in Konopacky et al. (2016) fits the recent measurements listed in Tables 2, 3, and 5. We found that this model visually and significantly deviates from the updated data set, especially for planet HR 8799b. Therefore, we refined the PO model using the same parametrization as for the merit function; see Eq. (1) in GM2020. In the formulation presented by these latter authors, the primary parameters of the astrometric model consist of all planet masses mi, i = 1, . . . , 4, the osculating period ratio κ for the two innermost planets – which selects the particular four-body Laplace resonance chain– , as well as the reference epoch τ, three Euler angles (I, ωrot, Ω) rotating the coplanar resonant configuration to the sky (observer) plane, and the system parallax Π. This means 11 free parameters. We note that all remaining orbital parameters are constrained through the N-body dynamics confined to the manifold of strictly periodic solutions. 4.1. Dynamical constraints on the astrometry We also implied Gaussian priors Nm = [8.7 ± 1.7, 8.7 ± 1.0, 8.7 ± 1.0, 6 ± 0.7] MJup for the masses of the innermost to the outermost planet, respectively, based on the hot-start cooling models by Baraffe et al. (2003), following discussion in Wang et al. (2018) and confirmed by our estimates collected in Table 4. The mass ranges for BB_H are consistent with the priors in Wang et al. (2018), and D_H3, D_K1 overlap with the determinations from the earlier astrometric model in GM2020. As also demonstrated by GM2020 and confirmed below, there are many discrepancies regarding the mass hierarchy; according to the resonant model, HR 8799d is the most massive one, and the masses of HR 8799e and HR 8799c appear strongly anti-correlated. Masses for D_K2 seem to be too far into the high-end range regarding the dynamical stability, especially that of planet HR 8799b. 4.1. Dynamical constraints on the astrometry Given the literature regarding astrometric fits of the HR 8799 system (e.g., Wang et al. 2018; Go´zdziewski & Migaszewski 2020, and references therein), it is now widely recognized that the present observation time-window does not make it possible to determine long-term stable orbital solutions of HR HR 8799 without invoking particular dynamical constraints. Such con- straints may arise from the coplanarity of the planets’ orbits and their relatively small eccentricities, which implies a ratio of orbital periods close to 2:1 for successive pairs of planets, that is, 2:1 mean-motion resonances (MMRs). This assumption can be further supported by the likely origin of the system from planetary migration (e.g., Wang et al. 2018); see also Sect. 5 in this paper. Also, the recent analysis of high-contrast images and resulting orbital solutions in Wahhaj et al. (2021) indicate that planets HR 8799e and HR 8799c are most consistent with co-planar and resonant orbits. The dynamical multiple MMR scenario therefore seems to be well justified and documented p p Regarding the stellar mass, we considered two fixed values of m⋆= 1.52 M⊙determined by Baines et al. (2012) as m⋆= 1.516+0.038 −0.024 M⊙for the age of ≃33 Myr, and m⋆= 1.47+0.12 −0.17 M⊙, following the most recent dynamical estimate by Sepulveda & Bowler (2022). The same value was used by Wang et al. (2018) in their earlier work. Kervella et al. (2022) determined m⋆= 1.50 ± 0.08 M⊙, which overlaps with the two estimates. We note here that the stellar mass is fixed in our astrometric N-body model because it also constrains the parallax Π of the whole system. These two parameters are strongly correlated through the near-III Keplerian law. Moreover, the parallax tends to be systematically tightly bounded in subsequent Gaia catalogs. The most recent Gaia eDR3 estimate of Π = 24.460 ± 0.045 mas is accurate to 0.1%, and it is a meaningful prior for the MCMC sampling. We note that the parallax did not change in the final Gaia DR3 catalog; now it is listed as Π = 24.462 ± 0.046 mas. A133, page 4 of 25 A133, page 4 of 25 A. Zurlo et al.: Dynamical analysis of HR 8799 from SPHERE and LUCI astrometry. Table 4. Values for the mass of each planet calculated using the evolutionary models in the different IRDIS filters. 4.3. The exact Laplace resonance revisited We performed the MCMC sampling from the posterior defined with the merit function in Eq. (1). This merit function is based on a numerical procedure for computing periodic solu- tions in a co-planar N-planet system, as described in GM2020 and implemented by Cezary Migaszewski (priv. comm.). To con- duct the MCMC sampling, we used the affine sampler in emcee As the parallax prior, we defined the most recent Gaia eDR3/DR3 estimate of Π = 24.46 ± 0.05 mas. This value is A133, page 5 of 25 A133, page 5 of 25 A&A 666, A133 (2022) Table 6. Osculating, heliocentric elements of the best-fitting solutions at the epoch of 1998.830. 4.3. The exact Laplace resonance revisited Model IVPO: m⋆= 1.52 M⊙, Π = 24.2585686 mas (24.26 ± 0.05) mas, χ2 ν = 4.32, ν = 441, dim p = 11 m (MJup) a (au) e I (deg) Ω(deg) ϖ (deg) M (deg) HR 8799e 8.1 ± 0.7 16.28 ± 0.03 0.147 ± 0.001 110.8 ± 0.5 336.8 ± 0.5 8.24465258 16.2817072 0.147508399 110.765660 336.793683 HR 8799d 9.5 ± 0.1 26.78 ± 0.08 0.115 ± 0.001 29.4 ± 0.9 59.8 ± 0.8 9.48438158 26.7888442 0.01154150 29.4676779 59.6448288 HR 8799c 7.7 ± 0.5 41.40 ± 0.10 0.055 ± 0.002 26.9 ± 0.2 62.2 ± 0.4 92.1 ± 0.5 145.6 ± 0.8 7.64047381 41.3945527 0.05485344 26.8923597 62.2416412 92.1126122 145.7203489 HR 8799b 6.0 ± 0.4 71.95 ± 0.13 0.017 ± 0.001 44.4 ± 3.0 309.2 ± 2.6 6.01476193 71.9477733 0.017597398 44.6384383 309.291519 Model IVPO: m⋆= 1.47 M⊙, Π = 24.5256617 mas (24.53 ± 0.04) mas, χ2 ν = 4.32, ν = 441, dim p = 11 m (MJup) a (au) e I (deg) Ω(deg) ϖ (deg) M (deg) HR 8799e 7.6 ± 0.9 16.10 ± 0.03 0.148 ± 0.001 110.8 ± 0.5 336.8 ± 0.3 7.4060918 16.1038912 0.147675953 110.816707 336.828050 HR 8799d 9.2 ± 0.1 26.46 ± 0.09 0.115 ± 0.002 29.1 ± 1.0 60.0 ± 1.0 9.18999484 26.45172667 0.114600334 29.0594738 60.1752106 HR 8799c 7.7 ± 0.7 40.97 ± 0.10 0.054 ± 0.002 26.9 ± 0.2 62.2 ± 0.4 92.3 ± 0.6 145.5 ± 0.7 7.79611845 40.9861801 0.05380902 26.8734271 62.1852189 92.3879462 145.5347082 HR 8799b 5.8 ± 0.4 71.14 ± 0.15 0.017 ± 0.002 43.50 ± 3.0 310.35 ± 3.0 5.80235941 71.1427851 0.01667073 42.9704380 310.895065 Model IV∆n: m⋆= 1.47 M⊙, Π = 24.5256617 mas (24.49 ± 0.07) mas, χ2 ν = 3.9, ν = 429, dim p = 23 m (MJup) a (au) e I (deg) Ω(deg) ϖ (deg) M (deg) HR 8799e 7.5 ± 0.6 16.00 ± 0.10 0.150 ± 0.004 108.8 ± 1.3 336.00 ± 1.5 7.40609179 16.1038912 0.147675954 110.816707 336.828050 HR 8799d 9.3 ± 0.5 26.55 ± 0.12 0.114 ± 0.004 28.66 ± 1.2 60.0 ± 1.0 9.18999484 26.45172667 0.11460033 29.05947383 60.1752106 HR 8799c 7.8 ± 0.6 41.00 ± 0.15 0.050 ± 0.003 26.5 ± 0.5 63.1 ± 0.5 91.8 ± 1.0 145.0 ± 1.0 7.79611845 40.9861801 0.05380902 26.8734271 62.1852189 92.3879462 145.5347082 HR 8799b 5.8 ± 0.8 71.3 ± 0.2 0.017 ± 0.002 42.3 ± 1.6 310.8 ± 2.0 5.80235941 71.1427851 0.016670734 42.9704380 310.895065 es. 4.3. The exact Laplace resonance revisited The stellar mass is m⋆= 1.52 M⊙or m⋆= 1.47 M⊙. Uncertainties for parameters in models IVPO and IV∆n are determined as the 16th h percentiles of the samples. Parameter values with 8–9 significant digits are provided in order to reproduce the results of particular integrati ameter ν = Nobs −dim p is for the degrees of freedom, where Nobs denotes the number of (α, δ) measurements, and dim p is the number of ameters. Notes. The stellar mass is m⋆= 1.52 M⊙or m⋆= 1.47 M⊙. Uncertainties for parameters in models IVPO and IV∆n are determined as the 16th and 86th percentiles of the samples. Parameter values with 8–9 significant digits are provided in order to reproduce the results of particular integrations. Parameter ν = Nobs −dim p is for the degrees of freedom, where Nobs denotes the number of (α, δ) measurements, and dim p is the number of free parameters. appears strongly correlated with geometric parameters of the system, i.e., the Euler angles and the parallax. These parameters show mutually weaker but still significant correlations.i package (Foreman-Mackey et al. 2013). We initiated 1536 walk- ers in a small hyper-ball in the parameter space around the initial condition in Table 6 determined with a search with the Powell non-gradient minimization algorithm. Since the auto- correlation time appears relatively short, of ∼100 iterations only, we ended up with 1000 iterations that make it possible to derive the posterior distribution, given the large number of walkers. i Parameters of the best-fitting solutions with their formal uncertainties and particular solutions from the posterior that may be useful for future numerical studies are collected in Table 6, referred to as Model IVPO for the two stellar masses of m⋆= 1.52 M⊙and m⋆= 1.47 M⊙, respectively. The derived posterior is illustrated in 2-dim projections and 1-dim histograms of the MCMC samples for the primary param- eters (Fig. 1). This experiment reveals significant correlations between different parameters already reported in GM2020 that still cannot be reduced with the new data. Besides a strong me−mc anti-correlation, there is also me−κ correlation and md −κ anti-correlation. The remaining two masses are free from sig- nificant mutual correlations, however, the mass of HR 8799b For a stellar mass of m⋆= 1.47M⊙, the HR 8799d mass seems to be constrained to ≃9.2 MJup within a relatively very small uncertainty of ≃0.1 MJup. 4.3. The exact Laplace resonance revisited This mass estimate is slightly larger than that found by GM2020. The mass of the outer- most planet HR 8799b may be determined as ≃5.8 MJup which is similar to ≃5.5 MJup found by these latter authors, yet with also smaller uncertainty of ≃0.3 MJup. Moreover, for the larger stellar A133, page 6 of 25 A133, page 6 of 25 A. Zurlo et al.: Dynamical analysis of HR 8799 from SPHERE and LUCI astrometry. me = 7.64+0.89 0.91 9.00 9.15 9.30 9.45 md[MJ] md = 9.18+0.08 0.08 6.4 7.2 8.0 8.8 mc[MJ] mc = 7.63+0.64 0.63 5.2 5.6 6.0 6.4 6.8 mb[MJ] mb = 5.84+0.30 0.30 1.968 1.976 1.984 1.992 = 1.98+0.01 0.00 24.45 24.50 24.55 24.60 24.65 [mas] = 24.53+0.03 0.03 26.50 26.75 27.00 27.25 27.50 Inc[deg] Inc = 26.90+0.16 0.17 6 7 8 9 10 me[MJ] 61.6 62.0 62.4 62.8 [deg] 9.00 9.15 9.30 9.45 md[MJ] 6.4 7.2 8.0 8.8 mc[MJ] 5.2 5.6 6.0 6.4 6.8 mb[MJ] 1.968 1.976 1.984 1.992 24.45 24.50 24.55 24.60 24.65 [mas] 26.50 26.75 27.00 27.25 27.50 Inc[deg] 61.6 62.0 62.4 62.8 [deg] = 62.25+0.30 0.32 Fig. 1. MCMC posterior for the best-fitting, strictly resonant model derived for m⋆= 1.47 MSun. Parameters included in the diagram are for th planet masses and orbital period ratio κ = Pd/Pe = κ, and parallax Π, inclination I, and the nodal angle Ω(the orbital scale ρ, PO epoch shift t and the common rotation angle ωrot in the orbital planet are skipped). The crossed lines mark the best-fitting PO solution in terms of the smalles χ2 that was used as a starting point to initiate the emcee walkers; see Tables 6 and 7 (Model 1) for parameters of this solution. Uncertainties fo the parameters are determined as the 16th and 86th percentile of the samples around the median values. A. Zurlo et al.: Dynamical analysis of HR 8799 from SPHERE and LUCI astrometry. A. Zurlo et al.: Dynamical analysis of HR 8799 from SPHERE and LUCI astrometry. mb[MJ] mc[MJ] Fig. 1. MCMC posterior for the best-fitting, strictly resonant model derived for m⋆= 1.47 MSun. Parameters included in the diagram are for the planet masses and orbital period ratio κ = Pd/Pe = κ, and parallax Π, inclination I, and the nodal angle Ω(the orbital scale ρ, PO epoch shift t0 and the common rotation angle ωrot in the orbital planet are skipped). 4.3. The exact Laplace resonance revisited The crossed lines mark the best-fitting PO solution in terms of the smallest χ2 that was used as a starting point to initiate the emcee walkers; see Tables 6 and 7 (Model 1) for parameters of this solution. Uncertainties for the parameters are determined as the 16th and 86th percentile of the samples around the median values. Fig. 1. MCMC posterior for the best-fitting, strictly resonant model derived for m⋆= 1.47 MSun. Parameters included in the diagram are for the planet masses and orbital period ratio κ = Pd/Pe = κ, and parallax Π, inclination I, and the nodal angle Ω(the orbital scale ρ, PO epoch shift t0 and the common rotation angle ωrot in the orbital planet are skipped). The crossed lines mark the best-fitting PO solution in terms of the smallest χ2 that was used as a starting point to initiate the emcee walkers; see Tables 6 and 7 (Model 1) for parameters of this solution. Uncertainties for the parameters are determined as the 16th and 86th percentile of the samples around the median values. mass of 1.52 M⊙, we derived slightly larger masses of HR 8799d ≃9.6 ± 0.1 MJup and HR 8799b ≃6.0 ± 0.4 MJup. This relation confirms the predicted strong stellar mass–parallax correlation and indirectly favors m⋆= 1.47 M⊙. The dynamical parallax determinations appear to be very accurate and mean- ingful, overlapping closely with the independent geometric Gaia measurements. p p The astrometric model for the updated data window yields Π ≃24.26 ± 0.05 mas, which is consistent with the earlier esti- mate by GM2020. The parallax estimate of Π ≃24.53±0.04 mas derived for 1.47 M⊙overlaps to 1σ with Π ≃24.460 ± 0.045 mas in Gaia eDR3/DR3. Moreover, as noted above, when applying the parallax correction of the Lindegren et al. (2021) eDR3 catalog, Kervella et al. (2022) obtained Π = 24.50 ± 0.05 mas which is even closer to the value derived from the PO model. Regarding the HR 8799e–HR 8799d mass anti-correlation, we conducted additional MCMC sampling for the primary parameters in the PO model for fake data series. We prepared two or three synthetic observations per year, extending the real observations window by ≃20 yr around the best-fitting periodic A133, page 7 of 25 A&A 666, A133 (2022) Fig. 2. 4.4. Near 8:4:2:1 MMR model (3) To characterize the system near the 2:1 MMR chain, we rely on the notion of the proper mean motions as the fundamental fre- quencies fi in the framework of conservative N-body dynamics. We resolve these frequencies with the refined Fourier frequency analysis (e.g., Laskar 1993; Nesvorny & Ferraz-Mello 1997, Numerical Analysis of Fundamental Frequencies, NAFF). To perform the NAFF, we consider the time series if ∆f(k) accounts only for the proper mean motions according to the d’Alembert rule. The resonant configuration may therefore be called the generalized Laplace resonance (Papaloizou 2015). This type of MMR, which possibly drives the orbital evolu- tion of HR 8799, is characterized with the proper mean motions fulfilling the following relation (e.g., GM2020): Sλ,i = { ai(t j) exp iλi(t j) }, SM,i = { ai(t j) exp iMi(t j) }, ∆f(k) = n1 −2n2 + n3 −2n4. (4) (4) where j = 0, 1, . . ., t j = j∆t, and ai(t), λi(t) and Mi are the canonical osculating semi-major axis, the mean longitude, and the mean anomaly for planet i, i = 1, 2, 3, 4, respectively, and ∆t is the sampling interval (i is the imaginary unit). These canon- ical elements inferred in the Jacobi or Poincaré reference frame make it possible to account for mutual interactions between the planets to the first order in the masses. Similarly to the classic Laplace resonance in the Galilean moons system, we consider the generalized MMR as a chain of two- body MMRs, Similarly to the classic Laplace resonance in the Galilean moons system, we consider the generalized MMR as a chain of two- body MMRs, 2ni+1 −ni + gj ≃2ni+1 −ni ≃0, i = 1, 2, 3, j = i, i + 1, for subsequent pairs of planets. Here, gj are for the proper fre- quencies (mode) associated with the pericenter rotation. In the exact resonance, which we consider as the PO in the reference frame rotating with a selected planet (Hadjidemetriou 1977), the apsidal lines of all planets rotate synchronously, with the same angular velocity relative to the inertial frame. The condition in Eq. (4) for the exact resonance (periodic motion in the rotating frame) may be expressed through the proper mean motions of the planets (e.g., Delisle 2017): for subsequent pairs of planets. Here, gj are for the proper fre- quencies (mode) associated with the pericenter rotation. 4.3. The exact Laplace resonance revisited Temporal evolution of the osculating orbital elements in the astrocentric (blue curves) and Jacobian (yellow curves) frames, respectively, for the same N-body initial condition implying a stable, near 8:4:2:1 MMR system. We note that the elements overlap for the first planet according to the construction of the Jacobian reference frame. Fig. 2. Temporal evolution of the osculating orbital elements in the astrocentric (blue curves) and Jacobian (yellow curves) frames, respectively, for the same N-body initial condition implying a stable, near 8:4:2:1 MMR system. We note that the elements overlap for the first planet according to the construction of the Jacobian reference frame. zeroth-order mean-motion resonance implies one of the possible linear combinations of the fundamental frequencies (the proper mean-motions), model with deviations and uncertainties of ∼1 mas. It appears that all correlations except those between the masses and κ would be almost eliminated. A probable cause of that effect may be the almost perfectly aligned gravitational tugs of these planets in the present particular time window of the observa- tions. If the PO model is correct, then likely only highly precise data similar to the most accurate GRAVITY measurement in GRAVITY Collaboration (2019) could break this degeneracy. This was indicated by GM2020. We note that the GRAVITY measurement is depicted with a star in all figures illustrating orbital solutions. ∆f(k) ≡ N X i=1 ki fi, (2) (2) where k ≡[k1, . . . , kN] is a vector of non-zero integers, and PN i=1 \|ki\| , 0, which yields small \|∆f\|. Simultaneously, we may determine the critical angle corresponding to η = arg min \|∆f(n)\|: where k ≡[k1, . . . , kN] is a vector of non-zero integers, and PN i=1 \|ki\| , 0, which yields small \|∆f\|. Simultaneously, we may determine the critical angle corresponding to η = arg min \|∆f(n)\|: θη(t) ≡ N X i=1 ηiλi, (3) 4.5. Astrometric fits to near-resonance configurations To sample the posterior defined with Eq. (1), we adopted priors described in Sect. 4.2. We did not imply any other particu- lar limits on the anticipated near-resonant N-body solutions, besides uniform priors for orbital parameters in sufficiently wide ranges. In this sense, the assumed prior set is minimal. Moreover, the N-body model is parameterized with (mi, ai, xi ≡ ei cos ϖi, yi ≡ei sin ϖi, Mi), i = 1, . . . , 4, that is, mass, semi- major axis, Poincaré elements composed of eccentricity and argument of pericenter, and the mean anomaly at the osculat- ing epoch for each orbit, as well as two angles (I, Ω) determining the orbital plane of the system, and the astrometric parallax Π. This means 23 free parameters.i In the top-right panel Fig. 4, for HR 8799c, blue circles repre- senting measurements in Konopacky et al. (2016) and red circles and yellow hexagons for SPHERE measurements are slightly shifted with respect to each other, and relative to the orbital model arcs. This is further visible in Figs. 5 and 6, which illustrate residuals of the best-fitting model in Table 6 in the ∆RA–∆Dec-plane as well as individual ∆RA(t) and ∆Dec(t) panels. While the SPHERE data are roughly uniformly clustered around the origin (0, 0), the blue circles exhibit systematic pat- terns, especially for planets HR 8799b and HR 8799c, besides a much larger spread. This effect is curious once we recall the two data sets consisting of uniformly reduced, essentially homoge- neous measurements in Konopacky et al. (2016) and this work, respectively. It is likely that the instruments and/or reduction pipelines are not fully compatible. The effect is quite subtle but still noticeable. It should be noted that the implicitly defined frequency prior (Eq. (5)) makes it difficult to derive the posterior distribution. In many experiments performed with a different number of MCMC walkers of up to 256 and up to 256 000 iterations, we could obtain an acceptance ratio as low as 0.1. Therefore, we understand the MCMC sampling with the ∆n prior as dynami- cally constrained optimization (rejection sampling) which helps to determine parameter ranges for the best-fitting, stable solu- tions. Also, due to implicit frequency prior definition, which has to be calibrated consistently with the expected dynamical stability of the system, the method has a heuristic character. 4.4. Near 8:4:2:1 MMR model In the exact resonance, which we consider as the PO in the reference frame rotating with a selected planet (Hadjidemetriou 1977), the apsidal lines of all planets rotate synchronously, with the same angular velocity relative to the inertial frame. The condition in Eq. (4) for the exact resonance (periodic motion in the rotating frame) may be expressed through the proper mean motions of the planets (e.g., Delisle 2017): i We note that the canonical orbital elements evolve differently from the common astrocentric elements. This is illustrated in Fig. 2. Subsequent panels are for the semi-major axis and eccen- tricity of HR 8799d,c,b, respectively, computed for the interval of a few tens of outermost orbits, and marked with different col- ors. It turns out that these elements span much wider ranges in the common astrocentric Keplerian frame. This effect is particu- larly noticeable for the two outermost planets. A small change in the N-body initial condition may result in substantial displace- ment of the orbit in the Keplerian (a, e)–plane. Therefore, the geometric elements should be understood as a formal representa- tion of the Cartesian N-body initial conditions, and the canonical elements are more suitable for the qualitative characterization of the orbits. ∆n ≡ 3 X i=1 ni ni+1 −2 ! = 0, (5) (5) given the proper mean-motions resolved from the time series S M,i. Numerically, we find that the exact resonance yields ∆n ≃ 10−13 rad d−1 and ∆f ≃10−15 rad d−1 for 3 × 8192 samples of ∆t = 2048 days. Considering a particular four-body MMR chain that can explain astrometric observations of the HR 8799 system, the A133, page 8 of 25 A. Zurlo et al.: Dynamical analysis of HR 8799 from SPHERE and LUCI astrometry A. Zurlo et al.: Dynamical analysis of HR 8799 from SPHERE and LUCI astrometry. We used the resonance constraint in Eq. (5) as one of the pri- ors for the MCMC sampling of the posterior defined through the maximum likelihood function in Eq. (1). This indirect frequency prior is Gaussian with a small variance σ∆n to be determined later. Laplace resonance center, which is selected as the starting PO model and that may explain missing mass correlations found for the PO model. 4.4. Near 8:4:2:1 MMR model While the fit quality in terms of ln L or χ2 may be somewhat improved with the quasi-periodic model, the RMS of the best-fitting models remains almost the same in the two approaches. It may be concluded that the quasi-periodic model does not lead to any qualitative change in the dynamical status of the system, apart from making it possible to systematically explore long-term stable solutions, which are not necessarily regular. We discuss this further in Sect. 4.6.i We determined the frequency variance σ∆n based on exten- sive numerical experiments that relied on calibrating the ∆n range with the Lagrange (geometric) stability interval, which should not be shorter than the stellar age of 40–50 Myr, as pre- dicted in the most recent work (Brandt et al. 2021). These authors also rule out ages for HR 8799 of greater than ≈300 Myr. The proper choice of the ∆n prior is crucial to bound (resolve) a long- term stable system in a very short integration time that spans merely a few hundred of the outermost orbits, ≃2 × 105 yr, which is also the instability timescale outside the Laplace resonance. We find, as described below in Sect. 4.6, that the astrometric HR 8799 solutions are long-term stable if \|∆n\| is roughly less than 10−5–10−6 rad d−1, as described above. Figure 4 illustrates the best-fitting orbital solutions (red curves) over-plotted for models within 1σ (grey curves) ran- domly selected from the MCMC samples such that their RMS varies between 7 and 9 mas. The best-fitting models with RMS ≃7.6 mas are marked as red curves. All of those solutions are depicted for roughly one osculating period for each planet. Clearly, the orbits do not close for all companions. This effect is best visible for the outermost ( top-middle panel) and HR 8799d ( bottom-right panel) planet, respectively. In the latter case, the range of the orbit splitting can be compared to a relatively large uncertainty on the HST measurements; for more accurate mea- surements, the splitting would be even more significant. This effect illustrates strong, short-term mutual interactions. Here, we follow Go´zdziewski & Migaszewski (2018), who indicated that Keplerian orbits are already inadequate for representing the proper astrometric solutions despite limited observational orbital arcs. 4.5. Astrometric fits to near-resonance configurations Nevertheless, we may gain much insight into the parameter ranges of stable near-resonance models consistent with the astrometric observations. Another trend of residuals is revealed in Fig. 6 for planet HR 8799e. The SPHERE data seem to be clearly arranged along a curve in the RA coordinate, revealing a systematic deviation in time with the ephemeris. This effect is especially clear for IRDIS data and may be noted in the lower right ∆RA–∆Dec panel and the ∆RA(t) panel, as the yellow hexagons spread along the ∆RA = 0 axis. This may indicate a systematic effect in IRDIS data reduction because it does not seem to appear for IFS data. However, the time–∆RA(t) panel is suggestive of this trend for both sets of measurements. If it has no instrumental origin, then it may indicate an unmodeled component of the astromet- ric model, such as the presence of a yet unknown object (e.g., a distant moon) or a different curvature of the orbit due to dif- ferent parameters and geometry (e.g., noncoplanarity). We note here the perfect position of the GRAVITY measurement, but this one point is insufficient to dismiss the trend. We performed many MCMC experiments varying σ∆n within the range of 10−7–10−9 rad d−1 and parameters of the emcee samplers. The example posterior for σ∆n = 10−8 rad d−1 is illustrated in Fig. 3 for parameters selected consistently with the posterior for PO shown in Fig. 1. During the sampling, we recorded all elements of the tested models, and the semi- amplitude of the critical angles of the Laplace resonance for the integration time spanning ≃200 outermost periods. Fig. 3 shows a collection of samples with \|∆n\| < 10−5 rad d−1, and the semi-amplitude of Laplace resonance argument \|∆θ\| < 60◦. This choice is explained in the following Sect. 4.6, in which we analyze the orbital evolution of particular selected solutions. Overall, when comparing the posterior distributions in Fig. 1 derived for the PO model, and in Fig. 4 for the quasi-periodic model, we may notice similar ranges for the parameters. A basic distinction relies on the different character of the solutions: while the PO posterior covers strictly stable, resonant solutions, the ∆n posterior also involves weakly chaotic solutions around the 4.6. Frequency priors versus the dynamical stability 4.6. Frequency priors versus the dynamical stability Regarding the dynamical character of solutions derived in Sect. 4.5, we analyzed representative examples of the best-fitting models marked in Fig. 4. We selected MCMC samples in Fig. 3 A133, page 9 of 25 A133, page 9 of 25 A&A 666, A133 (2022) − − Fig. 3. Posterior samples for stable solutions characterized with \|∆n\| < 10−5 rad d−1, and the semi-amplitude of the libration of the Laplac resonance argument \|∆θ\| < 60◦. The starting solution that initiated nearby MCMC walkers in the emcee samplers is the periodic configuratio in Tables 6 and 7 (Model 1). Uncertainties for the parameters are determined as the 16th and 86th percentiles of the samples around the media values. A&A 666, A133 (2022) − − Fig. 3. Posterior samples for stable solutions characterized with \|∆n\| < 10−5 rad d−1, and the semi-amplitude of the libration of the Laplace resonance argument \|∆θ\| < 60◦. The starting solution that initiated nearby MCMC walkers in the emcee samplers is the periodic configuration in Tables 6 and 7 (Model 1). Uncertainties for the parameters are determined as the 16th and 86th percentiles of the samples around the median values for m⋆= 1.47 M⊙and from other MCMC sampling for m⋆= 1.52 M⊙. These initial conditions were integrated with the IAS15 integrator from the REBOUND package (Rein & Spiegel 2015) for 1 Gyr or up to disruption of the system when a collision or ejection of a planet occurs. The results are shown in a sequence of plots in Figs. 7 and 8. argument is for the outermost pair of planets and librates around the center at ≃180◦. As it comes to the critical argument of the Laplace resonance, it librates with a very small amplitude of just ≃5◦around the resonance center at ≃15◦. The periodic, perfectly regular character of this solution manifests as time evolution of the eccentricities. The middle column (Model 2) in Fig. 7 is for a model slightly displaced from the exact resonance, as measured by \|∆n\| ≃ 10−7 rad d−1, yet it yields slightly better fit quality. The critical arguments evolve in the same manner, as for the exact resonance, but their amplitudes and eccentricities become noticeably larger. This solution also yields a fully stable configuration of the plan- ets. It is regular in the sense of the Lyapunov exponent, as it Systems illustrated in Fig. 4.6. Frequency priors versus the dynamical stability 7 are for long-term stable solutions that survived for at least 1 Gyr. The left column is for exactly res- onant Model 1 and its elements displays Table 7 and also Table 6. This solution is stable forever “by design” in the framework of the N body dynamics. All but one critical angle of the two- body MMRs between subsequent pairs of planets librate around centers in the [−90◦, 90◦]–range. The exceptional critical A133, page 10 of 25 A. Zurlo et al.: Dynamical analysis of HR 8799 from SPHERE and LUCI astrometry. Fig. 4. Best-fitting solutions to the four-planet model in Table 6 for m⋆= 1.47 M⊙, illustrated on the sky plane as randomly selected MCMC samples from the ∆n posterior. The y-axis corresponds to the north (N), and the x-axis corresponds to the east (E) direction, respectively (we note that the numerical values of ∆α are opposite in sign with respect to the formal left-hand direction of the RA α). Red-filled circles are for the new or re-reduced IRDIS measurements in this paper, yellow hexagons are for the IFS measurements, green hexagons are for the LUCI points, light-blue filled circles are for measurements in Konopacky et al. (2016), and dark-blue circles are for data in other references collected in Tables 2, 3, and 5 and summarised in Table A.1. Diamonds are for the GPI data, and a star symbol is for the most accurate GRAVITY point. Red curves mark stable solutions with the lowest RMS ≃7.6–8 mas, randomly selected from the MCMC samples, and darker grey curves are for other orbital arcs derived for stable models up to RMS ≃9–10 mas. All model orbits have been derived for all measurements available to date, and cover roughly one osculating orbital period for every planet, respectively. The osculating epoch is 1998.830. We also labeled the observational epochs encompassing the orbital arcs with data. The upper left panel is for the global view of the system on the sky plane, and subsequent panels are for its close-ups and interesting regions. Fig. 4. Best-fitting solutions to the four-planet model in Table 6 for m⋆= 1.47 M⊙, illustrated on the sky plane as randomly selected MCMC samples from the ∆n posterior. 4.6. Frequency priors versus the dynamical stability The y-axis corresponds to the north (N), and the x-axis corresponds to the east (E) direction, respectively (we note that the numerical values of ∆α are opposite in sign with respect to the formal left-hand direction of the RA α). Red-filled circles are for the new or re-reduced IRDIS measurements in this paper, yellow hexagons are for the IFS measurements, green hexagons are for the LUCI points, light-blue filled circles are for measurements in Konopacky et al. (2016), and dark-blue circles are for data in other references collected in Tables 2, 3, and 5 and summarised in Table A.1. Diamonds are for the GPI data, and a star symbol is for the most accurate GRAVITY point. Red curves mark stable solutions with the lowest RMS ≃7.6–8 mas, randomly selected from the MCMC samples, and darker grey curves are for other orbital arcs derived for stable models up to RMS ≃9–10 mas. All model orbits have been derived for all measurements available to date, and cover roughly one osculating orbital period for every planet, respectively. The osculating epoch is 1998.830. We also labeled the observational epochs encompassing the orbital arcs with data. The upper left panel is for the global view of the system on the sky plane, and subsequent panels are for its close-ups and interesting regions. left column in Fig. 8), 600 Myr (Model 5, the middle col- umn), and just 200 Myr (Model 6, the right column). Despite this, for the initial few hundred million years, extending safely beyond most approximations of the stellar age, which range between 30–160 Myr (Baines et al. 2012; Sepulveda & Bowler 2022), the systems are bounded and locked in the resonance. Simultaneously, Models 3 and 4 yield masses of the inner planet in the ≃10 MJup range, and Model 4 is especially inter- esting given its low χ2 compared to the initial starting PO configuration. These models yield a declining mass hierarchy that resembles that of the outer Solar System, and a mass of HR 8799e is consistent with the dynamical estimate in Brandt et al. (2021). can be seen in Fig. 9 showing dynamical maps in terms of the MEGNO fast indicator (Cincotta et al. 2003). 4.6. Frequency priors versus the dynamical stability The mean expo- nential growth factor of nearby orbits (MEGNO; also known as ⟨Y⟩) is a numerical technique designed to efficiently character- ize the stability of the N-body solutions in terms of the maximal Lyapunov exponent (MLE). We implemented MEGNO for the planetary problem in Go´zdziewski et al. (2001) and in our CPU- parallelized µFARM numerical package. It is also clear that this solution lies close to the edge of the resonance island (dark blue color). Finally, the right column (Model 3) in Fig. 7 is for a marginally stable solution, in the sense of nonzero MLE, with \|∆n\| ≃10−5 rad d−1. Although it survived for the 1 Gyr inte- gration interval, one of the critical angles in the outermost 2:1 MMR rotates. This solution is peculiar in the sense that, despite rotations of one of the 2:1 MMR critical arguments, the semi-amplitudes of other critical angles are similar to those for quasi-periodic Model 2. These examples are to justify that the system may be dynami- cally long-term stable in the planet mass range of ≃10 MJup, even if detuned from the exact resonance and mildly chaotic. In all unstable cases, one of the critical arguments of the 2:1 MMR of the two outermost planets progressively increases its libration amplitude and eventually begins to rotate. In this sense, the outer- most pair HR 8799b–c is the weakest link in the resonance chain, provoking instability of the whole system displaced from the res- onance. The time for the onset of instability can be relatively Model 3 in Fig. 7 is also remarkable when we compare it with a sequence of solutions in Fig. 8 illustrating chaotic, yet still long-term stable models characterised by \|∆n\| ≃10−6 rad d−1. 4.6. Frequency priors versus the dynamical stability All these models self-destruct between 800 Myr (Model 4, the A133, page 11 of 25 A133, page 11 of 25 A&A 666, A133 (2022) −40 −20 0 20 40 ΔRA [mas] −40 −30 −20 −10 0 10 20 30 40 ΔDEC [mas] HR8799b 1995 2000 2005 2010 2015 2020 2025 time [yr] −40 −20 0 20 40 ΔRA [mas] 1995 2000 2005 2010 2015 2020 2025 time [yr] −40 −20 0 20 40 ΔDEC [mas] −40 −20 0 20 40 ΔRA [mas] −40 −30 −20 −10 0 10 20 30 40 ΔDEC [mas] HR8799c 1995 2000 2005 2010 2015 2020 2025 time [yr] −40 −20 0 20 40 ΔRA [mas] 1995 2000 2005 2010 2015 2020 2025 time [yr] −40 −20 0 20 40 ΔDEC [mas] Fig. 5. Residuals to the selected best-fitting, near-resonant four-planet Model 2 in Table 7 for planets HR 8799b and HR 8799c; stellar mass is m⋆= 1.47 M⊙. The y-axis corresponds to the north (N), and the x-axis corresponds to the east (E) direction, respectively (we note that the numerical values of ∆RA are sign-opposite to regarding the formal left-hand direction of the RA axis). Red filled circles, and yellow and green hexagons are for the IRDIS, IFS, and LUCI measurements reported here, respectively, and dark-blue and light-blue (light-grey) filled circles are for measurements in previous papers and in Konopacky et al. (2016), respectively. Yellow diamonds are for GPI, the grey diamonds are for the early HST data. 1995 2000 2005 2010 2015 2020 2025 time [yr] −40 −20 0 20 40 ΔRA [mas] 1995 2000 2005 2010 2015 2020 2025 time [yr] −40 −20 0 20 40 ΔDEC [mas] −40 −20 0 20 40 ΔRA [mas] −40 −30 −20 −10 0 10 20 30 40 ΔDEC [mas] HR8799b [ ] −40 −20 0 20 40 ΔRA [mas] −40 −30 −20 −10 0 10 20 30 40 ΔDEC [mas] HR8799c 1995 2000 2005 2010 2015 2020 2025 time [yr] −40 −20 0 20 40 ΔRA [mas] 1995 2000 2005 2010 2015 2020 2025 time [yr] −40 −20 0 20 40 ΔDEC [mas] Fig. 5. Residuals to the selected best-fitting, near-resonant four-planet Model 2 in Table 7 for planets HR 8799b and HR 8799c; stellar mass is m⋆= 1.47 M⊙. 4.6. Frequency priors versus the dynamical stability The y-axis corresponds to the north (N), and the x-axis corresponds to the east (E) direction, respectively (we note that the numerical values of ∆RA are sign-opposite to regarding the formal left-hand direction of the RA axis). Red filled circles, and yellow and green hexagons are for the IRDIS, IFS, and LUCI measurements reported here, respectively, and dark-blue and light-blue (light-grey) filled circles are for measurements in previous papers and in Konopacky et al. (2016), respectively. Yellow diamonds are for GPI, the grey diamonds are for the early HST data. very long, as shown by the evolution of Models 4 and 5. In any case, the libration amplitudes of the critical angles seem to be a weak indicator of instability, because there is no clear rela- tionship between these amplitudes and the time of instability. Also, Model 5 illustrates the difficulty in predicting the system behavior based on the variation of critical angles, especially if the system stability is tested for a limited period of time. The apparently regular, quasi-periodic, and bounded evolution of the critical angles for ≃200 Myr does not prevent the system from eventually becoming unstable after ≃400–500 Myr. A similar effect may be observed for Model 6, although in this case, the critical argument of the Laplace resonance varies irregularly at the beginning of the integration. 4.7. Resonant structure of the inner debris disk Based on the updated orbital solutions in Table 6, we revised and extended simulations of the dynamical structure of the inner debris disk in Go´zdziewski & Migaszewski (2018). These experi- ments rely on the concept of the so-called ⟨Y⟩-model. We assume that the planets form a system of primaries in safely stable orbits robust to small perturbations. We then inject bodies with masses significantly smaller than those of the primaries and on orbits with different semi-major axes and eccentricities span- ning the interesting region, and randomly selected orbital angles. Next, we integrate the synthetic configuration and determine its stability with the MEGNO (⟨Y⟩) fast indicator. Calibra- tion experiments spanning orbital evolution of debris disks in HR 8799 for up to 70 Myr are described in detail in Go´zdziewski & Migaszewski (2018). A comparison of the results of direct numerical integrations with the outcomes of the ⟨Y⟩-model con- firms that these two approaches are consistent with one another, yet the ⟨Y⟩-based method is much more CPU efficient and there- fore makes it possible to obtain a clear representation of the structure of stable solutions. This algorithm is especially effec- tive for strongly interacting systems. Also, these simulations It is worth noting that all of the example solutions yield astro- metric fits that differ little in quality in terms of χ2 and RMS. The models are difficult to distinguish statistically and visually from each other. This may mean that for a coplanar, near-resonance, or resonance model we can hardly differentiate between per- fectly regular and chaotic evolution of the system, as long as the instability time is sufficiently long relative to the age of the star. However, these two types of configurations occur near the exact Laplace resonance. A133, page 12 of 25 A133, page 12 of 25 A. Zurlo et al.: Dynamical analysis of HR 8799 from SPHERE and LUCI astrometry. = Fig. 6. Residuals to the best-fitting, near-resonant four-planet Model 2 in Table 7 for m⋆= 1.47 M⊙, a continuation of Fig. 8 for planets HR 8799d and HR 8799e. See the caption of Fig. 5 for labels. The yellow star marks the GRAVITY measurement for HR 8799e. = Fig. 6. Residuals to the best-fitting, near-resonant four-planet Model 2 in Table 7 for m⋆= 1.47 M⊙, a continuation of Fig. 8 for planets HR 8799d and HR 8799e. See the caption of Fig. 5 for labels. 4.7. Resonant structure of the inner debris disk The yellow star marks the GRAVITY measurement for HR 8799e. = ing, near-resonant four-planet Model 2 in Table 7 for m⋆= 1.47 M⊙, a continuation of Fig. 8 for planets HR 8799d of Fig. 5 for labels. The yellow star marks the GRAVITY measurement for HR 8799e. the epoch 1998.830 up to the given final epoch t. For instance, t = 2009.575 (middle-right panel) is for the first detection of the innermost planet in Marois et al. (2010). revealed that the close proximity of the four primaries to the exact Laplace resonance makes the system stability robust even to apparently significant perturbations caused by probe masses as large as ≃2 MJup. We mark the model orbits of the two inner planets with red curves, and the astrometric measurements are over-plotted on them. Instant positions of the planets are marked with shaded large filled circles (for the first HST epoch), and large filled cir- cles for positions of the planets at the particular epoch labeled in the lower right corner of each panel. The time range of about 30 yr is more than half the orbital period of HR 8799e and roughly one orbital period of an asteroid involved in the 2:1e MMR with this planet. We considered two types of probe objects: Ceres-like aster- oids with a mass of 10−6 MJup and Jupiter-like planets with a mass of 1 MJup. To calculate the ⟨Y⟩values for the synthetic sys- tems, we integrated the N-body equations of motion and their variational equations with the GraggBulirsch–Stoer integrator (Hairer et al. 2000) for 3 Myr, which covers 6 × 104 orbital periods of HR 8799e and 7 × 103 orbital periods of HR 8799b, respectively. That integration time is consistent with the typical 104 outermost orbits required to achieve ⟨Y⟩convergence. We also note that the most significant interactions are exerted by the inner planets. We chose the integration time that is optimal from the CPU overhead point of view. Positions of the test particles are marked with different col- ors depending on their dynamical status: yellow and orange dots are for objects involved in 1:1e MMR with the innermost planet HR 8799e; green dots are for the 3:2e MMR, blue dots are for the 2:1e MMR, and gray dots are for the other stable asteroids. As can be seen in Fig. 4.7. Resonant structure of the inner debris disk The left column is for the exact Laplace resonance, the middle column is for a model displaced from the PO but rigorously stable, and the right column is for a >1 Gyr stable solution characterized by rotation of one critical angle of the 2:1 MMR in the outermost pair. The rows from the top to bottom are for all critical angles of the 2:1 MMR, the critical angle of the generalized Laplace MMR, and eccentricities for subsequent pairs of planets. See Table 7 for the initial conditions labeled from 1 to 3 at the top-left corner of each column. A&A 666, A133 (2022) Fig. 7. Orbital evolution of selected long-term-stable solutions with similar astrometric fit quality, but exhibiting qualitatively different stability signatures. The left column is for the exact Laplace resonance, the middle column is for a model displaced from the PO but rigorously stable, and the right column is for a >1 Gyr stable solution characterized by rotation of one critical angle of the 2:1 MMR in the outermost pair. The rows from the top to bottom are for all critical angles of the 2:1 MMR, the critical angle of the generalized Laplace MMR, and eccentricities for subsequent pairs of planets. See Table 7 for the initial conditions labeled from 1 to 3 at the top-left corner of each column. orbital phases, (a0, λe −λ0) (bottom panel). In both graphs, we mark the test particles with the same color scheme as in the previous plot, and the low-order resonances with HR 8799e are labeled. We also plot the geometrical collision curve (in gray) with planet HR 8799e constrained through the apocenter dis- tance of the inner orbit equal to the pericenter distance of the planet, a0(1 + e0) = ae(1 −ee). Also, the curve depicted in red is an image of the collision curve shifted by ∆a0 = 4 au towards the star and clearly marks a boundary of stable particles. the (a0, λe −λ0)-plane. Both graphs mark two families of aster- oids in 1:1e MMR – depicted in gold colour (e0 < 0.3) and in orange (e0 > 0.3). The low-eccentricity objects resemble clas- sic Trojan asteroids in the Solar System, relative by ±60◦to the planet. The second unusual family of highly eccentric particles in 1:1e MMR can be found on the sky plane far beyond the orbit of HR 8799e. 4.7. Resonant structure of the inner debris disk 10, the outer edge of the disk is highly nonsymmetric and quickly evolves in time. Its temporal struc- ture strongly changes even for a relatively very short interval of 25 years spanned by the astrometric observations of the system. The structure of the disk has a clear resonant structure that was also noted by Go´zdziewski & Migaszewski (2018). This is demonstrated in Fig. 11, which shows the distribution of canoni- cal osculating elements inferred in the Jacobi reference frame, in the (a0, e0)-plane (top panel), and on the plane of relative Positions of the test particles are marked with different col- ors depending on their dynamical status: yellow and orange dots are for objects involved in 1:1e MMR with the innermost planet HR 8799e; green dots are for the 3:2e MMR, blue dots are for the 2:1e MMR, and gray dots are for the other stable asteroids. As can be seen in Fig. 10, the outer edge of the disk is highly nonsymmetric and quickly evolves in time. Its temporal struc- ture strongly changes even for a relatively very short interval of 25 years spanned by the astrometric observations of the system. The results for the less massive Ceres-like asteroids are illus- trated in Fig. 10 and Fig. 11. In this experiment, we sampled the semi-major axis a0 ∈[4, 18] au and eccentricity e0 ∈[0, 0.8] of these objects. We collected 3 × 105 N-body initial conditions with \|⟨Y⟩−2\| < 0.05 that represent the structure of long-term- stable orbits in the inner debris disk. Subsequent panels in Fig. 10 illustrate snapshots of the disk at other epochs following the first observation (t0 =1998.830) as seen on the sky plane. To obtain the snapshots, we numerically integrated the whole set of ini- tial conditions for planets and the Ceres-like objects defined at The structure of the disk has a clear resonant structure that was also noted by Go´zdziewski & Migaszewski (2018). This is demonstrated in Fig. 11, which shows the distribution of canoni- cal osculating elements inferred in the Jacobi reference frame, in the (a0, e0)-plane (top panel), and on the plane of relative A133, page 13 of 25 A133, page 13 of 25 A&A 666, A133 (2022) Fig. 7. Orbital evolution of selected long-term-stable solutions with similar astrometric fit quality, but exhibiting qualitatively different stability signatures. A133, page 14 of 25 4.7. Resonant structure of the inner debris disk At some epochs (e.g., 2011.83), these appear closer to the orbit of HR 8799d, which may be counter-intuitive. Given the 1:1e MMR dynamical classification, they would be expected to share the same orbit as the planet. y y p The bottom graph shows objects involved in low-order MMRs with HR 8799e that are apparently widely spread on the sky plane, but they appear in narrow and well-localized islands on the planes of orbital elements; this is particularly clear on p This simulation reveals the dynamical structure composed of long-term-stable but mutually noninteracting asteroids in the system, on the same orbit as the innermost planet, whose orbital A. Zurlo et al.: Dynamical analysis of HR 8799 from SPHERE and LUCI astrometry. A. Zurlo et al.: Dynamical analysis of HR 8799 from SPHERE and LUCI astrometry. Fig. 8. Orbital evolution of selected long-term-stable chaotic solutions with similar astrometric fit quality. The rows from the top to bottom are or all critical angles of the 2:1 MMR, the critical angle of the generalized Laplace MMR, and eccentricities for subsequent pairs of planets. See Table 7 for the initial conditions labeled from 5 to 8 at the top of each column. y y Fig. 8. Orbital evolution of selected long-term-stable chaotic solutions with similar astrometric fit quality. The rows from the top to bottom are for all critical angles of the 2:1 MMR, the critical angle of the generalized Laplace MMR, and eccentricities for subsequent pairs of planets. See Table 7 for the initial conditions labeled from 5 to 8 at the top of each column. the warm disk is estimated down to 610 au (e.g., Su et al. 2009; Matthews et al. 2014; Chen et al. 2014), which coincides with the 2:1e MMR (Fig.11); see also Sect 5. Given the topic is complex and somewhat out of the scope of the present work, we postpone in-depth analysis of the dynamical structure of the warm dust disk to a future paper. evolution is governed by the gravitational interactions with the massive Jovian companions. Such objects should not be signif- icantly affected by nongravitational forces, such as Poynting- Robertson drag or the Yarkovsky effect. However, such forces may be crucial for the investigation and modeling of the actual emission profile of dust produced by collisional dynamics. The results indicate that the asteroid breakup events may be frequent and violent. 4.7. Resonant structure of the inner debris disk Given the large eccentricities and the instant mix- ing of different resonant fractions of these objects in the wide inner region between 4 au (and below) and 18 au, including wide Lagrange clumps of planet HR 8799e, their orbital velocity dis- persion may be significant. The interplay of all these factors – and especially the clearing rate of the dust due to radiation of the young and bright host star – determines the emission profile. The present observational evidence is limited, and the position of 4.8. Putative fifth Jupiter-like planet We performed a very similar experiment for test particles of mass 1 MJup, a hypothetical planet below the current detection level that may exist in the inner part of the HR 8799 system. The results are illustrated in Fig. 12 in a similar way to in Figs. 10–11. We collected 105 stable solutions. Again, the positions of the hypothetical planet are marked at characteristic epochs; for A133, page 15 of 25 A133, page 15 of 25 A&A 666, A133 (2022) Fig. 9. Dynamical maps in the Keplerian, osculating astrocentric semi-major-axis–eccentricity plane for the quasi-periodic, near-resonant config- uration described by Model 2. All masses and elements but the map coordinates are fixed at their best-fitting values in Table 7. The osculating epoch is 1998.830. Stable solutions are determined with \|⟨Y⟩−2\| ≃0 and marked with blue color. Fig. 9. Dynamical maps in the Keplerian, osculating astrocentric semi-major-axis–eccentricity plane for the quasi-periodic, near-resonant config- uration described by Model 2. All masses and elements but the map coordinates are fixed at their best-fitting values in Table 7. The osculating epoch is 1998.830. Stable solutions are determined with \|⟨Y⟩−2\| ≃0 and marked with blue color. 7 and 16 au lies above roughly three Jupiter masses, as also con- cluded here; see Fig. 13 and discussion in Sect. 6. Therefore, we believe that steadily improved imaging techniques, gaining better contrast and lower detection limits, combined with dynam- ical simulations similar to those conducted in this section may eventually reveal the fifth planet. However, if a relatively massive planet of mass ≃1 MJup exists in the inner part of the system, the outer edge of the debris disk carved out by this planet would have a much smaller radius than predicted at ≃10 au under the current observational configuration of four planets (Fig. 11), and related to the above-mentioned detections of warm dust at 6–10 au (e.g., Su et al. 2009; Hughes et al. 2011; Matthews et al. 2014; Chen et al. 2014). We defer the analysis of this situation to a future work as well, because of its complexity arising from different MMR scenarios. example, near the first epoch of the HST detection, up to the last epoch of measurements in this work, and one epoch ahead of that time. Compared to the previous case, the distribution of stable objects on the plane of the sky is much narrower and more restricted. 4.8. Putative fifth Jupiter-like planet As in the case of low-mass asteroids, the positions of the putative planets can be seen to change rapidly relative to the background model orbits and astrometric measurements. 7 and 16 au lies above roughly three Jupiter masses, as also con- cluded here; see Fig. 13 and discussion in Sect. 6. Therefore, we believe that steadily improved imaging techniques, gaining better contrast and lower detection limits, combined with dynam- ical simulations similar to those conducted in this section may eventually reveal the fifth planet. However, if a relatively massive planet of mass ≃1 MJup exists in the inner part of the system, the outer edge of the debris disk carved out by this planet would have a much smaller radius than predicted at ≃10 au under the current observational configuration of four planets (Fig. 11), and related to the above-mentioned detections of warm dust at 6–10 au (e.g., Su et al. 2009; Hughes et al. 2011; Matthews et al. 2014; Chen et al. 2014). We defer the analysis of this situation to a future work as well, because of its complexity arising from different MMR scenarios. We note that the stability limit (red curve) in the bottom plot for the (a0, e0)-plane is offset by ∆a0 = 6 au with respect to the collision curve with planet HR 8799e shown in gray. In addition to the clear dependence of the structure of stable solutions on the probe mass, this simulation indicates that the hypothetical planet may be located only on very narrow islands in the orbital param- eter space, limited to low-order resonances with the innermost planet. The temporal evolution of these islands in the sky can be useful for analyzing AO images and provides clues as to where an additional, Jovian planet might be expected. If such objects exist beyond ≃7 au, they should be involved in a 2:1e, 3:1e, or 5:2e MMR with the inner planet, respectively. These low-order reso- nances may be preferred if the system has undergone a migration in the past. We discuss this further in Sect. 5.ii 5. Possible history of the system Convergent migration due to tidal interaction with the nesting circumstellar disk is thought to be the most reliable mechanism for producing resonance trapping in multiple-planet systems (Masset & Snellgrove 2001; Lee & Peale 2002; Moorhead & Adams 2005; Thommes 2005; Beaugé et al. 2006; Crida et al. 2008; D’Angelo & Marzari 2012). The different masses of the planets and progressive inside-out depletion of gas in the disk due to the presence of the planet itself and photo-evaporation lead to different migration speeds for the planets, which may end up in resonance. Numerical modeling by Hands et al. (2014) and Szuszkiewicz & Podlewska-Gaca (2012) shows that during inward migration, planets are often trapped in resonances like the 2:1 and 3:2 (the most frequent). Attempts to find the hypothetical innermost fifth planet have so far been unsuccessful. Very recently, Wahhaj et al. (2021), using SPHERE measurements also reported here, did not detect planet HR 8799f at the most plausible locations, namely 7.5 and 9.7 au, down to mass limits of 3.6 and 2.8 MJup, respectively. Neither did these authors detect any new candidate companions at the smallest observable separation, of namely 0.1′′ or ≃4.1 au, overlapping with the semi-major axes range in Figs. 10–12. Wahhaj et al. (2021) conclude that the planet may still exist with a mass of 2–3.6 MJup at 7.5 au (3:1e MMR) or 1.5–2.8 MJup at 10 au, which is in the region we closely investigated in this sec- tion. The contrast curve in the interesting zone between roughly While in most resonant cases, such as Gliese 876, HD 82943, and HD 37124 (Wright et al. 2011), the planets are close to the A133, page 16 of 25 A133, page 16 of 25 A. Zurlo et al.: Dynamical analysis of HR 8799 from SPHERE and LUCI astrometry. e 7. Osculating, heliocentric elements of the best-fitting solutions at the epoch of 1998.83 for models illustrated in Fig. 4 and in Figs. 5. Possible history of the system 7– Model 1: m⋆=1.47 M⊙, Π = 24.525662 mas, χ2 = 1593.22, RMS = 7.41 mas, log \|∆n[rad d−1]\| = –12.0 m (MJup) a (au) e I (deg) Ω(deg) ω (deg) M (deg) HR8799e 7.4060918 16.1038912 0.1476760 26.8734271 62.1852189 110.8167067 -23.1719500 HR8799d 9.1899948 26.4517267 0.1146003 26.8734271 62.1852189 29.0594738 60.1752106 HR8799c 7.7961185 40.9861801 0.0538090 26.8734271 62.1852189 92.3879462 145.5347082 HR8799b 5.8023594 71.1427851 0.0166707 26.8734271 62.1852189 42.9704380 -49.1049354 Model 2: m⋆=1.47 M⊙, Π = 24.451807 mas, χ2 = 1568.19, RMS = 7.66 mas, log \|∆n[rad d−1]\| = –6.2 m (MJup) a (au) e I (deg) Ω(deg) ω (deg) M (deg) HR8799e 7.8680000 15.9753973 0.1524708 26.2394739 63.7770393 108.3445443 335.6210695 HR8799d 9.6490000 26.6396632 0.1145654 26.2394739 63.7770393 28.3126706 59.7394429 HR8799c 7.3081300 40.9406369 0.0517422 26.2394739 63.7770393 91.3551907 145.0637721 HR8799b 5.6590700 71.3289844 0.0173411 26.2394739 63.7770393 41.6627885 310.5199311 Model 3: m⋆=1.52 M⊙, Π = 24.347839 mas, χ2 = 1624.73, RMS = 7.66 mas, log \|∆n[rad d−1]\| = –4.5 m (MJup) a (au) e I (deg) Ω(deg) ω (deg) M (deg) HR8799e 8.9817200 16.1163303 0.1548634 27.3470617 62.9005413 108.0955330 336.2997163 HR8799d 9.0680400 26.8768634 0.1068892 27.3470617 62.9005413 29.2629241 60.0277429 HR8799c 7.2212900 41.0832702 0.0627005 27.3470617 62.9005413 91.9708727 144.7587008 HR8799b 6.1950900 71.7822044 0.0192741 27.3470617 62.9005413 44.4483815 308.9107410 Model 4: m⋆=1.52 M⊙, Π = 24.305464 mas, χ2 = 1564.70, RMS = 7.64 mas, log \|∆n[rad d−1]\| = –6.2 m [MJup] a [au] e I (deg) Ω(deg) ω (deg) M (deg) HR8799e 10.6577500 16.1274692 0.1496852 26.9747476 63.6149718 107.6133926 336.6560551 HR8799d 9.5739000 26.8852716 0.1169394 26.9747476 63.6149718 27.5720349 60.0200173 HR8799c 6.8413200 41.2618982 0.0555455 26.9747476 63.6149718 93.1349542 143.0235158 HR8799b 5.7085500 71.9446080 0.0217920 26.9747476 63.6149718 45.1149621 307.6876430 Model 5: m⋆=1.47 M⊙, Π = 24.466345 mas, χ2 = 1591.33, RMS = 7.63 mas, log \|∆n[rad d−1]\| = –6.9 m [MJup] a [au] e I (deg) Ω(deg) ω (deg) M (deg) HR8799e 7.1130900 15.9976012 0.1545297 26.5969851 62.8989533 108.8687608 336.1919760 HR8799d 9.8864900 26.5290674 0.1122973 26.5969851 62.8989533 27.8297522 61.2151462 HR8799c 7.7520500 41.1188833 0.0497536 26.5969851 62.8989533 92.4070918 144.9763772 HR8799b 5.5380200 71.3013150 0.0172797 26.5969851 62.8989533 44.6821435 308.5634968 Model 6: m⋆=1.47 M⊙, Π = 24.539234 mas, χ2 = 1568.59, RMS = 7.63 mas, log \|∆n[rad d−1]\| = –6.2 m [MJup] a [au] e I (deg) Ω(deg) ω (deg) M (deg) HR8799e 7.3387300 15.9246991 0.1553777 26.5990408 62.7197878 108.3820177 336.0139713 HR8799d 10.0516400 26.5092120 0.1117405 26.5990408 62.7197878 29.2671129 59.8339479 HR8799c 8.0973100 40.9565104 0.0509428 26.5990408 62.7197878 91.8212168 145.6257159 HR8799b 5.8932800 71.1073028 0.0167537 26.5990408 62.7197878 41.9648749 311.2692413 Table 7. 5. Possible history of the system Osculating, heliocentric elements of the best-fitting solutions at the epoch of 1998.83 for models illustrate ntric elements of the best-fitting solutions at the epoch of 1998.83 for models illustrated in Fig. 4 and in Figs. 7–8. star, in the case of HR 8799 the planets are significantly far away. This implies that the primordial disk from which they formed should have extended beyond 100 au which is compatible with the mass of the star being ∼1.5 M⊙. The most robust scenario is that in which the planets were fully formed further out in the disk and then migrated inwards until they became progressively trapped in the multiple resonances. This scenario raises the ques- tion of whether these planets were formed by core accretion (Mizuno 1980; Pollack et al. 1996) or by gravitational instability (Cameron 1978; Boss 1997). The resonance trapping in this system at that distance from the star is confirmed by hydrodynamical simulations performed with the FARGO code (Masset 2000). Fully radiative models have been adopted where the energy equation contains viscous heating and radiative cooling through the disk surface. A polar grid with 682 × 512 elements is used to cover the disk, extend- ing from 1 to 120 au. The initial surface gas density is given by Σ = Σ0r−1/2 with Σ0 = 50 g cm−2. This low density is moti- vated by the evolved state of the disk when the planets are fully formed. To test the sequential resonance trapping, we start the star, in the case of HR 8799 the planets are significantly far away. This implies that the primordial disk from which they formed should have extended beyond 100 au which is compatible with the mass of the star being ∼1.5 M⊙. The most robust scenario is that in which the planets were fully formed further out in the disk and then migrated inwards until they became progressively trapped in the multiple resonances. This scenario raises the ques- tion of whether these planets were formed by core accretion (Mizuno 1980; Pollack et al. 1996) or by gravitational instability (Cameron 1978; Boss 1997). A133, page 17 of 25 A133, page 17 of 25 A&A 666, A133 (2022) Fig. 10. Evolution of the inner debris disk as seen on the plane of the sky. 5. Possible history of the system The subsequent panels show the instantaneous positions of two inner planets and small asteroids in stable orbits (small colored dots) with a mass of 10−6 MJup at the epoch labeled in the lower-left corner of each graph. We collected 3.3 × 105 particles. Their distribution in the (a0, e0)-plane of osculating, canonical elements at the initial epoch 1998.830 is shown in Fig. 11. The largest filled circles correspond to the positions of the planets HR 8799e and HR 8799d at the snapshot epoch (large filled circles) and the initial osculating epoch (shaded filled circles), respectively. The asteroid colors are for the lowest order MMR types, as indicated in the bottom two plots, or those belonging to the innermost quasi-homogeneous disk (gray points). The 2024.83 epoch covers roughly one orbital period in a 2:1e MMR resonance (dark blue circles) with the innermost planet. A&A 666, A133 (2022) A&A 666, A133 (2022) Fig. 10. Evolution of the inner debris disk as seen on the plane of the sky. The subsequent panels show the instantaneous positions of two inner planets and small asteroids in stable orbits (small colored dots) with a mass of 10−6 MJup at the epoch labeled in the lower-left corner of each graph. We collected 3.3 × 105 particles. Their distribution in the (a0, e0)-plane of osculating, canonical elements at the initial epoch 1998.830 is shown in Fig. 11. The largest filled circles correspond to the positions of the planets HR 8799e and HR 8799d at the snapshot epoch (large filled circles) and the initial osculating epoch (shaded filled circles), respectively. The asteroid colors are for the lowest order MMR types, as indicated in the bottom two plots, or those belonging to the innermost quasi-homogeneous disk (gray points). The 2024.83 epoch covers roughly one orbital period in a 2:1e MMR resonance (dark blue circles) with the innermost planet. inner planets on already resonant orbits; they are not affected by the disk, and so do not migrate. The outer planet instead feels the disk perturbations and migrates inward until it is trapped in the 2:1 resonance and stops migrating. This behavior is illustrated in Fig. 14 where in the top panel the semi–major axis of the fourth planet is shown while in the middle and bottom panels the crit- ical argument of the 2:1 resonance between the third and fourth planet and the Laplace resonance argument is illustrated. 6. Planets–disk interaction Along with the four giant planets detected so far, the architecture of HR 8799 is enriched by the presence of an extended debris disk with two components. The cold Kuiper-like component was resolved in the FIR (Matthews et al. 2014) and at millimeter wavelengths (Booth et al. 2016; Wilner et al. 2018) and was detected up to 450 au from the star, with a halo extending to thousands of astronomical units (au). The warm component was instead never fully resolved and its inferred position of 9.3 au is given by modeling the IR excess at 155 K (Chen et al. 2014) in the SED of the star under the assumption of dust particles behaving like black bodies. The two components are separated by a huge dust-free gap that extends from 109 au (Wilner et al. 2018) to regions interior to the orbit of HR 8799e. p Figure 13 shows a summary of the results discussed above. The four detected planets are represented as pink circles together with the extension of their chaotic zone (pink shaded area). As it emerges from the image, the inner extension of the clearing zone gets very close to the inner belt (blue vertical line) whereas it stops roughly 20 au from the outer belt. Even a very low mass planet could carve the remaining part of the gap and, consistently with our observations, it would be too small to be detected with an instrument such as SPHERE, as proven by the detection lim- its (red curve; IRDIS data taken on 2017 October 12) shown in the figure. Simulations of the inner debris disk in the framework of the N-body resonant model of the system in Sect. 4.7 indicate possible localizations of such a planet. We want to stress that this is only a preliminary analysis of the planet–disk interaction. More detailed studies will have to consider the dynamical inter- action of the fifth planet with all four of the detected ones as well as the possibility of having a larger object locked in MMR with at least one of the other known planets, as discussed in the following. 5. Possible history of the system simulation, we increased Σ0 to 100 g/cm−2 in order to speed up the migration. Figure 15 shows the gas density distribution after 8 Kyr from the beginning of the simulation. The planets create a common gap where some overdensities are still present in the corotation regions of each planet. The semi-major axes of the planets are illustrated in the bottom panel, rescaled to be shown in a single plot. They migrate inwards while trapped in resonance, suggesting that the present position of the planets is not necessarily the one where they became trapped in resonance. The capture in the mutual resonance may have occurred farther out, followed by an inward migration until the disk dissipates. This may push the location where the planet initially grew even It is noteworthy that, once all the planets are trapped in res- onance, they share a common gap and migrate inwards. We performed an additional simulation where all four planets are affected by the disk perturbations and can freely migrate. In this A133, page 18 of 25 A133, page 18 of 25 A. Zurlo et al.: Dynamical analysis of HR 8799 from SPHERE and LUCI astrometry. Fig. 11. Structure of the inner debris disk composed of 3 × 105 Ceres-like asteroids of mass 10−6 MJup on stable orbits, seen in the (a0, e0)-plane of Jacobian (canonical) osculating elements at the initial 1998.830 epoch. The colors of the asteroids indicate the lowest order MMR in which they are involved with HR 8799e, as indicated in the diagrams, or belong to the innermost, quasi-homogeneous disk (gray points) according to Fig. 10. The light gray curve indicates the collision of the orbits with HR 8799e and the red curve is an image of the collision curve shifted by ∆a0 ≃4 au toward the star. We note that loose points above the collision curve in the top panel represent immediately scattered asteroids on regular but hyperbolic (open) orbits. Fig. 11. Structure of the inner debris disk composed of 3 × 105 Ceres-like asteroids of mass 10−6 MJup on stable orbits, seen in the (a0, e0)-plane of Jacobian (canonical) osculating elements at the initial 1998.830 epoch. The colors of the asteroids indicate the lowest order MMR in which they are involved with HR 8799e, as indicated in the diagrams, or belong to the innermost, quasi-homogeneous disk (gray points) according to Fig. 10. 5. Possible history of the system The light gray curve indicates the collision of the orbits with HR 8799e and the red curve is an image of the collision curve shifted by ∆a0 ≃4 au toward the star. We note that loose points above the collision curve in the top panel represent immediately scattered asteroids on regular but hyperbolic (open) orbits. farther out. It is noteworthy that the wider oscillations observed in the critical arguments of the resonances and the semi-major axis of the planets compared to a pure N-body problem are related to the presence of the perturbations of the disk on the planets. the inner edge of the gap. On the other hand, the chaotic zone for HR 8799b can clear the orbit of the planet up to 91.4 au. Given the position of the outer edge of the gap at 109 au, we can try to infer the characteristics of a fifth planet able to extend the chaotic zone up to the detected position of the edge. Following a very simple approach, we can use the same analytical tools to estimate the mass and semi-major axis of a planet able to carve a gap extending from 91.4 au to 109 au, that is, the free dynamical region left beyond the orbit of HR 8799b. As a result, we obtain a 0.12 MJup planet orbiting at 100 au. 6. Planets–disk interaction As the presence of the gap is tightly correlated with the plan- ets in it, we can assess the planets–disk interactions and infer whether or not the planets detected are responsible for carving the entire gap or whether or not there is further dynamical space for hosting other undetected planets. For this analytical study, the only planets taken into account are the two closest to the edges of the gap, HR 8799 b and e. The orbital parameters and masses adopted are the ones listed in Tables 6 and 7 for Model 1. To calculate the extension of the region from which dust particles are scattered away from the orbit of the planet (chaotic zone), we used Eqs. (9) and (10) of Lazzoni et al. (2018) for HR 8799b and HR 8799e, respectively. As a result, we obtained that the chaotic zone of the inner planet extends down to 9.8 au, which is con- sistent with the direct N-body simulations in Sect. 4.7. Given the uncertainties on the position of the inner belt and on its width, we can safely state that HR 8799e is likely responsible for shaping Indeed, the results of numerical N-body simulations in Go´zdziewski & Migaszewski (2018) based on the ⟨Y⟩-model described in Sect. 4.7 partially confirm the analytical predictions and address the likely real resonant configuration of the planets. These latter authors simulated the inner edge of the outer debris disk, also accounting for the presence of a hypothetical fifth planet with a mass between 0.1 and 1.66 MJup. These simula- tions are based on a quasi-periodic, near-resonant orbital model A133, page 19 of 25 A133, page 19 of 25 A&A 666, A133 (2022) A&A 666, A133 (2022) A&A 666, A133 (2022) Fig. 12. Possible instant positions (small gray and colored dots) of a hypothetical and still undetected planet of mass 1 MJup on the sky plane, at the epoch labeled in the lower-left corner of each graph. We collected 105 initial conditions. The bottom plot shows their distribution in the (a0, e0)– plane of canonical osculating elements at the initial epoch of 1998.830, similarly to Fig. 11. As in Fig. 10, the largest filled circles correspond to the positions of the planets HR 8799e and HR 8799d at the snapshot epoch (large filled circles) and the initial osculating epoch (shaded filled circles), respectively. 6. Planets–disk interaction Colors mark the lowest order MMR types, as labeled in the bottom plot. The light-gray curve marks the collision zone with HR 8799e, the thinner curve marks the stability zone for m0 = 10−6 MJup (see Fig. 10), and the red curve is the image of the geometric collision curve shifted by ∆a0 ≃6 au toward the star. A133, page 20 of 25 Fig. 12. Possible instant positions (small gray and colored dots) of a hypothetical and still undetected planet of mass 1 MJup on the sky plane, at the epoch labeled in the lower-left corner of each graph. We collected 105 initial conditions. The bottom plot shows their distribution in the (a0, e0)– plane of canonical osculating elements at the initial epoch of 1998.830, similarly to Fig. 11. As in Fig. 10, the largest filled circles correspond to the positions of the planets HR 8799e and HR 8799d at the snapshot epoch (large filled circles) and the initial osculating epoch (shaded filled circles), respectively. Colors mark the lowest order MMR types, as labeled in the bottom plot. The light-gray curve marks the collision zone with HR 8799e, the thinner curve marks the stability zone for m0 = 10−6 MJup (see Fig. 10), and the red curve is the image of the geometric collision curve shifted by ∆a0 ≃6 au toward the star. A133, page 20 of 25 A. Zurlo et al.: Dynamical analysis of HR 8799 from SPHERE and LUCI astrometry. Fig. 13. HR 8799 architecture: two belts (blue lines), four detected plan- ets (pink circles), and a putative fifth planet (light-blue circle). The pink shaded area represents the extension of the chaotic zone whereas the gray shaded zone represents the extension of the outer disk. The con- trast curve for the system is shown in red. Fig. 14. Resonance trapping of the HR 8799 system during the migra- tion phase. The upper panel shows the evolution of the semi-major axis of the outer planet at the moment of capture in resonance with the inner three bodies. After the resonance trapping, the semi-major axis remains constant. The middle panel shows the critical argument of the resonance between the two outer planets. After the capture in resonance, the criti- cal argument librates. The same occurs for the Laplace critical argument shown in the bottom panel. Fig. 13. 7. Conclusions Fig. 14. Resonance trapping of the HR 8799 system during the migra- tion phase. The upper panel shows the evolution of the semi-major axis of the outer planet at the moment of capture in resonance with the inner three bodies. After the resonance trapping, the semi-major axis remains constant. The middle panel shows the critical argument of the resonance between the two outer planets. After the capture in resonance, the criti- cal argument librates. The same occurs for the Laplace critical argument shown in the bottom panel. The system around HR 8799 is a unique laboratory with which to study the mutual gravitational interactions between the planets and their relation with the circumstellar disk in the early evolu- tionary stages of the system. To better understand the dynamics of this system, we followed up HR 8799 with SPHERE at the Very Large Telescope and with LUCI at the Large Binocu- lar Telescope to refine the orbital parameters of the system. We reduced the new data with state-of-the-art algorithms that apply the ADI technique, and for consistency, we also repeated the reduction of published SPHERE data from open-time pro- grams. Precise astrometry for the four planets was obtained for 21 epochs from SPHERE and one epoch from LUCI. million years), even if they are ∼2 MJup bigger than the masses proposed in the previous dynamical studies. We find masses of 8–9 MJup for planets HR 8799 e, d, and c, while for the exte- rior planet HR 8799b we estimate a smaller mass of ≃6 MJup. Moreover, the dynamical parallax is consistent with 1σ uncer- tainty with the corrected, independently determined Gaia eDR3 value of 24.50 ± 0.05 mas, reinforcing both the adopted mass of the host star m⋆= 1.47 M⊙and the assumed resonant or close-to-resonant configuration of the system. We performed a detailed exploration of the orbital parame- ters for the four planets with dynamical constraints imposed by the 8:4:2:1 Laplace resonance. This model was updated using 68 epochs from our reduction and the literature. As a result, we re- derived the dynamical masses of the planets and the parallax of the system with minimal prior information. 6. Planets–disk interaction HR 8799 architecture: two belts (blue lines), four detected plan- ets (pink circles), and a putative fifth planet (light-blue circle). The pink shaded area represents the extension of the chaotic zone whereas the gray shaded zone represents the extension of the outer disk. The con- trast curve for the system is shown in red. of the four known planets derived through migration simula- tions. However, given that Go´zdziewski & Migaszewski (2018) adopted the larger mass for the star of m⋆= 1.52M⊙and a larger parallax Π = 25.4 mas, the whole system appears more compact than predicted at present – these authors found the osculating astrocentric semi-major axis of HR 8799b ≃67.1 au, compared to ≃71 au in the present models. The updated values of the par- allax and the stellar mass translate to orbits expanded by a few au. According to the simulations, the inner edge of the outer disk is globally highly nonsymmetric, and planets with a mass of between 0.1 and 1.66 MJup may be present beyond the clear- ing zone, with semi-major axis >90 au. These planets could be involved in low-order resonances, such as 3:2b, 5:3b, 2:1b, or 5:2b with the outermost planet HR 8799b and in low and moder- ate eccentricity orbits; see Fig. 9 in Go´zdziewski & Migaszewski (2018). The fifth planet, even if it had a small mass, would be strongly affecting the shape of the inner parts of the outer debris disk. 7. Conclusions The derived masses are consistent with the prediction from the evolutionary models and allow long-term stability of the orbits (over a few hundred Regarding the quality of the N-body astrometric models con- sidered in this work, we did not find any significant or qualitative improvement of multi-parameter near-resonant configurations A133, page 21 of 25 A133, page 21 of 25 A&A 666, A133 (2022) Fig. 15. Orbital evolution of the HR 8799 system during the migration phase. The upper panel shows the gas density of the disk. The bottom panel shows the semi-major axes of the planets rescaled with constant values for ease of visualization in the same plot. planet HR 8799e, and there is no need for an additional inner planet to explain it. However, we also determined the dynami- cal structure of this region with direct N-body simulations. The results may be useful for predicting the positions of small-mass objects below the present detection limits of ≃3 MJup. Acknowledgements. We are thankful to the referee for the constructive report. A.Z. acknowledges support from the FONDECYT Iniciación en investigación project number 11190837 and ANID – Millennium Science Initiative Program – Center Code NCN2021_080. K.G. is very grateful to Dr Cezary Migaszewski for sharing a template C code for computing periodic orbits in a few planet sys- tems and explanations regarding the PO approach. K.G. thanks the staff of the Pozna´n Supercomputer and Network Centre (PCSS, Poland) for the generous long-term support and computing resources (grant No. 529). K.G. also thanks the staff of the Tricity Supercomputer Centre, Gda´nsk (Poland) for comput- ing resources on the Tryton cluster that were used to conduct some numerical experiments and for their great and professional support. A.V. acknowledges funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme, grant agreements no. 757561 (HiRISE). The LBTO AO group would like to acknowledge the assistance of R.T. Gatto with night observations. We would also like to thank J. Power for assisting in preparation of the observations using ADI mode. For the purpose of open access, the authors have applied a Creative Commons Attribution (CC BY) licence to any author accepted manuscript version arising from this submis- sion. 7. Conclusions SPHERE is an instrument designed and built by a consortium consisting of IPAG (Grenoble, France), MPIA (Heidelberg, Germany), LAM (Marseille, France), LESIA (Paris, France), Laboratoire Lagrange (Nice, France), INAF – Osservatorio di Padova (Italy), Observatoire de Genève (Switzerland), ETH Zurich (Switzerland), NOVA (Netherlands), ONERA (France), and ASTRON (Netherlands), in collaboration with ESO. SPHERE was funded by ESO, with additional contributions from CNRS (France), MPIA (Germany), INAF (Italy), FINES (Switzerland), and NOVA (Netherlands). SPHERE also received funding from the European Commission Sixth and Seventh Framework Programmes as part of the Optical Infrared Coordination Network for Astronomy (OPTICON) under grant number RII3-Ct-2004-001566 for FP6 (2004–2008), grant number 226604 for FP7 (2009–2012) and grant number 312430 for FP7 (2013–2016). References Fig. 15. Orbital evolution of the HR 8799 system during the migration phase. 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(2016) 2007.81 1500 7 836 7 -678 7 676 7 -175 10 -589 10 – – – – Konopacky et al. (2016) 2008.52 1527 4 799 4 -658 4 701 4 -208 4 -582 4 – – – – Marois et al. (2008) 2008.61 1527 2 801 2 -657 2 706 2 -216 2 -582 2 – – – – Marois et al. (2008) 2008.71 1516 4 818 4 -663 3 693 3 -202 4 -588 4 – – – – Konopacky et al. y Masset, F. 2000, A&AS, 141, 165 (2016) 2008.89 1532 20 796 20 -654 20 700 20 -217 20 -608 20 – – – – Hinz et al. (2010) 2009.02 – – – – -612 30 665 30 – – – – – – – – Hinz et al. (2010) 2009.58 1526 4 797 4 -639 4 712 4 -237 3 -577 3 -306 7 -211 7 Konopacky et al. (2016) 2009.62 1536 10 785 10 – – – – – – – – – – – – Currie et al. (2011) 2009.70 1538 30 777 30 -634 30 697 30 -282 30 -590 30 – – – – Hinz et al. (2010) 2009.76 1535 20 816 20 -636 40 692 40 -270 70 -600 70 – – – – Bergfors et al. (2011) 2009.77 1532 7 783 7 -627 7 716 7 -241 7 -586 7 -306 7 -217 7 Currie et al. (2011) 2009.83 1524 10 795 10 -636 9 720 9 -251 7 -573 7 -310 9 -187 9 Konopacky et al. (2016) 2009.84 1540 19 800 19 -630 13 720 13 -240 14 -580 14 -304 10 -196 10 Galicher et al. (2011) 2010.53 1532 5 783 5 -619 4 728 4 -265 4 -576 4 -323 6 -166 6 Konopacky et al. (2016) 2010.55 1547 6 757 9 -606 6 725 6 -269 6 -580 6 -329 6 -178 6 Currie et al. (2014) 2010.83 1535 15 766 15 -607 12 744 12 -296 13 -561 13 -341 16 -143 16 Konopacky et al. (2016) 2011.55 1541 5 762 5 -595 4 747 4 -303 5 -562 5 -352 8 -130 8 Konopacky et al. (2016) 2011.79 1579 11 734 11 -561 13 752 13 -299 13 -563 13 -326 13 -119 13 Esposito et al. (2013) 2011.86 1546 11 725 11 -578 13 767 13 -320 13 -549 13 -382 16 -127 16 Esposito et al. (2013) 2012.55 1545 5 747 5 -578 5 761 5 -339 5 -555 5 -373 8 -84 8 Konopacky et al. (2016) 2012.82 1549 4 743 4 -572 3 768 3 -346 4 -548 4 -370 9 -76 9 Konopacky et al. (2016) 2012.83 1558 6 729 9 -557 6 763 6 -343 6 -555 6 -371 6 -80 6 Currie et al. 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(2011) 2002.54 1481 23 919 17 – – – – – – – – – – – – Fukagawa et al. (2009) 2004.53 1471 6 884 6 -739 6 612 6 – – – – – – – – Konopacky et al. (2016) 2005.54 1496 5 856 5 -713 5 630 5 -87 10 -578 10 – – – – Currie et al. (2012) 2007.58 1504 3 837 3 -683 4 671 4 -179 5 -588 5 – – – – Konopacky et al. (2016) 2007.81 1500 7 836 7 -678 7 676 7 -175 10 -589 10 – – – – Konopacky et al. (2016) 2008.52 1527 4 799 4 -658 4 701 4 -208 4 -582 4 – – – – Marois et al. (2008) 2008.61 1527 2 801 2 -657 2 706 2 -216 2 -582 2 – – – – Marois et al. (2008) 2008.71 1516 4 818 4 -663 3 693 3 -202 4 -588 4 – – – – Konopacky et al. (2016) 2008.89 1532 20 796 20 -654 20 700 20 -217 20 -608 20 – – – – Hinz et al. (2010) 2009.02 – – – – -612 30 665 30 – – – – – – – – Hinz et al. y Masset, F. 2000, A&AS, 141, 165 (2010) 2009.58 1526 4 797 4 -639 4 712 4 -237 3 -577 3 -306 7 -211 7 Konopacky et al. (2016) 2009.62 1536 10 785 10 – – – – – – – – – – – – Currie et al. (2011) 2009.70 1538 30 777 30 -634 30 697 30 -282 30 -590 30 – – – – Hinz et al. (2010) 2009.76 1535 20 816 20 -636 40 692 40 -270 70 -600 70 – – – – Bergfors et al. (2011) 2009.77 1532 7 783 7 -627 7 716 7 -241 7 -586 7 -306 7 -217 7 Currie et al. (2011) 2009.83 1524 10 795 10 -636 9 720 9 -251 7 -573 7 -310 9 -187 9 Konopacky et al. (2016) 2009.84 1540 19 800 19 -630 13 720 13 -240 14 -580 14 -304 10 -196 10 Galicher et al. (2011) 2010.53 1532 5 783 5 -619 4 728 4 -265 4 -576 4 -323 6 -166 6 Konopacky et al. (2016) 2010.55 1547 6 757 9 -606 6 725 6 -269 6 -580 6 -329 6 -178 6 Currie et al. (2014) 2010.83 1535 15 766 15 -607 12 744 12 -296 13 -561 13 -341 16 -143 16 Konopacky et al. (2016) 2011.55 1541 5 762 5 -595 4 747 4 -303 5 -562 5 -352 8 -130 8 Konopacky et al. (2016) 2011.79 1579 11 734 11 -561 13 752 13 -299 13 -563 13 -326 13 -119 13 Esposito et al. (2013) 2011.86 1546 11 725 11 -578 13 767 13 -320 13 -549 13 -382 16 -127 16 Esposito et al. (2013) 2012.55 1545 5 747 5 -578 5 761 5 -339 5 -555 5 -373 8 -84 8 Konopacky et al. (2016) 2012.82 1549 4 743 4 -572 3 768 3 -346 4 -548 4 -370 9 -76 9 Konopacky et al. (2016) 2012.83 1558 6 729 9 -557 6 763 6 -343 6 -555 6 -371 6 -80 6 Currie et al. (2014) 2013.79 1545 22 724 22 -542 22 784 22 -382 16 -522 16 -373 13 -17 13 Konopacky et al. (2016) 2013.81 1562 8 713 13 -538 6 784 13 -377 7 -538 11 -394 11 -36 17 Maire et al. (2015) 2013.88 – – – – -537 1 782 2 -370 1 -539 1 -381 2 -30 0 Wang et al. y Masset, F. 2000, A&AS, 141, 165 (2018)1 2016.88 – – – – – – – – -464 1 -486 2 -382 2 94 6 IFS, This work 2016.88 1589 2 666 1 -464 2 824 2 -454 2 -489 2 -378 4 90 2 IRDIS, This work 2017.45 – – – – – – – – -473 2 -476 2 -377 1 115 3 IFS, This work 2017.45 1591 1 653 1 -449 1 835 1 -472 2 -482 2 -373 3 118 2 IRDIS, This work 2017.78 – – – – – – – – -480 2 -478 2 -372 1 129 2 IFS, This work 2017.78 1595 1 647 1 -441 1 839 1 -480 1 -477 1 -369 1 128 1 IRDIS, This work 2017.78 – – – – – – – – -492 5 -463 6 -370 2 135 3 IFS, This work 2017.78 1595 1 647 1 -441 1 839 1 -480 1 -477 1 -371 2 128 2 IRDIS, This work 2018.46 – – – – – – – – -495 1 -460 2 -360 2 162 2 IFS, This work 2018.46 1601 1 635 1 -424 1 848 1 -497 2 -463 2 -358 2 156 2 IRDIS, This work 2018.63 – – – – – – – – -509 2 -452 3 -361 2 166 2 IFS, This work 2018.63 1601 2 632 3 -421 1 850 1 -502 1 -461 1 -357 1 162 2 IRDIS, This work 2018.63 – – – – – – – – -503 2 -456 2 -359 1 167 2 IFS, This work 2018.63 1600 1 632 1 -421 1 851 1 -502 2 -458 1 -358 2 163 1 IRDIS, This work 2018.66 – – – – – – – – – – – – -358 0 163 0 GRAVITY Collaboration (2019) 2019.83 – - – – – – – – -527 3 -432 3 -337 3 210 3 IFS, This work 2019.83 1606 2 615 2 -392 2 875 2 -532 3 -425 2 -338 2 215 2 IRDIS, This work 2019.84 – – – – – – – – -528 1 -435 2 -337 2 208 2 IFS, This work 2019.84 1611 1 611 1 -388 2 870 2 -530 2 -430 1 -335 1 210 1 IRDIS, This work 2020.75 1620 1 591 3 -364 2 883 1 -551 1 -415 4 -315 3 242 5 LUCI, This work 2021.64 – – – – – – – – -569 1 -390 2 -292 2 276 2 IFS, This work 2021.64 1626 1 578 2 -339 2 890 2 -563 2 -391 2 -287 4 272 2 IRDIS, This work ( ) 2014.93 1574 4 701 3 -512 3 798 4 -400 4 -523 4 -390 7 12 4 IRDIS, This work 2014.93 1573 3 701 3 -512 3 797 4 -403 4 -524 4 -383 8 11 4 IRDIS, This work 2015.51 – – – – – – – – -424 4 -509 3 -391 1 33 2 IFS, This work 2015.51 1579 1 694 1 -498 1 806 1 -417 1 -517 1 -383 9 33 5 IRDIS, This work 2015.58 1580 5 689 3 -495 2 806 2 -419 2 -516 1 -386 1 36 1 IRDIS, This work 2015.65 1569 11 666 7 -482 5 813 6 -436 11 -510 12 – – – – Sepulveda & Bowler (2022) 2015.74 – – – – – – – – -420 4 -513 4 -392 1 39 3 IFS, This work 2015.74 1580 1 688 1 -494 1 811 1 -426 1 -512 1 -382 9 50 5 IRDIS, This work 2016.72 – – – – -466 1 821 2 -453 1 -499 1 -376 2 81 0 Wang et al. y Masset, F. 2000, A&AS, 141, 165 (2016) 2013.81 1562 8 713 13 -538 6 784 13 -377 7 -538 11 -394 11 -36 17 Maire et al. (2015) 2013.88 – – – – -537 1 782 2 -370 1 -539 1 -381 2 -30 0 Wang et al. (2018)a 2014.53 – – – – – – – – -400 4 -512 4 -389 1 -22 2 IFS, This work 2014.53 1570 3 704 3 -521 3 790 9 -391 2 -530 2 -387 2 -10 3 IRDIS, This work 2014.54 1560 13 725 13 -540 13 799 13 -400 11 -534 11 -387 11 3 11 Konopacky et al. (2016) 2014.62 – – – – – – – – -396 1 -524 2 -389 1 -17 2 IFS, This work 2014.70 1569 4 707 2 -519 1 794 2 -397 1 -530 2 – – – – Wang et al. (2018)1 2014.93 1575 2 702 4 -511 2 799 2 -400 2 -523 2 -385 3 12 2 Wertz et al. (2017) 2014.93 1574 3 701 2 -514 3 798 4 -399 4 -525 4 -389 8 11 4 IRDIS, This work A133, page 24 of 25 A. Zurlo et al.: Dynamical analysis of HR 8799 from SPHERE and LUCI astrometry. Table A.1: (continued) 2014.93 1574 4 701 3 -512 3 798 4 -400 4 -523 4 -390 7 12 4 IRDIS, This work 2014.93 1573 3 701 3 -512 3 797 4 -403 4 -524 4 -383 8 11 4 IRDIS, This work 2015.51 – – – – – – – – -424 4 -509 3 -391 1 33 2 IFS, This work 2015.51 1579 1 694 1 -498 1 806 1 -417 1 -517 1 -383 9 33 5 IRDIS, This work 2015.58 1580 5 689 3 -495 2 806 2 -419 2 -516 1 -386 1 36 1 IRDIS, This work 2015.65 1569 11 666 7 -482 5 813 6 -436 11 -510 12 – – – – Sepulveda & Bowler (2022) 2015.74 – – – – – – – – -420 4 -513 4 -392 1 39 3 IFS, This work 2015.74 1580 1 688 1 -494 1 811 1 -426 1 -512 1 -382 9 50 5 IRDIS, This work 2016.72 – – – – -466 1 821 2 -453 1 -499 1 -376 2 81 0 Wang et al. y Masset, F. 2000, A&AS, 141, 165 (2018)1 2016.88 – – – – – – – – -464 1 -486 2 -382 2 94 6 IFS, This work 2016.88 1589 2 666 1 -464 2 824 2 -454 2 -489 2 -378 4 90 2 IRDIS, This work 2017.45 – – – – – – – – -473 2 -476 2 -377 1 115 3 IFS, This work 2017.45 1591 1 653 1 -449 1 835 1 -472 2 -482 2 -373 3 118 2 IRDIS, This work 2017.78 – – – – – – – – -480 2 -478 2 -372 1 129 2 IFS, This work 2017.78 1595 1 647 1 -441 1 839 1 -480 1 -477 1 -369 1 128 1 IRDIS, This work 2017.78 – – – – – – – – -492 5 -463 6 -370 2 135 3 IFS, This work 2017.78 1595 1 647 1 -441 1 839 1 -480 1 -477 1 -371 2 128 2 IRDIS, This work 2018.46 – – – – – – – – -495 1 -460 2 -360 2 162 2 IFS, This work 2018.46 1601 1 635 1 -424 1 848 1 -497 2 -463 2 -358 2 156 2 IRDIS, This work 2018.63 – – – – – – – – -509 2 -452 3 -361 2 166 2 IFS, This work 2018.63 1601 2 632 3 -421 1 850 1 -502 1 -461 1 -357 1 162 2 IRDIS, This work 2018.63 – – – – – – – – -503 2 -456 2 -359 1 167 2 IFS, This work 2018.63 1600 1 632 1 -421 1 851 1 -502 2 -458 1 -358 2 163 1 IRDIS, This work 2018.66 – – – – – – – – – – – – -358 0 163 0 GRAVITY Collaboration (2019) 2019.83 – - – – – – – – -527 3 -432 3 -337 3 210 3 IFS, This work 2019.83 1606 2 615 2 -392 2 875 2 -532 3 -425 2 -338 2 215 2 IRDIS, This work 2019.84 – – – – – – – – -528 1 -435 2 -337 2 208 2 IFS, This work 2019.84 1611 1 611 1 -388 2 870 2 -530 2 -430 1 -335 1 210 1 IRDIS, This work 2020.75 1620 1 591 3 -364 2 883 1 -551 1 -415 4 -315 3 242 5 LUCI, This work 2021.64 – – – – – – – – -569 1 -390 2 -292 2 276 2 IFS, This work 2021.64 1626 1 578 2 -339 2 890 2 -563 2 -391 2 -287 4 272 2 IRDIS, This work A. Zurlo et al.: Dynamical analysis of HR 8799 from SPHERE and LUCI astrometry. Table A.1: (continued) A133, page 25 of 25
https://openalex.org/W2267513543	http://revistabiomedica.mx/index.php/revbiomed/article/download/426/437	es		Infección persistente por papillomavirus humano 18: estudio de caso.	Revista biomédica/Revista biomédica(en línea)	2,005	cc-by	1,527	medigraphic Artemisa en línea 255 Rev Biomed 2005; 16:255-257. Persistent infection of human papillomavirus 18: case report. Clinical Case 2 elaborado por medigraphic María del R. González-Losa1, Nina Valadez-González1,pdf Iván Rosado-López , Marylin Puerto-Solís1. 1 Laboratorio de Virología Centro de Investigaciones Regionales “Dr. Hideyo Noguchi”, Universidad Autónoma de Yucatán. 2Clínica de Displasias, Hospital General "Dr. Agustín O´Horán", Secretaria de Salud. Mérida, Yucatán, México. SUMMARY. Objective. This report shows that the presence of human papillomavirus (HPV) DNA in the genital tract could be the result of a persistent and not a recent infection. Study design. This case is part of a cross sectional study to determine the prevalence and genotypes of HPV in women with cervical cancer and precursor lesions. HPV diagnosis was done by polymerase chain reaction using MY09/MY11/ HMB01 primers; for genotyping line blot hybridization was used. Results. A biopsy of a woman with diagnosis of cervical cancer, who had sexual intercourse for the last time 30 years before, was positive to type 18 HPV. Conclusions. A new diagnosis of HPV in the genital tract is not necessarily the result of a recent infection. (Rev Biomed 2005; 16:255-257) Key words: Papillomavirus, chronic infection, sexually transmitted diseases. RESUMEN. Infección persistente por papillomavirus humano 18: estudio de caso. Objetivo. Este reporte tiene como objetivo demostrar que la presencia de ADN de papilomavirus humano (PVH) en el tracto genital puede ser el resultado de una infección persistente y no de un reciente contagio. Diseño del estudio. Este caso es parte de un estudio transversal, cuyo objetivo fue determinar la prevalencia y genotipos de PVH en mujeres con cáncer cervical y sus lesiones precursoras. El diagnóstico se realizó por medio de la técnica de reacción en cadena de la polimerasa, utilizando iniciadores universales MY09/MY11/HMB01; para genotipificar se utilizó hibridación en línea. Resultados. La biopsia de una paciente con diagnóstico de cáncer cervical invasor, que refirió no haber tenido vida sexual en los últimos 30 años, fue positiva PVH genotipo 18. Conclusiones. El diagnóstico de PVH en el tracto genital no implica necesariamente una infección Corresponding address: Dra. María del R. González-Losa. Centro de Investigaciones Regionales “Dr. Hideyo Noguchi”, Avenida Itzaez No. 490 x Calle 59, Centro, C.P. 97000, Mérida, Yucatán, México. Tel. 011 52 999 9 24 64 12 Ext. 157, 137 Fax. 011 52 999 9 23 61 20 E-mail: glosa@tunku.uady.mx Received March 14, 2005; Accepted May 11, 2005. This paper is also available at http://www.uady.mx/sitios/biomedic/revbiomed/pdf/rb051645.pdf Vol. 16/No. 4/Octubre-Diciembre, 2005 256 M del R González-Losa, N Valadez-González, I Rosado López, M Puerto-Solís. reciente. (Rev Biomed 2005; 16:255-257) Palabras clave: Papilomavirus, infección crónica, enfermedades de transmisión sexual. INTRODUCTION. To date HPV is the most frequent sexually transmitted infection. Its occurrence is associated with sexual activity, and in fact the prevalence of HPV peaks soon after initiation of sexual activity among young women (1). HPV infections are usually self-limited and revert spontaneously, with only a small group of women developing cervical cancer. The evolution of HPV infection to cancer is dependent on several factors, many of which are still unclear. However, persistent infections have been associated with the development of cervical cancer (2). Certain types HPV called "high risk": 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82 are the principal cause of invasive cervical cancer (3). In this case report we describe the presence of HPV type 18 in a cervical cancer of a woman who denied having had sexual activity during the previous 30 years. Case report. A 73 year old female patient was referred to the colposcopy clinic of hospital O´Horan in Merida, Yucatan, Mexico, with a Pap smear of invasive cervical cancer. Past history revealed first intercourse at 19 years, five pregnancies, four deliveries and one spontaneous abortion, menopause at age 50, last delivery occurring 36 years before the interview. She never used any contraceptive methods before or smoked, only had one sexual partner, and her last intercourse was 30 years before, at age 43, shortly before her husband died. A first pap smear was taken one month before the colposcopy. On that occasion the main reason for consultation was spontaneous vaginal bleeding. In the colposcopy an exophytic, friable, and bleeding lesion and dense aceto-white epithelium Revista Biomédica with diffuse border and abnormal vessels was seen. Two biopsies were taken from the lesion; one of which was placed in PBS and stored at -20ºC. DNA was extracted by phenol-chloroform separation. The presence of HPV DNA in the biopsies was determined by PCR L1 consensus MY09/MY11 primers. The samples were co-amplified with biotinylated MY09/MY11/HMB01 and βglobin primers GH20/PC04. HPV types were determined by reverse line blot hybridization that contained 29 probe lines detecting 27 individual HPV genotypes pdf por medigraphic andelaborado two concentrations of βglobin control probe. Samples were considered adequate if they hybridized with βglobin probe and HPV positive if they hybridized with any papillomavirus probe. DNA of SiHa was used as a positive control; PCR mix without DNA was used as negative control; the samples were positive for HPV 18 (figure 1). Histopatologic diagnosis was invasive squamouscell carcinoma. HPV 18 βGlobin Figure 1.- Results of the reverse line blot hybridization. Number nine is for the patient, the line at the top is specific for HPV 18; the two lines in the middle are two different concentrations of βglobin. Strip marked with + is positive control (DNA of SiHA). Strip marked – is negative control (PCR mix without DNA). 257 Persistent infection by HPV. DISCUSSION. Before we address the implication of this case, it is important that we consider its limitation. Persistent infection could not be determined with a cohort study indicating that the patient had DNA of the same HPV type in subsequent samples. Persistent infection was defined based on the patient´s history; the finding of HPV DNA in a patient without sexual activity in the previous three decades indicates the presence of an infection which has lasted for at least that time. Actually, HPV infection is the most common sexually transmitted infection. In 80% of young women the infection is transitory, with the virus being eliminated in a period of between 12 and 18 months (2). Persistent viral infection is a prerequisite for the development of cervical cancer. However, little has been written about the factors that promote or facilitate persistent viruses. In so far as the agent, it has been clear that high risk viruses shows greater tendency for persistence than those of low risk (2). Recently, smoking and low consumption of vegetables as well as a low concentration of seric cis-lycopene have been associated with persistent infection (4, 5). Epidemiological studies have shown that the number of sexual partners of women is directly related to the risk of developing cervical cancer. The present case is that of a monogamic woman, which is relevant in that it makes us think about the role the male plays in the development of the disease. The risk of monogamic women increases when there are signs of HPV in their partner (6). Nowadays HPV infection is an important reason for a visit to the gynecologist. Diagnosis of any sexually transmitted infection causes anxiety and stress in the patients (7). HPV diagnosis in women has psychological and emotional repercussions, since it creates doubts with respect to the fidelity of the partner, as it can be assumed that HPV infection is the result of a recent extramarital relation. Therefore, this often causes deteriorations in the couple´s relationship. One of the principal objectives in presenting this case is to show that finding HPV DNA in the genital tract does not necessarily have to be the result of a recent infection; it can be a persistent viral infection. We hope this information will allow medical doctors to face this diagnosis with the necessary tools, in order to give adequate information and support for couples who need it, and to reduce the psychological stress and the repercussions thatmedigraphic HPV diagnosis implies. pdf elaborado por REFERENCES. 1.- Lazacano-Ponce E, Herrero R, Muñoz N, Cruz A, Shah KV, Alonso P, et al. Epidemiology of HPV infection among Mexican women with normal cervical cytology. Int J Cancer 2001; 91:412-20. 2.- Ferenczy A, Franco E. Persistent human papillomavirus infection and cervical neoplasia. Lancet Oncol 2002; 3:116. 3.- Muñoz N, Bosch FX, Sanjosé S, Herrero R, Castellsagué X, Shah KV, et al. Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med 2003; 348:518-27. 4.- Giuliano AR, Sedjo RL, Roe DJ, Harri R, Baldwi S, Papenfuss MR, et al. Clearance of oncogenic human papillomavairus (HPV) infection: effect of smoking (United States). Cancer Causes Control 2002; 13:839-46. 5.- Sedjo RL, Roe DJ, Abrahamsen M, Harris RB, Craft N, Baldwin S, et al. Vitamin A, carotenoids, and risk of persistent oncogenic human papilomavirus. Cancer Epidemiol Biomarkers Prev 2002; 11:876-84. 6.- Castellsague X, Bosch X, Muñoz N, Meijer CJ, Shah KV, de Sanjosé S, et al. Male circumcision, penile human papillomavirus infection, and cervical cancer in female partners. N Engl J Med 2002; 346:1105-12. 7.- Linnehan MJ, Groce NE. Counseling and educational interventions of women with genital human papillomavirus infection. AIDS Patient Care STDS 2000; 14:439-45. Vol. 16/No. 4/Octubre-Diciembre, 2005
W2988265309.txt	https://iris.unimore.it/bitstream/11380/1192729/1/manfredi%20IndianJRheumatol144271-5933531_162855.pdf	en		Factors predicting early discontinuation of methotrexate as a first-line treatment for rheumatoid arthritis in Italy: Results from the GISEA registry	Indian journal of rheumatology/Indian Journal of Rheumatology	2,019	cc-by	4,191	[Downloaded free from http://www.indianjrheumatol.com on Thursday, February 13, 2020, IP: 155.185.104.10] Original Article Factors Predicting Early Discontinuation of Methotrexate as a First‑Line Treatment for Rheumatoid Arthritis in Italy: Results from the GISEA Registry Andreina Manfredi, Marco Sebastiani, Florenzo Iannone1, Elisa Gremese2, Alessandra Bortoluzzi3, Ennio Favalli4, Roberto Gorla5, Fausto Salaffi6, Enrico Fusaro7, Rosario Foti8, Luca Cantarini9, Roberto Caporali10, Alberto Cauli11, Stefano Alivernini2, Francesco Paolo Cantatore12, Antonio Carletto13, Fabrizio Conti14, Salvatore D’Angelo15, Oscar Epis16, Roberta Ramonda17, Antonio Marchesoni4, Gianfranco Ferraccioli2, Giovanni Lapadula1; On Behalf of GISEA (Italian Group for the Study of Early Arthritis) Department of Medicine, Rheumatology Unit, Policlinico Hospital of Modena, University of Modena and Reggio Emilia, Modena, 1Department of Medicine, Rheumatology Unit, University of Bari, Bari, 2Department of Medicine, Rheumatology Unit, Catholic University of the Sacred Heart, 14 Department of Internal Medicine, Sapienza University of Rome, Rome, 3Department of Medicine, Rheumatology Unit, Clinical and Experimental Medicine, Sant’Anna Hospital, University of Ferrara, Ferrara, 4Department of Rheumatology, Orthopedic Institute Gaetano Pini, 16Department of Medicine, Rheumatology Unit, ASST Grande Ospedale Metropolitano Niguarda, Milan, 5Department of Medicine, Rheumatology and Clinical Immunology Unit, Spedali Civili Di Brescia, Brescia, 6Department of Clinical and Molecular Sciences, Rheumatology Unit, Università Politecnica Delle Marche, Jesi, 7Department of Medicine, Rheumatology Unit, Città della Salute e della Scienza Hospital, Turin, 8Department of Medicine, Rheumatology Unit, A.O.U. Policlinico Vittorio Emanuele, Catania, 9Department of Medicine, Rheumatology Unit, University of Siena, Siena, 10Department of Medicine, Rheumatology Unit, IRCCS Policlinico San Matteo, University of Pavia, Pavia, 11Department of Medical Sciences, Rheumatology Unit, Policlinico of the University of Cagliari, Cagliari, 12Department of Medical and Surgical Sciences, Rheumatology Clinic, University of Foggia Medical School, Foggia, 13 Department of Medicine, Rheumatology Unit, University of Verona, Verona, 15Department of Medicine, Rheumatology Unit, “San Carlo” Hospital of Potenza and “Madonna delle Grazie” Hospital of Matera, Potenza, 17Department of Medicine, Rheumatology Unit, University of Padua, Padova, Italy Received: May, 2019 Accepted: August, 2019 Published: November, 2019 Address for correspondence: Dr. Andreina Manfredi, Rheumatology Unit, Policlinico Hospital of Modena, University of Modena and Reggio Emilia, Via Università, 4, 41124 Modena, Italy. E‑mail: andreina.manfredi@gmail. com Abstract Objective: Despite the well‑established efficacy of methotrexate (MTX) in rheumatoid arthritis (RA), monotherapy is not sufficient in almost half of patients. The aim of this registry‑based study was to detect possible predictive factors for the early failure of MTX as a first‑line treatment in early RA patients. Materials and Methods: Five‑hundred and ninety RA patients beginning MTX as the first‑line treatment were included. Persistence on therapy was re‑evaluated after 12 months. Baseline features of disease were evaluated by means of univariate Cox regression, and parameters significantly associated to the outcome were included in multivariate model. Results: One hundred and forty‑nine patients (25.3%) failed MTX during the 1st year, for inefficacy in 43.6% and adverse events in 37.5% of cases, respectively. At univariate analysis, patients who discontinued or failed treatment showed lower mean age, higher prevalence of anti‑citrullinated peptide antibodies (ACPAs), and higher number of tender/swollen joints. The dose of MTX was correlated with the efficacy and the tolerance of the drug. In particular, patients treated with 7.5 mg of MTX weekly showed a higher rate of discontinuation for inefficacy than adverse events, and the contrary was detected for higher doses. On multivariate analysis, age, ACPA, and number of tender joints were directly associated with MTX discontinuation or failure. Conclusions: More than 25% of RA patients treated with MTX as a first‑line therapy failed treatment at 12 months. ACPA positivity, age, and number of tender joints were associated with early withdrawal of MTX in RA patients, while the dose of MTX was correlated to the efficacy and safety of the drug. Key Words: Anti‑citrullinated protein antibodies, methotrexate, rheumatoid arthritis, treatment failure Access this article online Website: www.indianjrheumatol.com DOI: 10.4103/injr.injr_60_19 271 Quick Response Code This is an open access journal, and articles are distributed under the terms of the Creative Commons Attribution‑NonCommercial‑ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non‑commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms. For reprints contact: reprints@medknow.com How to cite this article: Manfredi A, Sebastiani M, Iannone F, Gremese E, Bortoluzzi A, Favalli E, et al. Factors predicting early discontinuation of methotrexate as a first-line treatment for rheumatoid arthritis in Italy: Results from the GISEA registry. Indian J Rheumatol 2019;14:271-6. © 2019 Indian Journal of Rheumatology \| Published by Wolters Kluwer - Medknow [Downloaded free from http://www.indianjrheumatol.com on Thursday, February 13, 2020, IP: 155.185.104.10] Manfredi, et al.: Early failure to methotrexate in rheumatoid arthritis Introduction Methotrexate (MTX) is considered to be the anchor drug for patients with rheumatoid arthritis (RA), recommended as the first‑line treatment in both early and long‑standing diseases.[1,2] This is mainly based on strong data about its efficacy, safety, and the possibility to adjust the dose and route of administration. According to the last American College of Rheumatology (ACR) and European League Against Rheumatism (EULAR) recommendations for the management of RA with synthetic and biologic disease‑modifying antirheumatic drugs (DMARDs), MTX should represent part of the first‑treatment strategy in RA patients, both in monotherapy or in combination.[2,3] Despite this crucial role of MTX in RA, it is well defined that a percentage of patients fail the treatment.[4‑12] To our knowledge, few studies have investigated the causes of early (within the 1st year) discontinuation or failure of MTX; in one of few available studies, a 2‑year retention rate of about 66% is described for RA patients treated with MTX, showing a lower age and a longer disease duration as independent predictors for discontinuation.[13] The aim of this study was to identify possible predictive factors for the early discontinuation or failure of MTX prescribed as the first‑line treatment in early RA patients enrolled in the Italian Group for the Study of Early Arthritis (GISEA) registry. Materials and Methods Italian Group for the Study of Early Arthritis registry The nationwide GISEA registry was launched in 2003 to record and monitor patients with early RA on the basis of the standard of clinical care. The registry involves hospital and community‑based rheumatology units throughout Italy. Moreover, the GISEA registry included early RA patients with disease duration from diagnosis lower than 6 months. Patients aged >18 years are enrolled after giving their written informed consent, and the registry has been approved by the local Ethics Committee of Modena. Patient data are recorded at baseline and every 6 months thereafter. RA is diagnosed on the basis of the 1987 or 2010 ACR criteria.[14,15] The data collected include age, sex, disease duration, the time from diagnosis to beginning of treatment with a biological drug (latency), the intake of glucocorticoids and DMARD, smoking status, body mass index (BMI), the 28‑joint disease activity score (DAS28), C‑reactive protein, erythrocyte sedimentation rate (ESR; mm/h), rheumatoid factor (RF), anti‑citrullinated peptide antibodies (ACPAs), and side effects. Comorbidities are recorded by monitoring the following: anemia, anxiety/ depression, cardiopathy, cerebrovascular diseases, diabetes, gastropathies, hypertension, liver diseases, lung diseases, neoplasia, nephropathy, and peripheral vasculopathy. Extra‑articular manifestations included Raynaud’s phenomenon, rheumatoid involvement, and sicca syndrome. nodules, lung Patients Patients with early RA who began MTX as the first‑line treatment were included in the study. For all patients, age, sex, disease duration, smoking status, the intake of glucocorticoids, clinical and serological data, comorbidities, and extra‑articular manifestations were collected at baseline, before starting MTX. For all enrolled patients, clinical status was re‑evaluated after 12 months, focusing on disease activity and the persistence with MTX therapy. When MTX was discontinued or a biologic drug was added, the time and the causes of the therapy change were also recorded. Failure was defined according to the EULAR nonresponse criteria after 12 months[16] or the association of a biologic DMARD during the 1st year of follow‑up. Discontinuation was defined as other cause of withdrawal of MTX, both intolerance and adverse events, requiring the suspension of the drug. Statistical analysis The differences among patients stopping or continuing at the 1st year of therapy with MTX were analyzed using the Mann–Whitney U‑test for the continuous variables (median values and interquartile ranges [IQR]) and the Chi‑square test for the categorical variables (absolute numbers and percentages) regarding baseline characteristics. The univariate analysis was performed using binomial Cox regression model to select parameters to be included in multiple Cox regression analysis (only parameters significantly different in univariate analysis were included in multiple model). The baseline variables considered were sex, disease duration, ESR, DAS28, the concurrent use of glucocorticoids and DMARDs, comorbidities, extra‑articular manifestations, BMI, and smoking habit. All analyses were made using SAS version 9.2 (SAS Institute Inc., Cary, NC, USA), and a P ≤ 0.05 was considered statistically significant. The data are expressed as percentages or median values and IQR unless otherwise indicated.[17] Results The registry included 1123 patients with early RA. Five hundred and eleven were excluded because they were treated with a DMARD other than MTX or because they were lost to follow‑up. Twenty‑two patients who stopped MTX during the 1st year of treatment for disease remission were excluded to avoid possible cases of self‑limiting arthritis mimicking RA. For the inclusion in the registry, the percentage of missing data had to be <10%. Indian Journal of Rheumatology ¦ Volume 14 ¦ Issue 4 ¦ December 2019 272 [Downloaded free from http://www.indianjrheumatol.com on Thursday, February 13, 2020, IP: 155.185.104.10] Manfredi, et al.: Early failure to methotrexate in rheumatoid arthritis We analyzed 590 RA patients (female/male 458/132, mean age 55.78 ± 14.5 years; mean DAS28 5.35 ± 1.2). RF was positive in 65.1%, and ACPA was positive in 39%. Comorbidities were observed in 19% of patients, mainly cardiovascular diseases, while extra‑articular RA manifestations were recorded in 1.5%. Sixty‑eight percent of subjects used low doses of steroids. MTX was administered by parenteral route (577/590, 97.8%) and only 13 patients took oral MTX. A summary of demographic and serological data, comorbidities, and treatment is presented in Table 1. One hundred and forty‑nine patients (25.3%) discontinued or failed MTX during the 1st year (mean period of treatment 4.7 ± 3.4 months). The causes were inefficacy for 65/149 patients (43.6%; namely 31.5% for primary inefficacy and 12.1% for loss of efficacy, respectively) and adverse events in 56/149 (37.5%, mainly liver function Table 1: Demographic, clinical and serological features of rheumatoid arthritis patients treated with methotrexate as first‑line therapy When we compared RA patients who continued MTX (Group A) with patients who discontinued or failed treatment during the 1st year (Group B), the latter group showed a lower mean age (P = 0.05) and a higher number of tender and swollen joints on 28 joints (P = 0.001 and P = 0.031, respectively) at baseline; the rate of discontinuation of MTX (P = 0.041) was reduced by the association of MTX with another DMARDs during the 1st year in 19 patients (hydroxychloroquine in 17 patients and leflunomide in 2); finally, DAS28 was higher in patients in Group B than Group A, but the difference was not significant. No differences were observed regarding the association with steroids [Table 2]. Stratifying the causes of failure or discontinuation according to the baseline dosage of MTX, we observed a different impact for adverse events or inefficacy [Table 3]; Nr 590 Male/female ratio 132/458 (0.29) Age (median, IQR) 57 (20) Smoke (%) 183 (31) ACPA (%) 361 (61.2) Rhematoid factor (%) 383 (64.9) ACPA + RF 165 (27.9) Isolated RF 218 (36.9) Isolated ACPA 176 (29.8) None 31 (5.2) Comorbidities (%) 112 (18.8) Cardiovascular diseases 99 (16.8) Lung diseases 37 (6.2) Liver diseases 11 (1.9) Skin disorders 68 (11.6) Diabetes 24 (4.1) Anemia 57 (9.7) Hyperuricemia 54 (9.2) Depression 40 (6.8) Other/not reported 199 (33.7) Extra‑articular manifestations (%) 9 (1.5) Steroid therapy (%) 405 (68.6) Mean methotrexate dose 10 (5) ERS 31 (32) CRP >5 mg/L (%) 406 (68.8) Tender joints count 28 7 (9) Swollen joints count 28 5 (7) DAS28 5.45 (1.61) BMI 42.6 (4.78) Continuous values are reported as median (IQR) dicotomic values are reported as percentage. ACPA: Anti‑citrullinated peptide antibodies, RF: Rheumatoid factor, ERS: Erythrosedimentation rate, DAS: Disease activity index, BMI: Body mass index, IQR: Interquartile range, CRP: C‑reactive protein 273 alteration, while only one patient discontinued the treatment for an infection of the lower respiratory tract); other reasons/not recorded in 28/149 (18.8%). Table 2: Demographic, clinical and serological features of rheumatoid arthritis patients treated with methotrexate after 1‑year therapy Continuing MTX Failure MTX 441 (74.7%) 149 (25.3%) 57 (20) 57 (19) 24.7 (4.9) 24.8 (5.9) 33 (32) 28 (36) 8 (8) 9 (9) 5 (6) 7 (8) 5.4 (1.5) 5.7 (1.8) 10 (5) 15 (5) 99 (22.4) 33 (22.1) 131 (29.7) 52 (35) 284 (64.4) 99 (66.3) 283 (64.1) 78 (52.3) 131 (29.7) 34 (23.2) 153 (34.7) 65 (43.6) 131 (29.7) 45 (29.1) 26 (5.9) 5 (3.3) 79 (17.9) 32 (21.6) 5 (1.1) 4 (2.7)% P Age 0.037 BMI 0.5576 ESR 0.0458 Tender joints 28 ≤0.001 Swollen joints 28 0.027 DAS28 0.1146 Mean dosage MTX 0.024 Male gender (%) 0.88 Smoke (%) 0.32 Rheumatoid factor (%) 0.558 ACPA (%) 0.016 RF + ACPA + 0.108 RF alone ACPA alone None Comorbidities (%) 0.255 Extra‑articular 0.182 manifestations (%) Steroid therapy (%) 306 (69.4) 99 (66.4) 0.641 Combination therapy (%) 18 (4.1) 1 (0.7) 0.041 Reactive C protein >5 312 (70.8) 94 (63.1) 0.135 mg/L (%) Continuous values are reported as median and IQR, dicotomic values are reported as percentage. Mann–Whitney U‑test was performed to evaluate association between continuous variables and MTX failure. Contingency table with Chi‑square test was used to evaluate associations between dicotomic variables and MTX failure. IQR: Interquartile range, MTX: Methotrexate, ACPA: Anti‑citrullinated peptide antibodies, DAS: Disease activity index, RF: Rheumatoid factor, ESR: Erythrosedimentation rate, BMI: Body mass index Indian Journal of Rheumatology ¦ Volume 14 ¦ Issue 4 ¦ December 2019 [Downloaded free from http://www.indianjrheumatol.com on Thursday, February 13, 2020, IP: 155.185.104.10] Manfredi, et al.: Early failure to methotrexate in rheumatoid arthritis in particular, patients treated with 7.5 mg of MTX weekly did not have adverse events, but inefficacy was the cause of failure or discontinuation in 91.7% of cases at this dosage. On the contrary, adverse events were recorded in 68.4% and inefficacy only in 5.3% of patients with a dosage of 20 mg weekly or higher. During the 1st year, 30% of patients increased the dose of MTX, without significant changes in 1 year efficacy or safety of the drug. We performed a multivariate analysis including tender joints, age, the baseline dosage of MTX, the presence of ACPA, and the combination therapy. Since both tender and swollen joints are correlated with MTX discontinuation or failure at univariate analysis, and considering that these data clearly influence each other, we decided to include in the model only the tender joints, showing a higher significance than swollen joints with respect to MTX failure. ACPA, age, and number of tender joints at baseline were confirmed to be directly associated with early MTX discontinuation or failure, also when corrected for the gender [Table 4]. Discussion Our study aimed to reveal possible predictive factors of early failure or discontinuation of MTX used as the first‑line treatment in RA patients during the 1st year of therapy. This topic has been investigated in only a few studies, mainly with long‑term treatments, without conclusive data. In 1991, Scully et al. reported a discontinuation rate of 69% for MTX after 5 years, in 124 RA patients;[8] patients who persisted in treatment were younger and with a shorter disease duration. The main causes of discontinuation were the poor efficacy and the adverse drug reactions.[4] The percentage of discontinuation of MTX varies largely in literature, from a maximum of 55% after 48 weeks to 50% after 50 months.[5‑7] In particular, Keysser showed Table 3: Causes of therapy failure according to dosage of methotrexate Adverse events (%) Inefficacy (%) ≤7.5 mg 0.0 91.7 10 mg 28.0 48.0 15 mg 41.4 44.9 ≥20 mg 68.4 5.3 Table 4: Multivariate analysis Parameter OR 95% CI P Age 0.98 0.971-0.998 0.025 ACPA 1.531 1.019-2.300 0.04 Combination therapy 0.148 0.019-1.138 0.066 MTX weekly dosage 1.039 0.985-1.095 0.16 Tender joint count 1.035 1.001-1.071 0.044 Factors associated to early discontinuation of methotrexate. ACPA: Anti‑cyclic citrullinated peptide antibodies, MTX: Methotrexate, CI: Confidence interval, OR: Odd ratio a drop‑out rate due to toxicity of 15.9% for MTX, mainly within the 1st year of treatment.[9] In 2016, a Cochrane meta‑analysis compared the efficacy of MTX in monotherapy or combination therapy, including 158 randomized controlled trials (over 37,000 patients) with a duration of at least 12 weeks. The authors concluded that monotherapy was less effective in controlling disease and in preventing joint damage than triple therapy (MTX, sulfasalazine, and hydroxychloroquine) or the association with biologic DMARDs or tofacitinib.[12] Finally, the PRESERVE trial aimed to analyze features predicting the loss of remission in patients with moderately active RA who received full‑dose combination etanercept plus MTX induction therapy, followed by a reduced‑dose of etanercept or etanercept withdrawal. Although in a subset of patients with a different clinical history (no early RA, no MTX as first‑line therapy), the authors detected, in the MTX monotherapy subgroup, predictive factors for the loss of remission similar to our study, namely the number of tender or swollen joints, other than DAS28, and low ESR levels.[11] In our study, the majority of patients discontinued treatment with MTX because of inefficacy, followed by adverse events. At univariate analysis, MTX monotherapy, lower age, positivity of ACPA, a higher number of tender and swollen joints, and surprisingly a higher dose of MTX were associated with the failure or early discontinuation of MTX during the 1st year of treatment. These results have not previously been reported. Furthermore, it is noted that some previous studies relate back to a period in which the measurement of ACPA was not used in clinical practice. Evaluating the possible correlation between the dose of the drug and the causes of discontinuation, we have observed that a lower dosage was more frequently associated with discontinuation for inefficacy than adverse events and the contrary was detected for higher doses. Interestingly, 49 patients (9.1%) were treated with 7.5 mg of MTX weekly. Intuitively, a low dosage of drug can contribute to early discontinuation due to inefficacy. It is surprising that about 10% of early RA patients are still treated with a very low dosage of MTX. This topic was previously investigated in the MTX for RA in Italy study, highlighting that in Italy, the first dose of MTX is often lower than or equal to 10 mg weekly and, in this regard, an initial low dose of MTX could contribute to an increase drug discontinuation for inefficacy. The authors argued that in common practice, rheumatologists tend to prescribe the lowest possible dose, even when complete remission is not achieved.[18] Both EULAR and ACR recommend an initial therapeutic dose of 15 mg/week, although it is recognized that Indian Journal of Rheumatology ¦ Volume 14 ¦ Issue 4 ¦ December 2019 274 [Downloaded free from http://www.indianjrheumatol.com on Thursday, February 13, 2020, IP: 155.185.104.10] Manfredi, et al.: Early failure to methotrexate in rheumatoid arthritis the average tolerable effective dose ranges between 15 and 20 mg/week.[2,3,19,20] In GISEA registry, a titration of initial dose of MTX is not reported; the absence of a titration of MTX dose could contribute to the development of gastrointestinal side effects and therefore to MTX discontinuation. Finally, ACPA positivity in younger patients and the higher count of tender joints regardless of the value of acute‑phase reactant levels could be helpful in the identification of a cluster of patients to be strictly evaluated at the early stage of the disease to consider the addition of other drugs. In case of MTX failure, and in the presence of poor prognostic factors, the addition of another DMARDs is not recommended, and a targeted synthetic or a biologic DMARDs should always be considered. Conclusions During the year of treatment, about a quarter of patients with early RA failed MTX therapy, mainly for inefficacy (43.6% of cases) or adverse events (37.5%). The presence of ACPA, the age of the patient, and the number of tender joints at baseline were directly associated with early MTX failure, while the dose of MTX was correlated to the efficacy and safety of the drug. Acknowledgments 5. 6. 7. 8. 9. 10. 11. 12. Editorial support was provided by Ray Hill on behalf of Health Publishing and Services Srl and was funded by Pfizer. The GISEA/OEG Group received an honorarium from Pfizer in connection with the development of this manuscript. The individual authors did not receive an honorarium from Pfizer in connection with the development of this manuscript. Financial support and sponsorship 13. 14. Nil. Conflicts of interest 15. There are no conflicts of interest. References 1. 2. 3. 4. 275 Combe B, Landewe R, Daien CI, Hua C, Aletaha D, Álvaro‑Gracia JM, et al. 2016 update of the EULAR recommendations for the management of early arthritis. Ann Rheum Dis 2017;76:948‑59. Smolen JS, Landewé R, Bijlsma J, Burmester G, Chatzidionysiou K, Dougados M, et al. EULAR recommendations for the management of rheumatoid arthritis with synthetic and biological disease‑modifying antirheumatic drugs: 2016 update. Ann Rheum Dis 2017;76:960‑77. Singh JA, Saag KG, Bridges SL Jr., Akl EA, Bannuru RR, Sullivan MC, et al. 2015 american college of rheumatology guideline for the treatment of rheumatoid arthritis. Arthritis Care Res (Hoboken) 2016;68:1‑25. Lie E, van der Heijde D, Uhlig T, Heiberg MS, Koldingsnes W, Rødevand E, et al. Effectiveness and retention rates of methotrexate in psoriatic arthritis in comparison with 16. 17. 18. methotrexate‑treated patients with rheumatoid arthritis. Ann Rheum Dis 2010;69:671‑6. Aletaha D, Neogi T, Silman AJ, Funovits J, Felson DT, Bingham CO 3rd, et al. 2010 rheumatoid arthritis classification criteria: An American College of Rheumatology/European League Against Rheumatism Collaborative Initiative. Arthritis Rheum 2010;62:2569‑81. Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, Cooper NS, et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 1988;31:315‑24. Altman DG. Practical Statistics for Medical Research. 1st ed.. London, UK: Chapman and Hall/CRC; 1991. Scully CJ, Anderson CJ, Cannon GW. Long‑term methotrexate therapy for rheumatoid arthritis. Semin Arthritis Rheum 1991;20:317‑31. Keysser M, Keysser G, Keysser C. Long‑term application of disease modifying antirheumatic drugs (DMARD). A single‑center, observational study of 1681 patients with rheumatoid arthritis (RA). Z Rheumatol 1999;58:267‑76. De La Mata J, Blanco FJ, Gómez‑Reino JJ. Survival analysis of disease modifying antirheumatic drugs in Spanish rheumatoid arthritis patients. Ann Rheum Dis 1995;54:881‑5. Barrera P, van der Maas A, van Ede AE, Kiemeney BA, Laan RF, van de Putte LB, et al. Drug survival, efficacy and toxicity of monotherapy with a fully human anti‑tumour necrosis factor‑alpha antibody compared with methotrexate in long‑standing rheumatoid arthritis. Rheumatology (Oxford) 2002;41:430‑9. Qiu Q, Huang J, Lin Y, Shu X, Fan H, Tu Z, et al. Polymorphisms and pharmacogenomics for the toxicity of methotrexate monotherapy in patients with rheumatoid arthritis: A systematic review and meta‑analysis. Medicine (Baltimore) 2017;96:e6337. Uribarri M, Ruiz‑Larrañaga O, Arteta D, Hernández L, Alcaro MC, Martínez A, et al. Influence of MTHFR C677T polymorphism on methotrexate monotherapy discontinuation in rheumatoid arthritis patients: Results from the GAPAID European project. Clin Exp Rheumatol 2015;33:699‑705. Acurcio FA, Machado MA, Moura CS, Ferre F, Guerra AA Jr., Andrade EI, et al. Medication persistence of disease‑modifying antirheumatic drugs and anti‑tumor necrosis factor agents in a cohort of patients with rheumatoid arthritis in Brazil. Arthritis Care Res (Hoboken) 2016;68:1489‑96. Smolen JS, Szumski A, Koenig AS, Jones TV, Marshall L. Predictors of remission with etanercept‑methotrexate induction therapy and loss of remission with etanercept maintenance, reduction, or withdrawal in moderately active rheumatoid arthritis: Results of the PRESERVE trial. Arthritis Res Ther 2018;20:8. van Gestel AM, Prevoo ML, van ‘t Hof MA, van Rijswijk MH, van de Putte LB, van Riel PL, et al. Development and validation of the European league against rheumatism response criteria for rheumatoid arthritis. Comparison with the preliminary American college of rheumatology and the World Health Organization/ International league against rheumatism criteria. Arthritis Rheum 1996;39:34‑40. Hazlewood GS, Barnabe C, Tomlinson G, Marshall D, Devoe DJ, Bombardier C, et al. Methotrexate monotherapy and methotrexate combination therapy with traditional and biologic disease modifying anti‑rheumatic drugs for rheumatoid arthritis: A network meta‑analysis. Cochrane Database Syst Rev 2016; 8:CD010227. Manara M, Bianchi G, Bruschi E, Azzolini V, Belai Beyene N, Corbanese S, et al. Adherence to current recommendations on the use of methotrexate in rheumatoid arthritis in Italy: Results Indian Journal of Rheumatology ¦ Volume 14 ¦ Issue 4 ¦ December 2019 [Downloaded free from http://www.indianjrheumatol.com on Thursday, February 13, 2020, IP: 155.185.104.10] Manfredi, et al.: Early failure to methotrexate in rheumatoid arthritis from the MARI study. Clin Exp Rheumatol 2016;34:473‑9. 19. Visser K, Katchamart W, Loza E, Martinez‑Lopez JA, Salliot C, Trudeau J, et al. Multinational evidence‑based recommendations for the use of methotrexate in rheumatic disorders with a focus on rheumatoid arthritis: Integrating systematic literature research and expert opinion of a broad international panel of rheumatologists in the 3E initiative. Ann Rheum Dis 2009;68:1086‑93. 20. Goodman SM, Cronstein BN, Bykerk VP. Outcomes related to methotrexate dose and route of administration in patients with rheumatoid arthritis: A systematic literature review. Clin Exp Rheumatol 2015;33:272‑8. Indian Journal of Rheumatology ¦ Volume 14 ¦ Issue 4 ¦ December 2019 276
https://openalex.org/W3176880777	https://www.nature.com/articles/s41467-021-24214-5.pdf	English	null	A genetically encoded anti-CRISPR protein constrains gene drive spread and prevents population suppression	Nature communications	2,021	cc-by	11,006	ARTICLE NATURE COMMUNICATIONS \| (2021) 12:3977 \| https://doi.org/10.1038/s41467-021-24214-5 \| www.nature.com/naturecommunications A genetically encoded anti-CRISPR protein constrains gene drive spread and prevents population suppression Chrysanthi Taxiarchi 1, Andrea Beaghton1, Nayomi Illansinhage Don1, Kyros Kyrou Dammy Shittu1, Scott P. Collins2, Chase L. Beisel 2,3,4, Roberto Galizi 5✉& Andre Chrysanthi Taxiarchi 1, Andrea Beaghton1, Nayomi Illansinhage Don1, Kyros Kyrou 1, Matthew Gribble1, Dammy Shittu1, Scott P. Collins2, Chase L. Beisel 2,3,4, Roberto Galizi 5✉& Andrea Crisanti1,6✉ CRISPR-based gene drives offer promising means to reduce the burden of pests and vector- borne diseases. These techniques consist of releasing genetically modiﬁed organisms car- rying CRISPR-Cas nucleases designed to bias their inheritance and rapidly propagate desired modiﬁcations. Gene drives can be intended to reduce reproductive capacity of harmful insects or spread anti-pathogen effectors through wild populations, even when these confer ﬁtness disadvantages. Technologies capable of halting the spread of gene drives may prove highly valuable in controlling, counteracting, and even reverting their effect on individual organisms as well as entire populations. Here we show engineering and testing of a genetic approach, based on the germline expression of a phage-derived anti-CRISPR protein (AcrIIA4), able to inactivate CRISPR-based gene drives and restore their inheritance to Mendelian rates in the malaria vector Anopheles gambiae. Modeling predictions and cage testing show that a single release of male mosquitoes carrying the AcrIIA4 protein can block the spread of a highly effective suppressive gene drive preventing population collapse of caged malaria mosquitoes. 1 Department of Life Sciences, Imperial College London, London, UK. 2 Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC, USA. 3 Helmholtz Institute for RNA-based Infection Research (HIRI), Helmholtz-Centre for Infection Research (HZI), Würzburg, Germany. 4 Medical Faculty, University of Würzburg, Würzburg, Germany. 5 Centre for Applied Entomology and Parasitology, School of Life Sciences, Keele University, Keele, UK. 6 Department of Molecular Medicine, University of Padova, Padova, Italy. ✉email: r.galizi@outlook.com; acrs@imperial.ac.uk 1 NATURE COMMUNICATIONS \| (2021) 12:3977 \| https://doi.org/10.1038/s41467-021-24214-5 \| www.nature.com/naturecommunicatio ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-24214-5 homing gene drive constructs exploit the germline-speciﬁc action of the ribonucleoprotein complex (e.g., Cas9–gRNA), which is designed to recognise and cleave a DNA sequence homologous to the one where the drive-encoding construct is inserted. Double-strand breaks generated in premeiotic cells are preferentially repaired using the uncleaved homologous chro- mosome as a template through the homology-directed repair process, thus resulting in the drive-containing DNA strand being copied into the wild-type chromosome. A genetically encoded anti-CRISPR protein constrains gene drive spread and prevents population suppression T g p The ﬁrst example of gene drive reversal systems was inspired by naturally occurring resistance to gene drives in the form of cleavage-refractory modiﬁcation of the DNA sequence targeted by the driving endonuclease. Resistant alleles can pre-exist in the population as polymorphisms, or be generated de novo through non-homologous end-joining (NHEJ) repair of endonuclease- induced cleavage10–13. Anti-drive individuals could be genetically engineered to carry similar “drive-refractory alleles” and used to rescue the target population8,10. However, refractory alleles rely on a selective advantage conferred by the higher ﬁtness compared to the drive, and therefore will have little effect on gene drives with low ﬁtness cost (e.g., population-replacement drives)14. In addition, there are cases where tight functional constraints at the gene drive target sequence may hinder the development of this type of reversal approach, such as for the dsx-targeting gene drive that we recently developed in the malaria vector Anopheles gambiae2. Alternative reversal strategies involve the use of CRISPR components to cleave and replace DNA sequences spe- ciﬁc to the gene drive construct with15,16 or without17–19 the use of an additional Cas9 expression cassette. Recently, guide RNA- only systems developed in Drosophila showed capacity to inac- tivate or replace gene drives in caged populations19. Although these strategies may offer the option to replace the drive with one or few “refractory alleles” or even restore the wild-type popula- tion, there are several complications attributable to the DNA- cleaving nature of the reversal that remain to be addressed, such as the formation and selection of resistant alleles and/or genomic rearrangement at the drive locus targeted by the reversal nuclease. In this work we describe the development and validation of a Germline expression of AcrIIA4 imposes Mendelian trans- mission of gene drive constructs. To test the AcrIIA4 inhibitory activity of gene drive homing in the mosquito germline, we crossed female mosquitoes hemizygous for the anti-drive con- struct (vasa:A4+/−) to the three most effective A. gambiae gene drive lines currently available. The zpg:dsxF line (previously identiﬁed as dsxFCRISPRh2) carries a driving construct, where the Cas9 from Streptococcus pyogenes (herein identiﬁed as Cas9) is expressed under the zpg regulatory elements, inserted within the sex-determining gene AgdsxF (AGAP004050-RB) to disrupt the production of the female splicing variant. Functional constraint of the targeted sequence allowed the driving construct to rapidly spread and eliminate caged mosquito populations by progressively reducing the reproductive capacity of females2. A genetically encoded anti-CRISPR protein constrains gene drive spread and prevents population suppression The zpg:7280 and nos:7280 lines (previously named zpgCRISPRh and nosCRISPRh 26) showed a dramatic improvement of the ﬁrst generation of sup- pressive gene drives3 by tuning the temporal expression of the Cas9 through the use of the zpg and nos promoters. In both lines, homing of the CRISPR cassette disrupts the haplosufﬁcient female fertility gene AGAP00728026. Male and female mosquitoes carry- ing one copy of both drive and anti-drive constructs (zpg:dsxF+/−; vasa:A4+/−, zpg:7280+/−;vasa:A4+/− and nos:7280+/−; vasa:A4+/−) were crossed to wild-type individuals to assess ferti- lity and analyse transmission frequencies of drive (D) and anti- drive (A) alleles to their progenies by screening for expression of the linked RFP (drive) and GFP (anti-drive) ﬂuorescent markers. Individuals carrying only the drive or the anti-drive construct were also crossed to the wild-type counterpart as a control (Fig. 2a). Inheritance of the anti-drive allele from vasa:A4+/− individuals was close to the expected Mendelian rates (average of 56.5% GFP positive from females and 57% from males), while all drive+/−individuals transmitted the drive allele to most of the progeny as consequence of homing (averages ranging from 88.5% up to 100% of RFP positives found in the progeny). Notably, individuals carrying both drive and anti-drive transgenes showed a g g y In this work, we describe the development and validation of a widely applicable genetic tool to counteract CRISPR-based gene drives by expressing a Cas9-inhibiting protein in a spatial and temporal manner coinciding with the gene drive activity, thereby circumventing problems related to additional cleavage of genomic sequences. We generated an A. gambiae transgenic line expres- sing the AcrIIA4 protein from the Listeria monocytogenes prophage20–23, and assessed its ability to both inhibit super- Mendelian inheritance of different driving constructs and prevent gene drive-mediated suppression of caged malaria mosquito populations. A genetically encoded anti-CRISPR protein constrains gene drive spread and prevents population suppression As a consequence of germline maturation and subsequent fertilisation of a wild- type individual, the gene drive element is transmitted to the majority of the progeny, resembling a homozygous trait, and therefore increases its frequency in the population over gen- erations. Aiming to inhibit this process, we genetically engi- neered an anti-drive transgenic line where the nuclear localisation signal (NLS)-tagged AcrIIA4 protein is expressed under the vasa promoter, transcriptionally active in the germ- line of both A. gambiae male and females24. As conﬁrmed in vitro, fusion to various NLS tags does not perturb inhibition of Cas9 activity by AcrIIA4 (Supplementary Fig. 1). The anti- drive construct was inserted via embryo microinjection in a pre- existing docking line25. The identiﬁcation of successful genomic integrations was based on the expected combination of ﬂuor- escent marker expression: the eGFP linked to anti-drive con- struct, and the eCFP already present at the docking site (Fig. 1). The progeny derived from a single eGFP (and eCFP) positive individual was used to establish the anti-drive transgenic line (identiﬁed as vasa:A4) after molecular conﬁrmation of correct insertion of the construct (Supplementary Fig. 2a, b). T he management of vector and pest populations using nuclease-based gene drives is becoming a realistic possi- bility, particularly after the recent proof-of-principle demonstrations of genetic control technologies based on the broadly applicable CRISPR–Cas nucleases1. These technologies rely on the release of genetically engineered individuals that can rapidly propagate genetic constructs through wild populations together with the linked genetic modiﬁcations (e.g., knockout of sex determination2 or fertility genes3) or the introduction of genetic cargos (e.g., pathogen-killing molecules designed to block the development of parasites within the vector4). Several gene drive systems have been proposed and a few potential candidate strains have already been developed in the laboratory for the control of several organisms, including invasive rodents5, agri- cultural pests6,7 and disease vectors2–4,8,9. Access to effective ways to counteract the spread of gene drive elements is highly valued for risk mitigation and management alongside the implementa- tion of these strategies: e.g., as a possible intervention in the case of unintended releases. This is particularly relevant for self- sustaining strategies showing high potential of spread, especially when these are set to control non-conﬁned populations dispersed in large areas across multiple countries. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-24214-5 The fraction of mated females recorded in these experiments also showed a possible impairment in mating competence that appeared more pro- nounced in A+/+ males, able to fertilise only 60% of the wild-type females tested compared to the 89% found in wild-type male crosses (Fisher’s exact test P = 0.0469). A+/−males were instead able to fertilise 80% of the females (Fisher’s exact test P = 0.4924). Although not statistically signiﬁcant, anti-drive females crossed to wild-type males showed a reduction in mating with 70% of A+/+ and 81% of A+/−females able to mate in these settings (Supplementary Table 1). striking reduction of inheritance of the drive allele down to Mendelian rates (averaging from 46.3 to 51.1% of RFP positives), in line with complete blockage of Cas9–gRNA cleavage activity and consequent inhibition of germline homing (Fig. 2b). Fitness analysis of the vasa:A4 anti-drive line. Fertility assays did not show any substantial reduction in progeny outputs from indi- viduals carrying one copy of the anti-drive construct (D+/−;A+/−or A+/−) compared to the respective gene drive controls (D+/−), giving a minimum normalised value (NV) of larvae produced per females of 0.77 from nos:7280+/−;vasa:A4+/−males (Welch’s t test P = 0.2544) and zpg:7280+/−;vasa:A4+/−females (Welch’s t test P = 0.0732). On the other hand, a higher number of larvae (NV = 1.57) was obtained from zpg:dsxF+/−;vasa:A4+/−females compared to zpg:dsxF+/−females. Although this difference was not statistically signiﬁcant when comparing independent oviposition counts from each cross (Welch’s t test P = 0.1035), the difference in overall number of larvae hatched was signiﬁcant for all three crosses cited above (Fisher’s exact test). Notwithstanding that this type of phenotype assays may be affected by interindividual or batch variability, previous studies showed that parental deposition of Cas9 in the embryo can cause reduction of fertility in D+/−females by generating embryonic mosaicisms for somatic null mutations at the targeted site, which are produced via NHEJ repairs10,26. These studies also indicated that the severity of this reduction varies among the different genes targeted and according to the promoter used to express the Cas9 protein, reﬂecting high levels of maternal depositions when the Cas9 expression is driven by the vasa pro- moter, compared to other regulatory sequences10,26,27. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-24214-5 Speciﬁcally, the gene drive constructs tested in this work are inserted at target sites within the AGAP007280 or AgdsxF (AGAP004050-RB) gene-coding sequences and contain: the Streptococcus pyogenes Cas9 nuclease (Cas9), under the transcriptional control of the male and female germline-speciﬁc promoters zpg or nos; the gRNA, targeting the respective insertion site, transcribed by the RNA polymerase III responsive promoter (U6), and the DsRed ﬂuorescent protein under the 3xP3 promoter (3xP3:DsRed) for the identiﬁcation of larvae carrying the drive. The anti-drive construct carries the Listeria monocytogenes anti- CRISPR protein (AcrIIA4) expressed under the vasa male and female germline-speciﬁc promoter with the N-terminus addition of a nuclear localisation signal (NLS) and the eGFP ﬂuorescent protein under the 3xP3 promoter (3xP3:eGFP) used for the screening of anti-drive positive larvae. The construct was inserted in a pre-existing docking line carrying the 3xP3:eCFP marker25. As a result the AcrIIA4 protein is expected to interact and inhibit the Cas9–gRNA complex, when co-expressed in the mosquito germline cells. striking reduction of inheritance of the drive allele down to Mendelian rates (averaging from 46.3 to 51.1% of RFP positives), in line with complete blockage of Cas9–gRNA cleavage activity and consequent inhibition of germline homing (Fig. 2b). Further phenotypic characterisation of anti-drive individuals showed a moderate reduction in larval output (NV = 0.79, Welch’s t test P = 0.1398) from males carrying two copies of the anti-drive construct (A+/+) compared to the wild-type control tested in parallel. A similar reduction was also seen in A+/−females (NV larvae per female = 0.79, Welch’s t test P = 0.0277); however, this was not reﬂected in A+/+ females that gave an average number of larvae comparable to wild-type (NV = 1.07, Welch’s t test P = 0.4168), indicating that this fertility impair- ments may not be directly caused by a double dose of AcrIIA4 in A+/+ individuals (Supplementary Fig. 3b, c). Furthermore, fertility assays from trans-hemizygous individuals, carrying one copy of the anti-drive construct and a marker construct inserted at the corresponding homologous locus (vasa:A4+/mars+) also showed similar reductions in larvae outputs compared to the wild-type control (NV = 0.67 and Welch’s t test P = 0.0147 from females, NV = 0.84 and Welch’s t test P = 0.1446 from males), indicating a moderate ﬁtness cost likely associated with the locus of insertion (Supplementary Fig. 3c). Results Generation of the vasa:A4 (anti-drive) line expressing the AcrIIA4 protein in the mosquito germline. CRISPR-based NATURE COMMUNICATIONS \| (2021) 12:3977 \| https://doi.org/10.1038/s41467-021-24214-5 \| www.nature.com/naturecommunications 2 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-24214-5 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-24214-5 eGFP 3xP3 attL eCFP 3xP3 attL Inactive Cas9:gRNA + 3xP3 DsRed U6 sgRNA Active Cas9:gRNA attL Target gene 5’ AgdsxF AGAP007280 attR Target gene 3’ AgdsxF AGAP007280 Germline promoter Cas9 zpg nos Gene Drive Anti-Drive AcrIIA4 Vasa NLS AcrIIA4 anti-Cas9 protein eGFP 3xP3 attL eCFP 3xP3 attL Inactive Cas9:gRNA + 3xP3 DsRed U6 sgRNA Active Cas9:gRNA attL Target gene 5’ AgdsxF AGAP007280 attR Target gene 3’ AgdsxF AGAP007280 Germline promoter Cas9 zpg nos Gene Drive Anti-Drive AcrIIA4 Vasa NLS AcrIIA4 anti-Cas9 protein Fig. 1 Schematic representation of gene drive and anti-drive constructs. Gene drive and anti-drive constructs respectively as inserted in the genome of the gene drive lines previously generated (zpg:dsxF2; zpg:7280 and nos:7280, ref. 26) and the newly generated anti-drive (vasa:A4) line. Speciﬁcally, the gene drive constructs tested in this work are inserted at target sites within the AGAP007280 or AgdsxF (AGAP004050-RB) gene-coding sequences and contain: the Streptococcus pyogenes Cas9 nuclease (Cas9), under the transcriptional control of the male and female germline-speciﬁc promoters zpg or nos; the gRNA, targeting the respective insertion site, transcribed by the RNA polymerase III responsive promoter (U6), and the DsRed ﬂuorescent protein under the 3xP3 promoter (3xP3:DsRed) for the identiﬁcation of larvae carrying the drive. The anti-drive construct carries the Listeria monocytogenes anti- CRISPR protein (AcrIIA4) expressed under the vasa male and female germline-speciﬁc promoter with the N-terminus addition of a nuclear localisation signal (NLS) and the eGFP ﬂuorescent protein under the 3xP3 promoter (3xP3:eGFP) used for the screening of anti-drive positive larvae. The construct was inserted in a pre-existing docking line carrying the 3xP3:eCFP marker25. As a result the AcrIIA4 protein is expected to interact and inhibit the Cas9–gRNA complex, when co-expressed in the mosquito germline cells. Gene Drive Active Cas9:gRNA Inactive Cas9:gRNA Fig. 1 Schematic representation of gene drive and anti-drive constructs. Gene drive and anti-drive constructs respectively as inserted in the genome of the gene drive lines previously generated (zpg:dsxF2; zpg:7280 and nos:7280, ref. 26) and the newly generated anti-drive (vasa:A4) line. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-24214-5 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-24214-5 100 75 50 25 0 25 50 75 100 56.5 % ±3.1 57.0% ±3.0 a b Inheritance from A+/- parents Inheritance from D+/-;A+/- parents A Parental genotype mated with wild-type Progeny genotype A D A A D NO homing D D Homing in the germline D D ♀ ♂ vasa:A4+/- Percentage of gene drive (RFP positive, ) and anti-drive (GFP positive, ) genotypes found in the progeny nos:7280+/-;vasa:A4+/- zpg:7280+/-;vasa:A4+/- zpg:dsxF+/-;vasa:A4+/- ♀ ♂ ♀ ♂ ♀ ♂ e p yt o n e g la t n e r a P dli w o t d e s s o r c -type Inheritance from D+/- parents ♀ ♂ ♀ ♂ ♀ ♂ nos:7280+/- zpg:7280+/- zpg:dsxF+/- 0 25 50 75 100 100 % 99.9 % ±0.1 94.8 % ±2.7 88.5% ±3.6 90.8% ±7.0 97.5% ±0.9 51.9 % ±4.5 52.8 % ±1.8 51.7% ±2.1 47.4% ±1.9 46.3 % ±5.1 50.0 % ±2.1 50.9 % ±2.1 51.1% ±1.2 50.0% ±3.9 49.8 % ±5.3 55.9% ±2.3 50.8% ±2.3 0 25 50 75 100 Drive locus Anti-drive locus Drive locus Anti-drive locus Drive locus Anti-drive locus a Parental genotype mated with wild-type Progeny genotype D D Homing in the germline D D Drive locus Anti-drive locus a D A A D NO homing Drive locus Anti-drive locus A A Drive locus Anti-drive locus Anti-drive locus b e p yt o n e g la t n e r a P dli w o t d e s s o r c -type Inheritance from D+/- parents ♀ ♂ ♀ ♂ ♀ ♂ nos:7280+/- zpg:7280+/- zpg:dsxF+/- 0 25 50 75 100 100 % 99.9 % ±0.1 94.8 % ±2.7 88.5% ±3.6 90.8% ±7.0 97.5% ±0.9 b 100 75 50 25 0 25 50 75 100 Inheritance from D+/-;A+/- parents nos:7280+/-;vasa:A4+/- zpg:7280+/-;vasa:A4+/- zpg:dsxF+/-;vasa:A4+/- ♀ ♂ ♀ ♂ ♀ ♂ 51.9 % ±4.5 52.8 % ±1.8 51.7% ±2.1 47.4% ±1.9 46.3 % ±5.1 50.0 % ±2.1 50.9 % ±2.1 51.1% ±1.2 50.0% ±3.9 49.8 % ±5.3 55.9% ±2.3 50.8% ±2.3 Inheritance from D+/- parents Inheritance from A+/- parents Inheritance from A+/- parents Inheritance from D+/-;A+/- parents Percentage of gene drive (RFP positive, ) and anti-drive (GFP positive, ) genotypes found in the progeny Fig. 2 Germline expression of anti-CRISPR protein AcrIIA4 causes complete inhibition of gene drive homing. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-24214-5 a Schematic representation of gene drive homing in the germline of individuals carrying one copy of the drive allele (D+/−): Cas9–gRNA-directed cleavage of the insertion site on the homologous wild-type chromosome is repaired via homology-directed repair (HDR), using the drive-carrying chromosome as template, resulting in the D allele being copied (the new drivecopy is indicated as dashed magenta rectangle) and transmitted to most of the progeny (left). Illustration of gene drive homing inhibition in individual carrying one drive and one anti-drive copy (D+/−;A+/−) as consequence of AcrIIA4-directed Cas9–gRNA blockage, resulting in Mendelian inheritance of the D allele (middle). Mendelian inheritance of the anti-drive from A+/−individuals (right). b Scatter plots showing the percentage of larvae carrying the gene drive (RFP positive) and/or the anti-drive (GFP positive) constructs from wild-type mosquitoes crossed to transgenic females or males carrying: only the gene drive construct, conﬁrming high transmission rates (up to 100%) of the D allele from each of the transgenic lines tested (left); both gene drive and anti-drive constructs, showing Mendelian inheritance of both D and A alleles (middle); only the anti-drive construct, showing expected Mendelian rates of the A allele (right). Vertical dashed lines indicate the 50% Mendelian inheritance. Error bars indicate mean percentage values and standard error of the mean of transmission rates from all biological samples assessed for each cross. A minimum of seven biologically independent samples (ovipositing females) were examined over two independent experiments for each cross, with the exception of zpg:dsxF crosses, which were examined only once. The respective raw data are provided in the Source data ﬁle. frequencies (over discrete generations) after a single release of vasa:A4 males into a deﬁned population, where individuals car- rying the zpg:dsxF gene drive (previously developed by Kyrou et al.2) were introduced. We tested our model predictions by releasing 20% of vasa:A4 males, after ﬂuorescence microscope- based enrichment for homozygotes (Supplementary Fig. 2c, d), into a population of 600 mosquitoes in total, including 50% heterozygous zpg:dsxF (males and females) and 30% wild-type individuals. Two experimental replicates were performed in parallel with two control cages, where 300 heterozygotes for the zpg:dsxF drive were added to 300 wild-type individuals. Inheri- tance of drive and/or anti-drive constructs and total egg output was measured at each generation from the four cages. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-24214-5 Consistent with these ﬁndings, maternally deposited AcrIIA4 from the vasa promoter may likely buffer somatic nuclease activity in D+/−;A+/− embryos and therefore mitigate the impact of null mosaicisms on the fertility of the developed individuals, as evidenced by the increased zpg:dsxF+/−;vasa:A4+/−female fertility compared to zpg: dsxF+/−females (Supplementary Fig. 3a). Single release of anti-drive males prevents elimination of caged mosquito populations by the suppressing CRISPR–Cas9 gene drive zpg:dsxF. Fitness values and transmission rates of the respective alleles were used to generate deterministic and sto- chastic models to simulate drive and anti-drive genotype NATURE COMMUNICATIONS \| (2021) 12:3977 \| https://doi.org/10.1038/s41467-021-24214-5 \| www.nature.com/naturecommunications 3 3 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-24214-5 As expected, the frequency of zpg:dsxF genotypes increased rapidly in all cages, leading to elimination of both control populations after the 6th and 11th generation, as a consequence of the pro- gressive reduction of egg output. Consistent with model predic- tions, after an initial increase, the zpg:dsxF spread was halted in the cages where vasa:A4 males were released, maintaining fre- quencies between 98.1 and 76.6% after generation 3, and an average of RFP positive individuals of 87.3% ± 0.05 SD from G3 to G16. Despite not having an inbuilt homing machinery like the zpg:dsxF drive, the frequency of vasa:A4 carrying individuals also increased gradually from the ~10% at G1 up to a maximum of 68.1% (Fig. 3a). As also shown by the model simulation, the gradual increment of anti-drive frequency is determined by the higher overall ﬁtness linked to the vasa:A4 alleles compared to the drive (Supplementary Table 2). On the other hand, the egg output of the two experimental cages was maintained above one- ﬁfth of the generation after release, with values (relative to G1) of 0.27 and 0.43 at generation 16, when the last measurement was performed (Fig. 3b). To determine the contribution of nuclease- resistant alleles over generations, we performed amplicon sequencing of pooled samples collected at generations 1, 5, 10 (from experimental and control cages) and 15 (for experi- mental cages). In line with modelling prediction, a progressive accumulation of mutations at the dsx-target site was observed in the control cages, reaching a total frequency of 0.047 and 0.062 (among non-drive alleles) in the generations prior to population collapse. On the contrary, mutations measured from the cages containing vasa:A4 carrying mosquitoes remained at low levels and gradually decreased reaching minimum total frequency at frequencies (over discrete generations) after a single release of vasa:A4 males into a deﬁned population, where individuals car- rying the zpg:dsxF gene drive (previously developed by Kyrou et al.2) were introduced. We tested our model predictions by releasing 20% of vasa:A4 males, after ﬂuorescence microscope- based enrichment for homozygotes (Supplementary Fig. 2c, d), into a population of 600 mosquitoes in total, including 50% heterozygous zpg:dsxF (males and females) and 30% wild-type individuals. Two experimental replicates were performed in parallel with two control cages, where 300 heterozygotes for the zpg:dsxF drive were added to 300 wild-type individuals. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-24214-5 Inheri- tance of drive and/or anti-drive constructs and total egg output was measured at each generation from the four cages. As expected, the frequency of zpg:dsxF genotypes increased rapidly in all cages, leading to elimination of both control populations after the 6th and 11th generation, as a consequence of the pro- gressive reduction of egg output. Consistent with model predic- tions, after an initial increase, the zpg:dsxF spread was halted in the cages where vasa:A4 males were released, maintaining fre- quencies between 98.1 and 76.6% after generation 3, and an average of RFP positive individuals of 87.3% ± 0.05 SD from G3 NATURE COMMUNICATIONS \| (2021) 12:3977 \| https://doi.org/10.1038/s41467-021-24214-5 \| www.nature.com/naturecommunications 4 4 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-24214-5 Although not accounting for spatial dispersal, our models show a long-term equilibrium between drive, anti-drive and wild-type alleles (Supplementary Fig. 5) that is primarily, although not exclusively, dependent on their relative ﬁtness (Supplementary Fig. 6), as described pre- viously for other reversal strategies28–30. Allele dynamics over an extended number of generations shows that a single release of vasa:A4 anti-drive males can sensibly constrain the gene drive spread, preventing it from reaching high frequencies and drastic reproductive load according to the drive/anti-drive relative fre- quency at release, even when the ﬁtness of females heterozygous NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-24214-5 anti-drive males constrains gene drive spread preventing population suppression in caged mosquitoes. Two cages Fig. 3 A single release of AcrIIA4 anti-drive males constrains gene drive spread preventing population suppression in caged mosquitoes. Two cages were initiated with a starting population of 600 A. gambiae mosquitoes of which 150 males and 150 females heterozygous for the zpg:dsxF driving allele (initial drive allele frequency of 25%), 120 homozygous-enriched males for the vasa:A4 allele (initial anti-drive allele frequency of 20%) and 180 wild-type of which 30 males and 150 females to maintain equal sex ratio (left). In parallel, two control cages were established by releasing the same proportion of drive alleles (150 zpg:dsxF+/−males and 150 zpg:dsxF+/−females) and 300 wild-type mosquitoes (150 males and 150 females). a The frequency of RFP positive drive (D+, dark purple solid lines), GFP positive anti-drive (A+, dark green solid lines) and non-transgenic individuals (NT, black solid lines) was recorded for each generation by screening larvae for the expression of the respective ﬂuorescent markers. b Absolute number of eggs produced each generation (dark grey solid lines). Genotype frequencies (D+, A+ and NT) and egg output (EO) values were overlapped to the respective deterministic (dotted lines) and stochastic (light-coloured lines) model simulation based on the parameters provided in Supplementary Table 2. The respective raw data are provided in the Source data ﬁle. for the drive is considered equal to wild-type, thereby facilitating the spread of the drive (Supplementary Fig. 6). G15 (0.013 and 0.019; Supplementary Fig. 4a). This result is consistent with no signiﬁcant end-joining resistance being gen- erated from mosquitoes carrying both the drive and anti-drive constructs (Supplementary Fig. 4b). Although not accounting for spatial dispersal, our models show a long-term equilibrium between drive, anti-drive and wild-type alleles (Supplementary Fig. 5) that is primarily, although not exclusively, dependent on their relative ﬁtness (Supplementary Fig. 6), as described pre- viously for other reversal strategies28–30. Allele dynamics over an extended number of generations shows that a single release of vasa:A4 anti-drive males can sensibly constrain the gene drive spread, preventing it from reaching high frequencies and drastic reproductive load according to the drive/anti-drive relative fre- quency at release, even when the ﬁtness of females heterozygous G15 (0.013 and 0.019; Supplementary Fig. 4a). This result is consistent with no signiﬁcant end-joining resistance being gen- erated from mosquitoes carrying both the drive and anti-drive constructs (Supplementary Fig. 4b). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-24214-5 0 10000 20000 30000 Gene Drive only (control cages) Generation t u p t u o g g e e t ulo s b A a b y c n e u q e rf e p yt o n e G Gene Drive + Anti-Drive (exp. cages) NT D+ EO Cage 3 (C): Cage 4 (C): D+ A+ NT Cage 1: Cage 2: EO 0 2 4 6 8 10 12 14 16 0 0.25 0.5 0.75 1 0 2 4 6 8 10 12 14 16 0 0.25 0.5 0.75 1 0 2 4 6 8 10 12 14 16 0 10000 20000 30000 Generation 0 2 4 6 8 10 12 14 16 Fig. 3 A single release of AcrIIA4 anti-drive males constrains gene drive spread preventing population suppression in caged mosquitoes. Two cages were initiated with a starting population of 600 A. gambiae mosquitoes of which 150 males and 150 females heterozygous for the zpg:dsxF driving allele (initial drive allele frequency of 25%), 120 homozygous-enriched males for the vasa:A4 allele (initial anti-drive allele frequency of 20%) and 180 wild-type of which 30 males and 150 females to maintain equal sex ratio (left). In parallel, two control cages were established by releasing the same proportion of drive alleles (150 zpg:dsxF+/−males and 150 zpg:dsxF+/−females) and 300 wild-type mosquitoes (150 males and 150 females). a The frequency of RFP positive drive (D+, dark purple solid lines), GFP positive anti-drive (A+, dark green solid lines) and non-transgenic individuals (NT, black solid lines) was recorded for each generation by screening larvae for the expression of the respective ﬂuorescent markers. b Absolute number of eggs produced each generation (dark grey solid lines). Genotype frequencies (D+, A+ and NT) and egg output (EO) values were overlapped to the respective deterministic (dotted lines) and stochastic (light-coloured lines) model simulation based on the parameters provided in Supplementary Table 2. The respective raw data are provided in the Source data ﬁle. Gene Drive only (control cages) a y c n e u q e rf e p yt o n e G Gene Drive + Anti-Drive (exp. NATURE COMMUNICATIONS \| (2021) 12:3977 \| https://doi.org/10.1038/s41467-021-24214-5 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-24214-5 cages) 0 2 4 6 8 10 12 14 16 0 0.25 0.5 0.75 1 0 2 4 6 8 10 12 14 16 0 0.25 0.5 0.75 1 a y c n e u q e rf e p yt o n e G Gene Drive + Anti-Drive (exp. cages) 0 2 4 6 8 10 12 14 16 0 0.25 0.5 0.75 1 0 0 0.25 0.5 0.75 1 Gene Drive only (control cages) 6 0 2 4 6 8 10 12 14 16 0 0.25 0.5 0.75 1 Gene Drive + Anti-Drive (exp. cages) Gene Drive only (control cages) 100 200 300 t u p t u o g g e e t ulo s b A b 0 2 4 6 8 10 12 14 16 0 10000 20000 30000 Generation 0 10000 20000 30000 Generation t u p t u o g g e e t ulo s b A b 0 2 4 6 8 10 12 14 16 0 10000 20000 30000 Generation 0 2 4 6 8 10 12 14 16 b Generation Fig. 3 A single release of AcrIIA4 anti-drive males constrains gene drive spread preventing population suppression in caged mosquitoes. Two cages were initiated with a starting population of 600 A. gambiae mosquitoes of which 150 males and 150 females heterozygous for the zpg:dsxF driving allele (initial drive allele frequency of 25%), 120 homozygous-enriched males for the vasa:A4 allele (initial anti-drive allele frequency of 20%) and 180 wild-type of which 30 males and 150 females to maintain equal sex ratio (left). In parallel, two control cages were established by releasing the same proportion of drive alleles (150 zpg:dsxF+/−males and 150 zpg:dsxF+/−females) and 300 wild-type mosquitoes (150 males and 150 females). a The frequency of RFP positive drive (D+, dark purple solid lines), GFP positive anti-drive (A+, dark green solid lines) and non-transgenic individuals (NT, black solid lines) was recorded for each generation by screening larvae for the expression of the respective ﬂuorescent markers. b Absolute number of eggs produced each generation (dark grey solid lines). Genotype frequencies (D+, A+ and NT) and egg output (EO) values were overlapped to the respective deterministic (dotted lines) and stochastic (light-coloured lines) model simulation based on the parameters provided in Supplementary Table 2. The respective raw data are provided in the Source data ﬁle. Microinjection of embryos and selection of transformed mosquitoes. All Microinjection of embryos and selection of transformed mosquitoes. All mosquitoes used in this work were reared under standard conditions of 80% relative humidity and 28 °C. Adult mosquitoes of a previously generated A. gam- biae attP docking line25 were blood-fed by Hemotek and freshly laid embryos were aligned for microinjections, as described previously34. The injected solution con- tained 50 ng/μL of the vasa:AcrIIA4 construct and 400 ng/μL of a helper plasmid expressing the φC31 integrase under the vasa promoter3. Hatched larvae were screened for transient expression of the eGFP marker and crossed to wild-type mosquitoes to obtain transgenic individuals expressing both eGFP and eCFP. Expression of ﬂuorescent markers was analysed on a Nikon inverted microscope (Eclipse TE200). Molecular conﬁrmation of insertion and zygosity assessment. Vasa:A4 and wild-type mosquitoes were used for genomic DNA extraction, using Qiagen blood and tissue kit (Qiagen) followed by PCR ampliﬁcations at the insertion locus to conﬁrm the correct integration of the transgene and zygosity of the vasa:A4 released in the cage trial. The ϕC31-mediated integration of the vasa:A4 construct was conﬁrmed using primers binding the integrated construct and the neigh- bouring genomic locus, using the RG1044 and RG187 primers (PCR A; Supple- mentary Fig. 2a, b). The proportion of heterozygous (vasa:A4+/−) and homozygous (vasa:A4+/+) anti-drive males released in the cage trial was determined, using the RG1044-5R1 primers binding the transgene and the ﬂanking genomic region (PCR B) and primers RG1047–RG1044 binding either side of the transgene insertion site (PCR C). A total of 37 male individuals were assessed for cage 1 and 25 male individuals for cage 2 (Supplementary Fig. 2a, c, d and Supplementary Table 3). pp g p p y As for gene drives, anti-drive technologies such as the one developed here will also require careful experimental and math- ematical evaluations before these can be considered as an effective and ﬁeld-applicable countermeasure. The effectiveness of this technology in blocking gene drive spread will ultimately depend on the relative ﬁtness of drive and anti-drive constructs, as well as rates and ﬁtness cost associated with genetic resistance against the drive or anti-drive, although the latter is expected to be reduced for our protein-based inhibitor compared to DNA-cleaving approaches. The anti-CRISPR tool described here will also be of great use to expedite laboratory husbandry of CRISPR–Cas expressing transgenic lines, including suppressive gene drives, which usually require continuous backcrossing to wild-type strains. Microinjection of embryos and selection of transformed mosquitoes. All Finally, this work provides an additional technology for genetic regulation of CRISPR activity that can be of great use for a broad range of applications in A. gambiae mosquitoes, as well as other medically relevant insects, including the model insect Drosophila melanogaster. Mosquito genetic crosses. Vasa:A4 males carrying one copy of the anti-drive construct (vasa:A4+/−) were crossed to heterozygous females of each gene drive line (zpg:dsxF+/−, zpg:7280+/−or nos:7280+/−). Larvae carrying one copy of the drive (RFP positive), one copy of the anti-drive (GFP positive) or both (RFP and GFP positive) were selected and crossed to wild-type individuals for phenotypic assays (Supplementary Fig. 3a). Vasa:A4 males were crossed to virgin females carrying a 3xP3:DsRed marker in the same locus (mars transgenic line25) to generate individuals carrying either both transgenes (vasa:A4+/mars+), and therefore homozygous for the disruption of the genetic locus (GFP and RFP positive) or either transgene in heterozygosity (GFP positive vasa:A4+/−and RFP positive mars+/−). For each genotype, transgenic males and females were crossed to wild-type individuals for phenotypic characterisation (Supplementary Fig. 3b). Transgenic individuals carrying both transgenes (vasa:A4+/mars+) were also crossed to each other to generate individuals homozygous either for the vasa:A4 (vasa:A4+/+) or the mars (mars+/+) construct, as well as siblings carrying one copy of each construct (vasa:A4+/mars+). Males and females of each genotype were crossed to wild-type for phenotypic characterisation (Supplementary Fig. 3c). Phenotypic assays. For each genotype tested, 30 transgenic male or female adults were crossed to an equal number of wild-type mosquitoes for 5 days, blood-fed and a minimum of 15 females allowed to lay individually. The entire egg and larval progeny were counted for each lay (Supplementary Fig. 3). Females that failed to give progeny and had no evidence of sperm in their spermathecae were excluded from fertility analysis, but considered for mating analysis (Supplementary Table 1). To conﬁrm parental zygosity of the vasa:A4 alleles, progenies were also screened for the presence of WT individuals (negative to ﬂuorescence screening). I h it f d i (RFP iti ) d ti d i (GFP iti ) Discussion I hi k For instance, in the case a gene drive- targeting gRNA17 may become genetically linked to a mutated Cas9 sequence that remains functionally active, but resistant to further cleavage by the reversal gRNA19. Another aspect to be considered is that broadly active anti-CRISPR proteins, con- ferring low ﬁtness costs to the carriers, have the potential to persist in a population over time, effectively generating a genetic barrier against newly introduced gene drives or self-limiting genetic control strategies, which rely on CRISPR–Cas with overlapping spatiotemporal activity. previously engineered for population suppression, and achieved proof-of-concept of propulation rescue in caged insects. Here, gene drive inhibition is achieved via protein–protein interaction, rather than DNA cleavage of drive-speciﬁc sequences, bypassing potential unintended effects of genome editing altera- tions caused by alternative Cas9–gRNA or gRNA-only reversal strategies15,17–19. Repair of CRISPR-mediated cleavage of genetic components encoding for the gene drive may alter either the activity or the speciﬁcity of action with unpredictable con- sequence in the long term; for example, upon modiﬁcation of the targeting sequence of the driving gRNA. Moreover, expression of anti-CRISPR proteins can be easily restricted to the cell types where the gene drive is active by using tissue-speciﬁc pol II promoters (e.g., the vasa promoter used here), while gRNA-based methods generally require ubiquitously active pol III promoters. The broad spectrum of DNA mimic proteins31,32 expands dra- matically the application range of this system, which in principle could be used to counteract any genetic control technology employing CRISPR–Cas9 tools, without requiring ad hoc engi- neering for different genetic constructs. This aspect may be par- ticularly advantageous in the case of synonymous or silent mutations arising at the driving genetic sequence (naturally or induced by DNA-cleaving reversal construct) that may prevent the effect of counteracting systems involving DNA cleavage. In addition, the generation and selection of resistance to the anti- drive is reduced compared to DNA-cleaving methods, for which creation of resistant alleles at the drive target is expected at each generation as shown for homing gene drive constructs10–13. It should also be considered that risks related to remobilisation of the anti-drive protein inhibitor are theoretically reduced com- pared to other strategies. For instance, in the case a gene drive- targeting gRNA17 may become genetically linked to a mutated Cas9 sequence that remains functionally active, but resistant to further cleavage by the reversal gRNA19. Discussion I hi k In this work, we demonstrate that a genetically encoded anti- CRISPR protein can be highly effective in inhibiting potent CRISPR-based gene drives engineered to suppress malaria mos- quitoes by restoring their inheritance to Mendelian rates. Our experiments show that a single low frequency release of males carrying the anti-CRISPR construct is highly effective in pre- venting population suppression, despite a reduced ﬁtness asso- ciated with the anti-drive. We successfully tested wide applicability of the AcrIIA4- based anti-drive technology against different gene drive systems, TURE COMMUNICATIONS \| (2021) 12:3977 \| https://doi.org/10.1038/s41467-021-24214-5 \| www.nature.com/naturecommunications 5 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-24214-5 previously engineered for population suppression, and achieved proof-of-concept of propulation rescue in caged insects. Here, gene drive inhibition is achieved via protein–protein interaction, rather than DNA cleavage of drive-speciﬁc sequences, bypassing potential unintended effects of genome editing altera- tions caused by alternative Cas9–gRNA or gRNA-only reversal strategies15,17–19. Repair of CRISPR-mediated cleavage of genetic components encoding for the gene drive may alter either the activity or the speciﬁcity of action with unpredictable con- sequence in the long term; for example, upon modiﬁcation of the targeting sequence of the driving gRNA. Moreover, expression of anti-CRISPR proteins can be easily restricted to the cell types where the gene drive is active by using tissue-speciﬁc pol II promoters (e.g., the vasa promoter used here), while gRNA-based methods generally require ubiquitously active pol III promoters. The broad spectrum of DNA mimic proteins31,32 expands dra- matically the application range of this system, which in principle could be used to counteract any genetic control technology employing CRISPR–Cas9 tools, without requiring ad hoc engi- neering for different genetic constructs. This aspect may be par- ticularly advantageous in the case of synonymous or silent mutations arising at the driving genetic sequence (naturally or induced by DNA-cleaving reversal construct) that may prevent the effect of counteracting systems involving DNA cleavage. In addition, the generation and selection of resistance to the anti- drive is reduced compared to DNA-cleaving methods, for which creation of resistant alleles at the drive target is expected at each generation as shown for homing gene drive constructs10–13. It should also be considered that risks related to remobilisation of the anti-drive protein inhibitor are theoretically reduced com- pared to other strategies. Discussion I hi k Another aspect to be considered is that broadly active anti-CRISPR proteins, con- ferring low ﬁtness costs to the carriers, have the potential to persist in a population over time, effectively generating a genetic barrier against newly introduced gene drives or self-limiting genetic control strategies, which rely on CRISPR–Cas with overlapping spatiotemporal activity. templates, GamS (Arbor Biosciences, Cat: 501024) was added to the 75 µL TXTL reaction master mix at a ﬁnal concentration of 2 µM (ref. 33). The anti-CRISPR protein AcrIIa4 (SPC gb013) and Cas9 sgRNAs were expressed from linear tem- plate DNA at 1 and 4 nM concentrations, respectively. Cas9 (pCB843) and deGFP (pCB556) were expressed from plasmid DNA templates at 1 and 0.5 nM con- centrations, respectively. The reactions were mixed by brief vortexing and collected using a benchtop centrifuge. Each reaction was split into two aliquots, each of 5 µL, and loaded into a 96-well V-bottom plate (Corning Costar 3357) and covered with a cap mat. The 96-well plate with TXTL droplets was loaded into a BioTek Synergy H1 plate reader at 29 °C without shaking. Fluorescence of TXTL reaction was measured at Exc. 485 nm, Em. 528 nm every 3 min, for 16 h. Only the ﬂuorescence from the endpoint of the reaction was reported (Supplementary Fig. 1b). Plasmid construction. The L. monocytogenes AcrIIA4-coding sequence, synthe- tised and codon-optimised for A. gambiae (ATUM), was ampliﬁed using primers containing the XhoI cleavage site followed by a NLS at the N-terminus side and the PacI site after the C-terminus (RG247–RG248; Supplementary Table 3). The fragment was digested and ligated into a pre-existing vector containing the vasa promoter and terminator sequences24 ﬂanking the XhoI and PacI sites, the eGFP- coding sequence under the control of the 3xP3 promoter separated by the ϕC31 attB recombination sequence, resulting in the ﬁnal plasmid named C77 (Genbank accession code MZ172909). Methods In parallel, two control cages were initiated by releasing an equal number of gene drive mosquitoes (150 zpg:dsxF+/− males and 150 zpg:dsxF+/−females) with 150 wild-type males and 150 wild-type females (corresponding to 25% allele frequency of the gene drive). where i and j are each summed such that {1, 2, 3} corresponds to {W, D, R} and k and l such that {1, 2} corresponds to {W, A}. The coefﬁcients cm;n ijjkl correspond to the proportion of the gametes from female individuals of type (ijjkl) that carry alleles (mjn). For example, assuming no linkage, for a female of genotype WD\|WA, the coefﬁcient for alleles of type mjn ¼ WjW; WjA; DjW and DjA is =¼, since inheritance of the drive is Mendelian due to the presence of anti-drive in that genotype, and is zero for alleles of type RjW and RjA, since it is assumed that no end-joining resistance is generated with anti-drive present. An analogous expression is used for sperm: where i and j are each summed such that {1, 2, 3} corresponds to {W, D, R} and k and l such that {1, 2} corresponds to {W, A}. The coefﬁcients cm;n ijjkl correspond to g y g Each generation, mosquitoes were left to mate for 5 days before they were blood-fed on anaesthetised mice. Two days later, egg bowls ﬁlled with water and lined with ﬁlter paper were added in the cages to allow for overnight oviposition. The following day, eggs laid in the egg bowl were dispersed using gentle water spraying to homogenise the population, and 650 eggs were randomly selected to seed the next generation. The remaining eggs were photographed and counted using JMicroVision V1.27 to obtain the overall egg output from each cage (Fig. 3b and Source data ﬁle). Larvae hatching from the 650 eggs were counted and reared at a density of 200 per tray (in ∼0.5 litre of water). L2/L3 larvae were screened for the presence of the RFP and GFP marker to measure gene drive and anti-drive genotype frequencies (Fig. 3a and Source data ﬁle). All the pupae obtained from the 650 eggs were used to seed the following generation. Methods These were subsequently mixed at L1 stage and reared in parallel with the offspring of vasa:A4+/−males crossed to vasa:A4+/− Discrete-generation (non-overlapping) cage trial. To minimise possible par- ental bias of Cas9–gRNA deposition and consequent generation of alleles resistant to the drive, the gene drive individuals released in the cage trial were obtained from both zpg:dsxF males crossed to wild-type females and zpg:dsxF females crossed to wild-type males in equal numbers. These were subsequently mixed at L1 stage and reared in parallel with the offspring of vasa:A4+/−males crossed to vasa:A4+/− females, and wild-type insects. RFP positive gene drive and GFP positive anti-drive larvae were screened at L3 stage, and the developing male and female pupae were sexed and allowed to emerge in individual cages in parallel with wild-type males and females. Vasa:A4+/+ individuals used for the release were selected based on higher intensity of the eGFP signal from larval progeny of vasa:A4 heterozygous parents. Adult mosquitoes were mixed only when all the pupae had emerged. We ﬁrstly consider the gamete contributions from each genotype. The proportions EmjnðtÞ with allele m ¼ fW; D; Rg at the gene drive locus, and n ¼ fW; Ag at the anti-drive locus in eggs produced by females participating in reproduction are given in terms of the female genotype frequencies Fijjkl tð Þ: females, and wild-type insects. RFP positive gene drive and GFP positive anti-drive larvae were screened at L3 stage, and the developing male and female pupae were sexed and allowed to emerge in individual cages in parallel with wild-type males and females. Vasa:A4+/+ individuals used for the release were selected based on higher intensity of the eGFP signal from larval progeny of vasa:A4 heterozygous parents. Adult mosquitoes were mixed only when all the pupae had emerged. Emjn tð Þ ¼ ∑3 i¼1∑3 j¼i∑2 k¼1∑2 l¼kcm;n ijjklwijjklFijjkl tð Þ ∑3 i¼1∑3 j¼i∑2 k¼1∑2 l¼kwijjklFijjkl tð Þ jn tð Þ ¼ ∑3 i¼1∑3 j¼i∑2 k¼1∑2 l¼kwijjklFijjkl tð Þ Two experimental cages were initiated by releasing 150 zpg:dsxF+/−males and 150 zpg:dsxF+/−females (corresponding to a 25% allele frequency at the gene drive locus) together with 120 anti-drive males enriched for homozygous (~20% allele frequency of anti-drive alleles), 30 wild-type males and 150 wild-type females (contributing 30% to the total of ~80% allele frequency of wild-type alleles at the anti-drive locus and 75% for the drive locus). LðtÞ ¼ 1 2FðtÞwf ðtÞwmðtÞ Assuming very large population sizes gives results for the genotype frequencies that are indistinguishable from the deterministic model. For the deterministic egg count, we use the large population limit of the stochastic model. Modelling. Discrete-generation recursion equations were used for genotype fre- quencies, with males and females treated separately as in refs. 2,11,25. Here, we model two loci: the gene drive locus, where we consider three alleles, W (wild-type), D (drive) and R (non-functional nuclease-resistant), and the anti-drive site with two alleles W (wild-type) and A (anti-drive). FijjklðtÞ and Mijjkl tð Þ denote the genotype frequency of females (or males) in the total population, where the ﬁrst set of indices denotes alleles at the target locus ij ¼ fWW; WD; WR; DD; DR; RRg, and the second set denotes the anti-drive locus, kl ¼ fWW; WA; AAg. For simplicity, we assume full recombination and no linkage between the loci. There are 18 female genotypes and 18 male genotypes (see list in Supplementary Table 2); 6 types of eggs in proportions EWjW; EWjA; EDjW, EDjA, ERjW, ERjA, where the ﬁrst index refers to the target site allele and the second to the anti-drive; and similarly 6 types of sperm, SWjW; SWjA; SDjW; SDjA, SRjW, SRjA. \| g heterozygotes and 150 of wild-type adults. Females may fail to mate, or mate once in their life, with a male of a given genotype according to its frequency in the male population times its mating ﬁtness (relative to wild-type), chosen randomly with replacement such that males may mate multiple times. The number of eggs from each mated female is multiplied by the egg production of the male relative to wild- type. To start the next generation, 650 eggs are randomly selected, and their hatching probability depends on the product of larval hatching values from the mother and father. The probability of subsequent survival to adulthood is assumed to be equal across genotypes. Assuming very large population sizes gives results for the genotype frequencies that are indistinguishable from the deterministic model. For the deterministic egg count, we use the large population limit of the stochastic model. Homing of the gene drive is assumed to occur only when the anti-drive is not present. Methods Smjn tð Þ ¼ ∑3 i¼1∑3 j¼i∑2 k¼1∑2 l¼kcm;n ijjklwijjklMijjkl tð Þ ∑3 i¼1∑3 j¼i∑2 k¼1∑2 l¼kwijjklMijjkl tð Þ To model cage experiments, the initial frequency of heterozygote drive females and males is FWDjWW ¼ MWDjWW ¼ 0:25, of anti-drive males MWWjAA ¼ 0:2, and of wild-type female and males FWWjWW ¼ 0:25 and MWW;WW ¼ 0:05. For release of gene drive only, MWW;WW ¼ MWD;WW ¼ 1=4 and FWW;WW ¼ FWD;WW ¼ 1=4: Assuming random mating, we obtain the following recursion equations for the female genotype frequencies in the next generation ðt þ 1Þ: Fijjkl t þ 1 ð Þ ¼ 1 2 1 δij 2 1 δkl 2 Eijk tð ÞSjjk tð Þ þ Ejjk tð ÞSijk tð Þ þ Eijl tð ÞSjjl tð Þ þ Ejjl tð ÞSijl tð Þ Animal husbandry. Mosquitoes in the cage trials were fed using anaesthetised 2–6 month old female mice (CD1 strain). CD1 mice were kept in social-housing caged units with 12/12 light–dark cycles, 45–65% relative humidity and at temperature of 20–24 °C. All animal work was conducted according to UK Home Ofﬁce Regulations and approved under Home Ofﬁce License PPL 70/8914 by the AWERB at Imperial College London. where δij is the Kronecker delta. The factors ð1 δij 2 Þ; ð1 δkl 2 Þ account for the factor of 1/2 for homozygosity at the drive target site (for ij ¼ {WW, DD, RR}) and at the anti-drive site (for {WW; AA}). Similar equations may be written for the male genotype frequencies Mijjklðt þ 1Þ. Amplicon sequencing analysis. Adult mosquitoes were collected at G1, G5, G10 and G15 from each of the four cages after obtaining the respective progenies (Fig. 3). DNA extraction from pooled individuals, PCR ampliﬁcation and amplicon sequencing were performed for each of the 14 samples as previously described2 using the 4050-Illumina-F and 4050-Illumina-R primers. Raw ampli- con sequencing data were deposited in the EBI-ENA database (accession code PRJEB44729). The CRISPResso v1.0.8 software35 was used to analyse the frequency of wild-type and mutated sequences at the zpg:dsxF gene drive target as previously described2, accounting for all indels and substitutions present at the target sequence and/or any of the two invariable nucleotides of the corresponding PAM sequence (-GG). Exogenous contaminant alleles were removed bioinformatically2 (Supplementary Fig. 4a). Methods Anti-CRISPR testing in cell-free reactions. Escherichia coli cell-free reaction mixture was sourced from Arbor biosciences (Arbor Biosciences, Cat: 507024). Each 75 µL, 1.25×-concentrated MyTXTL reaction was loaded with the necessary DNA expression templates and ultimately divided into 5-µL individual reaction droplets for incubation, expression and ﬂuorescence monitoring, as described in ref. 31 (Supplementary Fig. 1a). To prevent the degradation of linear DNA Inheritance of gene drive (RFP positive) and anti-drive (GFP positive) transgenes was measured by screening the entire larval progeny obtained from each Inheritance of gene drive (RFP positive) and anti drive (GFP positive) transgenes was measured by screening the entire larval progeny obtained from each NATURE COMMUNICATIONS \| (2021) 12:3977 \| https://doi.org/10.1038/s41467-021-24214-5 \| www.nature.com/naturecommunications 6 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-24214-5 oviposition. Females that produced less than ten larvae were excluded from the analysis of transgenic inheritance rates (Fig. 2b). rates of transmission of the driver allele in the two sexes, and uf and um are the fractions of non-drive gametes at the target site that are repaired by meiotic end- joining and are non-functional and resistant to the drive (R). If the anti-drive is present (WD\|WA and WD\|AA), drive inheritance is Mendelian. In all other genotypes, inheritance at the target site is also Mendelian. In the deterministic model, ﬁtness effects are manifested as differences in the relative ability of female or male genotypes to participate in mating and reproduction. We let wijjkl ≤1 represent the ﬁtness of genotype ijjkl relative to wWWjWW ¼ 1 for the wild-type homozygote (see “overall ﬁtness” in Supplementary Table 2). We assume the dsx-target gene is needed for female fertility, thus females with DD, DR and RR at the gene drive locus are sterile. Statistical differences against selected reference crosses tested in parallel were assessed using Welch’s unpaired t test, for both larval and egg output averages, and Fisher’s exact test for the total number of larvae hatched from each cross (Supplementary Fig. 3). Discrete-generation (non-overlapping) cage trial. To minimise possible par- Discrete-generation (non-overlapping) cage trial. To minimise possible par- ental bias of Cas9–gRNA deposition and consequent generation of alleles resistant to the drive, the gene drive individuals released in the cage trial were obtained from both zpg:dsxF males crossed to wild-type females and zpg:dsxF females crossed to wild-type males in equal numbers. LðtÞ ¼ 1 2FðtÞwf ðtÞwmðtÞ where wf tð Þ ¼ ∑18 k¼1 wkFk tð Þ=∑18 k¼1 Fk tð Þ is the average female ﬁtness and wm tð Þ ¼ ∑18 k¼1 wkMk tð Þ=∑18 k¼1 Mk tð Þ is the average male ﬁtness (here, k is summing over the 18 genotypes). F tð Þ ¼ ∑18 k¼1 Fk tð Þ is the proportion of females in the population (¼ 1=2 except for the zeroth generation). The load is zero when only wild-types are present. In the stochastic version of the model, as in refs. 2,5, probabilities of mating, egg production, hatching and emergence from pupae are estimated from experiments (Supplementary Table 2), and random numbers for these events are taken from the appropriate multinomial distributions. To model the cage experiments, 150 female and 30 male wild-type adults along with 120 male drive homozygotes (WD\|AA), and 150 each female and male heterozygous for the drive (WD\|WW) are initially present (600 individuals in total). For experiments with gene drive only and no anti-drive, there are 150 each of female and male WD\|WW gene drive In the stochastic version of the model, as in refs. 2,5, probabilities of mating, egg production, hatching and emergence from pupae are estimated from experiments (Supplementary Table 2), and random numbers for these events are taken from the appropriate multinomial distributions. To model the cage experiments, 150 female and 30 male wild-type adults along with 120 male drive homozygotes (WD\|AA), and 150 each female and male heterozygous for the drive (WD\|WW) are initially present (600 individuals in total). For experiments with gene drive only and no anti-drive, there are 150 each of female and male WD\|WW gene drive heterozygotes and 150 of wild-type adults. Females may fail to mate, or mate once in their life, with a male of a given genotype according to its frequency in the male population times its mating ﬁtness (relative to wild-type), chosen randomly with replacement such that males may mate multiple times. The number of eggs from each mated female is multiplied by the egg production of the male relative to wild- type. To start the next generation, 650 eggs are randomly selected, and their hatching probability depends on the product of larval hatching values from the mother and father. The probability of subsequent survival to adulthood is assumed to be equal across genotypes. Methods In the deterministic model, the load on the population incorporates reductions in female and male fertility and at time t is deﬁned as: LðtÞ ¼ 1 2FðtÞwf ðtÞwmðtÞ NATURE COMMUNICATIONS \| (2021) 12:3977 \| https://doi.org/10.1038/s41467-021-24214-5 \| www.nature.com/naturecommunications References 1. Jinek, M. et al. 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The authors received funding from Defense Advanced Research Projects Agency Safe Genes programme (HR0011-17-2-0042) for this research. The views, opinions and/or ﬁndings expressed should not be interpreted as representing the ofﬁcial views or policies of the Department of Defense or the U.S. Government. The salaries of A.B., N.I.D., K.K. and M.G. were supported by Target Malaria. 6. Simoni, A. et al. Development of synthetic selﬁsh elements based on modular nucleases in Drosophila melanogaster. Nucleic Acids Res. 42, 7461–7472 (2014). 7. Gantz, V. M. & Bier, E. Genome editing. The mutagenic chain reaction: a method for converting heterozygous to homozygous mutations. Science 348, 442–444 (2015). 8. Burt, A. Site-speciﬁc selﬁsh genes as tools for the control and genetic engineering of natural populations. Proc. Biol. Sci. 270, 921–928 (2003). The authors declare no competing interests. The authors declare no competing interests. 12. Unckless, R. L., Clark, A. G. & Messer, P. W. 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Cell 69, 146–157.e3 (2018). Competing interests The authors declare no competing interests. LðtÞ ¼ 1 2FðtÞwf ðtÞwmðtÞ Adults of genotype WDjWW (i.e., with no anti-drive) produce gametes at meiosis in the ratio WjW : DjW : RjW as follows: ð1 df Þð1 uf Þ : df : ð1 df Þuf in females, 1 dm 1 um : dm : 1 dm um in males. Here, df and dm are the 7 7 TURE COMMUNICATIONS \| (2021) 12:3977 \| https://doi.org/10.1038 Reprints and permission information is available at http://www.nature.com/reprints Reprints and permission information is available at http://www.nature.com/reprints 16. DiCarlo, J. E., Chavez, A., Dietz, S. L., Esvelt, K. M. & Church, G. M. Safeguarding CRISPR-Cas9 gene drives in yeast. Nat. Biotechnol. 33, 1250–1255 (2015). Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. 17. Gantz, V. M. & Bier, E. The dawn of active genetics. Bioessays 38, 50–63 (2016). 18. Wu, B., Luo, L. & Gao, X. J. Cas9-triggered chain ablation of cas9 as a gene drive brake. Nat. Biotechnol. 34, 137–138 (2016). Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. 19. Xu, X.-R. S. et al. Active genetic neutralizing elements for halting or deleting gene drives. Mol. Cell 80, 246–262.e4 (2020). 20. Pawluk, A. et al. Naturally occurring off-switches for CRISPR-Cas9. Cell 167, 1829–1838.e9 (2016). 21. Rauch, B. J. et al. Inhibition of CRISPR-Cas9 with bacteriophage proteins. Cell 168, 150–158.e10 (2017). 22. Dong, D. et al. Structural basis of CRISPR–SpyCas9 inhibition by an anti- CRISPR protein. Nature 546, 436–439 (2017). p Yang, H. & Patel, D. J. Inhibition mechanism of an anti-CRIS 23. Yang, H. & Patel, D. J. Inhibition mechanism of an anti-CRISPR suppressor AcrIIA4 targeting SpyCas9. Mol. Cell 67, 117–127.e5 (2017). 24. 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https://openalex.org/W2120073387	https://europepmc.org/articles/pmc3513719?pdf=render	English	null	Risk Factors for Development of Acute Kidney Injury in Critically Ill Patients: A Systematic Review and Meta-Analysis of Observational Studies	Critical care research and practice	2,012	cc-by	8,517	Hindawi Publishing Corporation Critical Care Research and Practice Volume 2012, Article ID 691013, 15 pages doi:10.1155/2012/691013 Hindawi Publishing Corporation Critical Care Research and Practice Volume 2012, Article ID 691013, 15 pages doi:10.1155/2012/691013 Rodrigo Cartin-Ceba,1 Markos Kashiouris,1 Maria Plataki,1 Daryl J. Kor,2 Ognjen Gajic,1 and Edward T. Casey3 Rodrigo Cartin-Ceba,1 Markos Kashiouris,1 Maria Plataki,1 Daryl J. Kor,2 Ognjen Gajic,1 and Edward T. Casey3 1Division of Pulmonary and Critical Care Medicine, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA 2Department of Anesthesia, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA 3Division of Nephrology and Hypertension, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA 1Division of Pulmonary and Critical Care Medicine, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA 2Department of Anesthesia, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA 3Division of Nephrology and Hypertension, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA Correspondence should be addressed to Rodrigo Cartin-Ceba, cartinceba.rodrigo@mayo.edu Received 22 June 2012; Accepted 29 August 2012 Academic Editor: Gemma Seller-P´erez Copyright © 2012 Rodrigo Cartin-Ceba et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background. Acute kidney injury (AKI) is a frequent complication of critically ill patients. The impact of diﬀerent risk factors associated with this entity in the ICU setting is unknown. Objectives. The purpose of this research was to assess the risk factors associated with the development of AKI in critically ill patients by meta-analyses of observational studies. Data Extraction. Two reviewers independently and in duplicate used a standardized form to collect data from published reports. Authors were contacted for missing data. The Newcastle-Ottawa scale assessed study quality. Data Synthesis. Data from 31 diverse studies that enrolled 504,535 critically ill individuals from a wide variety of ICUs were included. Separate random-eﬀects meta-analyses demonstrated a signiﬁcantly increased risk of AKI with older age, diabetes, hypertension, higher baseline creatinine, heart failure, sepsis/systemic inﬂammatory response syndrome, use of nephrotoxic drugs, higher severity of disease scores, use of vasopressors/inotropes, high risk surgery, emergency surgery, use of intra-aortic balloon pump, and longer time in cardiopulmonary bypass pump. Conclusion. The best available evidence suggests an association of AKI with 13 diﬀerent risk factors in subjects admitted to the ICU. Predictive models for identiﬁcation of high risk individuals for developing AKI in all types of ICU are required. 2. Methods The report of this protocol-driven systematic review and meta-analysis adheres to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) standards for reporting systematic reviews and meta-analysis studies [17]. 2.1. Study Eligibility. Eligible studies were observational studies (prospective and retrospective cohorts or case- control studies) where speciﬁc risk factors for AKI were investigated in critically ill adult (≥18) patients with no AKI at the time of ICU admission. Studies were required to have a control group with no AKI for comparison and the main outcome of interest was development of AKI by any deﬁnition during the ICU stay (deﬁnition must include criteria based on change in creatinine levels). Study inclusion was not limited by publication status or language. Exclusion criteria included studies assessing speciﬁc causes of AKI such as rapidly progressive glomerulonephritis, hemolytic-uremic syndrome, or hepatorenal syndrome, and also specialized ICUs (burn and transplant ICUs). Studies must have excluded end stage kidney disease patients. 2.2. Search Strategy. An expert medical librarian with exten- sive meta-analytical experience collaborated to design the search strategies. The following databases were searched since inception of the database until the third week of January of 2010: Ovid MEDLINE, Ovid EMBASE, Cochrane Library, Web of Science, and Scopus. Database-speciﬁc controlled language and terms that describe the key concepts were utilized: acute kidney injury, acute renal failure, acute renal insuﬃciency, risk factors, intensive care unit, critically ill, outcomes, and observational studies. The detailed search strategy is described in Appendix 1, in supplementary material available online at doi:10.1155/2012/691013. We also reviewed the reference sections of identiﬁed reviews, published guidelines, and published manuscripts known to the authors. In addition, we reviewed the reference sections of eligible studies. 2.5. Quality Assessment. Independent reviewers working in duplicate determined the quality of each study based on the Newcastle-Ottawa quality assessment scale (NOS) for cohorts and case control studies [21]. Disagreements were harmonized by consensus. When this was not possible, a third reviewer adjudicated the quality of the questionable study. 2.6. Statistical Analysis. The main outcome of assessment was the development of AKI. The risk estimate analyzed for every risk factor in the studies was the odds ratio (OR) for dichotomous variables, and the mean diﬀerence for the continuous variables with its corresponding 95% conﬁdence interval (CI). When available, we used the adjusted risk estimates (adjusted OR) from multivariate models. 1. Introduction the impact and the association of diﬀerent risk factors with AKI is of paramount importance for designing predictive models of high risk patients, and also to create preventive strategies that might beneﬁt patients from developing this lethal condition. Predictive models for development of AKI already exist in cardiac-surgery critically ill patients [12– 14]; however, reﬁnements are still required to modify these studies into clinically applicable tools, and there is lack of meaningful predictive models in mixed and medical ICUs where most of the prediction models have focused on the impact on mortality of AKI in ICU patients [15, 16]. Given this situation, how can we identify patients at risk for AKI and are there interventions to mitigate this risk? To answer the former question in order to be able to respond the latter one, we set out to conduct a systematic review of the risk factors associated with the development of AKI in the ICU. Acute kidney injury (AKI) is a common and highly lethal problem faced in the intensive care unit (ICU) [1, 2], with a reported incidence of 1 to 67% [3–6], and a mortality that ranges from 28 to 90% [5, 7–10]. This wide range in incidence and mortality is in part due to the near 35 diﬀerent deﬁnitions of AKI [11]. Diﬀerent risk factors for the development of AKI in the intensive care unit (ICU) have been assessed in diverse populations, including port-surgical, trauma, and medical patients. A wide variety of risk factors have been described but there is no clear understanding of what risk factors confer the highest risk for development of AKI. In addition, risk factors identiﬁed in some studies have not been conﬁrmed in subsequent studies or the eﬀect may diﬀer depending on the clinical setting. Better understanding Critical Care Research and Practice 2 characteristics of the individual studies (sample size, gender distribution), clinical setting (surgical (trauma, cardiac, or general), mixed or medical ICU) and country, number of centers involved in the study, type of study (prospective or historical cohort, case-control study), years of enrollment, deﬁnition of AKI utilized, incidence of AKI in the study, and risk factors for AKI. 1. Introduction Because of signiﬁcant inconsistency in reporting risk factors among the studies, we assessed the following risk factors deemed to be of signiﬁcant importance in the developing of AKI in all the eligible studies: age, diabetes, hypertension, heart failure, baseline serum creati- nine (as a measure of evidence of chronic kidney disease), sepsis/systemic inﬂammatory response syndrome (SIRS), nephrotoxic drugs (including intravenous contrast dye, aminoglycosides, amphotericin B, vancomycin, nonsteroidal anti-inﬂammatory drugs, angiotensin converting enzyme inhibitors, and angiotensin receptor blockers), severity of disease as measured by diﬀerent severity scores such as the acute physiologic and chronic health evaluation (APACHE) [18, 19] or the injury severity score (ISS) [20], hypotension or shock, use of vasopressors and/or inotropes, high risk surgery, and emergency surgery. High risk surgery and emergency surgery were not assessed in medical ICUs for obvious reasons. For cardiosurgical ICUs, we also assessed the use of intra-aortic balloon pump (IABP) and the time on the cardiopulmonary bypass pump in minutes. 3. Results 3.1. Search Results and Study Inclusion. Our initial search identiﬁed 829 unique publications in the form of titles and abstracts, which were narrowed by preliminary review to 108 potentially relevant original articles that were examined in full text. The interobserver agreement in this phase was κ = 0.90 (95% CI, 0.88–0.93). The search of references from the 108 retrieved papers identiﬁed 3 additional articles. Figure 1 describes the ﬂow diagram of the process of study selection and the reasons for exclusion of studies. Ultimately, our systematic review and meta-analysis included 31 studies [4–6, 27–54] that evaluated a total of 504,535 individuals. 3.4. Subgroup Analyses. Preplanned subgroup analyses were performed for every risk factor according to the type of ICU (surgical, mixed, or medical). Table 4 demonstrates that signiﬁcant interaction was only observed in diabetes and heart failure between mixed ICUs, and high risk/emergency surgery in surgical and mixed ICUs. The point estimate asso- ciation of diabetes and heart failure in the development of AKI in mixed ICUs remained signiﬁcant but lower than the pooled estimate. Patient characteristics’ diﬀerences regarding the inclusion of both medical and surgical patients in the mixed ICUs might account for the observed discrepancy, and less likely the studies included in the subgroup analyses which presented low heterogeneity for both diabetes (I2 0%) [34, 49, 50, 53] and heart failure (I2 0%) [4, 34, 50, 53]. Furthermore, outcomes of patients that develop AKI in surgical ICUs tend to be worse than in medical ICUs [9]. Eleven studies were prospective cohorts, sixteen studies were historical cohorts, three studies were case-control studies, and one study was a nested case-control study. Most of these studies were done in surgical ICUs (n = 20; 14 of which were cardiothoracic ICUs); the remainder were mixed ICUs (n = 8), and medical ICUs (n = 3). There were a wide range of years of enrollment of the cohorts, from 1976 to as recent as 2008. A total of 26 diﬀerent deﬁnitions of AKI were identiﬁed in these 31 studies, all had in common a prespeciﬁed elevation of creatinine or an elevation of creatinine from baseline. The incidence of AKI in the diﬀerent ICUs as deﬁned by the multiple deﬁnitions ranged from 1.2 to 67%. Table 1 describes the studies’ characteristics in detail. 2. Methods For sever- ity of disease, because of diverse severity of disease scoring systems, we estimated the point estimate for each study using the eﬀect size (ES), which expresses the eﬀect of the risk factor in terms of the standard deviation of the measurement producing a unitless estimate that can be compared across studies [22]. We performed separate meta-analyses with the random eﬀects model [23] to obtain the pooled OR or pooled mean diﬀerence for the development of AKI for each risk factor with its corresponding 95% CI. We then used the I2 statistic to quantify the proportion of observed inconsistency across study results not explained by chance [24]. I2 values of less than 25%, 50%, and more than 75% represent low, moderate, and high inconsistency, respectively [24]. We proposed predeﬁned subgroup analyses based on our a priori hypotheses to explain potential heterogeneity across studies on the strength of association due to diﬀerent 2.3. Study Selection. Using a high threshold for exclusion, pairs of reviewers, working independently and in duplicate, identiﬁed potentially eligible studies for full-text retrieval from the titles and abstracts. Studies in which the reviewers disagreed were also retrieved in full text. Subsequently, disagreements were harmonized by consensus; when this was not possible, an independent reviewer reviewed and resolved the discordance by arbitration. We assessed interobserver agreement by the kappa statistics. Two independent native French speakers reviewed 2 studies published in French; similarly, 2 independent native Spanish speakers reviewed 2 studies published in Spanish. 2.4. Data Extraction. Two investigators independently used standardized forms to abstract descriptive, methodological, and outcome data from all eligible studies. The reviewers utilized a standardized data extraction form that include Critical Care Research and Practice 3 was signiﬁcant (OR 1.43 95% CI 1.08, 1.89), and there was also an improvement in heterogeneity between studies. Higher levels of creatinine at baseline and a higher severity of disease score (equivalent diﬀerence of 18 points in the APACHE III score, 9 points in the ISS, and 6 points in the APACHE II score) were also associated with AKI. Besides these associations, patients in cardiothoracic ICUs that developed AKI showed a signiﬁcant association with the use of IABP (OR 3.29, 95% CI 2.21, 4.91) and with longer time on the cardiopulmonary bypass pump (mean diﬀerence 27.92, 95% CI 14.41, 41.43 minutes). Table 3 also describes the reporting of the diﬀerent risks appraised. 2. Methods It is noticeable that with the exemption of age, “reporting bias” is very suggestive in the remainder risk factors. Very few risk factors were also reported as part of multivariate adjustment and, when available, these results were included in the analyses. Signiﬁcant heterogeneity existed between studies among the diﬀerent risk factors evaluated (I2 > 75%), with the only exemption of nephrotoxic drugs that presented moderate heterogeneity with an I2 of 42%. ICU types (surgical, mixed, and medical), considering a signiﬁcant interaction when P < 0.05. In addition, in order to evaluate the impact of studies with poor methodological quality, we conducted sensitivity analyses excluding studies in the lower 2 quartiles of the NOS quality assessment scale scores. Comparisons of risk estimates between subgroups and sensitivity analyses were made with a test of interaction [25]. The presence of publication bias was investigated graphically by the method of Sterne and Egger [26]. All analyses were performed with Review Manager Software (RevMan Analyses Version 5.0.4 Copenhagen; The Nordic Cochrane Center, The Cochrane Collaboration, 2008). ICU types (surgical, mixed, and medical), considering a signiﬁcant interaction when P < 0.05. In addition, in order to evaluate the impact of studies with poor methodological quality, we conducted sensitivity analyses excluding studies in the lower 2 quartiles of the NOS quality assessment scale scores. Comparisons of risk estimates between subgroups and sensitivity analyses were made with a test of interaction [25]. The presence of publication bias was investigated graphically by the method of Sterne and Egger [26]. All analyses were performed with Review Manager Software (RevMan Analyses Version 5.0.4 Copenhagen; The Nordic Cochrane Center, The Cochrane Collaboration, 2008). 3. Results g The eﬀect of high risk/emergency surgery between surgical and mixed ICUs likely represents opposite eﬀects of the two types of ICUs in the pooled point estimate. The estimate of association with AKI is not signiﬁcant for high risk/emergency surgery in mixed ICUs as opposed to surgical ICUs, where the point estimate is even higher. A possible explanation could be that the risk is likely diluted due to the inclusion of both “surgical” and “medical” patients in mixed ICUs. To conﬁrm this hypothesis, we excluded the study with higher reported number of medical patients as opposed to surgical patients in the mixed with ICUs [4] and the resulted OR was 1.52 (95% CI 1.10, 2.09), with a test for interaction that was not signiﬁcant (P = 0.09). 3.2. Methodological Quality. Table 2 summarizes the quality of the studies utilizing the NOS quality assessment scale. The median score of the studies was 6 (interquartile range, 6 to 8), where most of the studies failed to demonstrate that the outcome (AKI) was not present at start of the study. Moreover, only 14 studies performed a multivariate analysis for adjustment of baseline imbalances and confounders. 3.3. Meta-Analyses. Separate meta-analyses for each risk factor demonstrated that all assessed risk factors, with the exception of hypertension, were signiﬁcantly associated with the development of AKI in critically ill patients as depicted in Table 3. There was a trend of association of hypertension with AKI; however, it was not statistically signiﬁcant (OR 1.15, 95% CI 0.76, 1.74). After excluding 4 studies that assessed hypertension due to poor quality (see sensitivity analyses), no interaction was observed but the risk estimate 3.5. Sensitivity Analyses. We performed sensitivity analyses to test how robust the results of the meta-analyses were in relation to the methodological quality of the studies. Studies with a NOS score <6 were excluded in our sensitivity analyses. Our results were not signiﬁcantly altered by the exclusion of studies with poor methodological quality, with mild inconsistency improvement, particularly in diabetes, Critical Care Research and Practice 4 Table 1: Description of studies included in the systematic review and meta-analysis. 3. Results First author, year Setting/country Study design/number of centers Years of enrollment Total sample, % men AKI deﬁnition Incidence of AKI in the study Severity of disease score, mean standard deviation (SD) Hilberman 1979 [43] Surgical-Cardiac/USA PC/1 1977-1978 226/NR Need for RRT of peritoneal dialysis or BUN > 70 mg/dL or GFR < 30 mL/min/m2 14.6% NR Wilkins and Faragher 1983 [53] Mixed/UK HC/1 1976–1979 475/51 Creatinine > 180 μmol/L and BUN > 8 mmol/L for >24 h 23% NR Corwin 1989 [37] Surgical-Cardiac/USA CC/1 1985 84/NR ↓Creatinine 50% of baseline N/A NR Boucher 1990 [32] Surgical-Trauma/USA CC/1 1988-1989 184/76 ↑Creatinine ≥0.5 mg/dL from baseline or ↑ creatinine > 50% baseline N/A ISS (28 ± 13) Groeneveld 1991 [42] Medical/Netherlands HC/1 1986-1987 487/52 ↑Creatinine ≥3.2 mg/dL or ↑creatinine more than 2 times the baseline 16% NR Tran 1994 [51] Surgical- Trauma/Netherlands HC/1 1985–1989 206/73 ↑Creatinine ≥3.2 mg/dL or ↑creatinine more than 2 times the baseline 15% ISS (39 ± 17.6) Zanardo 1994 [54] Surgical-Cardiac/Italy PC/1 1989–1999 734/68 ↑Creatinine ≥2.5 mg/dL 3.7% NR Ward 1996 [52] Medical/USA HC/1 NR 102/53 ↑Creatinine ≥0.5 mg/dL from baseline 17.6% APACHE II (18.7 ± 1.8) Vivino 1998 [5] Surgical-Trauma/Italy PC/1 1991–1994 153/80 ↑Creatinine ≥2 mg/dL or ↑creatinine > 20% of baseline 31% ISS (28.1 ± 11) Mangano 1998 [48] Surgical-Cardiac/USA PC/24 1991–1993 2,222/76 ↑Creatinine > 177 μmol/L or ↑creatinine > 62 μmol/L from baseline or need for RRT 7.7% NR Conlon 1999 [36] Surgical-Cardiac/USA PC/1 1995–1997 2,672/64 ↑Creatinine ≥1 mg/dL from baseline 8% NR De Mendonc¸a 2000 [4] Mixed/16 countries PC/40 May 1995 1411/NR ↑Creatinine ≥3.5 mg/dL and/or urine output < 500 mL/24 hours 24.7% NR Ficher 2002 [40] Surgical-Cardiac/Germany CC/1 1999-2000 143/73 ↑Creatinine ≥0.5 mg/dL from baseline or RRT N/A NR Clermont 2002 [35] Mixed/USA PC/1 NR 1530/NR ↑Creatinine ≥0.5 mg/dL if baseline ≤1.9 mg/dL, or ↑ creatinine ≥1 mg/dL if baseline ≥2 to ≤ 4.9 mg/dL, or ↑creatinine ≥1.5 mg/dL if baseline ≥ 5 mg/dL 17.2% APACHE III (64 ± 31.8) Critical Care Research and Practice 5 Table 1: Continued. 3. Results First author, year Setting/country Study design/number of centers Years of enrollment Total sample, % men AKI deﬁnition Incidence of AKI in the study Severity of disease score, mean standard deviation (SD) Bove 2004 [33] Surgical-Cardiac/Italy PC/1 1998–2002 5,068/67 ↑Creatinine ≥100% from baseline 3.4% NR Bahar 2005 [30] Surgical-Cardiac/Turkey HC/1 1991–2001 14,437 ↑Creatinine ≥100% from baseline 24 hours after surgery or sustained urine output < 20 mL/hour 1.2% NR Chawla 2005 [34] Mixed/USA PC/1 2002-2003 194/53 ↑Creatinine > 75% from baseline if creatinine ≤ 2 mg/dL or ↑creatinine > 50% from baseline if creatinine > 2 mg/dL 18% APACHE II (12.5 ± 5.6) Loef 2005 [46] Surgical- Cardiac/Netherlands HC/1 1991 843/73 ↑Creatinine ≥25% of baseline creatinine during ﬁrst week after surgery 17.2% NR Mataloun 2006 [49] Mixed, Brazil PC/1 1999-2000 221/46 ↑Creatinine ≥1.5 mg/dL 19% APACHE II (15.2 ± 7.7) Hoste 2006 [6] Mixed/USA HC/1 2000-2001 5,383/51 RIFLE 67% APACHE III Median (IQR) (56, 41–73) Landoni 2007 [45] Surgical-Cardiac/Italy PC/1 1998–2003 3,103/84 ↑Creatinine > 100% from baseline 2.2% NR Barrantes 2008 [31] Medical/USA HC/1 2005 381/57 ↑Creatinine ≥0.3 mg/dL or ≥50% from baseline and/or an episode of < 0.5 mL/Kg/hr urine output for > 6 hours despite ﬂuid challenge of ≥500 mL of normal saline 31.5% APACHE II (15 ± 5.85) Bagshaw 2008 [28] Mixed/Australia and New Zealand HC/57 2000–2005 120,123/59 RIFLE (only cases in ﬁrst 24 hours of admission), modiﬁed urine output criteria 36% APACHE II (16.9 ± 7.7) Bagshaw 2008 [29] Surgical-Trauma/Australia and New Zealand HC/57 2000–2005 9,449/70 RIFLE (only cases in ﬁrst 24 hours of admission), modiﬁed urine output criteria 18.1% APACHE II (17.5 ± 7.6) Dasta 2008 [38] Surgical- Cardiothoracic/USA NCC/1 1998–2002 3,741/63 RIFLE creatinine criterion 6.9% APACHE III (54 ± 17.3) 6 Critical Care Research and Practice 6 Table 1: Continued. 3. Results First author, year Setting/country Study design/number of centers Years of enrollment Total sample, % men AKI deﬁnition Incidence of AKI in the study Severity of disease score, mean standard deviation (SD) Abelha 2009 [27] Surgical- NonCardiac/Portugal HC/1 2006–2008 1,166/65 ↑Creatinine ≥0.3 mg/dL or ≥50% from baseline and/or an episode of < 0.5 mL/Kg/hr urine output for >6 hours despite a ≥500 ml ﬂuid challenge 7.5% APACHE II median (IQR) (8, 5 to 11) De Ara´ujo Brito 2009 [39] Surgical-Cardiac/Brazil HC/1 2003–2006 186/59 ↑Creatinine > 50% from baseline if creatinine > 1.3 mg/dL or ↑creatinine > 0.5 mg/dL from baseline if creatinine < 1.3 mg/dL, or need of RRT 30.6% NR Thakar 2009 [50] Mixed/USA HC/191 2001–2006 325,395/97 ↑Creatinine ≥0.3 mg/dL from baseline 22% NR Hobson 2009 [44] Surgical- Cardiothoracic/USA HC/1 1992–2002 2973/66 RIFLE creatinine criterion 43% NR Machado 2009 [47] Surgical-Cardiac/Brazil HC/1 2003–2008 817/70 ↑Creatinine ≥0.3 mg/dL or ≥50% from baseline 48.5% EuroSCORE, median (IQR) 1.8 (1.2–3) Gomes 2010 [41] Surgical-Trauma/Portugal HC/1 2001–2007 436/80 RIFLE 50% ISS (27.3 ± 11.4) AKI: acute kidney injury, SD: standard deviation, BUN: blood urea nitrogen, GFR: glomerular ﬁltration rate, HC: historical cohort, PC: prospective cohort, CC: case-control, NCC: nested case control, RRT: renal replacement therapy, NR: not reported, IQR: interquartile range, ISS: injury severity score, APACHE: acute physiologic and chronic health evaluation score, RIFLE: risk injury failure loss end stage kidney disease criteria, EuroSCORE: European System for Cardiac Operative Risk Evaluation. T l/d /d di id b Critical Care Research and Practice 7 Care Research and Practice Table 2: Quality of the studies utilizing the Newcastle-Ottawa quality assessment scale (maximum score of 9). 3. Results Cohort studies Selection Comparability Outcome Total score First author, year Representativeness of exposed cohort Selection of the nonexposed cohort Ascertainment of exposure Demonstration that outcome was not present at start of study Comparability on the basis of design or analysis Assessment of outcome Followup long enough Adequacy of followup of cohorts Hilberman 1979 [43] ∗ ∗ ∗ ∗ 4 Wilkins 1983 [53] ∗ ∗ ∗ ∗ ∗ ∗ 6 Groeneveld 1991 [42] ∗ ∗ ∗ ∗ ∗ ∗ ∗ 7 Tran 1994 [51] ∗ ∗ ∗ ∗ ∗ ∗ 6 Zanardo 1994 [54] ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ 8 Ward 1996 [52] ∗ ∗ ∗ 3 Vivino 1998 [5] ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ 8 Mangano 1998 [48] ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ 8 Conlon 1999 [36] ∗ ∗ ∗ ∗ ∗ ∗ 6 De Mendonc¸a 2000 [4] ∗ ∗ ∗ ∗ ∗ ∗ ∗ 7 Clermont 2002 [35] ∗ ∗ ∗ ∗ ∗ ∗ 6 Bove 2004 [33] ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ 8 Bahar 2005 [30] ∗ ∗ ∗ ∗ ∗ ∗ 6 Chawla 2005 [34] ∗ ∗ ∗ ∗ ∗ ∗ 6 Loef 2005 [46] ∗ ∗ ∗ ∗ ∗ ∗ 6 Mataloun 2006 [49] ∗ ∗ ∗ ∗ ∗ ∗ ∗ 7 Hoste 2006 [6] ∗ ∗ ∗ ∗ ∗ ∗ ∗ 7 Landoni 2007 [45] ∗ ∗ ∗ ∗ ∗ ∗ ∗ 7 Barrantes 2008 [31] ∗ ∗ ∗ ∗ ∗ ∗ 6 Bagshaw 2008 [28] ∗ ∗ ∗ ∗ ∗ 5 Bagshaw et al., 2008 [29] ∗ ∗ ∗ ∗ ∗ 5 Abelha 2009 [27] ∗ ∗ ∗ ∗ 4 De Ara´ujo Brito 2009 [39] ∗ ∗ ∗ ∗ 4 Thakar 2009 [50] ∗ ∗ ∗ ∗ 4 Hobson 2009 [44] ∗ ∗ ∗ ∗ ∗ ∗ ∗ 7 Machado 2009 [47] ∗ ∗ ∗ ∗ 4 Gomes 2010 [41] ∗ ∗ ∗ ∗ ∗ ∗ 6 Critical Care Research and Practice 8 8 Critical Care Research and Practi Table 2: Continued. 3. Results Case-control studies Selection Comparability Exposure Total score uthor, year Case deﬁnition adequate Representiveness of cases Selection of controls Deﬁnition of controls Comparability on the basis of designs or analysis Ascertainment of exposure Same ascertainment method Nonresponse rate n 1989 [37] ∗ ∗ ∗ 3 er 1990 [32] ∗ ∗ ∗ ∗∗ ∗ ∗ ∗ 8 2002 [40] ∗ ∗ ∗ ∗ 4 2008 [38] ∗ ∗ ∗ ∗ ∗ ∗ 6 Table 2: Continued. Case-control studies Selection Comparability Exposure Total score First author, year Case deﬁnition adequate Representiveness of cases Selection of controls Deﬁnition of controls Comparability on the basis of designs or analysis Ascertainment of exposure Same ascertainment method Nonresponse rate Corwin 1989 [37] ∗ ∗ ∗ 3 Boucher 1990 [32] ∗ ∗ ∗ ∗∗ ∗ ∗ ∗ 8 Ficher 2002 [40] ∗ ∗ ∗ ∗ 4 Dasta 2008 [38] ∗ ∗ ∗ ∗ ∗ ∗ 6 Table 2: Continued. Case-control studies Selection Comparability Exposure Total score First author, year Case deﬁnition adequate Representiveness of cases Selection of controls Deﬁnition of controls Comparability on the basis of designs or analysis Ascertainment of exposure Same ascertainment method Nonresponse rate Corwin 1989 [37] ∗ ∗ ∗ 3 Boucher 1990 [32] ∗ ∗ ∗ ∗∗ ∗ ∗ ∗ 8 Ficher 2002 [40] ∗ ∗ ∗ ∗ 4 Dasta 2008 [38] ∗ ∗ ∗ ∗ ∗ ∗ 6 9 Critical Care Research and Practice 9 Table 3: Risk factors for acute kidney Injury evaluated in 31 observational studies. Age (years) Diabetes Hypertension Baseline creatinine (mg/dL) Heart failure Sepsis/ SIRS Nephrotoxic drugs Severity of disease Hypotension/ shock Pressors/ inotropes High risk surgery/ emergency surgery Cardiopulmonary bypass time (minutes) IABP Hilberman 1979 [43] Wilkins 1983 [53] N/A N/A Corwin 1989 [37] Boucher 1990 [32] N/A N/A Groeneveld 1991 [42] ⊙ N/A N/A N/A Tran 1994 [51] N/A N/A Zanardo 1994 [54] ⊙ ⊙ ⊙ Ward 1996 [52] ⊙ ⊙ N/A N/A N/A Vivino 1998 [5] ⊙ ⊙ N/A N/A Mangano 1998 [48] ⊙ ⊙! 3. Results ⊙ ⊙ ⊙ ⊙ Conlon 1999 [36] ⊙ ⊙ ⊙ ⊙ De Mendonc¸a 2000 [4] ⊙ ⊙ ⊙ N/A N/A Ficher et al., 2002 [40] Clermont 2002 [35] N/A N/A Bove 2004 [33] ⊙ ⊙ ⊙ ⊙ ⊙ ⊙ Bahar 2005 [30] ⊙ ⊙ ⊙ Chawla 2005 [34] ⊙ N/A N/A Loef 2005 [46] Mataloun 2006 [49] ⊙ N/A N/A Hoste 2006 [6] ⊙ ⊙ ⊙ N/A N/A Landoni 2007 [45] ⊙ ⊙ ⊙ ⊙ Barrantes 2008 [31] N/A N/A N/A Bagshaw et al., 2008 [28] N/A N/A Bagshaw 2008 [29] N/A N/A 10 Critical Care Research and Practice Critical Care Resea Table 3: Continued. Age (years) Diabetes Hypertension Baseline creatinine (mg/dL) Heart failure Sepsis/ SIRS Nephrotoxic drugs Severity of disease Hypotension/ shock Pressors/ inotropes High risk surgery/ emergency surgery Cardiopulmonary bypass time (minutes) IABP Dasta 2008 [38] ∗ ∗ Abelha 2009 [27] ⊙ ⊙ N/A N/A De Ara´ujo Brito 2009 [39] Thakar 2009 [50] N/A N/A Hobson 2009 [44] Machado 2009 [47] Gomes 2010 [41] N/A N/A Number of studies pooled for Meta-analysis 26 15 9 10 14 8 7 10 8 7 10 9 11 Number of patients 172,710 359,163 347,484 31,740 346,503 122,240 2,341 133,668 5,046 18,418 147,384 27,984 33,198 OR (95% CI) 1.52 [1.27, 1.82] 1.15 [0.76, 1.74] 1.97 [1.65, 2.35] 3.56 [1.85, 6.85] 1.54 [1.14, 2.08] 2.89 [1.54, 5.42] 4.75 [2.51, 8.99] 2.34 [1.59, 3.43] 3.29 [2.21, 4.91] Mean diﬀerence (95% CI) 5.36 [3.72, 6.99] 0.22 [0.02, 0.41] 7.09 [4.86, 9.32] 27.92 [14.41, 41.43] I2 97% 80% 96% 99% 74% 95% 42% 100% 84% 88% 94% 94% 80% ∗Cases and controls were matched on age and APACHE III. !Only type 1 diabetes assessed. SIRS: systemic inﬂammatory response syndrome, IABP: intra-aortic balloon pump, CI: conﬁdence interval, OR: odds ratio. Risk assessed, no signiﬁcant diﬀerence between groups. 3. Results Risk assessed, signiﬁcant diﬀerence between groups in unadjusted comparison. ⊙Risk assessed, signiﬁcant diﬀerence between groups in adjusted comparison. N/A: not applicable (risk assessed only for cardiac surgery studies). Table 3: Continued. Age (years) Diabetes Hypertension Baseline creatinine (mg/dL) Heart failure Sepsis/ SIRS Nephrotoxic drugs Severity of disease Hypotension/ shock Pressors/ inotropes High risk surgery/ emergency surgery Cardiopulmonary bypass time (minutes) IABP Dasta 2008 [38] ∗ ∗ Abelha 2009 [27] ⊙ ⊙ N/A N/A De Ara´ujo Brito 2009 [39] Thakar 2009 [50] N/A N/A Hobson 2009 [44] Machado 2009 [47] Gomes 2010 [41] N/A N/A Number of studies pooled for Meta-analysis 26 15 9 10 14 8 7 10 8 7 10 9 11 Number of patients 172,710 359,163 347,484 31,740 346,503 122,240 2,341 133,668 5,046 18,418 147,384 27,984 33,198 OR (95% CI) 1.52 [1.27, 1.82] 1.15 [0.76, 1.74] 1.97 [1.65, 2.35] 3.56 [1.85, 6.85] 1.54 [1.14, 2.08] 2.89 [1.54, 5.42] 4.75 [2.51, 8.99] 2.34 [1.59, 3.43] 3.29 [2.21, 4.91] Mean diﬀerence (95% CI) 5.36 [3.72, 6.99] 0.22 [0.02, 0.41] 7.09 [4.86, 9.32] 27.92 [14.41, 41.43] I2 97% 80% 96% 99% 74% 95% 42% 100% 84% 88% 94% 94% 80% ∗Cases and controls were matched on age and APACHE III. !Only type 1 diabetes assessed. SIRS: systemic inﬂammatory response syndrome, IABP: intra-aortic balloon pump, CI: conﬁdence interval, OR: odds ratio. Risk assessed, no signiﬁcant diﬀerence between groups. Risk assessed, signiﬁcant diﬀerence between groups in unadjusted comparison. ⊙Risk assessed, signiﬁcant diﬀerence between groups in adjusted comparison. N/A: not applicable (risk assessed only for cardiac surgery studies). 11 Critical Care Research and Practice 11 Table 4: Subgroups analysis. Table 4: Subgroups analysis. 3. Results 829 potentially relevant studies identiﬁed by primary search 724 studies excluded after duplicate abstract screening 108 articles retrieved for full text review 77 studies were excluded for: - irrelevance to our review - no control or unexposed group - same cohort - case series - burn ICUs - transplant ICU (n = 1) (n = 1) (n = 4) (n = 4) (n = 45) (n = 22) 31 articles met the inclusion criteria and were included in the systematic review Figure 1: Flow diagram of the process of study selection. heart failure, nephrotoxic drugs, and cardiopulmonary bypass time as described in Table 5. limited studies, all point estimates remained signiﬁcant. Our ﬁndings demonstrated important methodological issues observed in several of the studies analyzed, particularly the potential impact of “reporting bias” and lack of adjustment for important covariates in observational studies. 3.6. Publication Bias. The funnel plots for every individual risk factor are presented in Appendix 2, in supplementary material available online at doi:10.1155/2012/691013. Most of the plots showed asymmetry, suggesting small-study bias (either the absence of or inability to ﬁnd studies with smaller or negative risk estimates) or unexplained heterogeneity. 4.1. Limitations/Strengths. Our results are limited to the extent that the observational studies included in this sys- tematic review yielded inconsistent and imprecise data about association eﬀects. One might argue the legitimacy of combining dissimilar patients from diﬀerent types of ICUs. Our analysis was subject to the limitation of diﬀerent deﬁnitions of AKI among the studies (Table 1), which in fact may have contributed to the unexplained heterogeneity that we have identiﬁed between our selected studies. This diﬀerential approach in deﬁning AKI could have potentially overestimated or underestimated some the risk factors identiﬁed in our analysis. However, the subgroup analysis looking at each type of ICU did not result in signiﬁcantly diﬀerent point estimates for the vast majority of the risk factors. Furthermore, in all systematic reviews, the usual key limitation is publication and reporting bias. Our review clearly showed evidence of signiﬁcant reporting bias and possible publication bias. The strengths of this systematic review stem from the exhaustive literature search, a thorough evaluation of the methodological quality of the studies, and a focused analysis with complete, prespeciﬁed subgroup, and sensitivity analyses. Furthermore, the most compelling evidence comes from our pooled analysis of studies that included thousands of patients. 3. Results Finally, our contact of the study authors to obtain missing data and their collaboration with our request further strengthen the quality of the review. 3. Results Risk Factor Number of studies Odds ratio or mean diﬀerence∗(95% CI) P value for interaction Inconsistency I2 Age Surgical 17 5.35 [2.84, 7.86] 0.99 96% Mixed 6 4.95 [2.27–7.63] 0.81 97% Medical 3 7.61 [4.77, 10.44] 0.17 0% Diabetes Surgical 11 1.61 [1.41, 1.83] 0.61 31% Mixed 4 1.09 [1.07, 1.11] <0.001 0% Hypertension Surgical 7 1.34 [1.04, 1.73] 0.53 68% Mixed 2 0.68 [0.45, 1.05] 0.08 49% Baseline creatinine∗ Surgical 8 0.20 [−0.02, 0.42] 0.89 99% Heart Failure Surgical 9 2.19 [1.92, 2.5] 0.34 8% Mixed 4 1.51 [1.47, 1.55] 0.003 0% Sepsis/SIRS Surgical 2 2.69 [1.28, 5.64] 0.57 47% Mixed 4 2.52 [1.36, 4.69] 0.45 87% Medical 2 8.58 [1.56, 47.22] 0.34 95% Nephrotoxic drugs Surgical 4 1.25 [0.92, 1.71] 0.34 0% Mixed 2 2.12 [1.04, 4.32] 0.41 64% Severity of disease∗ Surgical 5 5.69 [3.25, 8.14] 0.40 88% Mixed 3 10.66 [3.37, 17.95] 0.35 96% Medical 2 5.32 [3.98, 6.65] 0.18 0% Hypotension/shock Surgical 4 2.48 [1.3, 4.72] 0.74 57% Mixed 4 3.44 [1.09, 10.89] 0.79 92% Pressors/inotropes Surgical 5 5.36 [2.39, 12.03] 0.81 91% Mixed 2 3.41 [1.87, 6.20] 0.45 19% High risk surgery/emergency surgery Surgical 6 3.79 [2.91, 4.94] 0.04 33% Mixed 4 1.14 [0.64, 2.02] 0.04 91% CI: conﬁdence interval, SIRS: systemic inﬂammatory response syndrome. 12 Critical Care Research and Practice 829 potentially relevant studies identiﬁed by primary search 108 articles retrieved for full text review 724 studies excluded after duplicate abstract screening 31 articles met the inclusion criteria and were included in the systematic 77 studies were excluded for: - irrelevance to our review - no control or unexposed group - same cohort - case series - burn ICUs - transplant ICU 3 articles were added from bibliography review (n = 1) (n = 1) (n = 4) (n = 4) (n = 45) (n = 22) Figure 1: Flow diagram of the process of study selection. 4. Discussion This study found that the current evidence, drawn from a large number of observational studies that included more than half million individuals, indicates a signiﬁcantly increased risk of AKI in critically ill patients with older age, diabetes, higher baseline creatinine, heart failure, sep- sis/SIRS, use of nephrotoxic drugs, higher severity of disease scores, use of vasopressors/inotropes, high risk surgery, emergency surgery, and possibly hypertension. Additionally, cardiothoracic patients admitted to the ICU also presented increased risk of AKI with the use of IABP and longer time in cardiopulmonary bypass pump. We found that many of these observational studies were methodologically limited and presented high levels of heterogeneity. Multiple deﬁnitions of AKI, diﬀerences in populations studied, diﬀerences in the internal characteristics and practice of the diverse ICUs, diﬀerences in countries, and diﬀerences in processes of care over time might explain the inconsistencies identiﬁed in this review. Most of the deﬁnitions of AKI utilized in these studies possess high speciﬁcity for the diagnosis of AKI but poor sensitivity due to lack of urine output criteria [55]. With the exemptions already described, the risk factors described in this review that were associated with AKI presented similar point estimates across diﬀerent types of ICUs. It was reassuring that after exclusion of methodological 4.2. Clinical Implications and Future Research. Our ﬁndings should be considered for designing prediction models of AKI 13 Critical Care Research and Practice 13 Table 5: Sensitivity analysis. Risk factor Number of studies excluded Odds ratio or mean diﬀerence∗(95% CI) P value for interaction Inconsistency I2 Age 9 4.95 [3.79, 6.12] 0.69 80% Diabetes 4 1.58 [1.36, 1.84] 0.74 36% Hypertension 4 1.43 [1.08, 1.89] 0.39 73% Baseline creatinine∗ 3 0.14 [0.01, 0.27] 0.50 94% Heart failure 2 2.05 [1.77, 2.38] 0.73 27% Sepsis/SIRS 1 4.15 [2.36, 7.32] 0.72 83% Nephrotoxic drugs 1 1.53 [1.09, 2.14] 0.97 52% Severity of disease∗ 4 9.08 [4.57, 13.60] 0.43 94% Hypotension/shock 1 3.33 [1.70, 6.52] 0.76 84% Pressors/inotropes 2 4.52 [2.03, 10.05] 0.92 92% High risk surgery/emergency surgery 2 2.34 [1.23, 4.49] 0.99 92% Cardiopulmonary bypass time∗ 3 30.46 [23.41, 37.51] 0.74 59% IABP 3 3.76 [2.54, 5.57] 0.64 78% CI: conﬁdence interval, SIRS: Systemic inﬂammatory response syndrome, IABP: intra-aortic balloon pump. 4. Discussion EuroSCORE: European System for Cardiac Operative Risk Evaluation HC: Historical cohort study IABP: Intra-aortic balloon pump ICU: Intensive care unit IQR: Interquantile range ISS: Injury severity score N/A: Not applicable NCC: Nested case control study NOS: Newcastle-Ottawa scale NR: Not reported OR: Odds ratio PC: Prospective cohort study PRISMA: Preferred Reporting Items for Systematic Reviews and Meta-Analyses RIFLE: Risk injury failure loss end stage kidney disease criteria RRT: Renal replacement therapy SD: Standard deviation SIRS: Systemic inﬂammatory response syndrome. in diﬀerent ICUs with the overarching goal of implementing strategies to prevent this highly lethal and morbid condition. The results provided in this review are also applicable to clinical practice and counseling of patients at high risk of development of AKI. From the research perspective, the potential role of diabetes and heart failure in critically ill AKI patients from diﬀerent ICU types warrant further investigation. in diﬀerent ICUs with the overarching goal of implementing strategies to prevent this highly lethal and morbid condition. g g y The results provided in this review are also applicable to clinical practice and counseling of patients at high risk of development of AKI. From the research perspective, the potential role of diabetes and heart failure in critically ill AKI patients from diﬀerent ICU types warrant further investigation. 5. Conclusions While meta-analyses of observational studies carry signiﬁ- cant limitations inherent to the design of the studies, this review assessed and conﬁrmed the association of 13 diﬀerent risk factors in the development of AKI in critically ill adults from 31 studies of more than half million patients. This review demonstrated a signiﬁcantly increased risk of AKI in critically ill patients with older age, diabetes, hypertension, higher baseline creatinine, heart failure, sepsis/SIRS, use of nephrotoxic drugs, higher severity of disease scores, use of vasopressors/inotropes, high risk surgery, emergency surgery, use of IABP, and longer time in cardiopulmonary bypass pump. Early identiﬁcation of patients at risk of AKI may help to implement interventions to mitigate this highly lethal condition. Acknowledgments The authors thank the medical librarian Patricia J. Erwin for her assistance in performing the search for studies. They also thank Victor Montori for his comments and suggestions. [2] S. Uchino, “The epidemiology of acute renal failure in the world,” Current Opinion in Critical Care, vol. 12, no. 6, pp. 538–543, 2006. 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Crivellari et al., “Acute renal failure after isolated CABG surgery: six years of experience,” Minerva Anestesiologica, vol. 73, no. 11, pp. 559–565, 2007. [46] B. G. Loef, A. H. Epema, T. D. Smilde et al., “Immediate postoperative renal function deterioration in cardiac surgical patients predicts in-hospital mortality and long-term sur- vival,” Journal of the American Society of Nephrology, vol. 16, no. 1, pp. 195–200, 2005. [47] M. D. N. Machado, R. C. Miranda, I. T. Takakura et al., “Acute kidney injury after on-pump coronary artery bypass graft surgery,” Arquivos Brasileiros de Cardiologia, vol. 93, no. 3, pp. 230–252, 2009. [48] C. M. Mangano, L. S. Diamondstone, J. G. Ramsay, A. Aggarwal, A. Herskowitz, and D. T. Mangano, “Renal dysfunc- tion after myocardial revascularization: risk factors, adverse outcomes, and hospital resource utilization,” Annals of Internal Medicine, vol. 128, no. 3, pp. 194–203, 1998. [49] S. E. Mataloun, F. R. Machado, A. P. R. Senna, H. P. 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https://openalex.org/W3092076191	https://link.springer.com/content/pdf/10.1007/s11262-020-01795-9.pdf	English	null	Whole genome sequencing and phylogenetic classification of Tunisian SARS-CoV-2 strains from patients of the Military Hospital in Tunis	Virus genes	2,020	cc-by	2,850	Abstract In the present work, two complete genome sequences of SARS-CoV-2 were obtained from nasal swab samples of Tunisian SARS-CoV-2 PCR-positive patients using nanopore sequencing. The virus genomes of two of the patients examined, a Tunisian soldier returning from a mission in Morocco and a member of another Tunisian family, showed significant differ- ences in analyses of the total genome and single nucleotide polymorphisms (SNPs). Phylogenetic relationships with known SARS-CoV-2 genomes in the African region, some European and Middle Eastern countries and initial epidemiological conclusions indicate that the introduction of SARS-CoV-2 into Tunisia from two independent sources was travel-related. Keywords SARS-CoV-2 · COVID-19 · RT-qPCR · Sequencing · Tunisia · Morocco The current global spread of the newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID- 19), has led to worldwide social and economic disruption. The virus was first described in December 2019 in Wuhan, China, and has since spread into a pandemic. As a result, healthcare systems in almost all countries of the world are facing unprecedented challenges. A few clinically approved drugs have been shown to exhibit anti-SARS-CoV-2 activity, but none have yet been proven to be sufficiently effective in treating COVID-19 patients, nor are vaccines available [1]. By August 2020, the World Health Organization (WHO) had reported more than 21.99 million infected individuals and 775,893 deaths worldwide. In the African Region, 966,352 confirmed cases have been reported [2], but many cases remain undetected, probably due to insufficient diagnostic capacity, limited contact tracing and the oftentimes unspe- cific symptoms of affected patients. if Diagnostic tests for SARS-CoV-2 have been carried out in Tunisia since early February 2020. On 2 March 2020, the first case of COVID-19, a Tunisian patient from Italy, was reported [3, 4]. Since then, the number of positively-tested SARS-CoV-2 patients has been increasing. By 18 August 2020, a total of 117,086 tests were performed and 2427 SARS-CoV-2 positive cases were reported by the Tunisian Ministry of Health. Of these positive cases, 60 patients died [5]. So far, only a few whole genome sequences from Tuni- sia are available online. However, these few data already indicate various independent, travel-related introductions of SARS-CoV-2 into the country [6]. Whole genome sequencing and phylogenetic classification of Tunisian SARS‑CoV‑2 strains from patients of the Military Hospital in Tunis Received: 2 July 2020 / Accepted: 12 September 2020 / Published online: 9 October 2020 © The Author(s) 2020 * Susann Handrick SusannHandrick@bundeswehr.org 1 Bundeswehr Institute of Microbiology, Munich, Germany 2 Department of Virology, Military Hospital of Instruction of Tunis, Tunis, Tunisia Virus Genes (2020) 56:767–771 https://doi.org/10.1007/s11262-020-01795-9 Virus Genes (2020) 56:767–771 https://doi.org/10.1007/s11262-020-01795-9 SHORT REPORT Abstract Proper surveillance and monitoring of the SARS-CoV-2 epidemic in Tunisia and on the African continent, in order to guarantee faultless diagnostics, urgently requires further epidemiological and bioforensic data in addition to those currently available. Edited by Hartmut Hengel. Susann Handrick and Malena Bestehorn-Willmann have contributed equally. Roman Wölfel and Mohamed Ben Moussa are shared last authors. * Susann Handrick SusannHandrick@bundeswehr.org 1 Bundeswehr Institute of Microbiology, Munich, Germany 2 Department of Virology, Military Hospital of Instruction of Tunis, Tunis, Tunisia Edited by Hartmut Hengel. In the present work, two SARS-CoV-2 strains from Tuni- sian nationals were sequenced and phylogenetically com- pared with available SARS-CoV-2 genomes from Tunisia and other countries in Africa, Europe and the Middle East. In the present work, two SARS-CoV-2 strains from Tuni- sian nationals were sequenced and phylogenetically com- pared with available SARS-CoV-2 genomes from Tunisia and other countries in Africa, Europe and the Middle East. Four nasopharyngeal swab samples from Tunisian citi- zens were tested positive for SARS-CoV-2 RNA (Table 1) after viral RNA extraction with the QIAmp viral RNA Mini Kit (Qiagen, Hilden, Germany) using RT-qPCR according Roman Wölfel and Mohamed Ben Moussa are shared last authors. Four nasopharyngeal swab samples from Tunisian citi- zens were tested positive for SARS-CoV-2 RNA (Table 1) after viral RNA extraction with the QIAmp viral RNA Mini Kit (Qiagen, Hilden, Germany) using RT-qPCR according (0121 3456789) 3 3456789) 3 Virus Genes (2020) 56:767–771 768 Table 1 Patient information and sequencing results of MHT_1 and MHT_2 Sample ID PCR analyses AccNo. SNPs (AA exchanges) 31-year-old Tunisian soldier MHT_1 E Ct=29.48; RdRP Ct=33.14 MT559037 C3037T C14408T (ORF1b:P314L) C18877T A23403G (S:D614G) G25563T (ORF3a:Q57H) G29474T (N:D401Y) 59-year-old mother MHT_2 E Ct=27.18; RdRP Ct=31.17 MT559038 A361G C8782T G22468T T28144C (ORF8:L84S) G28878A (N:S202N) 62-year-old father MHT_4 E Ct=34.34; RdRP Ct=40.33 n.d. n.d. 23-year-old daughter MHT_3 E Ct=34.03; RdRP no Ct detectable n.d. n.d. 24-year-old daughter none negave n.a. n.a. 22-year-old son none negave n.a. n.a. n.d. not done, n.a. not applicable n.d. not done, n.a. not applicable to the protocol of Corman et al. [7] at the Military Hospi- tal in Tunis (MHT). The results of this investigation were confirmed at the Bundeswehr Institute of Microbiology in Munich (IMB) using an RT-qPCR protocol targeting the viral N gene [8]. Abstract An asymptomatic Tunisian soldier (31-year- old male, MHT_1) returning from a mission in Agadir, Morocco, tested positive immediately after his arrival at Tunis-Carthage Airport on 21 March 2020. In addition, three of five members of a Tunisian family from Ezzahra, 15 km south of Tunis, tested positive on 24 March 2020 (59-year- old mother, MHT_2; 62-year-old father, MHT_4) and 28 March 2020 (23-year-old daughter, MHT_3; Table 1). One of the patients, the father, showed symptoms such as fever, cough, arthralgia and headache one week before the first RT-qPCR test. The other two COVID-19 infections were without development of symptoms. All patients were tested negative for SARS-CoV-2 RNA in nasopharyngeal swabs after 14 days of home quarantine. the ARTIC pipeline [11], closed genomes lacking only the outermost nucleotides of the 5′UTR or 3′UTR regions were obtained. Both genomes were annotated by and submitted to GenBank (accession MT559037 and MT559038). SNP and phylogenetic analyses were performed using a local installation of the nextstrain.org pipeline [12]. Strains listed in GISAID belonging to the African sub- group (N = 1203 of 22 June 2020) and with available whole genome sequences were included in the initial analysis and further filtered phylogenetically to minimise redundancies and selected for relevance to possible travel and/or trade routes (N = 38). In comparison to the reference strain Wuhan-Hu-1, MHT_1 shows six SNPs, four of which lead to amino acid changes in two different open reading frames (ORF) as well as the S and N genes respectively. Compared to Wuhan- Hu-1, MHT_2 contains five SNPs, two of which lead to amino acid changes in the ORF8 and the N gene. When com- paring the two Tunisian SARS-CoV-2 sequences, no inser- tions or deletions and no mutual SNPs were found (Table 1). Phylogenetic analyses showed that the genomes in our study are grouped in two different branches (Fig. 1). Sample MHT_1 belongs to clade 20A (Nextstrain.org nomenclature [13] matches Pangolin lineage B.1 [14]) with the character- istic mutation D614G (SNP A23403G) of the SARS-CoV-2 spike protein assumed to enhance viral infectivity [15]; In comparison to the reference strain Wuhan-Hu-1, MHT_1 shows six SNPs, four of which lead to amino acid changes in two different open reading frames (ORF) as well as the S and N genes respectively. Compared to Wuhan- Hu-1, MHT_2 contains five SNPs, two of which lead to amino acid changes in the ORF8 and the N gene. Abstract When com- paring the two Tunisian SARS-CoV-2 sequences, no inser- tions or deletions and no mutual SNPs were found (Table 1). The four positively-tested RNA samples were further analysed at the IMB. For two of the four samples, having the highest virus loads (MHT_1/MHT_2; Table 1), whole genome sequencing of SARS-CoV-2 according to the nCoV- 2019 sequencing protocol [9] on a GridION instrument was successful. Briefly, after passing quality control, demulti- plexing, adapter trimming and consensus sequences genera- tion based on the reference strain Wuhan-Hu-1 [10] using Phylogenetic analyses showed that the genomes in our study are grouped in two different branches (Fig. 1). Sample MHT_1 belongs to clade 20A (Nextstrain.org nomenclature [13] matches Pangolin lineage B.1 [14]) with the character- istic mutation D614G (SNP A23403G) of the SARS-CoV-2 spike protein assumed to enhance viral infectivity [15]; 1 3 769 Virus Genes (2020) 56:767–771 ample MHT_2 is clustering within clade 19B (Pangolin neage A). While genomes of clade 19 were dominated by i with a focus on the northern and central countries of this continent, the existence of geographically-associated genetic g. 1 Phylogenetic tree showing the genomic epidemiology of a unisia-focused subsampling of 38 SARS-CoV-2 genomes sampled etween Dec 2019 and May 2020. Unpublished data are included with the permission of the data generators and does not affect their right to publish. A complete list of sequence authors is available at nextstrain.org with the permission of the data generators and does not affect their right to publish. A complete list of sequence authors is available at nextstrain.org Fig. 1 Phylogenetic tree showing the genomic epidemiology of a Tunisia-focused subsampling of 38 SARS-CoV-2 genomes sampled between Dec 2019 and May 2020. Unpublished data are included with a focus on the northern and central countries of this continent, the existence of geographically-associated genetic clusters can be observed. sample MHT_2 is clustering within clade 19B (Pangolin lineage A). While genomes of clade 19 were dominated by Asian samples, especially during the first weeks of the incip- ient pandemic outbreak, clade 20A was the group rapidly spreading worldwide in February/March/April 2020. Obser- vation of the genomes of both clones, which are present in North Africa, suggests multiple introductions at different points in time. References 1. Wu R, Wang L, Kuo H-CD, Shannar A, Peter R, Chou PJ, Li S, Hudlikar R, Liu X, Liu Z et al (2020) An update on current thera- peutic drugs treating COVID-19. Curr Pharmacol Rep. https://doi. org/10.1007/s40495-20-00216-7 Therefore, correct and sufficient epidemiological data remain the greatest need for case tracing and successful infection control. In multidisciplinary approaches that take into account both epidemiological and genetic data, phy- logeographic predictions can be verified on the basis of genetic analyses. While in epidemic or localised outbreaks with rapidly evolving pathogens, such as the Ebola virus, whole genomes can greatly assist in contact tracing, multiple introductions are difficult to monitor and track by phyloge- netic analysis during prolonged outbreaks and pandemics. Medical and scientific cooperation is, therefore, one of the main triggers for combating pandemics like COVID-19. g 2. WHO Coronavirus Disease (COVID-19) Dashboard Available online: https://covid19.who.int. Accessed on Aug 20, 2020 3. WHO Coronavirus disease (2019) (COVID-19) Situation Report—43. WHO, Geneva, p 2020 4. Louhaichi S, Allouche A, Baili H, Jrad S, Radhouani A, Greb D, Akrout I, Ammar J, Hamdi B, Added F et al (2020) Features of patients with 2019 novel coronavirus admitted in a pneumology department: the first retrospective Tunisian case series. Tunis Med 98:261–265 5. Tunesienexplorer Covid-19 Zahlen Tunesien von Dienstag, 15 Aug 2020. Tunesienexplorer 2020. 6. Fares W, Chouikha A, Ghedira K, Gdoura M, Rezig D, Boubaker SH, Dhifallah IB, Touzi H, Hammami W, Zi Meddeb et al (2020) First whole genome sequences and phylogenetic analysis of SARS-CoV-2 virus isolates during COVID-19 outbreak in Tuni- sia, North Africa. https://doi.org/10.22541/au.159137642.26983 355 Acknowledgements We gratefully acknowledge the Tunisian patients described in these case reports for participating in the study and for giving written consents for publication of the obtained data. We acknowledge the authors from originating and submitting laboratories of sequence data provided by GISAID on which the analysis is based. 7. Corman VM, Landt O, Kaiser M, Molenkamp R, Meijer A, Chu DK, Bleicker T, Brünink S, Schneider J, Schmidt ML et al (2020) Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Euro Surveill Bull Eur Sur Mal Transm Eur Commun Dis Bull 25(3):2000045. https://doi.org/10.2807/1560-7917. ES.2020.25.3.2000045 Author contributions All authors contributed to the conception and design of the study. Material preparation, data collection and analy- sis were performed by MBW, MCW, SH, SE, HN and MBM. References The first draft of the manuscript was written by SH, and all authors com- mented on earlier versions of the manuscript. All authors have read and approved the final manuscript. 8. Nao N, Shirato K, Katano H, Matsuyama S, Takeda M (2020) Detection of second case of 2019-nCoV infection in Japan (cor- rected version). National Institute of Infectious Diseases, Tokyo y 9. Quick J (2020) nCoV-2019 sequencing protocol. https://doi. org/10.17504/protocols.io.bbmuik6w Funding Open Access funding enabled and organized by Projekt DEAL. This work was supported by the Enable and Enhance Initia- tive of the German Government (Ertüchtigungsinitiative der Deutschen Bundesregierung, OR12-370.43 ERT TUN IMB). 10. Wu F, Zhao S, Yu B, Chen Y-M, Wang W, Song Z-G, Hu Y, Tao Z-W, Tian J-H, Pei Y-Y et al (2020) A new coronavirus associated with human respiratory disease in China. Nature 579:265–269. https://doi.org/10.1038/s41586-020-2008-3 p g 11. Lohman N, Rowe W, Rambaut A (2020) ARTIC nCoV-2019 novel coronavirus bioinformatics protocol v1.1 Available online: https ://artic.network/ncov-2019/ncov2019-bioinformatics-sop.html. Accessed on May 16, 2020i Abstract If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Abstract When comparing whole genomes from Africa Since the amount of genome sequences from the Afri- can continent is still small compared to European, Asian or American sequencing data, the interpretation of the results should be treated with caution. If more metadata are taken 1 3 3 770 Virus Genes (2020) 56:767–771 into account, possible epidemiological links become obvious and could explain genetic similarities. In pandemic situa- tions, however, not all genetic similarities can be explained, even if data is available, e.g. the Bav-MVP0225/2020 sample from Germany. The situation is similar in the case of sample MHT_1, where the genetically closest neighbour is the iso- late Morocco/6891/2020. This is conclusive, as this isolate originates from a Tunisian soldier who had returned from a stay in Morocco. The family isolate, on the other hand, is related to other virus strains, mainly from Central Afri- can countries such as Ghana, Uganda and the Democratic Republic of Congo (DRC). A direct link from Tunisia to these regions, e.g. on the basis of travel or trade, could not be established.fi into account, possible epidemiological links become obvious and could explain genetic similarities. In pandemic situa- tions, however, not all genetic similarities can be explained, even if data is available, e.g. the Bav-MVP0225/2020 sample from Germany. The situation is similar in the case of sample MHT_1, where the genetically closest neighbour is the iso- late Morocco/6891/2020. This is conclusive, as this isolate originates from a Tunisian soldier who had returned from a stay in Morocco. The family isolate, on the other hand, is related to other virus strains, mainly from Central Afri- can countries such as Ghana, Uganda and the Democratic Republic of Congo (DRC). A direct link from Tunisia to these regions, e.g. on the basis of travel or trade, could not be established.fi Open Access This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. g 15. Hu J, He C-L, Gao Q-Z, Zhang G-J, Cao X-X, Long Q-X, Deng H-J, Huang L-Y, Chen J, Wang K et al (2020) The D614G muta- tion of SARS-CoV-2 spike protein enhances viral infectivity and decreases neutralization sensitivity to individual convalescent sera. bioRxiv. https://doi.org/10.1101/2020.06.20.161323 for SARS-CoV-2 to assist genomic epidemiology. bioRxiv. https ://doi.org/10.1101/2020.04.17.046086 for SARS-CoV-2 to assist genomic epidemiology. bioRxiv. https ://doi.org/10.1101/2020.04.17.046086 for SARS-CoV-2 to assist genomic epidemiology. bioRxiv. https ://doi.org/10.1101/2020.04.17.046086 15. Hu J, He C-L, Gao Q-Z, Zhang G-J, Cao X-X, Long Q-X, Deng H-J, Huang L-Y, Chen J, Wang K et al (2020) The D614G muta- tion of SARS-CoV-2 spike protein enhances viral infectivity and decreases neutralization sensitivity to individual convalescent sera. bioRxiv. https://doi.org/10.1101/2020.06.20.161323 Compliance with ethical standards Conflict of interest The authors declare that they have no conflict of interest. 12. Hadfield J, Megill C, Bell SM, Huddleston J, Potter B, Callender C, Sagulenko P, Bedford T, Neher RA (2018) Nextstrain: real-time tracking of pathogen evolution. Bioinformatics 34:4121–4123. https://doi.org/10.1093/bioinformatics/bty407 Ethical approval The use of the biological material described in the underlying study was approved by the Ethics Committee of the Mili- tary Hospital in Tunis and written consent forms were signed by all included participants. p g y 13. Year-letter Genetic Clade Naming for SARS-CoV-2 on Nextstain. org Available online: https://nextstrain.org//blog/2020-06-02- SARSCoV2-clade-naming. Accessed on Jun 25, 2020 14. Rambaut A, Holmes EC, Hill V, O’Toole Á, McCrone JT, Ruis C, du Plessis L, Pybus OG (2020) A dynamic nomenclature proposal 1 Virus Genes (2020) 56:767–771 771 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 1 3 1 3 3
https://openalex.org/W3021847157	https://podest.ipu.hr/islandora/object/ipu:16/datastream/FILE0/download	Croatian	null	Bilješka o osječkoj galeriji: od Galerije slika do Muzeja likovnih umjetnosti	null	2,019	cc-by	4,899	likovnih umjetnosti Zec, Daniel Source / Izvornik: Institucije povijesti umjetnosti : zbornik 4. kongresa hrvatskih povjesničara umjetnosti, 2019, 43 - 50 Conference paper / Rad u zborniku Publication status / Verzija rada: Published version / Objavljena verzija rada (izdavačev PDF) https://doi.org/10.31664/z4khpu.06 Permanent link / Trajna poveznica: https://urn.nsk.hr/urn:nbn:hr:254:901876 Rights / Prava: Attribution 4.0 International / Imenovanje 4.0 međunarodna Download date / Datum preuzimanja: 2024-10-24 Repository / Repozitorij: PODEST - Institute of Art History Repository Zec, Daniel Uvod j ( 4 ) 2 JOVAN GOJKOVIĆ, Galerija slika u Osijeku, u: Bulletin Instituta za likovne umjetnosti JAZU, Zagreb, god. 1, 3 – 4 (1953.), 54; JOVAN GOJKOVIĆ, Galerija slika u Osijeku, u: Osječki zbornik, Osijek, Muzej Slavonije, 4 (1954.), 167 – 168; DANICA PINTEROVIĆ, O razvoju osječkog muzeja, u: Osječki zbornik, Osijek, Muzej Slavonije , 6 (1958.), 7 – 22; JELICA AMBRUŠ, Povijest Galerije likovnih umjetnosti u Osijeku, u: Vodič stalnog postava Galerije likovnih umjetnosti u Osijeku, Osijek, Galerija likovnih umjetnosti, 1978.; VESNA BURIĆ, Prof. dr. Bauer i Osijek, u: Glasnik slavonskih muzeja, Vukovar, 45 (1982.), 43 – 45.; VLASTIMIR KUSIK, Galerija likovnih umjetnosti, Osijek, u: Informatica Museologica, 1 – 4 (1986.), 78 – 80; VLASTIMIR KUSIK, Prvi stalni postav i dvije izložbe osječke Galerije slika, u: Galerija likovnih umjetnosti Osijek, monogra fija – zbornik, Osijek, GLUO, 1987., 31 – 34; OTO ŠVAJCER, Umetak XII (izložbeni prostori u Osijeku – Galerija), u: Oto Švajcer, Likovna kronika Osijeka 1850 – 1969. godine, Osijek, GLUO, 1991., 207 – 208; JELICA AMBRUŠ, Galerija likovnih umjetnosti u Osijeku, u: Muzeologija, 31 (1994.), 70 – 73. U tekstu što slijedi sažeto se prikazuje povijest Galerije, odno sno Muzeja likovnih umjetnosti u Osijeku,1 te se razmatra nje gova uloga institucije povijesti umjetnosti u gradu, regiji i u nacionalnim okvirima. O povijesti osječke Galerije do danas objavljeno je više članaka u stručnim časopisima i knjigama, a pisali su ih protagonisti njezina stvaranja i rada, poglavito njezini osnivači, kustosi i ravnatelji.2 Najviše tekstova o Galeriji napisao je i objavio Antun Bauer,3 a osobito obuhvatan tekst o njezinoj povijesti i djelovanju zahvaljujemo Stjepanu Brlošiću, dugogodišnjem ravnatelju ustanove.4 Daniel Zec Muzej likovnih umjetnosti, Osijek daniel.zec@mlu.hr http://orcid.org/0000-0001-6075-1453 1 Od osnutka do danas osječka je Galerija mijenjala ime: Galerija slika, odnosno Moderna galerija slika (1940./1941.); Galerija slika u Osijeku (1954.); Galerija likovnih umjetnosti u Osijeku (1966.); Muzej likov nih umjetnosti (2014.) 1 Od osnutka do danas osječka je Galerija mijenjala ime: Galerija slika, odnosno Moderna galerija slika (1940./1941.); Galerija slika u Osijeku (1954.); Galerija likovnih umjetnosti u Osijeku (1966.); Muzej likov nih umjetnosti (2014.) Muzeji, galerije i stvaranje kanona 4 STJEPAN BRLOŠIĆ, Kronika Galerije likovnih umjet nosti, Osijek, u: Galerija likovnih umjetnosti Osijek, monografija – zbornik, Osijek, GLUO, 1987., 11 – 30. 6 Komisija za sakupljanje historijskih spomenika, starina i knjižnica (KOMZA) formirana je nepo sredno po završetku Drugoga svjetskog rata pri Ministarstvu prosvjete NRH. Počeci: u okrilju Muzeja Slavonije Uz Bauera, koji je poduzeo konkretne korake na planu osnivanja Galerije, važnu ulogu u njezinu pokretanju imali su Franjo Buntak i nakon njega Josip Bösendorfer kao kustosi i ravnate lji osječkog Gradskog muzeja te Arheološki klub Mursa, odno sno Društvo prijatelja starina Mursa iz Osijeka. su zahvaljujući Baueru 1940. i 1941. ušle u osječki muzej. Uz Bauera, koji je poduzeo konkretne korake na planu osnivanja Galerije, važnu ulogu u njezinu pokretanju imali su Franjo Buntak i nakon njega Josip Bösendorfer kao kustosi i ravnate lji osječkog Gradskog muzeja te Arheološki klub Mursa, odno sno Društvo prijatelja starina Mursa iz Osijeka. Predlažući osnivanje Galerije u Osijeku, Antun Bauer imenovao ju je specifičnim nazivom Moderna galerija. Taj je naziv doista odgovarao prvotno zamišljenoj koncepciji njezina fundusa i postava, koji se sastojao većinom iz djela hrvatskih umjetnika moderne. Uz taj naziv uvriježio se i naziv Galerija slika, koji je ostao u primjeni kao službeni naziv ustanove sve do 1966. godine. Galerija je službeno otvorena u prostorijama Gradskoga muzeja 16. studenoga 1941., „kao posebni odjel Gradskoga muzeja”. Autor tog prvog postava Galerije bio je sam Antun Bauer, a njezin prvi kustos bio je osječki slikar Stjepan Macanić (1911. – 1945.). Bio je to prvi službeni i javni oblik muzealne ekspozi cije djela likovnih umjetnosti u gradu Osijeku. Trajanje izložbe zbog ratnih je okolnosti bilo kratko – zatvorena je već u proljeće 1943. godine. Bio je to početak niza seljenja gale rijske zbirke, što ih je Galerija proživjela s osječkim Muzejom unutar kojega je prvotno egzistirala, ali i poslije, nakon odva janja i osamostaljivanja. Tijekom ratnih i poratnih četrdesetih godina 20. stoljeća velik je broj umjetničkih djela u Muzej, odnosno u Galeriju u nje govu sklopu, ušao putem konfiskacije. U zbirku je 1941. ušao dio umjetnina koje su bile konfiscirane od imućnih osječkih židovskih obitelji, a djelovanjem KOMZA-e6 između 1945. i 1948. u Muzej je dopremljena velika količina umjetnina kon fisciranih iz slavonskih vlastelinskih dvoraca. Reprezentativne umjetnine iz vlasništva slavonskih plemićkih obitelji na taj su način spašene od pljačke i uništenja, i vrijedan su temelj današnjih muzejskih zbirki. S povećanjem fundusa u Muzeju je 1947. postavljen osječki slikar i kipar Josip Leović kao kustos za tzv. galerijski materijal, a 1949. kustos galerijske zbirke, odnosno zbirke slika i kipova postao je Jovan Gojković. Društvo povjesničara umjetnosti Hrvatske Institut za povijest umjetnosti Zbornik 4. kongresa hrvatskih povjesničara umjetnosti Počeci: u okrilju Muzeja Slavonije 3 ANTUN BAUER, Ad „Galerija slika u Osijeku”, Zagreb, vlastito izdanje, umnoženo, 1952. (MDC, knjižnica, Zbirka Bauer); 367; ANTUN BAUER, Novo otvorenje Galerije slika u Osijeku, u: Vijesti Društva muzej sko-konzervatorskih naučnih radnika NRH, Zagreb, vol. 1, 5/6 (1952.), 42; ANTUN BAUER, Osnivanje Galerije umjetnina u Osijeku: nakon referata na plenumu u Galeriji u Osijeku 26. IV. 56, 1956., MDC; ANTUN BAUER, Otvorenje Galerije likovnih umjet nosti u Osijeku, u: Vijesti muzealaca i konzervatora Hrvatske, Zagreb, vol. 14, 1 (1965.), 16; ANTUN BAUER, Osnivanje Galerije slika u sklopu Gradskog muzeja u Osijeku, u: Osječki zbornik, Osijek, Muzej Slavonije, 17 (1979.); ANTUN BAUER, Malvina Hermann Osijek: donator Gradskom muzeju za osnivanje Galerije u Osijeku 1939 – 41., 1982., MDC, knjižnica, Zbirka Bauer. Postanak Muzeja, donedavno Galerije likovnih umjetnosti u Osijeku, vezan je uz osječki Muzej Slavonije, osnovan 1877.,5 u okviru kojega je Galerija tijekom nastanka i formiranja admi nistrativno i fizički egzistirala jedanaest godina. No od sama početka ona nije bila u funkciji zbirke slika i skulptura Muzeja Slavonije, niti se takvom nazivala, već je imala stanovitu samo stalnost, o čemu svjedoči odluka o njezinu osnivanju iz 1940. godine, a konačno i sam njezin prvobitni naziv, koji je dugo vremena ostao u uporabi – Galerija slika. U institucionalnom okrilju osječkoga Muzeja razvijala se postupno, od 1941. pa do 1952. godine, kada se izdvojila prostorno, a 1954. i institucio nalno, u samostalnu muzejsku ustanovu. Jedan od inicijatora, pokretač i organizator – zapravo, istinski utemeljitelj osječke Galerije slika – bio je Antun Bauer (1911. – 2000.), povjesničar umjetnosti, osnivač i tadašnji ravnatelj Gipsoteke u Zagrebu. Na prijedlog Antuna Bauera Galerija je osnovana 1940. u sklopu Gradskoga muzeja u Osijeku, a temelj u procesu njezina stvaranja bila je vrijedna donacija umjet nina gradu Osijeku (za osnivanje Galerije) iz Zagreba, koje 4 STJEPAN BRLOŠIĆ, Kronika Galerije likovnih umjet nosti, Osijek, u: Galerija likovnih umjetnosti Osijek, monografija – zbornik, Osijek, GLUO, 1987., 11 – 30. 5 Muzej Slavonije od osnutka mijenjao je ime: Muzej sl. i kr. grada Osijeka (1877.); Hrvatski državni muzej (1942.); Muzej Slavonije (1947.). Daniel Zec 43 – 50 43 su zahvaljujući Baueru 1940. i 1941. ušle u osječki muzej. Počeci: u okrilju Muzeja Slavonije S obzirom na vrstu građe odnosno povijesno-stilske značajke umjetnina, prvotno zamišljenoj, Bauerovoj galeriji, doista je odgovarao naziv Moderna galerija, no taj naziv izgubio je smi sao s dolaskom umjetnina iz bogatih plemićkih kolekcija, koje su velikom većinom bile iz 18. i 19. stoljeća. Nakon 1943. Galerija slika otvarana je i zatvarana nekoliko puta, na različitim lokacijama u Osijeku: 1946. godine, kada je cjelokupni muzejski inventar preseljen u nekadašnju zgradu gradske vijećnice u Tvrđi, u zgradu u kojoj se Muzej Slavonije nalazi i danas; potom 1948. godine, prvi put u vla stitoj, zasebnoj zgradi – autor tog postava, obogaćena novim akvizicijama, bio je Josip Leović; 1950. godine u reduciranom obliku, ponovno je u prostoru Muzeja Slavonije, pod naslovom 100 godina osječkog slikarstva (1850. – 1950.). Tu izložbu posta vili su Josip Leović i Jovan Gojković, a izdan je i prvi katalog. 44 44 „Secesijsko razdoblje” daniel zec, Opaske uz poje dina djela Roberta Frangeša Mihanovića: primjer Zbirke kiparstva Muzeja likovnih umjetnosti u Osijeku, u: Imago, imaginatio, imaginabile: Zbornik u čast Zvonka Makovića, Lovorka Magaš Bilandžić; Dragan Damjanović (ur.), Zagreb, Filozofski fakultet Sveučilišta u Zagrebu, 2018., 366. U Galeriji održao je Jovan Gojković brojna predavanja o umjet nosti, a kroz galerijski postav vodio je skupine posjetitelja; već je ranije Josip Leović školskoj djeci pri posjetu muzeju tuma čio muzejske zbirke i vodio posjetitelje kroz postav – bio je to pionirski rad na području muzejske pedagogije u specijalizi ranom (umjetničkom) muzeju u gradu Osijeku i šire. Radno mjesto muzejskog pedagoga u Galeriji prvi put je otvoreno 1965. godine.8 U Galeriji održao je Jovan Gojković brojna predavanja o umjet nosti, a kroz galerijski postav vodio je skupine posjetitelja; već je ranije Josip Leović školskoj djeci pri posjetu muzeju tuma čio muzejske zbirke i vodio posjetitelje kroz postav – bio je to pionirski rad na području muzejske pedagogije u specijalizi ranom (umjetničkom) muzeju u gradu Osijeku i šire. Radno mjesto muzejskog pedagoga u Galeriji prvi put je otvoreno 1965. godine.8 Gojković je dao vrijedan doprinos u procesu pozicioniranja Galerije kao nove kulturne ustanove u gradu; bio je koautor njezina postava i priređivač kataloga, a likovno-pedagoškim radom pridonio je komunikaciji s publikom. Ipak, u procesu izdvajanja i osamostaljivanja Galerije ključnu je ulogu odigra la Danica Pinterović, tadašnja ravnateljica Muzeja Slavonije. Drugoga veljače 1954. Galerija slika u Osijeku izdvojila se iz sasta va Muzeja Slavonije i postala samostalnom muzejskom usta novom. Prvi vršitelj dužnosti ravnatelja bio je Jovan Gojković, do smrti 1957. godine. Naslijedio ga je, od 1958. do kraja 1960., povjesničar umjetnosti Drago Dodigović, a 1961. v. d. ravnatelja Galerije postao je akademski kipar Stjepan Brlošić.9 Drugoga veljače 1954. Galerija slika u Osijeku izdvojila se iz sasta va Muzeja Slavonije i postala samostalnom muzejskom usta novom. Prvi vršitelj dužnosti ravnatelja bio je Jovan Gojković, do smrti 1957. godine. Naslijedio ga je, od 1958. do kraja 1960., povjesničar umjetnosti Drago Dodigović, a 1961. v. d. ravnatelja Galerije postao je akademski kipar Stjepan Brlošić.9 Drugoga veljače 1954. Galerija slika u Osijeku izdvojila se iz sasta va Muzeja Slavonije i postala samostalnom muzejskom usta novom. Prvi vršitelj dužnosti ravnatelja bio je Jovan Gojković, do smrti 1957. godine. Naslijedio ga je, od 1958. do kraja 1960., povjesničar umjetnosti Drago Dodigović, a 1961. v. d. ravnatelja Galerije postao je akademski kipar Stjepan Brlošić.9 „Secesijsko razdoblje” Novo razdoblje u historijatu Galerije slika započinje fizičkim izdvajanjem iz Muzeja Slavonije 1952. te formalnim osamosta ljenjem 1954., a kulminira 1964. konačnim preseljenjem u nov muzejski prostor, gdje se nalazi i danas. Godine 1952. Muzeju Slavonije Grad Osijek ustupio je za Galeriju prostor na prvom katu Županijske zgrade, u današnjoj Županijskoj ulici broj 4. Postav izložbe, otvorene 24. kolovoza 1952. pod naslovom Razvoj slikarstva od kasne renesanse do po lovice 20. stoljeća, radili su Jovan Gojković i Josip Leović, uz stručnu pomoć Zdenke Munk.7 Bio je to reprezentativan, dotad najopsežniji izložbeni postav, s najvrednijim slikarskim djeli ma. Po Bauerovu mišljenju osječka galerija tada je ušla u red po fundusu najreprezentativnijih umjetničkih muzeja u zemlji. U prostoru Županijske zgrade Galerija je radila sve do 1. kolovoza 1955., a za nepune tri godine primila je oko 30.000 posjetilaca. Godine 1952. Muzeju Slavonije Grad Osijek ustupio je za Galeriju prostor na prvom katu Županijske zgrade, u današnjoj Županijskoj ulici broj 4. Postav izložbe, otvorene 24. kolovoza 1952. pod naslovom Razvoj slikarstva od kasne renesanse do po lovice 20. stoljeća, radili su Jovan Gojković i Josip Leović, uz stručnu pomoć Zdenke Munk.7 Bio je to reprezentativan, dotad najopsežniji izložbeni postav, s najvrednijim slikarskim djeli ma. Po Bauerovu mišljenju osječka galerija tada je ušla u red po fundusu najreprezentativnijih umjetničkih muzeja u zemlji. U prostoru Županijske zgrade Galerija je radila sve do 1. kolovoza 1955., a za nepune tri godine primila je oko 30.000 posjetilaca. 7 Za izložbu je tiskan i katalog, koji je priredio Jovan Gojković. 7 Za izložbu je tiskan i katalog, koji je priredio Jovan Gojković. j 8 STJEPAN BRLOŠIĆ (bilj. 4), 24. Š Ć j 8 STJEPAN BRLOŠIĆ (bilj. 4), 24. 9 STJEPAN BRLOŠIĆ (bilj. 4), 15 – 17. 9 STJEPAN BRLOŠIĆ (bilj. 4), 15 – 17. 10 Izgrađena u reprezentativnoj secesijskoj i histori cističkoj arhitektonsko-urbanističkoj cjelini grada Osijeka, zgrada Galerije podignuta je 1895. godine za Dragutina Neumanna, prema projektu arhitekta Josipa Vancaša. 11 STJEPAN BRLOŠIĆ, Galerija slika u Osijeku, u: Osječk zbornik, Osijek, Muzej Slavonije, 8 (1962.), 374. 12 Spomenik je 1946. demontiran sa svoje izvorne loka cije u Osijeku i pohranjen u Gipsoteci u Zagrebu, a 1964. je na zahtjev Galerije slika vraćen u Osijek i postavljen u njenom dvorištu. Odande je premje šten i 1996. postavljen ispred zgrade osječkog Doma hrvatskih branitelja. Bilješka o osječkoj galeriji: od Galerije slika do Muzeja likovnih umjetnosti Uloga institucije povijesti umjetnosti u gradu, regiji i nacionalnim okvirima Galerija, odnosno današnji Muzej likovnih umjetnosti, muzej ska je ustanova prvorazrednoga značenja ne samo za likovnu kulturu grada Osijeka kao regionalnog središta nego i za hrvat sku likovnu umjetnost. Prema tipu muzeja i vrsti fundusa može se usporediti s nekoliko srodnih specijaliziranih umjetničkih muzeja u Hrvatskoj: s Muzejom moderne i suvremene umjet nosti u Rijeci, Galerijom umjetnina u Splitu i Umjetničkom galerijom Dubrovnik, s kojima ujedno dijeli ulogu središnjih regionalnih specijalističkih umjetničkih muzeja. Nadalje, fundus Muzeja likovnih umjetnosti komparabilan je, u smi slu periodizacije pa i vrijednosti pojedinih djela, s predme tima u zbirkama drugih, većih, matičnih muzejskih ustanova nacionalnog značaja, primjerice Moderne galerije, Muzeja za umjetnost i obrt, Muzeja suvremene umjetnosti i Hrvatskoga povijesnog muzeja u Zagrebu. Kako je već istaknuo Vlastimir Kusik, „Osječki je muzej po nekim segmentima svoga fundusa ili pojedinačnim djelima superioran srodnim, pa i većim muze jima i galerijama, no jednako tako u pojedinim dijelovima fun dusa postoje inferiornosti. Ono što je bitno, jest činjenica da njegov materijal kao cjelina, ili niz vrijednih pojedinačnosti, ima nacionalnu vrijednost.”13 13 VLASTIMIR KUSIK, Slikarstvo i kiparstvo XX. stoljeća ili osječki postav hrvatske moderne umjetnosti, u: Tri stoljeća umjetnosti u Galeriji likovnih umjetnosti Osijek, katalog izložbe, Osijek, Galerija likovnih umjetnosti, 1998., 88. j Današnji Muzej likovnih umjetnosti jedini je muzej specijalistič kog tipa, odnosno jedini muzej koji se sustavno bavi prikuplja njem, istraživanjem, čuvanjem i prezentacijom isključivo djela likovnih umjetnosti od 18. stoljeća do suvremenosti, ne samo u Slavoniji kao najužoj regiji djelovanja nego u cijelom pro storu sjeveroistočne Hrvatske. Svojim fundusom, muzejskom djelatnošću i ulogom institucije povijesti umjetnosti, mlu je ponajprije usmjeren na likovnu baštinu regije, ali izlazi i izvan tih okvira. Temelj muzejskog fundusa čine djela koja imaju kapitalnu važnost za hrvatsku likovnu baštinu od sredine 18. stoljeća do danas, i po njima je osječki Muzej najpoznatiji i najpriznatiji. To se, ponajprije, odnosi na zbirke slika, koje su bile u vlasništvu slavonskih velikaških obitelji iz 18. i 19. sto ljeća – na djela stranih majstora kasnog baroka, klasicizma i romantizma, koja ulaze i u srednjoeuropski kulturni korpus. Muzej, nadalje, posjeduje djela prve generacije hrvatskih pej zažista i portretista sredine 19. stoljeća te djela na prijelazu u 20. stoljeće i iz njegove prve polovine, kada se utemeljuje hrvatsko moderno slikarstvo.14 Tu su i neka od antologijskih djela klasika hrvatske moderne (Vladimir Becić, Akt pred ogle dalom; Bela Čikoš Sesija, Saloma i drugi). Europska avenija broj 9 U jesen 1962. Galeriji je dodijeljena zgrada na tadašnjem Bulevaru JNA broj 9,10 današnjoj Europskoj aveniji, čime je riješen dugogodišnji problem njezina smještaja – tamo se Muzej likovnih umjetnosti nalazi i danas. Prostorni kapacitet prizemlja i prvoga kata od 755 m² bio je predviđen za stalne i povremene izložbe, za upravu i studijski rad, čuvaonicu za slikarsku, grafičku i kiparsku zbirku te za stolarsku radionicu i fotolaboratorij.11 Galerija slika otvorena je 20. prosinca 1964. godine, a u njezinom dvorišnom prostoru iste je godine inici jativom djelatnika Galerije postavljen Spomenik palim vojni cima 78. Šokčevićeve pukovnije Roberta Frangeša Mihanovića.12 Daniel Zec 43 – 50 45 45 U veljači 1966. godine Galerija slika u Osijeku promijenila je ime u Galerija likovnih umjetnosti u Osijeku. To će ime nositi sve do 2014., kada ga mijenja u Muzej likovnih umjetnosti. U veljači 1966. godine Galerija slika u Osijeku promijenila je ime u Galerija likovnih umjetnosti u Osijeku. To će ime nositi sve do 2014., kada ga mijenja u Muzej likovnih umjetnosti. Društvo povjesničara umjetnosti Hrvatske Institut za povijest umjetnosti Zbornik 4. kongresa hrvatskih povjesničara umjetnosti 13 VLASTIMIR KUSIK, Slikarstvo i kiparstvo XX. stoljeća ili osječki postav hrvatske moderne umjetnosti, u: Tri stoljeća umjetnosti u Galeriji likovnih umjetnosti Osijek, katalog izložbe, Osijek, Galerija likovnih umjetnosti, 1998., 88. Uloga institucije povijesti umjetnosti u gradu, regiji i nacionalnim okvirima U Muzeju su, nadalje, zastupljeni svi osječki umjetnici te najveći dio njihova likov nog stvaralaštva, od nositelja osječke risarske škole, Huga Conrada Hötzendorfa i Adolfa Waldingera, pa do umjetnika koji su djelovali u prvoj polovini 20. stoljeća. Razdoblje druge 14 BRANKA BALEN, VLASTIMIR KUSIK, JASMINKA NAJCER SABLJAK, Tri stoljeća umjetnosti u osječkoj Galeriji, deplijan izložbe, Osijek, Galerija likovnih umjetnosti, 2007. 15 DANICA PINTEROVIĆ, O razvoju osječkog muzeja, u: Osječki zbornik, Osijek, Muzej Slavonije, 6 (1958.), 7. polovine 20. stoljeća i suvremena umjetnost još su, kako je to istaknuo Vlastimir Kusik, „bez željena opsega, ali nedvojbeno vjerodostojne kvalitete”. No promišljenom politikom otkupa, po načelu otkupljivanja onoga što zaslužuje mjesto u stal nom postavu i onoga što se izlaže na povremenim izložbama, u nekoliko posljednjih godina Muzej se upotpunjuje djelima suvremene umjetnosti, čime je desetljećima oskudijevao. Danas muzejski fundus broji 5734 muzejska predmeta raspoređe nih u devet muzejskih zbirki: Zbirka slikarstva 18. i 19. stoljeća, Zbirka slikarstva prve polovine 20. stoljeća, Zbirka slikarstva druge polovine 20. stoljeća, Zbirka crteža i grafika 18. i 19. sto ljeća, Zbirka crteža i grafika prve polovine 20. stoljeća, Zbirka crteža i grafika druge polovine 20. stoljeća, Zbirka kiparstva, Zbirka plaketa i medalja i Zbirka novih medija. Sve zbirke regi strirane su kao kulturno dobro Republike Hrvatske. Muzejsko-izložbena djelatnost Izložbeni program, kao najvidljiviji oblik djelovanja Muzeja likovnih umjetnosti, uporište ima u predstavljanju likovnih stvaralaca grada i regije, nacionalne baštine i suvremene umjetnosti te njegovanju međunarodne suradnje, posebno sa susjednim zemljama i inozemstvom u najširem smislu. Uz sku pne i samostalne te tematske, monografske i retrospektivne izložbe Muzej kontinuirano, bijenalno i trijenalno, organizira tri velike likovno-izložbene manifestacije, koje su koncepcij ski, organizacijski i produkcijski njegov izvorni i samostalni proizvod, i koje su postale svojevrsnim zaštitnim znakom nje gova izložbenog programa: Slavonski biennale, Memorijal Ive Kerdića – Triennale hrvatskog medaljerstva i male plastike te Dani grafike. Egzistiranje Muzeja, odnosno Galerije, kao samostalne muzejske ustanove obilježila su i tri različito koncipirana stalna postava, u kojima je konstantu činila građa 18. i 19. stoljeća, a mijenama je bio podložan materijal 20. stoljeća: stalni postav iz 1952. pod naslovom Razvoj slikarstva od kasne renesanse do polovice 20. stoljeća, stalni postav iz 1964. godine formiran po useljenju u novi prostor, te stalni postav iz 1998. pod naslovom Tri stoljeća umjetnosti u osječkoj Galeriji. Uz prekide, taj je postav bio u funkciji sve do 2011. godine, kada je rasformiran. Uz izložbe, nezaobilazno je istaknuti i izdavačku djelatnost Muzeja likovnih umjetnosti – monografske publikacije i publi kacije koje su u obliku kataloga redovito pratile sve izložbene projekte i time postale važan prilog i dokument likovne povi jesti grada, regije ali i nacionalne povijesti umjetnosti. Stručni i znanstveni doprinos djelatnika „Značaj bilo kojeg muzeja ogledava se u strukturi njegovih zbirki i u organizaciji naučnog i prosvjetnog rada kustosa, kojima su zbirke povjerene”, napisala je 1958. jedna od najve ćih osječkih i hrvatskih muzealki, Danica Pinterović, u svom Daniel Zec 43 – 50 Bilješka o osječkoj galeriji: od Galerije slika do Muzeja likovnih umjetnosti Daniel Zec 43 – 50 47 uvodu u historijat Muzeja Slavonije.15 Doista, njezina je misao odmjerena i dostojanstvena maksima kojom bi se neki muzej trebao voditi. Uz već spomenute djelatnike osječke Galerije, od 1963. do danas svoj su obol stručnom, znanstvenom i pedagoškom radu ustanove dali, svatko na svoj način, ponaj prije povjesničari umjetnosti.16 Neki od njih u Galeriji su kao kustosi proveli cijeli radni vijek, a nekih više nema među nama. Kronologijom dolaska u Galeriju, to su Ljiljana Sudić, Branka Balen (†), Jelica Ambruš, Vlastimir Kusik (†), Jasminka Najcer Sabljak, Valentina Radoš, Daniel Zec. j U posljednjih nekoliko godina broj je djelatnih kustosa u Muzeju likovnih umjetnosti znatno reduciran. Odlascima u mirovinu ili na druga radna mjesta, uz nepromišljeno vođenu kadrov sku politiku, danas je zapravo prepolovljen – sveden je na tek tri povjesničara umjetnosti. Gubitak stručnoga kadra težak je gubitak kako za unutarnje profesionalno funkcioniranje muzeja tako i za njegovu ulogu institucije povijesti umjetno sti – kao jednoga od generatora i ishodišta iz kojega proizlazi rad povjesničara umjetnosti. U konačnici, to je gubitak i za povijest umjetnosti kao znanstvenu disciplinu koja u ovome dijelu Hrvatske raspolaže vrlo malim brojem relevantnih stručnjaka: muzej poput ovoga nije samo mjesto za pohranu, čuvanje i izlaganje – on bi trebao prerasti u regionalni znan stvenoistraživački centar za likovnu umjetnost. j Ono što je za stručni i znanstveni rad u Muzeju Slavonije značila Danica Pinterović, za Galeriju likovnih umjetnosti bio je Oto Švajcer (1907. – 2003.), iako nikada nije bio njezin kustos. Jer, ukoliko se osobama može pridati značenje institucije povijesti umjetnosti, onda je za grad Osijek i za Galeriju likovnih umjet nosti to bio upravo Oto Švajcer. Riječima Predraga Golla, „u lič nosti Ota Švajcera, najznačajnijeg istraživača kulture i likovne baštine, ponajviše slikarstva, grada i regije, Galerija je imala više od 50 godina svog istraživača, biografa i kroničara”.17 Ili, kako je napisao Vlastimir Kusik: „U njemu je likovni život Osijeka, kako je duhovito kazao dr. Bogdan Mesinger, našao ‘čovjeka instituciju’, svog kroničara, kritičara, istraživača i tumača. 16 Za specifično pedagoško djelovanje zaslužni su muzejski pedagozi: Marijanka Himelrajh, Mirjana Gorički i Ljiljana Habuš (od 1965. do 1970.), Leonilda Conti (od 2006.) te Kristina Delalić Vetengl (od 2015.). Društvo povjesničara umjetnosti Hrvatske Institut za povijest umjetnosti Zbornik 4. kongresa hrvatskih povjesničara umjetnosti 17 PREDRAG GOLL, Umjesto predgovora, u: Galerija li kovnih umjetnosti Osijek, monografija-zbornik, Osijek, GLUO, 1987., 7. 18 VLASTIMIR KUSIK, Oto Švajcer i osječka Galerija, u: Oto Švajcer, Domaći i strani slikari XVIII. i XIX. stoljeća u Galeriji likovnih umjetnosti Osijek, Osijek, Galerija likovnih umjetnosti, 1988., 3. Stručni i znanstveni doprinos djelatnika (…) Jer, ako je Galerija mjesto u koje je smještena baština likov nog života grada Osijeka i dijela regije, Oto Švajcer je taj koji joj je dao ime i prezime, pa i više od toga, on joj je oblikovao tijelo našavši joj mjeru i udahnuo mu dušu davši joj smisao.”18 Stopama Ota Švajcera išle su kustosice Galerije likovnih umjet nosti Branka Balen i Jasminka Najcer Sabljak – otišavši korak dalje od svoga prethodnika u istraživanju likovne baštine 18. i 19. stoljeća, otvorile su nova poglavlja naše povijesti umjetnosti. Jelica Ambruš monografski je obrađivala umjetnike Slavonije i Osijeka u 20. stoljeću. Drugim smjerom išao je Vlastimir Kusik; kao kustos i muzejski savjetnik Galerije dugogodišnjim je djelovanjem ostavio svoj pečat u osječkoj likovnoj kritici, pridonijevši tumačenju i poznavanju hrvatskoga likovnog moderniteta i nadasve suvremenosti te Osijeka kao jednoga njihova stjecišta i izvorišta. Od šezdesetih (i prije) naovamo, svi oni na različite su načine, na različitim poljima djelovanja i u različitom opsegu, no iz osječke Galerije kao logističkog (i logičnog) uporišta, utjecali na profiliranje fizionomije povijesti 48 umjetnosti ovog dijela Hrvatske i formiranje znanja o njego voj likovnoj baštini i suvremenoj umjetnosti – znanja kojim raspolažemo danas. O njihovu, kao i o stručno-znanstvenom i likovno-kritičkom djelovanju drugih kustosa ovoga muzeja, ali i brojnih uglednih povjesničara umjetnosti koji su kao vanjski suradnici surađivali s ovom ustanovom, rječito govore izložbe i katalozi, publicirani radovi i monografije, koje ustanova kao novo znanje proizvodi od 1960-ih do danas. Bilješka o osječkoj galeriji: od Galerije slika do Muzeja likovnih umjetnosti Daniel Zec 43 – 50 K novom muzeju Pored Daniel Zec 43 – 50 Bilješka o osječkoj galeriji: od Galerije slika do Muzeja likovnih umjetnosti 49 toga, Galerija likovnih umjetnosti ipak je bila prepoznatljivo ime i osječki kulturni brend, kao što je to, primjerice, Moderna galerija u Zagrebu, ime koje se prepoznaje i ističe u nizu drugih, privatnih galerija, koje nisu muzeji, a djeluju u gradu i nisu joj konkurencija. Uostalom, neki od najvećih i najstarijih svjetskih umjetničkih muzeja danas u svojem imenu nose riječ galerija; na našem brendu trebalo je tek malo više marketinški poraditi. toga, Galerija likovnih umjetnosti ipak je bila prepoznatljivo ime i osječki kulturni brend, kao što je to, primjerice, Moderna galerija u Zagrebu, ime koje se prepoznaje i ističe u nizu drugih, privatnih galerija, koje nisu muzeji, a djeluju u gradu i nisu joj konkurencija. Uostalom, neki od najvećih i najstarijih svjetskih umjetničkih muzeja danas u svojem imenu nose riječ galerija; na našem brendu trebalo je tek malo više marketinški poraditi. Dok promjena imena pripada ipak isključivo semantičkoj razini, u godinama što slijede trebalo bi doći do znatno obuhvatnije i konkretnije promjene, presudne za budući rad Muzeja likov nih umjetnosti: ona se tiče planirane izgradnje nove muzejske zgrade. g Još je prilikom otvaranja Galerije 1964. Antun Bauer konstatirao da je njezin novi prostor nedostatan za njezin fundus, značenje i potrebe, a danas je to više no očigledno. Prostorni kapaciteti zgrade u kojoj je Muzej smješten ne udovoljavaju više potre bama koje zahtijeva njezin današnji opsežan i dinamičan izlož beni program. Kustosi Muzeja danas se susreću s problemom nemogućnosti istodobnog funkcioniranja povremenih izložbi i stalnog postava, koji se morao skidati pri dolasku svake veće izložbe. Naposljetku je poglavito iz tih razloga Muzej i posve izgubio svoj stalni postav. g j p Nužno je obrazložiti intenciju gradnje dodatnog muzejskog pro stora, odnosno još jednom naglasiti da je prostor historicističke gradske palače Neumann, u kojoj je Muzej likovnih umjetnosti smješten, nedostatan i neadekvatan za sve sadržaje kojima MLU danas kao muzejska, ali i galerijska institucija raspolaže i svo jom djelatnošću komunicira. Već dugi niz godina taj je prostor s obzirom na muzejsku građu i izložbeni program prekapaci tiran te više ne udovoljava zahtjevima nužnim da bi muzejska ustanova primjereno funkcionirala. Dva su goruća problema: prostor za stalni postav te prepunjeni depoi u kojima više nema mjesta za adekvatan smještaj rastućeg broja predmeta iz svih muzejskih zbirki. K novom muzeju Oba problema bila bi riješena izgradnjom nove zgrade, kao interpolacije u dvorištu postojećeg objekta i s njom fizički povezane, a planirana je tijekom sljedećih neko liko godina. Tako će stalni postav naći svoje mjesto u staroj zgradi, dok će u novoj dio izložbenog prostora biti namijenjen segmentu stalnoga postava u kojemu se predstavljaju djela suvremene umjetnosti, a dio postavljanju povremenih izložbi iz godišnjeg programa. Jednako važno, u projektu nove muzej ske zgrade, koji udovoljava svim muzeološkim standardima, predviđeni su dovoljno veliki prostori za čuvaonice muzejske građe, ali i sve ostale muzejske sadržaje. Društvo povjesničara umjetnosti Hrvatske Institut za povijest umjetnosti Zbornik 4. kongresa hrvatskih povjesničara umjetnosti K novom muzeju Novi naziv Galerije na početku novoga stoljeća promjena je što simbolično uvodi u znatno temeljitije promjene koje usta novu očekuju u vrlo bliskoj budućnosti – promjenama što vode k novom muzeju, koji će djelovati u novom prostoru, s novim muzeološkim konceptom i s novim stalnim postavom. p p Odluka promjene naziva muzejske ustanove vrlo je važna i dale kosežna te mora imati opravdanje i obrazloženje. Razlozi iz kojih je 2014. donesena odluka promjene naziva, odnosno preimenovanja Galerije u Muzej likovnih umjetnosti čine se vrlo jednostavnima: u gradu Osijeku egzistira nekoliko gale rijskih ustanova i galerijskih prostora koji nisu muzejskog, već isključivo galerijskog karaktera, a s kojima se Galerija likovnih umjetnosti pogrešno izjednačavala. Takva pogrešna percepcija nije odgovarala njezinu statusu najveće i najvažnije muzejske ustanove umjetničkog tipa u istočnoj Hrvatskoj. Galerija se naime nije, ponajprije kad je riječ o muzejskoj publici i medi jima, zatim u krugovima izvan muzejske struke, a čak i unutar nje, percipirala kao muzejska ustanova, odnosno kao muzej umjetničkog tipa s vlastitim fundusom i misijom prikupljanja, čuvanja, zaštite, istraživanja i komunikacije likovne baštine. Poglavito zbog te specifične razlike, smatralo se da bi se pro mjenom naziva ustanove, odnosno zamjenom riječi galerija riječju muzej, instituciji pridao veći dignitet; točnije, da bi se time dokinulo identificiranje Galerije likovnih umjetnosti s drugim osječkim galerijama koje nemaju vlastitog fundusa, ali i utjecalo na promjenu stava i recepcije muzejske publike, odnosno društvene zajednice u cjelini, prema Galeriji i njezinu poslanju, a s konačnim ciljem veće posjećenosti. Osim pro mjene imena, sve drugo ostalo je isto; nije bilo strukturnih pro mjena ni preinaka muzejskog ustroja – novi muzej nije prešao u neki viši rang niti mu je povećan budžet. Posve je iskrivljena interpretacija da je Galerija promjenom imena prerasla u muzej ili postala muzejom. Kako je izloženo na početku, posta nak ovoga muzeja veže se uz 1954. godinu, kada on funkcionira kao samostalna muzejska ustanova, odnosno muzej. No upitno je koliko će promjena naziva doista pridonijeti promjeni svi jesti muzejske publike i lokalne društvene zajednice, njihovoj recepciji te konačno konzumiranju kulturnog proizvoda koji Galerija, odnosno Muzej, komunicira. Možda se više moglo postići promišljenijim aktivnostima u sferi odnosa s javnošću te edukacijom muzejske publike, dok promjena naziva ne mora, niti sama po sebi može, donijeti drastičnu promjenu. Bilješka o osječkoj galeriji: od Galerije slika do Muzeja likovnih umjetnosti / Daniel Zec / cc by / 4.0 doi: https://doi.org/10.31664/z4khpu.06 Zbornik 4. kongresa hrvatskih povjesničara umjetnosti Društvo povjesničara umjetnosti Hrvatske Institut za povijest umjetnosti 50
https://openalex.org/W2981743940	https://europepmc.org/articles/pmc6857096?pdf=render	English	null	Robustness of Radiomics for Survival Prediction of Brain Tumor Patients Depending on Resection Status	Frontiers in computational neuroscience	2,019	cc-by	8,580	Robustness of Radiomics for Survival Prediction of Brain Tumor Patients Depending on Resection Status Leon Weninger, Christoph Haarburger and Dorit Merhof Prediction of overall survival based on multimodal MRI of brain tumor patients is a difﬁcult problem. Although survival also depends on factors that cannot be assessed via preoperative MRI such as surgical outcome, encouraging results for MRI-based survival analysis have been published for different datasets. We assess if and how established radiomic approaches as well as novel methods can predict overall survival of brain tumor patients on the BraTS challenge dataset. This dataset consists of multimodal preoperative images of 211 glioblastoma patients from several institutions with reported resection status and known survival. In the ofﬁcial challenge setting, only patients with a reported gross total resection (GTR) are taken into account. We therefore evaluated previously published methods as well as different machine learning approaches on the BraTS dataset. For different types of resection status, these approaches are compared to a baseline, a linear regression on patient age only. This naive approach won the 3rd place out of 26 participants in the BraTS survival prediction challenge 2018. Previously published radiomic signatures show signiﬁcant correlations and predictiveness to patient survival for patients with a reported subtotal resection. However, for patients with reported GTR, none of the evaluated approaches was able to outperform the age-only baseline in a cross-validation setting, explaining the poor performance of approaches based on radiomics in the BraTS challenge 2018. BRIEF RESEARCH REPORT published: 08 November 2019 doi: 10.3389/fncom.2019.00073 Keywords: BraTS 2018, survival prediction, radiomics, brain tumor, machine learning, feature selection Edited by: Spyridon Bakas, University of Pennsylvania, United States Reviewed by: Hamed Akbari, University of Pennsylvania, United States Zhi-Cheng Li, Shenzhen Institutes of Advanced Technology (CAS), China Keywords: BraTS 2018, survival prediction, radiomics, brain tumor, machine learning, feature selection Correspondence: Leon Weninger leon.weninger@lfb.rwth-aachen.de 1. INTRODUCTION The high-grade glioma, a subtype of brain tumors, is one of the most aggressive and dangerous diseases worldwide. For the US, a 5-year survival rate of glioblastoma patients of only 5.6% was reported for 2000–2015 (Ostrom et al., 2018). Automatic analysis of these tumors is challenging, as their shape, location and extent can diﬀer substantially. Since 2012, the BraTS challenge (Menze et al., 2015) is held annually to allow an unbiased comparison of diﬀerent segmentation algorithms. Since 2017, an overall survival (OS) prediction task is included to assess whether quantitative image features based on these segmentations can provide further clinical insight. In the OS task, patients need to be classiﬁed in long-survivors (OS>15 months), short-survivors (OS <10 months), and mid- survivors (10 months <OS <15 months). While data is provided for patients with diﬀerent resection status, the oﬃcial evaluation is carried out only on patients with a reported gross total resection (GTR). A total of 41 teams took part in this survival prediction task in 2017 and 2018. Received: 25 April 2019 Accepted: 09 October 2019 Published: 08 November 2019 Citation: Weninger L, Haarburger C and Merhof D (2019) Robustness of Radiomics for Survival Prediction of Brain Tumor Patients Depending on Resection Status. Front. Comput. Neurosci. 13:73. doi: 10.3389/fncom.2019.00073 Received: 25 April 2019 Accepted: 09 October 2019 Published: 08 November 2019 Citation: Weninger L, Haarburger C and Merhof D (2019) Robustness of Radiomics for Survival Prediction of Brain Tumor Patients Depending on Resection Status. Front Comput Neurosci 13:73 November 2019 \| Volume 13 \| Article 73 Frontiers in Computational Neuroscience \| www.frontiersin.org Survival Prediction Depending on Resection Weninger et al. Using the age as sole feature with a linear regressor, we achieved an accuracy of 0.56 (n = 77) on the test set in the BraTS challenge 2018. In comparison, the ﬁrst placed approaches of 2017 (Shboul et al., 2018) and 2018 (Feng et al., 2019) achieved accuracies of around 0.58 and 0.62, respectively (Bakas et al., 2018b). Shboul et al. relied on automatic radiomic feature extraction combined with a Random Forest Regressor (RFR), while Feng et al. used geometric features in combination with a linear model. The developers of other top performing algorithms chose similar strategies of combining either hand- selected or automated radiomic features with a supervised machine learning algorithm: Radiomic feature extraction was used in combination with an RFR (Sun et al., 2019) or a Multilayer Perceptron (MLP) (Baid et al., 2019). Geometric features only were used with an MLP (Jungo et al., 2018), and ﬁnally atlas locations together with relative tumor sizes and an RFR were also employed (Puybareau et al., 2019). These teams achieved accuracies between 0.55 and 0.6. Further submitted approaches ranged from deep learning algorithms to radiomic feature analysis to handcrafted feature engineering, that achieved accuracies between 0.15 and 0.55. As three classes were equally subdivided, a random choice would result in an accuracy of 0.33. In this paper, we analyze diﬀerent radiomic-based approaches to survival prediction on the BraTS data. To be independent of segmentation inaccuracies, we only use the BraTS training data for all experiments. For this data, groundtruth segmentations are publicly available, approved by experts and reviewed by a single board-certiﬁed neuro-radiologist (Bakas et al., 2017c). The data can be subdivided by resection status into patients with reported GTR, subtotal resection (STR) and patients with unavailable resection status (NA). The oﬃcial evaluation was carried out only on the GTR subset. First, we re-evaluate previously published radiomic signatures on the diﬀerent resection status subsets. We show that these methods are predictive for OS on the STR subset. Second, diﬀerent machine learning tools are evaluated on the radiomic feature set. 2.1. Dataset In our evaluation, we discard the BraTS test- and validation datasets, as no groundtruth segmentations and no OS information are available, and use only the training dataset. All subjects of the BraTS 2018 dataset are included in the BraTS 2019 dataset; thus, the analysis is focused on the larger BraTS 2019 dataset. The BraTS survival data training dataset consists of data from 211 brain tumor patients from diﬀerent institutions. For each patient, the following data is available: • 4 MRI acquisitions: T1, T1 post contrast agent (T1CE), T2 and T2-FLAIR. All are resampled to an isotropic resolution of 1 × 1 × 1mm3, co-registered and skull stripped. 1 × 1 × 1mm3, co-registered and skull stripped. Radiomic feature extraction describes the process of automatically computing a variety of quantitative image features. By quantifying lesions, radiomics can not only be used for prognosis, but can also help increase precision in diagnosis. For example, radiomics has been successfully used to distinguish between high- and low-grade glioma (Cho et al., 2018) on the BraTS dataset. An overview of radiomics and its applications is given by Rizzo et al. (2018). For brain tumor analysis in particular, a review of radiomics-based techniques for quantitative imaging is given by Zhou et al. (2018). • Segmentation map: Edema (ED), enhancing tumor (ET), and non-enhancing / necrotic tumor core (NEC). • The age of the patient. • Resection status. The resection status is either reported as GTR, subtotal resection (STR), or unknown (NA). For a few subjects (n = 21), the resection status was given as STR in the BraTS 2018 dataset, but omitted for the 2019 dataset. These statuses were re-entered into the dataset. Next, two patients were reported as still alive. Their overall survival in the database was set to the maximum survival time in the dataset, 1,767 days. Radiomic features combined with a machine learning model is thus a natural choice for OS prediction. We initially evaluated diﬀerent radiomics-based machine learning techniques for the BraTS challenge, too. However, when thoroughly validating the results, all considered approaches could not outperform a linear regressor based on the patients age only. We thus decided to submit an age-only linear regressor (Weninger et al., 2019), and won the third place in the BraTS challenge 2018. Citation: Third, as the number of extracted radiomic features is very large and important features could remain undetected, two diﬀerent feature reduction methods are assessed. For the patients with GTR, neither previously published methods, nor diﬀerent machine learning models, nor unsupervised feature reduction techniques could establish a robust signature for patient survival prediction. Finally, the importance of thoroughly assessing the robustness of radiomic markers is discussed, and ideas on how to improve survival prediction based on MRI images even after tumor resection are provided. On other brain tumor datasets, encouraging results for OS prediction have been published. A successful radiomic-based brain tumor patient OS and progression-free survival prediction on a private dataset comprising 119 patients was described by Kickingereder et al. (2016). Positive ﬁndings with data-mining algorithms have also been reported when including Diﬀusion- MRI and relative cerebral blood volume data (Zacharaki et al., 2012) or Perfusion-MRI data (Jain et al., 2014) next to the MR- sequences used in the BraTS dataset. Deep learning based OS prediction has been successfully used on another, smaller (n = 93) private dataset (Nie et al., 2019). However, as the BraTS summary (Bakas et al., 2018b) indicates, deep learning techniques performed rather poorly on the open-access data. Quantitatively comparing deep learning to classical regression on radiomic features for OS on the BraTS data was also carried out by Suter et al. (2019). They concluded that radiomic feature are better suited, as features extracted from deep learning networks seemed to be unstable for this task. Frontiers in Computational Neuroscience \| www.frontiersin.org 3. METHODS Image intensity statistics consists of features such as minimum, maximum, mean, median, percentiles, standard deviation, skewness, kurtosis, and uniformity. In combination with diﬀerent modalities and ﬁlters, 1,944 features resulted from this category. Our OS prediction pipeline can be divided into the following substeps: (1) Image preprocessing, (2) extraction of radiomic features, (3) unsupervised feature reduction, and (4) statistical inference and out-of-sample prediction. These major substeps of the pipeline are visualized in Figure 2. For the BraTS challenge, only out-of-sample prediction is necessary. In order to determine whether radiologic features are appropriate for the given problem, we supplement out-of-sample prediction with classical hypothesis testing. Combined with the age, a total of 9871 features were obtained. In contrast, the total number of observations was 211—the number of variables p is much bigger than the number of samples n. Such a setting is actually common for pattern-learning methods in neuroscience (Bzdok, 2017), and is referred as wide data, in contrast to long data where the number of samples is bigger than the number of variables. Using such wide data directly for inference often leads to non-robust results and to overﬁtting on the training set. Consequently, before inference the number of features needs to be reduced as much as possible while maintaining the characteristics of the data. 2.2. Cohort Study Most data are provided either by the Center for Biomedical Image Computing and Analytics from University of Pennsylvania (CBICA, n = 128) or by The Cancer Imaging Archive (TCIA, n = 76) (Bakas et al., 2017a,b). A small amount of the data (n = 7) November 2019 \| Volume 13 \| Article 73 2 Survival Prediction Depending on Resection Weninger et al. 3.2. Feature Extraction originates from other sources. All subjects have a pathologically conﬁrmed diagnosis of primary de novo glioblastoma (Bakas et al., 2018b). Nevertheless, as population or diﬀerences in treatment could inﬂuence clinical outcome, an overview over diﬀerences and similarities of the diﬀerent provenances is given. originates from other sources. All subjects have a pathologically conﬁrmed diagnosis of primary de novo glioblastoma (Bakas et al., 2018b). Nevertheless, as population or diﬀerences in treatment could inﬂuence clinical outcome, an overview over diﬀerences and similarities of the diﬀerent provenances is given. Using the package PyRadiomics (van Griethuysen et al., 2017), shape features were extracted from the provided segmentation masks, and image intensity and texture features were extracted from the four diﬀerent image modalities for each segmentation mask. Image intensity and texture features were calculated for the original image and on wavelet decomposed images. In total, the following features were extracted: For all TCIA subjects, the resection status is unknown. In contrast, 94 of the 101 subjects with GTR as well as all subjects with STR originate from one institution, CBICA. In the dataset, there are no statistically signiﬁcant diﬀerences between age or survival for the diﬀerent data provenances or the diﬀerent types of resection status (ANOVA: p >0.05). However, the relative brain tumor volume, as determined as tumor volume divided by brain volume, is signiﬁcantly smaller in the TCIA data than in the CBICA data (p <0.0001). Between the resection status STR and GTR, in contrast, there is no signiﬁcant relative brain tumor volume diﬀerence (Figure 1). Shape features comprise volume, surface area, sphericity, maximum diameter, elongation, axis lengths and ﬂatness. These were extracted for the diﬀerent tumor classes, resulting in 42 features. Gray-level features include gray-level co-occurrence (glcm), gray-level run length (glrlm), gray-level dependence matrix (gldm), gray-level size zone, and neighboring gray tone diﬀerence features. As these were extracted for the original and wavelet transformed images and four image modalities, this resulted in 7,884 features. 3.1. Image Preprocessing The data was acquired with various MRI scanners and diﬀerent clinical protocols. In consequence, absolute image intensities, and, subsequently, radiomic features, can be strongly inﬂuenced. This was counteracted with a bias-ﬁled correction and subsequent normalization of the images. First, the ANTs N3 (Tustison et al., 2010) bias-ﬁeld correction was applied to all images, removing local diﬀerences in image intensities. Second, in order to harmonize the MRI acquisitions from diﬀerent institutions, all images were normalized with z-score normalization to zero mean and unit variance. Frontiers in Computational Neuroscience \| www.frontiersin.org 3.3. Preselection of Features Radiomic features are typically redundant (Rizzo et al., 2018), i.e., they are multicollinear. Diﬀerent techniques exist to reduce the number of features and thus the multicollinearity. For the present problem, a subset of features should be kept after feature reduction. In contrast to synthetic features obtained by a PCA, a feature selection method oﬀers more interpretable results. Further, in order to use the complete BraTS training dataset, the method should be unsupervised. With an unsupervised method, the complete BraTS OS training data (n = 211) can be used for feature selection, as features of preoperative images should be independent of resection status. In contrast, for this study, a supervised method could only be done on the speciﬁc resection status subset (GTR: n = 101). As splitting into train- and test set would further be necessary, an even smaller number of examples could be employed for feature selection. Histogram equalization was considered as alternative normalization technique, but discarded as it did not improve the results. This could be due to the properties of tumor tissue in MRI images: Parts of the brain tumor are often the brightest or darkest area in the acquisitions, while occupying only a small proportion of the brain. The contrast-enhancing part is especially bright in T1CE acquisitions while covering just a small single- digit percentage of the brain volume. Histogram equalization or other nonlinear brightness adaptation techniques will thus shrink the contrast for these outlier points, actually leading to less contrast in the examined regions. For a comparison of the results using histogram equalization, all evaluations relying not only on tumor shape and/or age were repeated with histogram equalization instead of z-score normalization. The results can be found in the Supplementary Materials. Thus, a method relying on correlation matrix clustering and Variance-Inﬂation-Feature (VIF) iterative reduction (James et al., 2014) was chosen as the most appropriate. As a ﬁrst step to reduce multicollinearity, single redundant features were discarded. For this purpose, each feature was linearly Frontiers in Computational Neuroscience \| www.frontiersin.org November 2019 \| Volume 13 \| Article 73 3 Survival Prediction Depending on Resection Weninger et al. FIGURE 1 \| Difference in age and tumor-to-brain volume by resection status, and survival by provenance. FIGURE 1 \| Difference in age and tumor-to-brain volume by resection status, and survival by provenance. Frontiers in Computational Neuroscience \| www.frontiersin.org 3.6. Evaluation of Previously Published Methods Previously reported relationships between radiomic signatures and survival time were evaluated on the BraTS dataset. Gutman et al. (2013) reported that the length of the lesion’s major axis and the proportion of contrast-enhanced tumor were negatively correlated with survival on the TCGA glioblastoma dataset. It should be noted that this dataset is included in the BraTS dataset with the resection status NA. It has also been shown that volumetric features of enhancing tumor, non-enhancing tumor core and necrosis, and edema normalized to brain volume are associated with shorter survival time on diﬀerent independent datasets (Zhang et al., 2014; Macyszyn et al., 2015). 3.3. Preselection of Features For each analysis, the minimum number of principal Frontiers in Computational Neuroscience \| www.frontiersin.org November 2019 \| Volume 13 \| Article 73 4 Survival Prediction Depending on Resection Weninger et al. one random forest classiﬁer (RFC) as estimator and one for the ﬁnal prediction was evaluated. The regression models were directly ﬁtted to the survival days, while the classiﬁer can only predict the classes. As classes, the three classes as proposed by the BraTS challenge (long-survivors (OS >15 months), short- survivors (OS <10 months), and mid-survivors (10 months <OS <15 months) were used. FIGURE 3 \| Reordered correlation matrix and obtained dendrogram of the radiomic features obtained from the normalized MRI images. Green designates strongly correlated features while red indicates an r2 close to zero. Diﬀerent radiomic features are represented by absolute values at very diﬀerent scales. Furthermore, outliers of single features may strongly inﬂuence the results. Consequently, the radiomic features were ﬁrst normalized: The feature median is subtracted, and the features were scaled by the interquartile range, i.e., the range between the 25th quantile and the 75th quantile. The diﬀerent machine learning models were ﬁrst employed on the complete feature set for the diﬀerent resection status. The same methods were then also tested on the VIF-based feature subset as well as on the PCA reduced feature set, in order to evaluate whether these models could improve robustness on GTR patients. All machine learning models were implemented with scikit- learn v0.21.2 (Pedregosa et al., 2011) or scikit-learn-contrib using default settings. Next to the methodology presented in this paper, we further evaluated the linear regressor on the age only as submitted during the BraTS challenge 2018, as well as a linear regression on age and the features remaining signiﬁcant after Benjamini–Hochberg correction. FIGURE 3 \| Reordered correlation matrix and obtained dendrogram of the radiomic features obtained from the normalized MRI images. Green designates strongly correlated features while red indicates an r2 close to zero. components explaining 95% of variance in the data were kept. The obtained features are ﬁnally concatenated, and the predictiveness for survival prediction can be evaluated via machine learning models. 3.3. Preselection of Features FIGURE 2 \| Methodology used to evaluate the predictiveness of radiomic features for OS with the available acquisitions and labels. FIGURE 1 \| Difference in age and tumor-to-brain volume by resection status, and survival by provenance. FIGURE 1 \| Difference in age and tumor-to-brain volume by resection status, and survival by provenance. FIGURE 2 \| Methodology used to evaluate the predictiveness of radiomic features for OS with the available acquisitions and labels. FIGURE 2 \| Methodology used to evaluate the predictiveness of radiomic features for OS with the available acquisitions and labe Multicollinear features were excluded in a second step. Those features can be identiﬁed by checking the VIF. Iteratively, by removing the feature with the highest VIF, the multicollinearity can be reduced until a predeﬁned threshold is obtained. A maximum VIF of 10 is chosen, as thresholds of either 5 or 10 are recommended for this method (James et al., 2014). The number of features retained with a threshold of 10 should not pose problems to the machine learning models, so we did not consider lower thresholds. regressed against every other single feature, thus obtaining the coeﬃcient of determination r2 and creating an r2 correlation matrix. This matrix was then reordered using a hierarchical clustering algorithm. For this, we relied on the Voor Hees Algorithm (Voorhees, 1986) implemented in SciPy (Jones et al., 2001) for linkage, and Euclidian distances between rows or columns of the correlation matrix. A visual impression of the obtained clustered correlation matrix is given in Figure 3. As proposed by Gillies et al. (2016), representative features can be chosen from each cluster to reduce redundant elements. For this, areas of high correlation (R2 > 0.95) were reduced to the element with the highest inter-patient variability. Using this method, only features having a pairwise collinear correlation can be identiﬁed and omitted. Multicollinearity, i.e., highly related associations between more than two features, is not taken into account. Next to the VIF-based feature preselection method, we evaluated a principal component analysis (PCA) based feature reduction pipeline. One PCA feature reduction was carried out independently for the shape features, gray-level features and image intensity features of the original image. A fourth PCA was performed on all features of wavelet decomposed images. 3.5. Multivariate Prediction The statistical hypothesis testing can only reveal if single features are signiﬁcantly predictive for OS. Nonlinear relationships of single predictors to the target variable as well as feature interactions cannot be detected. Diﬀerent machine learning models that are able to surpass this limitation are available, ranging in complexity from basic linear regressors to complex neural networks. 3.4. Statistical Hypothesis Testing on Single Features Null hypothesis testing with false discovery rate correction on the original dataset is not beneﬁcial, as there are too many correlated features. The subset selected by the VIF feature selection (section 3.3), however, is much smaller and hypothesis tests can now reveal if single features are actually predictive for OS. As multiple radiomic features remained, a false discovery rate correction still needs to be used. We relied on the Benjamini– Hochberg procedure (Benjamini and Hochberg, 1995), that controls the false discovery rate at a speciﬁc level α = 0.05. Kickingereder et al. (2016) proposed a supervised principal component analysis of radiomic features for glioblastoma patients. In this study, a set of MRI acquisitions also comprising diﬀusion and susceptibility-weighted MR imaging was used. Thus, compared to our analysis, the study relied on a diﬀerent set of radiomic features. Nevertheless, their statistical analysis pipeline with z-score feature normalization and supervised principal component analysis is directly applicable to the features described in section 3.2. Frontiers in Computational Neuroscience \| www.frontiersin.org 4.2. Multivariate Prediction All methods were cross-validated in a leave-one-out setting, e.g., 100 samples were used to infer the 101th sample for the GTR dataset. From the 101 obtained results, the major test statistics as used in the BraTS challenge were computed: Accuracy (based on the three diﬀerent time intervals described in section 1), mean squared error (MSE), median error, and Spearman rank correlation. For classiﬁers, all metrics are computed with respect to the class value (long-survivors: 824 days, mid-survivors: 379 days, short-survivors: 150 days). For the accuracy, we also assessed the statistical signiﬁcance of the result with a binomial test and provide the p-value. All results can be seen in Table 2. These sections show that a predictive radiomic signature can be extracted for STR patients. For these patients, radiomics based approaches to survival prediction outperform the age- only approach that can be seen in Figure 4. However, on the patients that underwent total resection of the tumor, the ﬁndings are diﬀerent: The established radiomic features as well as all considered machine learning models fail to improve the survival prediction. Regression on age-only, however, is signiﬁcantly correlated with shorter survival for GTR patients (Figure 4). Then, as the results of diﬀerent models show a high variability, it is assessed in section 4.3 whether models based on a selected subset of features can lead to more robust results for GTR patients. On the GTR subset, no model achieved better results than the age-only baseline. However, on the STR subset, most models were more predictive of survival than the age-only approach. 4. RESULTS We evaluated diﬀerent machine learning models: linear, lasso and ridge regressors, k-nearest neighbors (kNN), random forests regressors (RFR), support vector regressors (SVR), and support vector classiﬁers (SVC). Furthermore, the Boruta (Kursa and Rudnicki, 2010) feature selection algorithm in combination with First, the predictiveness of state-of-the art methods and machine learning model using radiomic features is evaluated for the diﬀerent types of resection status in sections 4.1 and 4.2. November 2019 \| Volume 13 \| Article 73 Frontiers in Computational Neuroscience \| www.frontiersin.org Survival Prediction Depending on Resection Weninger et al. FIGURE 4 \| Age-only regression for the different types of resection status on BraTS 2019 training dataset. The obtained regression line is plotted together with the 95% conﬁdence interval. This model is used as baseline against radiomics based approaches. Next, the statistical analysis pipeline for radiomic features of glioblastoma patients proposed by Kickingereder et al. (2016) was applied to the diﬀerent resection status subsets. In the original publication, MRI acquisitions that are not available in the BraTS data (e.g., diﬀusion MRI) and slightly diﬀerent radiomic features were used. Nevertheless, the proposed z-score feature normalization and supervised principal component analysis is directly applicable to the present dataset, and can give a good baseline model. Using the proposed model parameters, the analysis was repeated on the radiomic features described in section 3.2 in a leave-one-out cross-validation. The results are compared to the age-only baseline approach for the diﬀerent resections status in Table 1. It can be seen that the proposed supervised PCA approach achieves a higher accuracy and better mean square error than the age-only approach. In contrast, even as the age is included in the feature set, this approach fails on the GTR subset. FIGURE 4 \| Age-only regression for the different types of resection status on BraTS 2019 training dataset. The obtained regression line is plotted together with the 95% conﬁdence interval. This model is used as baseline against radiomics based approaches. Frontiers in Computational Neuroscience \| www.frontiersin.org 4.3. Feature Reduction Approaches 4.3. Feature Reduction Approaches Several publications have shown the predictiveness of radiomics for survival prediction on diﬀerent datasets (see section 1). On top, the re-implemented methods could reveal predictiveness of radiomic features for survival on patients with subtotal resection. However, these methods, as well as diﬀerent machine learning models presented in this paper and as well as the majority of radiomic approaches submitted in the BraTS challenge 2018 failed on patients that underwent GTR. Thus, in this subsection, we focus on the GTR patients. Dataset As a ﬁrst step, previously reported relationships between radiomic signatures and survival time (section 3.6) were evaluated on the BraTS dataset. For evaluation, the dataset was ﬁrst divided by the three diﬀerent reported types of resection status. The published radiomic signatures were evaluated on each subset individually. y The two features proposed by Gutman et al. (2013) and the volumetric features of ET and NEC normalized to brain volume proposed by Zhang et al. (2014), Macyszyn et al. (2015) can be seen in Figure 5. These ﬁndings can be reproduced on the STR, and the same trends can also be seen on the NA subset (Pearson’s r: p <0.05). Especially the ET volume and the lesions’ major axis achieve a high signiﬁcance (p ≈0.003) on the STR subset. Of the reported features, the only non-signiﬁcant relationships are ED volume, that shows a negative, but non- signiﬁcant (p >0.05) correlation on both subsets, and the ET tumor proportion, that shows a signiﬁcant negative correlation on the STR subset, but only a non-signiﬁcant negative correlation on the NA subset. However, on the subset with reported GTR, no correlation can be identiﬁed for any feature. Even as all presented models performed worse than the age- only regressor, it can also be observed that the results of diﬀerent machine models achieve strongly varying results. This could be due to the high number of radiomic features. Thus, it is evaluated whether the two proposed feature reduction techniques can produce more robust outcomes on the GTR dataset. The presented unsupervised feature subset selection has two subsequent steps: First, the correlation matrix clustering, which suppresses pairwise correlated features, reduced the number of radiomic features from 9,870 to 5,338. Then, the VIF-based feature reduction, that checks also for multicollinearity, further reduced the number of features to 94. Combined with the age, Frontiers in Computational Neuroscience \| www.frontiersin.org November 2019 \| Volume 13 \| Article 73 6 Survival Prediction Depending on Resection Weninger et al. FIGURE 5 \| Linear relationship between previously reported signiﬁcant features and the different resection status. FIGURE 5 \| Linear relationship between previously reported signiﬁcant features and the different resection status. TABLE 1 \| Age-only linear regression compared to the supervised PCA (sPCA) model for the different types of resection status. TABLE 1 \| Age-only linear regression compared to the supervised PCA (sPCA) model for the different types of resection status. Dataset Next, the unsupervised feature selection method based on PCA is considered. After applying PCA as explained in section 3.3, 15 principal components representing tumor shape were kept, 38 for the image intensity statistics features, 55 for the gray level features, and 98 for the wavelet features. The extracted features were concatenated together with the age in order to be used for multivariate prediction. Model Accuracy p (Binomial) MSE Median err. SpearmanR GTR Age-only 0.48 0.00 109966 159 0.47 sPCA 0.27 0.20 148421 208 −0.41 STR Age-only 0.23 0.40 186772 194 −0.64 sPCA 0.46 0.21 148028 205 0.31 NA Age-only 0.37 0.49 136866 196 0.29 sPCA 0.26 0.20 159227 231 −0.03 The metrics are explained in section 4.2. Model Accuracy p (Binomial) MSE Median err. SpearmanR These features, as well as the features selected by the VIF- analysis, were separately employed for survival prediction (see Table 4). Consistent to 4.2, all features were normalized with a robust scaler, subtracting the median and scaling by the by the interquartile range, and the same machine learning models were utilized. 5. DISCUSSION TABLE 3 \| Correlation analysis of VIF—selected features with OS for GTR patients. Feature Correlation with OS p-value Age −0.46 0.000001 Wavelet LHH ImageIntensity Kurtosis T2 NEC 0.39 0.00002 Wavelet LLL ngtdm Complexity T2 ET 0.22 0.03 Wavelet LHL ImageIntensity Kurtosis T1CE nec 0.22 0.03 Wavelet LLL ImageIntensity Minimum T1 ET −0.20 0.05 TABLE 4 \| Performance comparison of different feature selection methods and machine learning models for GTR patients. Model Accuracy p (Binomial) MSE Median err. SpearmanR VIF-BASED FEATURE SUBSET Regression 0.47 0.01 28154236838 1,112 0.18 Regr. BH 0.46 0.07 109618 148 0.46 Lasso 0.36 0.60 2293591760 557 0.05 Ridge 0.33 1.0 16655918658 672 −0.02 kNN 0.30 0.53 159553 223 −0.08 RFR 0.35 0.75 149299 207 0.15 SVR 0.27 0.20 140181 189 −0.77 SVC 0.40 0.17 194331 445 0.06 FEATURES EXTRACTED by PCA Regression 0.39 0.17 672193 478 0.03 Lasso 0.36 0.59 688037 559 0.04 Ridge 0.38 0.25 558488 457 0.02 kNN 0.34 0.92 149014 194 0.07 RFR 0.34 0.92 163826 218 0.02 SVR 0.27 0.20 140298 189 −0.79 SVC 0.40 0.17 198655 445 0.05 Regr. BH, Regression on all features that were signiﬁcant after Benjamini–Hochberg multiple test correction. TABLE 2 \| Performance comparison of different machine learning models for the different types of resection status. TABLE 4 \| Performance comparison of different feature selection methods and machine learning models for GTR patients. ImageIntensity Kurtosis T2 NEC, was statistically signiﬁcant after Benjamini–Hochberg correction. However, after leveraging this ﬁnding in a predictive regression model, no clear beneﬁt could be observed. Why radiomic features were not predictive on GTR patients remains unclear. It can only be hypothesized that survival for STR patients depends on the malignancy of the primary subtotally resected tumor, while survival for GTR patients relates to possible metastases that are not directly dependent on image features of the original tumor. It can nevertheless be concluded that OS of brain tumor patients given radiomic images is strongly dependent not only on the preoperative images themselves. Given a high number of features and strong inﬂuences that cannot be assessed with preoperative MRI images, survival prediction is an ill-posed problem on a limited dataset. Researchers need to pay attention to the problems that arise when using radiomics or other big data methods on wide data, i.e., datasets with much more features than observations. 5. DISCUSSION Speciﬁcally, challenge participants and other researchers in clinical data analysis need to be fully aware of overﬁtting pitfalls, not only on the training set, but even on the validation dataset. TABLE 3 \| Correlation analysis of VIF—selected features with OS for GTR patients. Feature Correlation with OS p-value Age −0.46 0.000001 Wavelet LHH ImageIntensity Kurtosis T2 NEC 0.39 0.00002 Wavelet LLL ngtdm Complexity T2 ET 0.22 0.03 Wavelet LHL ImageIntensity Kurtosis T1CE nec 0.22 0.03 Wavelet LLL ImageIntensity Minimum T1 ET −0.20 0.05 developed radiomic models could not identify any connection between image based features and survival that went beyond the predictiveness of patient age. In previously published ﬁndings, the resection status is often not known or not clearly stated (Gutman et al., 2013; Macyszyn et al., 2015; Kickingereder et al., 2016; Lao et al., 2017; Li et al., 2017), or radiomic features are not assessed dependent on resection status (Zhang et al., 2014; Nie et al., 2019). Patient age, a clinical marker that is not strongly predictive of survival for patient without total tumor resection (cf. Figure 4) seems to be the strongest predictor of patient survival after GTR. One single feature, the Wavelet LHH developed radiomic models could not identify any connection between image based features and survival that went beyond the predictiveness of patient age. In previously published ﬁndings, the resection status is often not known or not clearly stated (Gutman et al., 2013; Macyszyn et al., 2015; Kickingereder et al., 2016; Lao et al., 2017; Li et al., 2017), or radiomic features are not assessed dependent on resection status (Zhang et al., 2014; Nie et al., 2019). Patient age, a clinical marker that is not strongly predictive of survival for patient without total tumor resection (cf. Figure 4) seems to be the strongest predictor of patient survival after GTR. One single feature, the Wavelet LHH In radiomics, a very high number of features are extracted. In our case, a total of 9,871 features were initially considered. Combined with a limited dataset, as is often the case for medical applications, problems arise due to the curse of dimensionality. One problem encountered is the robustness of signiﬁcance: The features that are signiﬁcant on the whole dataset are not necessarily signiﬁcant on the training subset, and vice versa, features identiﬁed as signiﬁcant on a small dataset do not need to be signiﬁcant on larger datasets. 5. DISCUSSION Previous ﬁndings, especially those using volumetric features, could be reproduced for patients with subtotal resection. Furthermore, diﬀerent considered machine learning models also showed predictiveness of survival. Thus, even as the sample size was limited, and diﬀerent machine learning models show varying results, radiomic features seem to be correlated to patient survival for patients with subtotal resection. 95 features were obtained, which is slightly less than the number of examples. On this reduced feature set, hypothesis testing is feasible. Without any correction for false positives, 5 of the 95 features would have been considered signiﬁcant (p <0.05). However, after Benjamini–Hochberg correction, only the age of the patient (p = 5.8 × 10−5) and one radiomic feature, the Wavelet LHH ImageIntensity Kurtosis on the necrotic part in the T2 acquisition remained signiﬁcant. All statistically signiﬁcant features can be seen in Table 3. However, when applying these methods to patients that underwent GTR, no signiﬁcant relationship between radiomic features and overall survival could be identiﬁed. In eﬀect, for this subgroup, the considered previously published and newly November 2019 \| Volume 13 \| Article 73 Frontiers in Computational Neuroscience \| www.frontiersin.org 7 Survival Prediction Depending on Resection Weninger et al. TABLE 2 \| Performance comparison of different machine learning models for the different types of resection status. Model Accuracy p (Binomial) MSE Median err. SpearmanR GTR Regression 0.43 0.04 1778965427 394 0.11 Lasso 0.44 0.03 3070271 272 0.24 Ridge 0.42 0.07 1765220107 394 0.11 kNN 0.29 0.40 159451 248 −0.04 RFR 0.36 0.46 154447 190 0.14 SVR 0.27 0.20 140088 189 −0.77 SVC 0.41 0.11 175014 229 0.12 Boruta+RFC 0.45 0.02 133497 229 0.36 STR Regression 0.54 0.03 13748742 271 0.32 Lasso 0.46 0.20 415488 228 0.18 Ridge 0.54 0.03 13754344 272 0.32 kNN 0.50 0.09 173798 211 0.21 RFR 0.58 0.01 144744 125 0.43 SVR 0.23 0.40 175720 157 −0.65 SVC 0.31 0.99 221768 229 −0.43 Boruta+RFC 0.50 0.09 92744 229 0.45 NA Regression 0.32 0.91 1434873 412 −0.02 Lasso 0.39 0.25 533963 282 0.11 Ridge 0.32 0.91 1435941 412 −0.02 kNN 0.25 0.13 166072 254 −0.18 RFR 0.33 1.00 146459 247 0.20 SVR 0.25 0.13 155827 225 −0.78 SVC 0.40 0.16 169944 229 0.00 Boruta+RF 0.30 0.56 162734 229 −0.13 All features as described in section 3.5 including the age were used as input. For the age-only approach as comparison, see Table 1. November 2019 \| Volume 13 \| Article 73 5. DISCUSSION Although it is impossible to test all possible combinations of diﬀerent radiomic features and machine learning models, we think that our evaluation shows the Frontiers in Computational Neuroscience \| www.frontiersin.org November 2019 \| Volume 13 \| Article 73 8 Survival Prediction Depending on Resection Weninger et al. limitations of radiomic analysis on glioblastoma patients with GTR. To be as robust as possible against such subset biases, we used extensive cross-validation and determined an orthogonal subset of features. information from radiomic features to the age of the patient does not necessarily improve accuracy for this task. To show this, we evaluated diﬀerent published techniques as well as a sophisticated radiomic feature extraction combined with modern machine learning techniques. However, no helpful information could be extracted, and our baseline—a linear regression on the age of the patient—could not be consistently outperformed on this limited dataset. In contrast, on patients with a diﬀerent resection status—either where the resection status was not available or the tumor was subtotally resected—previously published ﬁndings could be reproduced, and diﬀerent machine learning techniques could extract information predictive for overall survival. Next to the diﬃculties encountered when applying radiomics to patients that underwent GTR, we believe one main limitation in the BraTS challenge 2018 was the small training dataset in combination with overﬁtted approaches. In the BraTS setting, a predeﬁned validation set was released by the organizers, and could be used by all contributors to evaluate their algorithms during development. Thus, if contributors test diﬀerent algorithms or hyperparameter settings on this left- out validation set, it has to be taken into account that one may accidentally overﬁt on this validation set. Such “result- peeking” invalidates accuracy scores on this left-out dataset, i.e. the developed approaches may generalize poorly to other samples. In fact, it seemed that this actually happened during the BraTS challenge. As can be seen on the oﬃcial BraTS challenge online leaderboard (Bakas et al., 2018a), a total of nine diﬀerent teams obtained accuracy scores at least as good as ours on the validation dataset. However, on the test set, our naïve algorithm scored the 3rd place out of 26 participants. On this dataset, segmentation and OS results could not be evaluated by participants, making this part of the data impossible to overﬁt. In contrast to private datasets, algorithm developers cannot—be it deliberately or accidentally—invalidate the obtained results by result-peeking in such a setting. ETHICS STATEMENT This work relies only on the BraTS dataset. For the use of this dataset, no ethics statement is necessary. FUNDING This work was supported by the International Research Training Group 2150 and the German Research Foundation (DFG) under grant no ME3737/3-1. 5. DISCUSSION In order to move from fundamental research to translational medicine, future research in brain tumor radiomics should focus on ﬁnding novel radiomic features that are applicable if the patient undergoes surgery. A possible set of features that was not assessed in this study are location based features. Location based features are not as established as shape or texture features in radiomics. However, they could be more promising for survival prediction even for patients that underwent GTR, as the position of the tumor in the brain could inﬂuence prognosis. DATA AVAILABILITY STATEMENT Publicly available datasets were analyzed in this study. This data can be found here: https://www.med.upenn.edu/sbia/brats2018/ registration.html. Thus, challenges such as the BraTS challenge are important for unbiased algorithm comparison and to assess whether ﬁndings from research are robust and can be applied to translational medicine. Here, it was assessed whether ﬁndings in radiomics of glioblastoma patients can be transferred to patients that underwent GTR. In this case, classical radiomic features seem not to be suited for robust results in survival prediction. In contrast, positive ﬁndings, with previously reported approaches as well as with diﬀerent machine learning techniques can be reported for patients with subtotal resection. AUTHOR CONTRIBUTIONS LW performed algorithm development and implementation and wrote the manuscript. CH assisted in algorithm conception and interpretation of the results. DM supervised the work and critically revised the manuscript. Nevertheless, the approaches presented in this paper are not exhaustive. We do not want to present the new “best” survival prediction algorithm. Default parameter settings were utilized for all machine learning techniques, as exhaustive hyperparameter tuning—as employed by most winning approaches in machine learning challenges—on a small dataset would invalidate the results. The approaches presented in this manuscript, especially those relying on orthogonal feature subset selection, were utilized to analyze the robustness of radiomic features. They may not be the “best” algorithms for survival prediction. Thus, C-index, hazard ratio, or KM analysis were not regarded, as the focus of this analysis lies on robustness of radiomic features, and not on a single survival prediction algorithm. REFERENCES Kickingereder, P., Burth, S., Wick, A., Götz, M., Eidel, O., Schlemmer, H.-P., et al. (2016). Radiomic proﬁling of glioblastoma: identifying an imaging predictor of patient survival with improved performance over established clinical and radiologic risk models. 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Jones, E., Oliphant, T., and Peterson, P. (2001). SciPy: Open Source Scientiﬁc Tools for Python. Weninger, L., Rippel, O., Koppers, S., and Merhof, D. (2019). “Segmentation of brain tumors and patient survival prediction: Methods for the brats 2018 challenge,” in Brainlesion: Glioma, Multiple Sclerosis, Stroke and Traumatic Brain Injuries, eds A. Crimi, S. Bakas, H. REFERENCES Multi-channel 3d deep feature learning for survival time prediction of brain tumor patients using multi-modal neuroimages. Sci. Rep. 9:1103. doi: 10.1038/s41598-018-3 7387-9 Bakas, S., Min Ha, S., and Bergman, M. (2018a). BraTS Validation Survival Leaderboard 2018. Available online at: https://web.archive. org/web/20191019144601/https://www.cbica.upenn.edu/BraTS18// lboardValidationSurvival.html Ostrom, Q. T., Gittleman, H., Truitt, G., Boscia, A., Kruchko, C., and Barnholtz-Sloan, J. S. (2018). 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(2019). “Brain tumor segmentation using an ensemble of 3d u-nets and overall survival prediction using radiomic features” in Brainlesion: Glioma, Multiple Sclerosis, Stroke and Traumatic Brain Injuries, eds A. Crimi, S. Bakas, H. Kuijf, F. Keyvan, M. Reyes, and T. van Walsum (Cham: Springer International Publishing), 279–288. Shboul, Z. A., Vidyaratne, L., Alam, M., and Iftekharuddin, K. M. (2018). “Glioblastoma and survival prediction,” in Brainlesion: Glioma, Multiple Sclerosis, Stroke and Traumatic Brain Injuries, eds A. Crimi, S. Bakas, H. 6. CONCLUSION The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fncom. 2019.00073/full#supplementary-material The BraTS survival prediction challenge focuses on glioblastoma patients that underwent GTR. This paper shows that adding November 2019 \| Volume 13 \| Article 73 Frontiers in Computational Neuroscience \| www.frontiersin.org 9 Survival Prediction Depending on Resection Weninger et al. Zacharaki, E., Morita, N., Bhatt, P., O’Rourke, D., Melhem, E., and Davatzikos, C. (2012). Survival analysis of patients with high-grade gliomas based on data mining of imaging variables. Am. J. Neuroradiol. 33, 1065–1071. doi: 10.3174/ajnr.A2939 Zhou, M., Scott, J., Chaudhury, B., Hall, L., Goldgof, D., Yeom, K., et al. (2018). Radiomics in brain tumor: image assessment, quantitative feature descriptors, and machine-learning approaches. Am. J. Neuroradiol. 39, 208– 216. doi: 10.3174/ajnr.A5391 Frontiers in Computational Neuroscience \| www.frontiersin.org November 2019 \| Volume 13 \| Article 73 REFERENCES Kuijf, F. Keyvan, M. Reyes, and T. van Walsum (Cham: Springer International Publishing), 3–12. Jungo, A., McKinley, R., Meier, R., Knecht, U., Vera, L., Perez-Beteta, J., et al. (2018). “Towards uncertainty-assisted brain tumor segmentation and survival prediction,” in Brainlesion: Glioma, Multiple Sclerosis, Stroke and Traumatic Brain Injuries, eds A. Crimi, S. Bakas, H. Kuijf, B. Menze, and M. Reyes (Cham: Springer International Publishing), 474–485. November 2019 \| Volume 13 \| Article 73 Frontiers in Computational Neuroscience \| www.frontiersin.org 10 Survival Prediction Depending on Resection Weninger et al. Zacharaki, E., Morita, N., Bhatt, P., O’Rourke, D., Melhem, E., and Davatzikos, C. (2012). Survival analysis of patients with high-grade gliomas based on data mining of imaging variables. Am. J. Neuroradiol. 33, 1065–1071. doi: 10.3174/ajnr.A2939 Conﬂict of Interest: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Copyright © 2019 Weninger, Haarburger and Merhof. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Zhang, Z., Jiang, H., Chen, X., Bai, J., Cui, Y., Ren, X., et al. (2014). Identifying the survival subtypes of glioblastoma by quantitative volumetric analysis of MRI. J. Neuro Oncol. 119, 207–214. doi: 10.1007/s11060-014-1478-2 Zhou, M., Scott, J., Chaudhury, B., Hall, L., Goldgof, D., Yeom, K., et al. (2018). Radiomics in brain tumor: image assessment, quantitative feature descriptors, and machine-learning approaches. Am. J. Neuroradiol. 39, 208– 216. doi: 10.3174/ajnr.A5391 November 2019 \| Volume 13 \| Article 73 Frontiers in Computational Neuroscience \| www.frontiersin.org 11
https://openalex.org/W1984640107	https://journals.uni-lj.si/ala/article/download/180/318	English	null	An Analysis of the Efficiency of Existing Kanji Indexes and Development of a Coding-based Index	Acta linguistica asiatica	2,012	cc-by-sa	11,694	Izvleček Pričujoči članek obravnava lastnosti 15 obstoječih kazal v slovarjih kitajskih pismenk. Da bi primerjali relativno učinkovitost teh kazal, definiramo koncept izbirnosti ter izračunamo in primerjamo koeficient izbirnosti teh kazal. Nadalje predlagamo in predstavljamo rabo in učinkovitost novih kazal, ki smo jih osnovali na kodiranju z latiničnimi črkami, simbolnem kodiranju, semantičnem kodiranju in kodiranju na osnovi pomenskega ključa in števila potez. Abstract Considering the problems faced by learners of Japanese from non-kanji background, the present paper discusses the characteristics of 15 existing kanji dictionary indexes. In order to compare the relative efficiency of these indexes, the concept of selectivity is defined, and the selectivity coefficient of the kanji indexes is computed and compared. Furthermore, new indexes developed by the present authors and based on an alphabet code, a symbol code, a semantic code, and a radical and stroke number code are presented and their use and efficiency are explained. Keywords kanji index; search; efficiency index; selectivity; kanji coding AN ANALYSIS OF THE EFFICIENCY OF EXISTING KANJI INDEXES AND DEVELOPMENT OF A CODING-BASED INDEX Galina N. VOROBЕVA* Kyrgyz National University g.vorobyova@yahoo.com Victor M. VOROBЕV Kyrgyz National University v.vorobyov@yahoo.com Acta Linguistica Asiatica, 2(3), 2012. ISSN: 2232-3317, http://revije.ff.uni-lj.si/ala/ DOI: 10.4312/ala.2.3.27-60 * Translated by Boštjan Bertalanič, Andrej Bekeš and Kristina Hmeljak Sangawa 28 Galina N. VOROBEVA, Victor M. VOROBEV 28 Galina N. VOROBEVA, Victor M. VOROBEV 1. Background of the study The most commonly known indexes used to look up kanji (Chinese characters) in character dictionaries are the radical index (部首索引 bushu-sakuin), the stroke- number index (総画数索引 sōkakusuu-sakuin) and the readings index (音訓索引 onkun-sakuin). The radical index is used when searching kanji by means of their radical; it consists of a list of all radicals arranged by increasing number of strokes, where each radical is followed by a list of characters belonging to this radical, and each list of characters with the same radical is usually arranged by increasing number of strokes. The stroke number index is used to look up characters when their number of strokes is known; it consists of all characters contained in a dictionary, arranged by increasing total number of strokes. The readings index is used to look up characters by their reading, and consists of a list of all readings of the characters contained in the dictionary, usually arranged in standard gojūon kana order. However, it is also generally known that learners from non-kanji background, i.e. learners who are not familiar with Chinese character writing, find it difficult to use traditional character dictionaries. When searching in a traditional radical index, it is sometimes difficult to determine which part of the character is to be considered its radical, as in the case of 巨 where 工 is the radical. Traditional ordering is complicated also because it does not classify characters consistently according to shape, but rather takes into account meaning, such as in the case of the characters 間 (“between”)，閉 (“close”) ，開 (“open”) which are indexed under radical 門 (“gate”), while the character 問 (“question”) is indexed under radical 口 (“mouth”) and 聞 (“listen”) under radical 耳 (“ear”). The user should therefore already know in advance the meaning of a character in order to look it up, which is seldom the case. Indexes ordered by number of strokes are also troublesome to use, since we tend to make mistakes when counting, and there are many characters with the same number of strokes. In order to use reading indexes, on the other hand, the user needs to know the reading of a given character in order to look it up, but most users from a non-kanji background generally look up characters exactly because they do not know how to read them. Ključne besede kazalo kitajskih pismenk; iskanje; indeks učinkovitosti; selektivnost; kodiranje kitajskih pismenk * Translated by Boštjan Bertalanič, Andrej Bekeš and Kristina Hmeljak Sangawa 4. Research method Given the variety of existing character indexes, we considered that an evaluation and comparative analysis of their efficiency was necessary. In order to compare the efficiency of the various character indexes, in this study we decided to use the concept of selectivity which expresses the processing efficiency of computer data. With reference to character indexes, we introduced the concept of selectivity coefficient and used it to compare and assess the efficiency of existing character indexes by calculating their selectivity coefficient. We then built some new types of character indexes on the basis of character codes which accurately express character form. We constructed a database with four kinds of character codes for all new Jōyō kanji, sorted the data according to the order used in dictionary compiling, and developed four indexes: an alphabet code index, a symbol code index, a semantic code index and a radical and stroke code index. Finally, we compared and evaluated the efficiency of these four code indexes. 2. Previous research Previous research has aimed at developing more efficient search methods. Both in the cultural sphere using Chinese characters and outside of it, diverse types of search methods have been developed and are being used besides the above mentioned and well known radical index, stroke index or readings index. To mention a few, other methods include the “five step arrangement kanji table” (五段排列漢字表 godan hairetsu kanji hyō) developed by the Russian researcher Rosenberg (1916), the Four corner method (四角号碼 Sì jiǎo hào mǎ) developed in China (Wang, 1925), the phonetic key index (音符 onpu) developed by Shiraishi (1971/1978), the index of katakana shapes, the initial stroke pattern index and the index of meaning symbols An Analysis of the Efficiency of Existing Kanji Indexes 29 developed by Kanō (1998), the index of stroke order patterns by Wakao and Hattori (1989), the index of character shapes by Sakano, Ikeda, Shinagawa, Tajima and Tokashiki (2009), the key words and primitive meanings index by Heisig (1977/2001), a radical index consistently based on shape by Hadamitzky and Spahn (1981), the system of kanji indexing by patterns (SKIP) developed by Halpern (1988), and the Kanji Fast Finder system by Matthews (2004). The authors of the present paper have also contributed to research on character indexing (Vorobeva, 2009, 2011). 3. Research aims The goal of this paper is to 1) describe existing character search methods and analyse their efficiency; and 2) develop an efficient character search method based on a coding of character which appropriately expresses character form. 5.1 Five step arrangement kanji table - 五段排列漢字表 The “graphic system” search method developed by the Russian researcher Vasil’ev (1867) and the “five step arrangement kanji table” developed by Rosenberg (1916) are generally known as the “Russian graphic system” (“Kod_Rozenberga”, 2012). This is a method of arranging characters by form. Users only need to remember some simple rules and can use this method even when they cannot determine which part is the radical, have problems counting the number of strokes and do not know how to read the character. The method was developed by professor Vasilij Pavlovič Vasil´ev, a Chinese language scholar at Kazan University in Russia, who extracted 19 kinds of graphic elements or strokes (see Figure 1) which compose Chinese characters, and classified each character according to its last stroke, i.e. the stroke which is written last. Figure 1: Vasile’v’s 19 graphic elements of Chinese characters 5. Types and characteristics of existing character indexes In the following paragraphs we introduce 12 existing types of character indexes, omitting the on-kun readings index, the radical index and the stroke-number index already described in the introduction. 30 Galina N. VOROBEVA, Victor M. VOROBEV 1 特に前代日本の文明，前代日本の文学芸術を知らんとする人に取りては，漢字の知識は，その主要科目たるべきなり。 2 非常なる不便と困難とを感じたり。それは主として漢字にアルファベットの如き順序なきによれるなり。 Figure 1: Vasile’v’s 19 graphic elements of Chinese characters On the basis of these graphic elements, he compiled a new type of character dictionary with a unique character ordering and search method: the first Chinese- Russian dictionary, entitled Графическая система китайских иероглифов. Опыт первого китайско-русского словаря [Grafičeskaâ sistema kitajskih ieroglifov. Opyt pervogo kitajsko-russkogo slovarâ. - “Graphic System of Chinese Characters. An attempt at the First Chinese-Russian Dictionary.”] (Vasil’ev, 1867, see figure 2). This was the beginning of a far-reaching reform in dictionary structuring and organisation. Figure 2: Vasil’ev’s First Chinese-Russian Dictionary Figure 2: Vasil’ev’s First Chinese-Russian Dictionary An Analysis of the Efficiency of Existing Kanji Indexes 31 Two decades later, professor D. A. Pešurov at Saint Petersburg University published a Chinese-Russian dictionary entitled Китайско-русский словарь. по графической системе (Kitajsko-russkij slovar´ po grafičeskoj sisteme - “Chinese- Russian Dictionary. Based on the Graphic System”, Pešurov, 1891) on the basis of Vasil’ev’s graphic method (see figure 3). Figure 3: Pešurov’s Chinese-Russian Dictionary Figure 3: Pešurov’s Chinese-Russian Dictionary The Russian graphic system was subsequently also used in a Japanese character dictionary, compiled by professor Otto Rosenberg of St. Petersburg University, who emphasised the importance of kanji learning stating as follows: “… especially for the person who is going to study the literary arts of Japan and Japanese civilisation of former ages, the knowledge of Chinese characters should become the core subject of learning”1 (Rosenberg, 1916, p. 7). At the same time, he also discussed the difficulties to be faced when learning Chinese characters and using character dictionaries, and wrote the following comment regarding characters being listed according to their radicals: “I feel considerable inconvenience and difficulty, because Chinese characters do not possess an ordering such as alphabets2” (Rosenberg, 1916, p. 1). On the basis of the graphic system developed by Vasil´ev, Rosenberg constructed a search system that was based on the shape of the characters and was completely different from the traditional indexes based on radicals, stroke number and readings, and used it to compile the 五段配列漢字典 (Godan hairetsu kanjiten - “Five Step 32 Galina N. VOROBEVA, Victor M. VOROBEV 32 Arrangement Character Dictionary”) a dictionary with a novel character arrangement and search system published in Japan (Rosenberg, 1916, see figure 4). Arrangement Character Dictionary”) a dictionary with a novel character arrangement and search system published in Japan (Rosenberg, 1916, see figure 4). Figure 1: Vasile’v’s 19 graphic elements of Chinese characters Figure 4: Rosenberg’s 五段配列漢字典 (Godan hairetsu kanjiten, 1916) Figure 4: Rosenberg’s 五段配列漢字典 (Godan hairetsu kanjiten, 1916) The basic idea of the five-step arrangement search system is that “One look at the shape of the character [...] is enough. It can then immediately be found rapidly and reliably3” (Rosenberg, 1916, Explanatory notes 1). Russian speakers are used to the notation in Cyrillic and Roman letters, and even in the case of Chinese characters they feel the need for a systematisation similar to the alphabet. Rosenberg took that into account when classifying and arranging characters according to the last written stroke. In contrast to Vasil’ev’s system with 19 strokes (Vasil’ev, 1867), Rosenberg used 24 strokes and divided them into 5 groups. In order to implement a Chinese character ordering system based on stroke order, Rosenberg extracted 24 types of strokes, and grouped them into 5 categories, according to the direction in which they are written. Finally, he selected one stroke to represent each of the five groups, as shown in table 1 (Rosenberg, 1916, p. 20). Table 1: Basic strokes in Rosenberg’s system Direction Basic strokes ↗ 〳 ↘ 乀 ↙ ノ ↓ ｜ → 一 3 文字（中略）の形を，一見したるのみにて，十分なり。而して直ちに迅速確實に検出することを得べし。 Table 1: Basic strokes in Rosenberg’s system Direction Basic strokes ↗ 〳 ↘ 乀 ↙ ノ ↓ ｜ → 一 Table 1: Basic strokes in Rosenberg’s system 3 文字（中略）の形を，一見したるのみにて，十分なり。而して直ちに迅速確實に検出することを得べし。 3 文字（中略）の形を，一見したるのみにて，十分なり。而して直ちに迅速確實に検出することを得べし。 An Analysis of the Efficiency of Existing Kanji Indexes 33 Rosenberg exemplified the 5 types of strokes using the 5 strokes of the character 本, as shown in figure 5. Rosenberg exemplified the 5 types of strokes using the 5 strokes of the character 本, as shown in figure 5. Figure 5: Rosenberg’s five types of strokes, exemplified by the character 本 Figure 5: Rosenberg’s five types of strokes, exemplified by the character 本 The Chinese character chart (漢字の字母表 kanji no jibohyō) in Rosenberg’s (1916) dictionary is shown in figure 6, while figure 7 shows one page of the Five step arrangement Chinese character index (五段配列漢字表 godan hairetsu kanjihyō), and figure 8 shows one page of the dictionary’s main entries. Figure 6: The Chinese character chart (漢字の字母表 kanji no jibohyō) in Rosenberg’s dictionary Figure 6: The Chinese character chart (漢字の字母表 kanji no jibohyō) in Rosenberg’s dictionary 36 Galina N. VOROBEVA, Victor M. VOROBEV 36 Galina N. VOROBEVA, Victor M. VOROBEV 34 Galina N. VOROBEVA, Victor M. VOROBEV The Chinese character type chart (see figure 6) includes the five basic strokes, beneath them all 24 strokes classified by shape as belonging to one of the basic five strokes, and finally, beneath them, 567 character types (Chinese characters and character patterns) classified according to the 24 strokes and divided into 60 columns. Characters listed in the Five step arrangement Chinese character index (五段配列漢字表 godan hairetsu kanjihyō, see figure 7) are arranged according to the order of their strokes in the character chart. Figure 8 shows an example dictionary page. Figure 7: First page of the Five step arrangement Chinese character index (五段配列漢字表 godan hairetsu kanjihyō) in Rosenberg’s dictionary Figure 7: First page of the Five step arrangement Chinese character ind (五段配列漢字表 godan hairetsu kanjihyō) in Rosenberg’s dictionar Figure 7: First page of the Five step arrangement Chinese character index (五段配列漢字表 godan hairetsu kanjihyō) in Rosenberg’s dictionary An Analysis of the Efficiency of Existing Kanji Indexes 35 Figure 8: Examples of main entries in Rosenberg’s dictionary Figure 8: Examples of main entries in Rosenberg’s dictionary In the 19th century Russian researchers focused on the form of characters and, adhering to the principle of coherent classification, proposed a novel character sequencing and search method. In the former Soviet Union, this “Russian graphic system” was the basis for the Большой китайско-русский словарь (Bol´šoj kitajsko- russkij slovar´ - “The Great Chinese-Russian Dictionary”, Panasyuk & Suhanov, 1983). Later, the “Russian graphic system” for arranging and searching characters according to their stroke characteristics was also influential in the creation of the “Four corner method” (四角号碼 Sì jiǎo hào mǎ, see figures 9 and 10), developed in China during the 1920s. (Wáng, 1934, p. 38). Figure 10: Introduction to 四角號碼檢字法附檢字表 Sì jiǎo hàomǎ jiǎnzì fǎ: fu jiǎnzì fǎ biao - “Four corner code based character search method: with a character look-up table appendix” (Wang, 1934, p. 38) To give an example, the character 法 is assigned the code 34131, by assigning these numbers to the strokes in each corner, in the following sequence: 3 4 法 1 1 3 3 4 法 1 1 3 The Four corner method was adopted in the compilation of 大漢和辞典 (Dai Kan- Wa Jiten, Morohashi, 1960), The Great Chinese Character - Japanese Dictionary in 13 volumes published in Japan. The coding rules used in this dictionary are described in Morohashi (1984, p. 1038). 5.2 The Four corner method The Four corner method (Chinese 四角號碼 sì jiǎo hàomǎ, Japanese 四角号碼 skikakugōma) is one of the Chinese character search systems and in the context of Japanese language means the “code based on four corners”. It was developed in China by Wáng Yún Wǔ, who published his 號碼檢字法 (Hàomǎ jiǎnzì fǎ - “Code based character search method”) in 1925, 四角號碼檢字法 (Sì jiǎo hàomǎ jiǎnzì fǎ - “Four corner code based character search method”) in 1926, and finally 四角號碼檢字法‧ 附檢字表 (Sì jiǎo hàomǎ jiǎnzì fǎ: fu jiǎnzì fǎ biao - “Four corner code based character search method: with a character look-up table appendix”) in 1934. Just like the “Russian graphical system”, the Four corner method does not depend on the radical, stroke number, stroke order, reading or meaning of a character, but rather allows for character look-up by means of a code which is based on the shape of the strokes in the four corners of a character. The stroke shapes in each of the four corners are assigned numbers from 0 to 9, and in order to distinguish between those Chinese characters which happen to end up with the same quadruplet of assigned digits, an extra “corner”, named 附角 (fujiǎo) is additionally assigned. Each Chinese character can thus be uniquely coded and ordered by a five digit number. In order to use this Chinese character index, it is not necessary to have any knowledge of traditional search systems based on radicals, stroke number or stroke order. Figure 9: Front page of 四角號碼檢字法附檢字表 (Sì jiǎo hàomǎ jiǎnzì fǎ: fu jiǎnzì fǎ biao - “Four corner code based character search method: with a character look-up table appendix”) (Wang, 1934) Figure 9: Front page of 四角號碼檢字法附檢字表 Figure 9: Front page of 四角號碼檢字法附檢字表 (Sì jiǎo hàomǎ jiǎnzì fǎ: fu jiǎnzì fǎ biao - “Four corner code based character search method: with a character look-up table appendix”) (Wang, 1934) Figure 9: Front page of 四角號碼檢字法附檢字表 (Sì jiǎo hàomǎ jiǎnzì fǎ: fu jiǎnzì fǎ biao - “Four corner code based character search method: with a character look-up table appendix”) (Wang, 1934) 37 An Analysis of the Efficiency of Existing Kanji Indexes Figure 10: Introduction to 四角號碼檢字法附檢字表 Sì jiǎo hàomǎ jiǎnzì fǎ: fu jiǎnzì fǎ biao - “Four corner code based character search method: with a character look-up table appendix” (Wang, 1934, p. 38) 5.3 Katakana shape based classification The Katakana shape based classification system (カタカナ字形分類索引 - katakana jikei bunrui sakuin, Kanō, 1998, p. 1007) sorts all the 1945 jōyō kanji according to the similarity of their component strokes to Japanese kana syllabary character shapes, with Chinese characters accordingly arranged as kana in the “a, i, u, e, o” order. Chinese characters are listed under the part that shares the same shape with some katakana character, as can be seen in table 2. The position of the katakana-like shape within each character is not questioned. 38 Galina N. VOROBEVA, Victor M. VOROBEV 38 Table 2: Examples of Chinese characters listed under each katakana character katakana form type examples of corresponding Chinese characters ア了, 子, 孔, 好, … イ仙, 代, 付, 休, … エ工, 功, 式, 江, … オ才, 材, 財, 閉, … Table 2: Examples of Chinese characters listed under each katakana character 5.4 Initial stroke pattern index The Initial stroke pattern index (書き出しパターン索引 kakidashi pataan sakuin, Kanō, 1998, p. 1020) shares similarities with the aforementioned Five step arrangement Chinese character table developed by Rosenberg, but while Rosenberg operates with the strokes written at the very end, the Initial stroke pattern index deals with the strokes that are written first. The Initial stroke pattern index defines the six initial stroke patterns given in table 3. Table 3: Initial stroke patterns in Kanō’s Initial stroke pattern index (1998) Table 3: Initial stroke patterns in Kanō’s Initial stroke pattern index (1998) 1 2 3 4 5 6 一丨ノ丶フレ Characters with the same initial stroke pattern are ordered by their number of strokes. For example, characters belonging to pattern 1 (一) appear in the following order, according to their number of strokes 一, 二,丁, 三, 天, … . 5.6 Key Words and Primitive Meanings Index The Key Words and Primitive Meanings Index (Heisig, 2001, p. 506) assigns a unique meaning or interpretation to each character or character component part. English words representing these meanings are ordered alphabetically, making it possible to look up any character according to these English translations of assigned meanings. In order to use this index, the user must first learn the assigned meanings of each component part. 5.5 Stroke order index The Stroke order index (筆順索引 hitsujun sakuin) was implemented by Wakao and Hattori (1989) in their dictionary for the decipherment of cursive style characters (くずし解読字典 Kuzusi kaidoku jiten). Characters in this dictionary are ordered according to their stroke order and stroke direction. Brush movement is represented by arrows pointing to eight directions, with each direction being assigned a number (code) ranging from 0 to 7 (Wakao & Hattori, 1989, p. 469), as shown in table 4. Table 4: Brush stroke direction patterns in Wakao & Hattori’s Stroke order index (1989) 0 1 2 3 4 5 6 7 ↑ ↗ → ↘ ↓ ↙ ← ↖ Table 4: Brush stroke direction patterns in Wakao & Hattori’s Stroke order index (1989) 0 1 2 3 4 5 6 7 ↑ ↗ → ↘ ↓ ↙ ← ↖ able 4: Brush stroke direction patterns in Wakao & Hattori’s Stroke order index (1989) 39 An Analysis of the Efficiency of Existing Kanji Indexes Each character is assigned a code, based on the direction of the first four strokes (起筆 kihitsu “first stroke”, 第二筆 dainihitsu “second stroke”, 第三筆 daisanhitsu “third stroke” and 第四筆 daiyonhitsu “fourth stroke”). For example, the first strokes of the character 仙 are written in the following directions in the cursive style: ↙ (5), ↗ (1), ↓ (4). Here the second stroke, ↗ (1), not visible in the printed form of this character, is included in cursive stroke counting because the brush is brought back to its starting point after the first stroke (Wakao & Hattori, 1989, p. 466). This coding system could easily be applied to standard character forms in general character dictionaries. In this case, the character 仙 would be coded according to its standard stroke order, ↙, ↓, ↓, ↓, resulting in the four digit code 5-4-4-4. 5.7 System of Kanji Indexing by Patterns (SKIP) Halpern (1988/1990, 1999), developing his System of Kanji Indexing by Patterns (SKIP), assigned to each character a numeric code. In order to do this he first divided characters into four patterns, numbered from 1 to 4: 1 - Characters that can be divided into left and right parts; 1 - Characters that can be divided into left and right parts; 2 - Characters that can be divided into top and bottom parts; 3 - Characters that can be divided by an enclosure element; 4 - Characters that cannot be classified under patterns 1, 2 or 3 These pattern numbers are used as the first digit in a code assigned to each characters. Characters of type 1 to 3 are divided into two parts. The number of strokes in each part of the character is then used as the second and third component of the character code. For example, the character 相 consists of a left and a right part and is thus categorised as type 1; the left part, 木, has 4 strokes and the right part, 目, has 5 strokes, resulting in its SKIP code 1-4-5. In the case of characters belonging to type 4, the second component of their code is the total number of strokes, while the third component is a code number, ranging from 1 to 4, assigned according to their shape. For details, readers can refer to the dictionary’s detailed front matter. Further examples of SKIP codes are given in table 5 below. 40 Galina N. VOROBEVA, Victor M. VOROBEV 40 Table 5: Examples of System of Kanji Indexing by Patterns (SKIP) coding Type Kanji Number of strokes SKIPcode 1 八 2 1-1-1 相 9 1-4-5 2 二 2 2-1-1 父 4 2-2-2 3 山 3 3-2-1 間 12 3-8-3 4 火 4 4-4-4 女 3 4-3-4 Table 5: Examples of System of Kanji Indexing by Patterns (SKIP) coding The dictionary contains a SKIP index constructed by ordering characters according to their SKIP codes in ascending order. The dictionary contains a SKIP index constructed by ordering characters according to their SKIP codes in ascending order. 5.8 Fast Finder Matthews (2004), in a way similar to Halpern (1988/1990) and Halpern (1999), assigns a pattern to each character, but does not create codes. Characters are divided into 8 patterns on the basis of their constituent components: left of the left-right, right of the left-right, top of the top-bottom, bottom of the top-bottom, three types of enclosures and non-divisible characters. Characters belonging to each pattern are listed together on pages beginning with the pattern itself, followed by all characters belonging to it. Lists of characters with complex shapes are further minutely subdivided and ordered according to the complexity of their shape, in ascending order of their number of strokes. 5.9 Index by Radicals Hadamitzky and Spahn (1981) adopted an index based on traditional radicals, but reduced the number of radicals in order to simplify character search. They selected 79 of the 214 generally used radicals, which are also standardised in Unicode (Unicode, 2012), and used them to construct their Index by Radicals. Those radicals whose shapes were deemed too complex were omitted and characters traditionally assigned to them were assigned to other radicals. Some examples are given in Table 6 below. As a result of the reduced number of radicals, the number of characters listed under each radical has increased. An Analysis of the Efficiency of Existing Kanji Indexes 4 41 Table 6: Examples of radical substitution in Hadamitzky and Spahn (1981) 214 radicals in general use 79 radicals proposed by Hadamitzky and Spahn (1981) Radical number Radical Radical number Radical 176 面 3s 囗 177 革 3k 艹 178 韋 3d 口 Table 6: Examples of radical substitution in Hadamitzky and Spahn (1981) 5.10 Index by meaning symbols The Index by meaning symbols (意味記号索引 imikigō sakui) lists characters according to 495 meaning symbols used by Kanō (1998), ordered according to their increasing number of strokes. Kanō (1998, p. 6) explains that “… We indicate the part that represents the meaning of a character as its ‘meaning symbol’… Some of the ‘meaning symbols’ have the same shape as radicals, but are, as radicals, called differently (e.g. 水 mizu ‘water’ and 氵sanzui ‘three [drops of] water’). [...] It also happens sometimes that a character does not contain a ‘meaning symbol’. In such cases we have shown the original character, on which the current character is based.” For example, the radical of 斎 (nowadays usually substituted by 斉) is 斉, and its ‘meaning symbol’ is 示 (the shape of an altar) (cf. Kanō, 1998, p. 952). 5.11 Index by character shapes The Index by character shapes (字形索引 jikei sakuin) is implemented in a text book including 512 Chinese characters (Banno, Ikeda, Shinagawa, Tajima & Tokashiki, 2009) and is similar to a radical index. However, the 215 “character shapes” (jikei) used include both radicals and other shapes which are traditionally not considered as radicals. The index lists “character shapes” ordered by their number of strokes, each followed by characters containing it, with their assigned numbers corresponding to the order in which they are introduced within the textbook. 42 Galina N. VOROBEVA, Victor M. VOROBEV 42 Galina N. VOROBEVA, Victor M. VOROBEV 6.1 Shared characteristics of existing character indexes A point shared by all aforementioned character indexes is that only one character element or property is selected as the basis upon which the index is built. Elements or properties employed for this purpose are the number of strokes, radicals, initial stroke pattern etc. Considering the necessity to evaluate and comparatively analyse each of these indexes, we introduced the notion of selectivity of character indexes and used it to compare and evaluate existing indexes. 6.2 Definition of the “coefficient of selectivity” To compare and evaluate the efficiency of character indexes, we will use a notion used to express computer processing efficiency, i.e., “selectivity” (Vorobeva, 2009, p. 72). The “selectivity” of an index has previously been defined as follows. “The ratio of the number of distinct values in the indexed column / columns to the number of records in the table represents the selectivity of an index. Example with good selectivity: a table having 100,000 records where one of its indexed columns has 88,000 distinct values, then the selectivity of this index is 88,000 / 100,000 = 0.88 Example with bad selectivity: if an index on a table of 100,000 records has only 500 distinct values, then the index’s selectivity is 500 / 100000 = 0.005 and in this case a query which uses the limitation of such an index will return 100000 / 500 = 200 records for each distinct value.” (Akadia, 2008) Based on the notion of selectivity, the “Coefficient of Selectivity” (hereafter CS) as a measure of index efficiency is defined as: 5.13 Final considerations The above survey revealed a great variety of different types of character indexes. Among those which are based on character constituent elements and strokes, there are also indexes which assign numerical codes to characters. In order to compare and evaluate these indexes, we introduce the notion of “selectivity” (選択性 sentakusei). A comparison and evaluation of indexes based on selectivity is presented in the next section. 5.12 Index by principal semantic determiners The Index by principal semantic determiners (意符 ifu) developed by Shiraishi (1971/1978) is similar to a radical index, but instead of radicals relies on 243 characters representing principal semantic determiners. These determiners also include radicals, characters and character constituent elements that are not radicals. Principal semantic determiners are ordered by stroke number. CS=V/N×100％ where V stands for the number of distinct values in the indexed column(s) and N is the total number of records in the table. An Analysis of the Efficiency of Existing Kanji Indexes 43 Here, in the case of an index based on character shape, N is the total number of characters in the index, and V is the number of different groups to which characters are assigned. A group is for example a group of all characters under the same radical or a group of all characters with the same number of strokes. For example, in the case of an index by stroke numbers, V is the number of groups containing characters with the same number of strokes. In the case of an index based on radicals, V is the number of different radicals. On the other hand, in a phonetic based on-kun (loan and native reading) index, N is the total number of all loan (onyomi) and native (kunyomi) readings associated with all characters covered by the index, while V is the number of all distinct loan (onyomi) and native (kunyomi) readings. To give an example of how to compute the coefficient of selectivity, let us use the old version of the jōyō kanji list (not the new shin jōyō kanji). The 1945 characters in the jōyō kanji list can be divided into 23 groups according to their number of strokes, or 201 groups of characters with distinct radicals (only 201 radicals are used in the 1945 jōyō kanji). Thus, for example, for the stroke number index and radical index we can compute the CS as follows: stroke number index: V=23, N=1945, CS=23/1945×100％=1.2％ radical index: V=201, N=1945, and CS＝201/1945×100％=10.3％ From this we can conclude that the radical index is about 10 times more efficient than the stroke number based index. In the following subsections we are going to compare and evaluate the existing indexes according to this notion of “selectivity”. 6.3 Evaluation of efficiency of existing character indexes In Vorobeva (2009), ten types of existing character indexes were compared, while in the present paper the object of comparison are 15 indexes. Existing indexes were compared on the basis of the CS (Table 7). In the table, comparison of indexes based on the character shape, character readings and character meaning is given. It is clear from the above analysis that the CS of character shape based indexes varies between 1.2% and 25.4%, and is thus generally low. The possible reason for this is that such indexes are generally based only on one character property or element. For example, for each character, the radical index relies only on one element - the radical, the stroke number index relies only on one property - the stroke number, the initial stroke index relies only on the initial stroke, etc. Groups thus defined, i.e. groups of characters with the same radical, the same number of strokes or the same type of initial stroke, each contain a large number of different characters. On the other hand, the CS of indexes based on character reading vary in the range of 27.6~40.6％, and are more efficient if compared to character shape based indexes. However, in order to use these indexes, one has to know the readings in advance. Galina N. VOROBEVA, Victor M. VOROBEV 44 Table 7: Coefficients of selectivity of various character indexes Index type CS (%) Indexes based on character form Stroke number index (Henshall, 1988) 1.2 Index by katakana shapes (Kanō, 1998) 2.6 Radical index (Hadamitzky & Spahn, 1981) 4.1 Initial stroke pattern index (Kanō, 1998) 6.1 Five step arrangement kanji table (Rosenberg, 1916) 7.1 Four corner method (Morohashi, 1984) 10.2 Radical index (Henshall, 1988) 10.3 Stroke order index (Wakao & Hattori, 1989) 10.7 Index by principal semantic determiners (Shiraishi, 1978) 12.4 Fast Finder (Matthews, 2004) 14.1 SKIP (Halpern, 1988) 15.4 Index by meaning symbols (Kanō, 1998) 25.4 Indexes based on character reading Index by principal phonetic determiners (Shiraishi, 1978) 27.6 On-kun reading index (Henshall, 1988) 40.6 Index based on character meaning Key Words Index and Primitive Meanings (Heisig, 2001) 100.0 Table 7: Coefficients of selectivity of various character indexes Indexes based on character meaning, such as the Key Words Index and Primitive Meanings (Heisig, 2001) have a CS reaching 100%, but require the user to know in advance the meaning of all characters. 6.3 Evaluation of efficiency of existing character indexes Based on these findings it can be said that indexes which rely on the coded total shape of a character, such as the two indexes above, are much more efficient then indexes relying on a single element or property. It can therefore be concluded that such an index is needed for efficient character searching. Thus, in the following section, a new efficient index will be proposed and developed. For this purpose, character were structurally decomposed and coded. 7. Structural decomposition and coding of characters Three coding systems based on the structural decomposition of characters and three character databases built on the basis of these systems are introduced by Vorobeva (2011). In the present paper, we will discuss four systems of character coding. All characters contained in the jōyō kanji list, and also those added in the shin jōyō kanji list were encoded, and four databases were built on the basis of the following codes: An Analysis of the Efficiency of Existing Kanji Indexes 45 (1) an alphabet code (2) a symbol code (containing roman letters and digits), (3) a semantic code (expressed by words), (4) a radical and stroke code. Structural decomposition of characters and semantic analysis of their constituent elements stimulate a deeper understanding of character meaning. Structural decomposition of characters can be achieved at two levels, i.e., decomposition into strokes (書記素 shokiso) and decomposition into constituent elements (the smallest meaningful constituent elements of Chinese characters, 構成要素 kōseiyōso). Decomposition into strokes follows the stroke order, as in the following example: 女 → { く, 丿, 一 } Decomposition into constituent elements also follows the stroke order, but to obtain the smallest meaningful constituent elements, as in the following example: 露 → ｛雨，足，夂，口｝ After having defined the alphabet and number codes of strokes and constituent elements of each character, and the alphabet and number codes for each whole character, the complete shin jōyō kanji list was encoded. JLQ） All characters in the shin jōyō kanji list were encoded according to this procedure, and the alphabetic order of the code thus obtained was used to arrange the entries in a new character dictionary index. In this way a new index of character entries could be developed. An excerpt of the index is given in section 8, table 12. 7.1.1 The alphabet code of strokes According to Zadoenko and Khuan (1993), Chinese characters used in China can be decomposed into strokes belonging to 24 different types. Fazzioli (1987) also uses almost the same strokes. In an analysis of the shapes of all characters in the shin jōyō kanji list, we found that the 24 types of strokes proposed by Zadoenko and Khuan (1993) are necessary and sufficient. We therefore coded these 24 shapes into Latin alphabet letters from A to Z based on the similarity of their shapes, so that for each stroke a corresponding letter could be guessed on the basis of its shape (cf. Table 8). Table 8: The 24 character strokes and their alphabet codes Table 8: The 24 character strokes and their alphabet codes 46 Galina N. VOROBEVA, Victor M. VOROBEV 7.2.1 Types of constituent elements There are two types constituent elements which can form characters: elements which are radicals and elements which are not traditionally considered radicals but are patterns which correspond to radicals in some aspects. We name the latter “graphemes”. Constituents combine in complex ways to form compound characters. 7.1.2 The alphabet coding of characters and its use An alphabet code was obtained for each character by following its stroke order and transforming each stroke into its corresponding alphabet code as given in table 8. Thus, the stroke order for each character could also be expressed by an alphabet code, as in the following examples: 7.2.2 Types and coding of radicals Traditionally, 214 types of graphic patterns have been considered as radicals, as mentioned in the previous sections. Unicode 6.1.0 (Unicode, 2012) includes a table of these 214 radicals, and each radical is assigned a unique number. Analysing both the jōyō kanji list and the shin jōyō kanji list, we found that of these traditional 214 radicals, 201 types could be found as constituent elements of characters in the jōyō kanji list and 202 types as constituent elements of characters in the shin jōyō kanji list. In the present study, we assigned three different codes to each radical: (1) an alphabetic code composed of the alphabetic codes of all strokes which make up the radical; (1) an alphabetic code composed of the alphabetic codes of all strokes which make up the radical; (2) a symbol code corresponding to the traditional numbering of radicals from 1 to 214; (3) a semantic code consisting of a word expressing the principal meaning of each radical. (3) a semantic code consisting of a word expressing the principal meaning of each radical. Examples of these codes are given in table 9. Examples of these codes are given in table 9. An Analysis of the Efficiency of Existing Kanji Indexes 47 An Analysis of the Efficiency of Existing Kanji Indexes Table 9: Examples of coded radicals Radical Alphabet code Symbol code (number) Semantic code (Principal meaning) 一 A 1 one 丨 B 2 stick 人 PO 9 man 山 BEB 46 mountain 馬 BABAAGLQQQ 187 horse Table 9: Examples of coded radicals 7.2.3 Types and coding of graphemes Vorobeva (2011, p. 11) structurally decomposed each character from the shin jōyō kanji list and extracted all graphemes included in them, resulting in 161 different types of graphemes. This extraction was based on an intuitive notion of what would constitute a grapheme. In the present study, we first defined some rules to determine what part of the character can be extracted as a grapheme. We then structurally decomposed each character in the shin jōyō kanji list according to these rules, extracted their graphemes and found 220 different types of graphemes. Combining this list with the 202 traditional radicals found in the previous analysis, we found that the 2136 characters in the shin jōyō kanji list are composed of 422 different constituent elements. The rules used to structurally decompose characters and extract graphemes are based on Stalph (1989, p. 69) and are given below. 1. Graphemes are limited to characters or character constituent elements included either in Unicode 6.1.0 (Unicode, 2012) or the “Mojikyō tankanji” list (Mojikyō Kenkyūkai, 2002). Unicode 6.1.0 contains 74,617 characters listed as CJK Unified Ideographs, and the “Mojikyō tankanji” list contains 110,000 characters. The reason for using these lists is that when characters are decomposed, the decomposition may result in shapes that do not appear in the shin jōyō kanji list. 2. When the decomposition of characters or constituent elements into smaller elements reaches a point where a stroke does not constitute an independent character, the decomposition is stopped and such a shape is considered a grapheme. For example: decomposing 及 further would give two elements: 乃 and 乀, but since 乀is not an independent element, the decomposition is stopped and 及 is considered itself a grapheme. In shorthand notation: 及 ≠ 乃＋乀 Attention is necessary in the case of 一 and 乙. Here they are considered as characters and not as strokes. 3. When the decomposition of a character or a constituent element reaches an element which by itself is not a radical but is a constant shape always used in Galina N. VOROBEVA, Victor M. VOROBEV 48 combination with a radical or other graphemes, this shape is considered a grapheme, without further decomposition. For example, the constituent element 辟 appears in characters such as 避, 壁, 癖, and 璧. It contains the element which by itself is not a radical and which regularly accompanies the radical 辛. 7.2.3 Types and coding of graphemes This constituent element 辟 is not further decomposed, i.e. 辟 ≠ ＋辛, and 辟 is considered as a grapheme. 4. When decomposing a character or a constituent element, if it is necessary to cut one stroke to obtain two independent shapes and if the total number of resulting strokes is then higher than in the original character or constituent element, then such character or constituent element is considered as a grapheme and is not decomposed further. For example: 4. When decomposing a character or a constituent element, if it is necessary to cut one stroke to obtain two independent shapes and if the total number of resulting strokes is then higher than in the original character or constituent element, then such character or constituent element is considered as a grapheme and is not decomposed further. For example: 出 ≠ 山 + 山, 重 ≠ 千 + 里. Thus, 出 and 重are considered as graphemes. 7.2.4 Symbol code, semantic code, radical and stroke code, and their use Symbol codes and semantic codes of characters are described in Vorobeva (2007, p. 22). The symbol code of each character is obtained by listing the symbol codes of each constituent element (cf. 7.2.2), following the stroke order. The semantic code of a character is defined as the semantic code of its first two constituent elements, following the stroke order. Only the first two elements are used in order to avoid long codes. Further, codes based on radicals and strokes are obtained by listing the numbers of elements which correspond to traditional radicals and the symbol codes (roman letters) assigned to each of the remaining strokes, following standard stroke order. Letters indicating strokes and numbers indicating radical shapes are divided by slashes, as in the following examples. 決 → {氵,ㄱ, 大} → 85 / H / 37，今 → { , 一, フ} → 9 / 1 / Y Numbers and letters used in the above code are, for radicals, standard radical numbers from Unicode 6.1.0 (Unicode, 2012), and for strokes, the alphabet codes assigned to basic strokes (cf. table 8). Following the above procedure, all characters in the shin jōyō kanji list were encoded and a database was constructed to include the alphabet codes, symbol codes, semantic codes, as well as radical and stroke codes. Examples of such coding are given in table 11 below. Table 11: Examples of alphabet, symbol, semantic and radical-stroke character codes character Alphabet code Symbol code Semantic code Radical and stroke code 九 PR 2PR nine 4/5 逸 PYBHBAPCQMO 8PYB/162 escape/road 18/30/2/10/162 新 SAQLAABPOPPAB 117/75/69 stand/tree 117/75/69 11: Examples of alphabet, symbol, semantic and radical-stroke character codes Thus, 出 and 重are considered as graphemes. 5. When decomposing a character or a constituent element, if it is necessary to insert a stroke into a decomposed element to make it complete and if the total number of resulting strokes of the two parts exceeds the number of strokes in the original character, then such a character or constituent element is considered a grapheme and not decomposed. For example: 雀 ≠ 少 + 隹 and thus 雀 is considered a grapheme. 5. When decomposing a character or a constituent element, if it is necessary to insert a stroke into a decomposed element to make it complete and if the total number of resulting strokes of the two parts exceeds the number of strokes in the original character, then such a character or constituent element is considered a grapheme and not decomposed. For example: 雀 ≠ 少 + 隹 and thus 雀 is considered a grapheme. 6. When decomposing a character or a constituent element, if it is necessary to split two crossing strokes to arrive at a grapheme or a radical, such a character or constituent element is considered a grapheme. For example: 必 ≠ 心 + ノ, and thus 必 is a grapheme. 7. A character or constituent element which is deemed impossible to further decompose is considered a grapheme. For example: 尞, 喬, 兼, etc, are considered graphemes. As the next step, all graphemes were assigned three types of codes according to the same procedure used for radicals. Details about encoding are given in Vorobeva (2011, p. 21). Examples of codes for graphemes which are component parts of characters in the shin jōyō kanji list are given in table 10 below. ble 10: Examples of coding of graphemes included in characters in the shin jōyō kanji list Table 10: Examples of coding of graphemes included in characters in the shin jōyō kanji list Grapheme Alphabet code Symbol code Semantic code 丁 AJ 2AJ street 丂 AN 2AN snare マ YQ 2YQ chop-seal 亼 POA 3POA meeting An Analysis of the Efficiency of Existing Kanji Indexes 49 8.1 Construction and use of a new type of character index Starting from the premise that it is necessary to develop a character index which would make search in character dictionaries more efficient and which would be suitable for learners not familiar with Chinese character writing, we developed a character code based on appropriate representations of character shapes (cf. Vorobeva Galina N. VOROBEVA, Victor M. VOROBEV 50 2007, 2009, p. 72). In order to develop such a new type of character index, we sorted the alphabet codes, symbol codes, semantic codes, and radical/stroke codes for all characters in the shin jōyō kanji list in standard dictionary order (alphabetical and numerical order). Searching for characters in such indexes should require the same amount of labour as searching for words in alphabetically ordered dictionaries to which learners from non-kanji background are accustomed. A symbol code index and a semantic code index were developed and implemented in two character textbooks including 518 characters, Kanji monogatari I [漢字物語Ⅰ] (Vorobeva, 2007) and Kanji monogatari II [漢字物語Ⅱ] (Vorobev & Vorobeva, 2007). Further, an alphabet code index, a symbol code index and a semantic code index were developed for the 1945 characters in the jōyō kanji list, selectivity coefficients were computed and compared with existing character indexes (Vorobeva, 2009). In 2010, after the new shin jōyō kanji list with 2136 characters was approved, an alphabet code index, a symbol code index and a semantic code index were compiled for the new list, and a new, easier to use radical and stroke code index was also developed. The next sections introduce each of these new indexes. 8.2 Alphabet code index The first part of the alphabet code index is shown in Table 12. In order to be able to use this index, one needs to memorise the 24 types of strokes and the rules governing stroke order of characters. Table 12: First part of the alphabet code index for all characters in the jōyō kanji list Alphabet coding of character character A 一 AA 二 AAA 三 AAABPQAAPB 耕 AAABPQPAAC 耗 Beginning learners are not yet accustomed to the constituent elements of characters. The alphabet code index is therefore expected to be easier to understand and use. An Analysis of the Efficiency of Existing Kanji Indexes 51 8.3 Symbol code index Most characters are compound characters, composed of multiple constituent elements. We coded all characters in our textbook using radical and grapheme codes, compiled a database of characters with their respective radical and grapheme codes, sorted the database according to the code column, in standard dictionary ascending order (i.e. alphanumeric order), and thus obtained a symbol code index. Users can thus search for characters in this index using codes based on character shape. For example, the symbol code for the character 親 is 117/75/147. The numbers in the code are the numbers of the radical shapes into which the character can be decomposed, i.e. 立 (117), 木 (75), and 見 (147). To use the symbol code index, users refer to the table of radical numbers and grapheme codes. After some time, users generally end up remembering the numbers and codes by using them. An excerpt from the symbol code index in Kanji monogatari I [漢字物語Ⅰ] (Vorobeva, 2007) and Kanji monogatari II [漢字物語Ⅱ] (Vorobev & Vorobeva, 2007) is shown in table 13. Table 13: First part of the symbol code index in the textbooks Kanji monogatari I and II character symbol code character character number in Kanji monogatari I,II 1 一 11 1/106 百 21 1/119 来 51 1/13/46 両 238 1/132/34 夏 189 Table 13: First part of the symbol code index in the textbooks Kanji monogatari I and II 8.4 Semantic code index Part of the semantic code index from the textbooks Kanji monogatari I and II is given in table 14. When using the semantic code index, users refer to the list of words representing the meaning labels of character constituent elements. The semantic code is obtained by listing the meaning labels of the first two constituent elements, following standard stroke order. For example, the semantic code for the character 新 is “stand/tree”, since the first two constituent elements and their corresponding meaning labels are 立 (stand), and 木 (tree). In order for the semantic code not to be too long, only the meaning labels of the first two constituent elements are used. Galina N. VOROBEVA, Victor M. VOROBEV 52 Table 14: First part of the semantic code index in the textbooks Kanji monogatari I and II character semantic code (Russian /English) character character number in Kanji monogatari I,II Азия/сердце (Asia / heart) 悪 140 бамбук/встреча (bamboo/ meeting) 答 447 бамбук/дерево (bamboo/tree) 箱 449 Table 14: First part of the semantic code index in the textbooks Kanji monogatari I and II 8.5 Radical and stroke index The special characteristic of this index is that users only need to remember the radicals and the basic strokes in order to use it. The first part of the radical and stroke code index is shown in Table 15. Table 15: First part of the radical and stroke index Radical and stroke code character 1 一 1/102/17 画 1/106 百 1/132/34 夏 1/25 下 1/30/6/1/30/6/76 歌 Table 15: First part of the radical and stroke index The nine types of strokes (A, B, C, F, J, P, Q, R, W), given in Table 8 are at the same time also constituent elements appearing in the table of radicals. In the compilation of the radical and stroke based index, these strokes were treated as radicals. By analysing all characters in the shin jōyō kanji list, we found that more than 90% of these characters are entirely composed of elements which can be found in the traditional list of radicals. Characters that include other strokes (i.e. D, E, G, H, K, L, M, N, O, R, S, T, V, Y, Z in table 8) are relatively few, less than 10% of all characters in the shin jōyō kanji list. The radical and stroke code, which is mainly based on radicals, is therefore presumably easier to acquire and use than the symbol code index and the semantic code index. Table 16 shows the frequency of use of stroke codes in the radical and stroke code index for all characters in the shin jōyō kanji list. An Analysis of the Efficiency of Existing Kanji Indexes 53 Table 16: Frequency of use of stroke codes in the radical and stroke code index for characters in the shin jōyō kanji list Stroke code D E G H K L M N O R S T V Y Z Frequency of use 0 25 20 19 5 42 4 16 51 2 0 9 19 0 4 Table 16: Frequency of use of stroke codes in the radical and stroke code index for characters in the shin jōyō kanji list Table 16: Frequency of use of stroke codes in the radical and stroke code index for characters in the shin jōyō kanji list 8.6 Comparison and evaluation of the new type of character indexes If the new character indexes introduced in the preceding sections are learned and used, the workload necessary for search is comparable to the search in alphabetic dictionaries, resulting in more efficient character search. At the same time it can be expected that the learners gain a better insight into the structure of characters and are thus freed from rote memorisation. Table 17 gives the coefficients of selectivity (CS) of these new indexes, computed for the 1945 characters the jōyō kanji list. CS for each index is obtained by dividing the number of distinct characters under each code by the total number of characters in the index, and multiplying it by 100, as explained in section 6.2. The 1945 characters in the jōyō kanji list were considered instead of the 2136 characters from the shin jōyō kanji list, in order to facilitate a comparison with existing indexes. Table 17: Coefficients of selectivity (CS) for the new types of indexes New type of index Coefficient of selectivity (%) Semantic code index 64.1 Alphabet code index 98.4 Symbol code index 99.4 Radical and stroke index 99.4 Table 17: Coefficients of selectivity (CS) for the new types of indexes The results of the analysis show that the alphabet code index, the symbol code index and the radical and stroke code index all have a CS close to 100%, implying better ease of search compared with existing indexes based on character shape. In other words, in the new indexes, different characters sharing the same code are few, and many characters have a unique code. For example, in the radical and stroke code index, there are only 11 characters sharing their code with other characters (cf. table 18). CS values for the radical and stroke code index for characters in the jōyō kanji list and shin jōyō kanji list are: shin jōyō kanji list: СS = (2136-11)/2136100 = 99.5% jōyō kanji list: СS = (1945-11)/1945100 = 99.4% shin jōyō kanji list: СS = (2136-11)/2136100 = 99.5% jōyō kanji list: СS = (1945-11)/1945100 = 99.4% shin jōyō kanji list: СS = (2136-11)/2136100 = 99.5% jōyō kanji list: СS = (1945-11)/1945100 = 99.4% 54 Galina N. VOROBEVA, Victor M. 8.7 Uses of the new index types At the beginning stage of learning to read and write Chinese characters, the alphabetic index is probably easier to use than other indexes, since learners do not yet know the constituent elements which make up characters. However, as learning progresses, characters to be learned become more complex, being made up of more strokes, and the alphabetic code becomes longer. At the same time, learners progressively learn to recognise different constituent parts within complex characters, and are able to use other indexes, choosing the one that best suits their learning style, be it the symbol code index, the semantic code index, or the radical and stroke code index. At this point, they usually start using mainly one of these indexes. The radical and stroke index is probably easier to learn and use than the symbol code and the semantic code index, but the latter two are probably more useful for deepening learners’ understanding of character structural composition. We used the symbol code index and the semantic code index in two textbooks we developed, Kanji monogatari I [漢字物語Ⅰ] (Vorobeva, 2007) and Kanji monogatari II [漢字物語Ⅱ] (Vorobev & Vorobeva, 2007). In the following paragraphs we explain how the use of these indexes was introduced to our learners. In order to look up an unknown character in the textbook, learners firstly write the first two constituent elements of the character and convert them into their respective codes. At first, when they are yet not accustomed to characters, they actually write down the first elements on paper, later they learn to just imagine writing the character and convert the first two elements into their codes. They then look through either the symbol code index or the semantic code index, where codes are arranged in alpha- numeric order, and look up the character as they would in a dictionary of alphabetically arranged words, until they find the number of the character in the index. Using this number, they can then find the character inside the textbook and read the textbook explanation about the character they were searching for. Searching in these two types of indexes is equivalent to searching through an alphabetically ordered dictionary. The procedure (algorithm) used when searching the aphanumeric symbol code index is described by Vorobeva (2007, p. 25). 8.6 Comparison and evaluation of the new type of character indexes An Analysis of the Efficiency of Existing Kanji Indexes 55 8.7 Uses of the new index types Through use, learners get progressively accustomed to this way of looking up characters, and after some practice are able to find a character in a few seconds. 8.6 Comparison and evaluation of the new type of character indexes VOROBEV Table 18: Characters from the shin jōyō kanji list sharing the same code in the radical and stroke code index radical and stroke code character 1 character 2 character 3 character 4 96 王玉 102 田申由甲 57/2 引弔 9/7/28 会伝 30/154 員唄 120/102 細紳 83 氏民 1/75 末未 64/102 押抽 Table 18: Characters from the shin jōyō kanji list sharing the same code in the radical and stroke code index The CS value for the semantic code index is 64.1%, lower than the CS for the alphabetic code index, the symbol code index and the radical and stroke index. This is because the semantic code only includes the codes of the first two elements in a character, resulting in relatively many sets of characters with the same code. In order to use character coding systems, some effort is required. For the new coding systems proposed above, it is especially important to accurately master the rules governing stroke order and the decomposition of characters into constituent elements and strokes. The alphabet code index is probably the easiest to learn among the new indexes proposed, since users only need to memorise 24 types of basic strokes and their corresponding alphabetic codes. This index can therefore be used from the beginning stages of character instruction. In order to use the radical and stroke index, learners need to memorise radicals, their numeric codes, and 24 basic strokes with their alphabetic codes. In order to use the symbol index and the semantic code index, learners need to memorise radicals with their respective numerical codes, and graphemes with their alphabetic codes. However, the symbol code index, the semantic code index, and the radical and stroke index have two merits if compared with the alphabetic code. (1) The code is short. For example, the alphabetic code for the character 高 is SABHABGBHA, while its symbol code and its radical and stroke code (identical in both cases) is 189. (1) The code is short. For example, the alphabetic code for the character 高 is SABHABGBHA, while its symbol code and its radical and stroke code (identical in both cases) is 189. (2) In order to use the radical and stroke code, users need to learn how to structurally decompose characters and to learn about their constituent parts, which enhances comprehension and memorisation of characters. 9. Selectivity of character indexes and learning burden In the present paper, we defined the Coefficient of Selectivity as an index of efficiency for character indexes, and compared existing indexes with our newly developed index types on the basis of this coefficient. However, there is one more factor to be considered when considering the efficiency of indexes, i.e. the learning burden required from users, the special knowledge they need to acquire in order to be Galina N. VOROBEVA, Victor M. VOROBEV 56 able to actually use each of these indexes, such as character radicals, types of strokes and their counting, readings and meanings associated with each character, their coding etc. When discussing the efficiency of indexes, such learning burden must also be considered, as discussed in Vorobeva (2011, p. 24). able to actually use each of these indexes, such as character radicals, types of strokes and their counting, readings and meanings associated with each character, their coding etc. When discussing the efficiency of indexes, such learning burden must also be considered, as discussed in Vorobeva (2011, p. 24). The index by total number of strokes is probably the easiest to master among the indexes presented in the previous sections. However, given its CS of 1.2%, it is clearly not an efficient index. On the other hand, the Key Words and Primitive Meanings Index (Heisig, 2001), with a CS of almost 100%, allows for very efficient search, but in order to use it, learners need to memorise approximately 2000 semantic keywords. We can therefore conclude that in future research it will be necessary to analyse the learning burden of character indexes, and further define a comprehensive index of efficiency which would reflect both selectivity and learning burden of character indexes. 10. Conclusion and further work In the present paper we discussed the difficulties faced by learners of Japanese not familiar with Chinese characters when they use character dictionaries, presented 15 types of existing character indexes and described their characteristics. We pointed out that for users who need to look up characters in a dictionary, in addition to the generally used radical index, stroke number index and readings index, many other different types of character indexes have been developed and used, including the five step arrangement kanji table (Rosenberg, 1916), the four corner method (Wang, 1925), the phonetic key index (Shiraishi, 1971/1978), the index of katakana shapes, the index of initial stroke patterns and the index of meaning symbols (Kanō, 1998), the stroke order index (Wakao & Hattori, 1989), the index of character shapes (Sakano, Ikeda, Shinagawa, Tajima, & Tokashiki, 2009), the key words and primitive meanings index (Heisig, 1977/2001), the index by radicals (Hadamitzky & Spahn, 1981), the system of kanji indexing by patterns - SKIP (Halpern, 1988), the kanji fast finder (Matthews, 2004) and others. In order to compare the effectiveness of different character indexes, we applied the concept of selectivity, a concept used to express the efficiency of computer data processing, to character indexes, and defined the concept of coefficient of selectivity (CS) as an index of efficiency of character indexes. We then computed CS for existing character indexes and compared their efficiency. We found that indexes based on character form or structure had a low CS in the range of 1.2% to 25.4%, probably because most indexes based on character form use only one structural element or characteristic of each character for indexing, such as the radical index using only the radical part of a character, the total stroke number index using only the number of strokes, or the initial stroke pattern index using only the form of the first stroke for indexing characters. 57 An Analysis of the Efficiency of Existing Kanji Indexes In order to improve the efficiency of character dictionary indexes, we considered it necessary to develop a new type of index with a hight selectivity coefficient that would be appropriate for the habits of learners not familiar with Chinese character writing. We therefore developed an alphabetic and a symbol code index, based on a code which accurately represents the form of Chinese characters, a semantic code index and a radical and stroke code index. 10. Conclusion and further work In this paper, we described the use of these indexes, and comparatively evaluated their efficiency. We found that the alphabetic code index, the symbol code index and the radical and stroke index have a coefficient of selectivity nearing 100%, while the semantic code index only has a coefficient of selectivity of 64.1%. The reason for such a low CS is probably that it only takes into account the first two structural elements of each character, which leads to a relatively high number of characters with the same code. We expect that learners using the above new types of indexes should be able to look up characters in dictionaries more efficiently, deepen their understanding of character structure, and be emancipated from rote learning. We implemented these new types of indexes in two introductory textbooks including 518 characters, Kanji monogatari I (Vorobeva, 2007) and Kanji monogatari II (Vorobev & Vorobeva, 2007). We plan to include these new types of indexes (an alphabetic code index, a symbol code index, a semantic code index, and a radical and stroke index) in a new textbook with 1006 characters for the initial and intermediate level that is under development, and to empirically investigate the efficiency of these indexes by surveying how learners use them in practice. Moreover, the usefulness of character indexes should be investigated by measuring not only their coefficient of selectivity, but also the learning burden associated with them, as suggested in Vorobeva (2011, p. 24). We are therefore planning to further investigate both factors and define a comprehensive measure of efficiency for character dictionaries. References Akadia. (2008). How to measure index selectivity. Retrieved July 27, 2012, from http://www.akadia.com/services/ora_index_selectivity.html Banno, E. [坂野永理], Ikeda, Y. [池田庸子], Shinagawa, C. [品川恭子], Tajima, K. [田嶋香織], & Tokashiki, K. [渡嘉敷恭子]. (2009). 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[ヴォロビヨフ・ヴィクトル], ＆ Vorobeva, G. [ヴォロビヨワ・ガリーナ] (2007). Kanji monogatari II [漢字物語Ⅱ]. Bishkek: Lakprint. Vorobeva, G. [ヴォロビヨワ・ガリーナ] (2009). Sentakusei ga takai kanji sakuin no kaihatsu [選択性が高い漢字索引の開発]. Nihongo kyōiku hōhō kenkyū kaishi [日本語教育方法研究会誌], 16 (1), 72-73. Vorobeva, G. [ヴォロビヨワ・ガリーナ] (2011). Kōzō bunseki to kōdoka ni motozuku kanji jitai jōhō shori shisutemu no kaihatsu [構造分析とコード化に基づく漢字字体情報処理システムの開発]. Nihongo kyōiku [日本語教育], (149), 16-30. Wakao, S.[若尾俊平], & Hattori D. [服部大超]. (1989). Kuzushi kaidoku jiten [くずし解読字典]. Tokyo: Kashiwa Shobō [栢書房]. Wáng, Yún Wǔ [王雲五]. (1925). Hào mǎ jiǎn zì fǎ [號碼檢字法]. Shanghai: Shāngwù yìnshūguǎn [商務印書館]. An Analysis of the Efficiency of Existing Kanji Indexes 59 Acknowledgments The present paper presents a part of the results of a research supported by the Hakuho Foundation and conducted under the guidance of prof. Yokoyama Shoichi at the Department of Linguistic Theory and Structure of the National Institute for Japanese Language and Linguistics. Prof. Takada Tomokazu of the same institute introduced us to valuable resources. The paper was translated from the Japanese by assist. dr. Boštjan Bertalanič, prof. dr. Andrej Bekeš and dr. Kristina Hmeljak Sangawa at Ljubljana University. We would like to extend our sincere thanks to all of them.
https://openalex.org/W4385411276	https://pure.eur.nl/files/98984691/fonc-13-1247808.pdf	English	null	Editorial: Risk factors in multiple myeloma identified before and during treatment: are we ready to personalize treatment?	Frontiers in oncology	2,023	cc-by	1,728	TYPE Editorial PUBLISHED 31 July 2023 DOI 10.3389/fonc.2023.1247808 TYPE Editorial PUBLISHED 31 July 2023 DOI 10.3389/fonc.2023.1247808 Mattia D’Agostino 1†, Carolina Terragna 2† and Mark van Duin 3† †These authors have contributed equally to this work 1SSD Clinical Trial in Oncoematologia e Mieloma Multiplo, Division of Hematology, University of Torino, Azienda Ospedaliero-Universitaria Città della Salute e della Scienza di Torino, Torino, Italy, 2IRCCS Azienda Ospedaliero-Universitaria di Bologna, Istituto di Ematologia “Seràgnoli”, Bologna, Italy, 3Department of Haematology, Erasmus Medical Center, Rotterdam, Netherlands KEYWORDS 1SSD Clinical Trial in Oncoematologia e Mieloma Multiplo, Division of Hematology, University of Torino, Azienda Ospedaliero-Universitaria Città della Salute e della Scienza di Torino, Torino, Italy, 2IRCCS Azienda Ospedaliero-Universitaria di Bologna, Istituto di Ematologia “Seràgnoli”, g g g Bologna, Italy, 3Department of Haematology, Erasmus Medical Center, Rotterdam, Netherlands D’Agostino M, Terragna C and van Duin M (2023) Editorial: Risk factors in multiple myeloma identiﬁed before and during treatment: are we ready to personalize treatment? Front. Oncol. 13:1247808. doi: 10 3389/fonc 2023 1247808 multiple myeloma, prognosis, risk assessment, minimal residual disease, liquid biopsy OPEN ACCESS Mattia D’Agostino 1†, Carolina Terragna 2† and Mark van Duin 3† Editorial on the Research Topic 10.3389/fonc.2023.1247808 10.3389/fonc.2023.1247808 The analysis of IgH rearrangement in cfDNA proved highly speciﬁc but not sensitive enough, as compared to the same analysis on BM aspirate (3). Nevertheless, a reliable comparison between the two approaches should be performed when multiple molecular targets will be employed to measure residual disease by ctDNA, as this expedient would increase the probability to detect low amount of ctDNA, suggestive of the residual presence of disease in the BM. Beyond ﬁrst-line treatment, prognosis prediction in relapsed and/ or refractory MM (RRMM) patients could be inﬂuenced by different factors, as compared to NDMM patients. Morabito et al. analyzed 919 RRMM patients receiving Elotuzumab-lenalidomide-dexamethasone or Carﬁlzomib-lenalidomide-dexamethasone. Interestingly, factors that can only be measured in RRMM like the interval from diagnosis to therapy, the number of previous lines and prior lenalidomide exposure were independently associated with the risk of death. Another type of prognostic factor is MRD, which describes the disease dynamics during treatment. MRD measurement in the bone marrow can provide insights into disease status and might represent a fast method to evaluate efﬁcacy in clinical trials. Flow cytometry is a solid MRD detection method, which is overall considered easy of use and returns quick results. A standardization of both ﬂow cytometry panels and number of cells to be measured resulted in the development of next generation ﬂow cytometry (NGF) by the Euroﬂow consortium. In addition to NGF, next generation sequencing (NGS) is considered a reference method for MRD detection. Both NGF and NGS can achieve sensitivity of <10-5 (1). The method commonly employed in the context of clinical trials for NGS-MRD detection has been the Adaptive ClonoSEQ™assay. Despite the usefulness of this assay and its widespread use, alternative NGS methods might be considered, particularly when quick results are needed and budget is limited. In this Research Topic, Ferla et al. described a LymphoTrack-MiSeq platform for NGS-MRD. MRD was evaluated in 24 patients at day +100 post-ASCT. Among 11 patients reaching stringent complete remission, 5 were MRD negative and 6 positive by NGS performed according to the LymphoTrack assay. Despite the small sample size, MRD relation with prognosis was conﬁrmed, as well as the feasibility of the method, including the use of a speciﬁc analysis software. MRD status can also be used to adapt treatment. Indeed, one report has indicated that patients with a positive MRD test during maintenance might beneﬁt from treatment intensiﬁcation (2). Editorial on the Research Topic Risk factors in multiple myeloma identiﬁed before and during treatment: are we ready to personalize treatment? © 2023 D’Agostino, Terragna and van Duin. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Thanks to a better knowledge of Multiple Myeloma (MM) biology, new prognostic tools are available. Moreover, we are now able to induce unprecedented rates of deep remissions, which can be dissected with very high sensitive techniques, developed to detect very low levels of minimal residual disease (MRD). However, treatment guidelines do not recommend risk-adapted approaches, stating that more evidence is needed to perform personalized medicine strategies. Thus, ﬁrst-line therapy is still largely tailored on the eligibility of the patients to undergo autologous stem cell transplantation (ASCT) rather on either the biological characteristics of the disease itself or the deepness of response achieved. In this Research Topic we tried to collect articles generating/reviewing data on factors detected both before and throughout treatment, that can enable a more precise prediction of MM patients’ outcome that would allow personalized approaches. The ﬁrst challenge has been considered the deﬁnition of high-risk disease, intended as disease with higher risk of relapse and/or death. Marcon et al. tried to reach a consensus on the deﬁnition of high-risk MM. A questionnaire was submitted to 6 Italian experts from the European Myeloma Network (EMN) and a consensus was reached, according to the Delphi method. Two rounds of the survey identiﬁed that the current risk stratiﬁcation algorithm at diagnosis (R-ISS, Revised International Staging System) can be improved by adding chromosome 1 abnormalities, TP53 mutation, circulating plasma cells and extramedullary plasmacytomas. Indeed, another work performed by You et al. supported the role of 1q21 gain/ ampliﬁcation as a high-risk marker in newly diagnosed MM patients. In this work, a multivariate analysis performed in 248 patients treated in a single center in China, showed that 1q21 was an independent adverse predictor of progression-free survival and overall survival. Frontiers in Oncology 01 frontiersin.org D’Agostino et al. D’Agostino et al. Author contributions All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication. Editorial on the Research Topic Many MRD-adapted trials are evaluating this kind of response-tailored approach. To date, the main added value of cfDNA analysis probably relies on the prognostic signiﬁcance of the presence of peripheral molecular markers. In particular, the amount of detected cfDNA at diagnosis has a prognostic relevance (3), even though it is worth to note that ctDNA, deriving directly from the BM tumour burden, is more signiﬁcantly associated to inferior OS. In addition, qualitative genomic information obtained from ctDNA might be more informative on disease distribution than the conventional single-site BM aspirate. Therefore, by pooling data from comparable studies, the present meta-analysis highlights the role of cfDNA as a good alternative to, or an important implementation of BM biopsies for monitoring disease dynamics in MM. In conclusion, the articles in this Research Topic showed how risk factors identiﬁed at baseline (both at diagnosis and at relapse) and during treatment (both in the bone marrow and in the peripheral blood) can identify myeloma patients with different outcomes, paving the way to the design of personalized therapeutic strategies. Conﬂict of interest However, the measure of MRD in the bone marrow is subjected to some limitation such as the invasiveness of the procedure and the spatial heterogeneity of the disease. The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Indeed, liquid biopsy might overcome the shortcoming of bone marrow (BM) sampling and has an added value over conventional “single-site” BM biopsies, by avoiding sampling biases due to the limited glance on the molecular proﬁle of the disease. MRD assessment by liquid biopsy in MM is indeed an innovative tool for disease characterization and disease dynamics monitoring. Publisher’s note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their afﬁliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. In this Research Topic, Ye et al. reported the results of a meta- analysis of data available so far on the clinical role of cell-free (cf) DNA in MM, derived from seven studies, including 235 patients. Main observations concern the relevance of cfDNA for Minimal Residual Disease (MRD) detection and the role of both cfDNA and cfDNA tumour fraction (ctDNA) as prognostic factors. 02 Frontiers in Oncology frontiersin.org D’Agostino et al. 10.3389/fonc.2023.1247808 in multiple myeloma. J Hematol Oncol (2021) 14(1):126. doi: 10.1186/s13045-021- 01135-w 3. Biancon G, Gimondi S, Vendramin A, Carniti C, Corradini P. Noninvasive molecular monitoring in multiple myeloma patients using cell-free tumor DNA. J Mol Diagnostics. (2018) 20(6):859–70. doi: 10.1016/j.jmoldx.2018.07.006 3. Biancon G, Gimondi S, Vendramin A, Carniti C, Corradini P. Noninvasive molecular monitoring in multiple myeloma patients using cell-free tumor DNA. J Mol Diagnostics. (2018) 20(6):859–70. doi: 10.1016/j.jmoldx.2018.07.006 in multiple myeloma. J Hematol Oncol (2021) 14(1):126. doi: 10.1186/s13045-021- 01135-w 1. Oliva S, D’Agostino M, Boccadoro M, Larocca A. Clinical applications and future directions of minimal residual disease testing in multiple myeloma. Front Oncol (2020) 10. doi: 10.3389/fonc.2020.00001 2. Martinez-Lopez J, Alonso R, Wong SW, Rios R, Shah N, Ruiz-Heredia Y, et al. Making clinical decisions based on measurable residual disease improves the outcome 10.3389/fonc.2023.1247808 1. Oliva S, D’Agostino M, Boccadoro M, Larocca A. Clinical applications and future directions of minimal residual disease testing in multiple myeloma. Front Oncol (2020) 10. doi: 10.3389/fonc.2020.00001 D’Agostino et al. cadoro M, Larocca A. Clinical applications and future sease testing in multiple myeloma. Front Oncol (2020) R, Wong SW, Rios R, Shah N, Ruiz-Heredia Y, et al. on measurable residual disease improves the outcome in multiple myeloma. J Hematol Oncol (2021) 14(1):126. doi: 10.1186/s13045-021- 01135-w 3. Biancon G, Gimondi S, Vendramin A, Carniti C, Corradini P. Noninvasive molecular monitoring in multiple myeloma patients using cell-free tumor DNA. J Mol Diagnostics. (2018) 20(6):859–70. doi: 10.1016/j.jmoldx.2018.07.006 References 1. Oliva S, D’Agostino M, Boccadoro M, Larocca A. Clinical applications and future directions of minimal residual disease testing in multiple myeloma. Front Oncol (2020) 10. doi: 10.3389/fonc.2020.00001 2. Martinez-Lopez J, Alonso R, Wong SW, Rios R, Shah N, Ruiz-Heredia Y, et al. Making clinical decisions based on measurable residual disease improves the outcome 03 03 Frontiers in Oncology frontiersin.org
https://openalex.org/W2789382740	https://hal.archives-ouvertes.fr/hal-02538219/file/2018_Charrier_ScienceAdvances_1.pdf	English	null	Drought will not leave your glass empty: Low risk of hydraulic failure revealed by long-term drought observations in world’s top wine regions	Science advances	2,018	cc-by	12,784	To cite this version: Guillaume Charrier, Sylvain Delzon, Jean-Christophe Domec, Li Zhang, Chloe E. L. Delmas, et al.. Drought will not leave your glass empty: Low risk of hydraulic failure revealed by long-term drought observations in world’s top wine regions. Science Advances , 2018, 4 (1), pp.1-9. 10.1126/sci- adv.aao6969. hal-02538219 Drought will not leave your glass empty: Low risk of hydraulic failure revealed by long-term drought observations in world’s top wine regions Guillaume Charrier, Sylvain Delzon, Jean-Christophe Domec, Li Zhang, Chloe E. L. Delmas, Isabelle Merlin, Déborah Corso, Andrew King, Hernan Ojeda, Nathalie Ollat, et al. Distributed under a Creative Commons Attribution 4.0 International License 1Bordeaux Science Agro, Institut des Sciences de la Vigne et du Vin, Ecophysiologie et Génomique Fonctionnelle de la Vigne, UMR 1287, F-33140 Villenave d’Ornon, France. 2Biodiversité Gènes et Communautés, Institut National de la Recherche Agronomique (INRA), Université Bordeaux, 33610 Cestas, France. 3Bordeaux Sciences Agro, UMR 1391 Interactions Sol Plante Atmosphère, INRA, 1 Cours du General de Gaulle, Gradignan Cedex 33175, France. 4Université Clermont Auvergne, INRA, Physique et Physiologie Intégratives de l’Arbre en environnement Fluctuant, F-63000 Clermont-Ferrand, France. 5Santé et Agroécologie du VignoblE, Bordeaux Sciences Agro, INRA, 33140 Villenave d’Ornon, France. 6Synchrotron Source Optimisée de Lumière d’Energie Intermédiaire du LURE, L’Orme de Merisiers, Saint Aubin-BP48, Gif-sur-Yvette Cedex, France. 7INRA, UE0999, Unité expérimentale de Pech Rouge, 11430 Gruissan, France. 8Estación Experimental Agropecuaria Mendoza, Instituto Na- cional de Tecnología Agropecuaria, Luján de Cuyo (5507), Mendoza, Argentina. 9Fru- ition Sciences SAS, MIBI 672 rue du Mas de Verchant, 34000 Montpellier, France. 10Terra Spase, 345 La Fata Street, Suite D, St. Helena, CA 94574, USA. Corresponding author. Email: guillaume.charrier@inra.fr on February 13, 2018 http://advances.sciencemag.org/ One thing is inescapable; as plant water status [that is, leaf water potential (Yleaf)] declines, plants regulate stomata to control transpira- tionalwaterloss. Stomatalclosure isone of the first responsesto drought stress (4), protecting against loss of hydraulic conductivity by maintain- ing xylem pressure above the onset of embolism (5, 6). Scientists have used a framework to describe variation in stomatal control where a spe- cies may be defined asmore isohydric or more anisohydric (7–10). Most commonly, iso/anisohydric behavior refers strictly to differences in the regulation of stomatal conductance in response to water deficit, Grapevines are crops for which different varieties supposedly exhibit contrasting behaviorswithin the iso/anisohydric paradigm [reviewed by Chaves et al. (15)]. The most classic examples are Grenache (isohydric) and Syrah [anisohydric (16)]. These contrasting varieties are often in- voked in a discussion of drought resistance, but it is not entirely clear which would be most beneficial in the context of a woody perennial crop where the iso/anisohydric paradigm constitutes a trade-off be- tween safety with respect to hydraulic failure and productivity. Further- more, the intraspecific iso/anisohydric behaviors among grapevine varieties can be difficult to separate (17), and individual grapevine vari- eties can exhibit contradictory behavior depending on the study (15). Stomatal closure and reductions in transpiration should occur before the onset of embolism (3). Recent work in Vitis vinifera suggests that this is indeed the case, where stem embolism occurs at lower (more negative) Y [Y inducing 50% loss of hydraulic conductivity (Y50) < −1.7 MPa] than those typically observed for stomatal closure [Yleaf < −1.0 MPa (18)]. The distance between the minimum stem water potential (Ystem) experienced in the field (Ymin) and the Y50 repre- sents the hydraulic safety margin (19, 20). The smaller this margin, Grapevines are crops for which different varieties supposedly exhibit contrasting behaviorswithin the iso/anisohydric paradigm [reviewed by Chaves et al. (15)]. The most classic examples are Grenache (isohydric) and Syrah [anisohydric (16)]. These contrasting varieties are often in- voked in a discussion of drought resistance, but it is not entirely clear which would be most beneficial in the context of a woody perennial crop where the iso/anisohydric paradigm constitutes a trade-off be- tween safety with respect to hydraulic failure and productivity. Further- more, the intraspecific iso/anisohydric behaviors among grapevine varieties can be difficult to separate (17), and individual grapevine vari- eties can exhibit contradictory behavior depending on the study (15). Drought will not leave your glass empty: Low risk of hydraulic failure revealed by long-term drought observations in world’s top wine regions Guillaume Charrier,1,2,3,4 Sylvain Delzon,2 Jean-Christophe Domec,3 Li Zhang,1 Chloe E. L. Delmas,5 Isabelle Merlin,1 Deborah Corso,2 Andrew King,6 Hernan Ojeda,7 Nathalie Ollat,1 Jorge A. Prieto,8 Thibaut Scholach,9 Paul Skinner,10 Cornelis van Leeuwen,1 Gregory A. Gambetta1 Guillaume Charrier,1,2,3,4* Sylvain Delzon,2 Jean-Christophe Domec,3 Li Zhang,1 Chloe E. L. Delmas,5 Isabelle Merlin,1 Deborah Corso,2 Andrew King,6 Hernan Ojeda,7 Nathalie Ollat,1 Jorge A. Prieto,8 Thibaut Scholach,9 Paul Skinner,10 Cornelis van Leeuwen,1 Gregory A. Gambetta1 Grapevines are crops of global economic importance that will face increasing drought stress because many varieties are described as highly sensitive to hydraulic failure as frequency and intensity of summer drought increase. We developed and used novel approaches to define water stress thresholds for preventing hydraulic failure, which were compared to the drought stress experienced over a decade in two of the world’s top wine regions, Napa and Bordeaux. We identified the physiological thresholds for drought-induced mortality in stems and leaves and found small intervarietal differences. Long-term observations in Napa and Bordeaux revealed that grapevines never reach their lethal water-potential thresholds under seasonal droughts, owing to a vulnerability segmentation promoting petiole embolism and leaf mortality. Our findings will aid farmers in reducing water use without risking grapevine hydraulic integrity. HAL Id: hal-02538219 https://hal.science/hal-02538219v1 Submitted on 26 May 2020 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License S C I E N C E A D V A N C E S \| R E S E A R C H A R T I C L E P L A N T S C I EN C ES Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to originalU.S.Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC). INTRODUCTION assuming that stomatal conductance is assessed under identical environmental conditions across decreasing Y [for example, see study of Martínez‐Vilalta et al. (9)]. But equally, iso/anisohydric behavior can also refer to the regulation of stomatal conductance in response to increasing vapor pressure deficit (D) (11–13). A more isohydric behav- ior is one where a plant maintains Y within a given range under water deficit and high evaporative demand through limiting transpiration. A more anisohydric behavior is one where the plant, to a certain extent, maintains transpiration under water deficit and high evaporative de- mand, tolerating increasingly negative Y. A more isohydric behavior avoids the immediate risk of xylem embolism formation by maintaining Y but consequently limits photosynthesis. In the ecological context, in- terspecific differences in iso/anisohydric behavior between forest trees correspond to differences in their performance and mortality under drought (14). However, this framework is currently debated even at the interspecific level (8–10), and to be of use for the breeding of agri- cultural crops, differences in iso/anisohydric behavior need to occur at the intraspecific (or intragenus) level. Water availability is one of the most important factors in determining plant survival and productivity in both ecological and agricultural contexts (1). The consumption of freshwater resources for agriculture is enormous, especially in dry environments; for example, 80% of freshwater resources are used for agriculture in California. Thus, reduc- ing crop water use is essential to increase agricultural sustainability. The need to reduce water use, paired with the increased likelihood of large-scale water deficits and extreme drought events (2), is driving the search for more drought-resistant crops. A cornerstone to this pursuit is integrated understanding of the physiological mechanisms driving the resilience of the plant hydraulic system, including water potential (Y) and stomatal regulation, vulnerability to embolism along the water column, and ability to recover from drought (3). Unraveling (an-)isohydric behavior by integrating responses to soil water deficit and vapor pressure deficit Unraveling (an-)isohydric behavior by integrating responses to soil water deficit and vapor pressure deficit p p Assigning a determined behavior in Y regulation is problematic in grapevine (15) and other species (22). V. vinifera cv. Grenache and Syr- ah have repeatedly been described as isohydric and anisohydric, respec- tively (16, 23). Thus, Grenache would be expected to maintain midday water potential (Ymdl) at a higher level than Syrah under similar soil water status [estimated via the relative water content (RWC)] and/or evaporative demand [estimated via the vapor pressure deficit (D)]. Using a large data set of Y monitoring across a wide range of drought conditions and combining field observations and greenhouse experiments, we explored the relationship between midday leaf (Ymdl) and predawn water potential (Ypd). These two varieties did not exhibit significant differences in the relationship between Ymdl and Ypd (where the slope s represents the relativesensitivity of the transpiration rate and plant hydraulic conductance to declining water availability) (9), under field conditions (P = 0.074; Fig. 1) nor during a greenhouse drydown experiment (P = 0.225). Additional comparisons also did not reveal significant differences in the relationship between midday stem water potential (Ymds) and Ypd among three varieties in the Langue- doc and two varieties in Saint-Emilion (P > 0.270 and 0.068, respec- tively; fig. S1). Finally, the water use envelope (24) or “hydroscape” (19) that corresponded to the limit of s did not exhibit significant dif- ferences across varieties (1.32 and 1.38 MPa2 for Grenache and Syrah, respectively; Fig. 1). f ( ) h d b h ll d d [f l on February 13, 2018 mag.org/ Neither the dynamics of sx (9) nor the hydroscape framework (10) necessitates a linear curve fit. We also explored the framework with nonlinear fits, which improved the statistical significance of the fit [see the estimated Akaike information criterion (AIC); fig. S2], better represented the minimum Y, and suggested a slightly larger hydro- scape for Syrah (1.42 MPa2) compared to Grenache (1.17 MPa2). This approach also revealed differences between Syrah and Gre- nache at high Y (Ypd > −0.8 MPa). If (an-)isohydric behavior was genetically determined [for example, see the study of Coupel-Ledru et al. 25], we would expect each variety to exhibit a consistent behavior regardless of the growing conditions. Under field conditions, both varieties exhibited extreme anisohydric behavior (s = 1.172 ± 0.034 MPa MPa−1, mean ± SE; Fig. S C I E N C E A D V A N C E S \| R E S E A R C H A R T I C L E S C I E N C E A D V A N C E S \| R E S E A R C H A R T I C L E Fig. 1. Ymdl depending on Ypd in different V. vinifera varieties. Syrah (A) and Grenache (B) were measured in the field [domain “Pech Rouge” (PR); dark colors] and during a drydown experiment in greenhouse (GH; light colors). The slope s of the linear regressions was not statistically different across varieties in the field (P = 0.074) nor in the greenhouse (P = 0.225). Top left insets represent the slope sx (sensitivity to declining water availability) depending on the range of Ypd (from 0 to x; x representing the lower limit of the range of Ypd) combining field and greenhouse data. Bottom right insets represent the correlation between Ypd and minimum Ymdl (that is, average value of three lowest Ymdl per 0.1-MPa wide class of Ypd) combining field and greenhouse data. This linear regression is used to define the lower range of the hydroscape (colored by a blue-to-red gradient representing increasing water stress on the main figure). the more susceptible a plant willbe to hydraulic failure. For example, the width of the hydraulic safety margin has been related to differences in drought-induced mortality in trees (21). In perennial crops, the hydrau- lic safety margin could be important in longevity and long-term productivity under drought. To date, the hydraulic safety margin has not been directly assessed in grapevine, a trait that could potentially dif- fer substantially between varieties exhibiting different strategies for reg- ulation of plant water status. The current study explores physiological differences in grapevine at the interspecies level by comparing the responses to extreme water deficit(thatis,untildeathof theabovegroundbiomass)acrossV.vinifera and rootstock varieties. We developed an integrative approach to char- acterize the dynamic hydraulic strategies of these varieties during increasingdroughtstressthatincludedtheregulationofYleaf(greenhouse versus field plants) and stomatal conductance (at the leaf and whole- plant scale in a greenhouse) in response to a decrease in soil water availability, to plant water status, and to an increase in evaporative de- mand while accounting for other confounding environmental para- meters such as light level. The stem vulnerability to embolism was characterized along the growing season using different techniques [in situ flow centrifuge– and bench-based vulnerability curves, con- firmed using high-resolution computed tomography (HRCT)]. S C I E N C E A D V A N C E S \| R E S E A R C H A R T I C L E For the first time, the safety margin of a range of grapevine varieties was quantified and compared to the in situ water stress experienced in the vineyard across more than a decade in two of the world’s most impor- tant wine regions. Fig. 1. Ymdl depending on Ypd in different V. vinifera varieties. Syrah (A) and Fig. 1. Ymdl depending on Ypd in different V. vinifera varieties. Syrah (A) and Grenache (B) were measured in the field [domain “Pech Rouge” (PR); dark colors] and during a drydown experiment in greenhouse (GH; light colors). The slope s of the linear regressions was not statistically different across varieties in the field (P = 0.074) nor in the greenhouse (P = 0.225). Top left insets represent the slope sx (sensitivity to declining water availability) depending on the range of Ypd (from 0 to x; x representing the lower limit of the range of Ypd) combining field and greenhouse data. Bottom right insets represent the correlation between Ypd and minimum Ymdl (that is, average value of three lowest Ymdl per 0.1-MPa wide class of Ypd) combining field and greenhouse data. This linear regression is used to define the lower range of the hydroscape (colored by a blue-to-red gradient representing increasing water stress on the main figure). partially isohydric behavior was observed in potted plants (s = 0.861 ± 0.025 MPa MPa−1, mean ± SE; P < 0.001). Synthesizing published studies on these varieties suggests a similar variability between field (16) and potted plants (26). RESULTS AND DISCUSSION l h d b The ratio between belowground and aboveground biomass was substantially higher in the field than in the greenhouse and likely re- sulted in differences in accessing soil water. Consequently, the apparent behavior in response to drought may be related to the range of Y explored by the plant. Compared to field conditions, greenhouse plants experienced much more negative Ypd (−1.4 and −2.9 MPa in the field and greenhouse, respectively). Therefore, we explored changes in s across an expanding range of Ypd:sx. The slope sx var- ied widely (top insets in Fig. 1). Under moderate stress (Ypd > −1.5 MPa), both varieties behaved as extremely anisohydric (sx > 1) and then anisohydric (sx = 1) for Ypd = −1.5 MPa, whereas they became partially isohydric (sx < 1) under more severe levels of stress (Ypd < −1.5 MPa). At moderate stress, Syrah exhibits a more pronounced anisohydric behavior according to sx, which converges with Grenache as stress levels increase. The current definition regarding the behavior of these varieties may be improved by representing the behavior as dy- namic across the (an-)isohydric spectrum, representing an anisohydric to isohydric transition as stress increases. on February 13, 2018 http://advances.sciencemag.org/ Stomatal closure and reductions in transpiration should occur before the onset of embolism (3). Recent work in Vitis vinifera suggests that this is indeed the case, where stem embolism occurs at lower (more negative) Y [Y inducing 50% loss of hydraulic conductivity (Y50) < −1.7 MPa] than those typically observed for stomatal closure [Yleaf < −1.0 MPa (18)]. The distance between the minimum stem water potential (Ystem) experienced in the field (Ymin) and the Y50 repre- sents the hydraulic safety margin (19, 20). The smaller this margin, 1 of 9 Charrier et al., Sci. Adv. 2018;4:eaao6969 31 January 2018 Unraveling (an-)isohydric behavior by integrating responses to soil water deficit and vapor pressure deficit Syr, Syrah; Gre, Grenache; 110R, 110 Richter. then it would be observed at the stomatal level as well. However, no significant differences across four contrasting varieties were observed in the decline of stomatal conductance in response to Ymdl at the leaf (gs) or whole-plant (Gs) scale (P = 0.558 and 0.164 for gs and Gs, respec- tively; Fig. 2, A and B). Equally, no difference was observed in response to Ypd (P = 0.836; fig. S3). Drawing firm conclusions about differ- ences in leaf-level stomatal conductance (Fig. 2A) is difficult because of the intrinsic variability in the measurement, but this parameter was as- sessed to complement our whole-plant data (Fig. 2B), which is extreme- ly robust across the full range of Y (approximately 5000 data points per variety).These findings are consistent with the lack of differences in leaf intrinsic water-use efficiency reported between isohydric and aniso- hydric genotypes (17). y g yp The transition from anisohydry to isohydry observed here would imply a stronger control of gs or Gs, especially to D, as drought stress increases. Using a lysimeter platform, we assessed the real-time whole- plant Gs over a 2-month period, exploring the dynamic change in stomatal sensitivity to D during increasing drought stress (fig. S4). To visualize this relationship between Gs and D as a function of plant water status, we used the framework established by Oren et al. (28) in which sensitivity to vapor pressure deficit [that is, dGs/Ln (D)] is plotted against Gs at1-kPavaporpressure deficit (Gsref) (Fig.2C). In allvarieties, Gs did not decrease with increasing D until Ypd was lower than −0.5 MPa (Fig. 2C, inset, and fig. S4). Dynamic changes in sensitivity revealed that the Y threshold at which Gs became sensitivitivity to D was similar acrossvarieties:Ypd = −0.72 ± 0.03MPa (−0.67, −0.69, −0.71, and −0.80 MPa in Grenache, Syrah, V. riparia, and 110 Richter, respectively). Furthermore, when drought stress increased, the ratio between Gsref and sens became constant, reaching the generic value of 0.6 that as- sumes tight stomatal regulation of Yleaf across isohydric species (28). Fig. 2. Response of stomatal conductance depending on decreasing Y in different V. vinifera varieties. (A) Midmorning (9:00 a.m. to 12:00 p.m.) stomatal conductance measured on individual leaves (gs), depending on Ymdl in four grapevine varieties [V. vinifera cv. Grenache (n = 58; blue), V. vinifera cv. Unraveling (an-)isohydric behavior by integrating responses to soil water deficit and vapor pressure deficit Syrah (n = 61; red), 110 Richter (n = 48; green), and V. riparia (n = 51; pink)] during a drydown experiment in greenhouse. (B) Midmorning (9:00 a.m. to 12:00 p.m.) stomatal conductance calculated at the whole-plant scale (Gs; obtained from 150 individuals over 2 months) depending on Yleaf. Symbol and bars represent the mean and SEs of 0.1-MPa classes. Lines repre- sent the best fit using sigmoid functions for each variety. (C) Sensitivity to vapor pres- sure deficit [that is, dGs/Ln(D)] depending on Gsref in four grapevine varieties (V. vinifera cv. Grenache and Syrah, 110 Richter, and V. riparia), along a water deficit gradient [Ymdl, from −0.8 (blue) to −2.3 MPa (red)]. Gsref and sensitivity were calculated as the inter- cept and slope of the logarithmic correlation between Gs and D (as presented in fig. S3) from a sliding frame of 500 consecutive values sorted by increasing drought stress. The line (slope = 0.6) represents the theoretical slope between stomatal con- ductance at D = 1 kPa and stomatal sensitivity to D, which is consistent with the role of stomata in regulating minimum Yleaf in isohydric species according to the hydraulic limitation theory (29). The inset represents the estimates (±SE) of Gsref and sensitivity at four different level of drought stress: well hydrated (>−0.5 MPa) in blue, mild stress (−0.5; −1.0 MPa) in green, moderate stress (−1.0; −1.5 MPa) in yellow, and severe stress (<−1.5 MPa) in red in four different varieties of grapevine. Syr, Syrah; Gre, Grenache; 110R, 110 Richter. These dynamics in stomatal regulation with respect to D under decreasing water availability may explain the continuum between ani- sohydric and isohydric behaviors (8). Well-watered plants exhibited wide variations in s because Ymdl is mainly driven by daily variations in transpiration, and thus, by environmental conditions [that is, light, temperature, and D (29)]. Although plants can exhibit differences under very moderate stress, these differences disappear as water stress in- creases (Ypd < −0.8 MPa). These dynamic relationships with respect to soil water and D likely explain the resulting confusion, with respect to either Y or stomatal regulation, in assigning a single behavior. Understanding a variety’s behavior under drought requires examining stomatal responses with respect to both soil water and D simultaneously across the full range of potential stress levels. Here, we present one pos- sible framework to visualize and quantify soil water deficit/D dynamics in an integrated fashion. Unraveling (an-)isohydric behavior by integrating responses to soil water deficit and vapor pressure deficit 1), whereas Traditionally, the stomatal response to drought stress has been used to assign the (an-)isohydric behavior (7, 14, 16). For instance, anisohydric plants could sustain longer periods of transpiration and photosynthesis under water scarcity, allowing them to be more drought-tolerant than isohydric species (27). If grapevine varieties would regulate Y differently, 2 of 9 Charrier et al., Sci. Adv. 2018;4:eaao6969 31 January 2018 S C I E N C E A D V A N C E S \| R E S E A R C H A R T I C L E Stem vulnerability is dynamic, closely following drought Fig. 2. Response of stomatal conductance depending on decreasing Y in different V. vinifera varieties. (A) Midmorning (9:00 a.m. to 12:00 p.m.) stomatal conductance measured on individual leaves (gs), depending on Ymdl in four grapevine varieties [V. vinifera cv. Grenache (n = 58; blue), V. vinifera cv. Syrah (n = 61; red), 110 Richter (n = 48; green), and V. riparia (n = 51; pink)] during a drydown experiment in greenhouse. (B) Midmorning (9:00 a.m. to 12:00 p.m.) stomatal conductance calculated at the whole-plant scale (Gs; obtained from 150 individuals over 2 months) depending on Yleaf. Symbol and bars represent the mean and SEs of 0.1-MPa classes. Lines repre- sent the best fit using sigmoid functions for each variety. (C) Sensitivity to vapor pres- sure deficit [that is, dGs/Ln(D)] depending on Gsref in four grapevine varieties (V. vinifera cv. Grenache and Syrah, 110 Richter, and V. riparia), along a water deficit gradient [Ymdl, from −0.8 (blue) to −2.3 MPa (red)]. Gsref and sensitivity were calculated as the inter- cept and slope of the logarithmic correlation between Gs and D (as presented in fig. S3) from a sliding frame of 500 consecutive values sorted by increasing drought stress. The line (slope = 0.6) represents the theoretical slope between stomatal con- ductance at D = 1 kPa and stomatal sensitivity to D, which is consistent with the role of stomata in regulating minimum Yleaf in isohydric species according to the hydraulic limitation theory (29). The inset represents the estimates (±SE) of Gsref and sensitivity at four different level of drought stress: well hydrated (>−0.5 MPa) in blue, mild stress (−0.5; −1.0 MPa) in green, moderate stress (−1.0; −1.5 MPa) in yellow, and severe stress (<−1.5 MPa) in red in four different varieties of grapevine. Charrier et al., Sci. Adv. 2018;4:eaao6969 31 January 2018 Stem vulnerability is dynamic, closely following drought intensity along the season In light of the data presented here, the traditional Grenache/Syrah iso/anisohydric contrast is more nuanced. The relationship between Gsref and sens (Fig. 2C) suggests a more anisohydric behavior for Syrah, but these differences are slight and are significant only when partic- ular frameworks are used (that is, Figs. 1 and 2A and fig. S2). In the field, Syrah experiences slightly more negative Y than Grenache (Fig. 1 and fig. S2), but at these levels of stress, Gs is nearly zero, suggesting that these differences have to be due to factors other than stomatal regulation per se (for example, differences in cuticular transpiration and/or root hydraulic conductance). Approaches to breeding drought resistance in Vitis based solely on phenotyping differences in stomatal regulation appear insufficient and may benefit from using a more detailed, holistic, and integrative approach like the one used in this study. Despite the fact that V. vinifera is adapted to environments exper- iencing seasonal drought, studies differ in concluding whether it is sensitive (30) or resistant to embolism (31). Because of the similarity in regulating Y and stomatal conductance described above, a variety’s hydraulic safety margin would ultimately depend on its xylem vul- nerability. Therefore, we may expect contrasting varieties to exhibit significant differences in this drought-related trait. Long stems from dif- ferent varietieswere centrifuged to mimic drought stressandinduce loss of hydraulic conductivity using the in situ flow centrifuge technique equipped with a 1-m-large rotor. Contrary to all expectations, no signif- icant differences were observed across V. vinifera varieties (P = 0.67 and 0.09 in July and September, respectively; Fig. 3, A and B). 3 of 9 S C I E N C E A D V A N C E S \| R E S E A R C H A R T I C L E These results were confirmed using the bench dehydration method combined with the gravimetric method (Fig. 3C). Different V. vinifera varieties consequently exhibit a similar hydraulic vulnerability. hydraulic conductivity was stable from June to October (P = 0.387), and the mean vessel lengths were shorter than 66 cm, changes in hydraulic vulnerability across the season can only be attributed to ontogenic changes. Increased lignification of stem tissues, along with increased thickness of pit membranes from spring to autumn, likely contributes to this increasing resistance to air seeding (33). Drought exposure (that is, minimum Ystem) is dynamic over the course of the growing season, although no compensatory seasonal changes in stem hydraulic vulnerability have been observed in tree species (32). Therefore, we could expect the safety margin to be dy- namic in relation with the change in drought exposure. Unexpectedly, we found that stem vulnerability was dynamic over the growing season (Y50 shifted from −2.07 ± 0.08 to −2.82 ± 0.03 MPa in July and Septem- ber, respectively; Fig. 3). Furthermore, the Y50 values observed here were lower than those observed at an earlier stage of plant development (May) reported using HRCT [that is, <−1.7 MPa (18)] and were almost identical with the Y50 values observed in Chardonnay of similar age via nuclear magnetic resonance (31), both noninvasive methods using intact plants. Because plants were continuously well watered from budburst, Stems originating from rootstocks appeared slightly but significantly more resistant than those of V. vinifera [lower Y50 (P < 0.001) and slope (P < 0.001; Fig. 3B)]. However, stem vulnerability did not differ across six different grafted combinations between the scions (Grenache or Syrah) and rootstocks [Selection Oppenheim 4 (SO4), Vitis riparia ‘Gloire de Montpellier’ (RGM), or 110 Richter] for Y50 nor for slope (fig. S5). Previous studies reported changes in drought tolerance conferred by the identity of the rootstock (34). Because no difference was ob- served across varieties grafted onto different rootstocks, it is unlikely that these observed differences were brought about by differences in scion stem vulnerability. plants. Because plants were continuously well watered from budburst, Fig. 3. Hydraulic vulnerability to drought of different grapevine varieties, seasons, and techniques. (A to C) Percent stem loss of hydraulic conductivity de- pending on applied pressure (A and B) or minimum Ystem experienced by the plant (C). S C I E N C E A D V A N C E S \| R E S E A R C H A R T I C L E Vulnerability curves were obtained either during the growing season [n = 6 to 8; July (A)] or after growth cessation [n = 6 to 8; September (B)], using a dedicated 1-m- diameter Cavitron device. FC, flow centrifuge. Loss of hydraulic conductivity was also measured using the bench dehydration (BD) technique combined with the gravimetric method after relaxation of the tension in the xylem sap [n = 15 to 20 (C)]. Lines and colored areas represent modeled vulnerability curves and the confidence interval at 95% for each model. Vineyards maintain a significant hydraulic safety margin Unrecoverable water potential [that is, the Y below which transpir tion cannot be restored to normal values after rewatering (Yrecov varies between plant taxa and has been observed at Y50 in conife (3) and at higher losses of conductivity in angiosperms [Y88 (35) even Y99 (36)]. We hypothesized that Yrecov for grapevine would similar to other angiosperms. Plants exposed to various levels drought stress during the greenhouse experiment were rewater to field capacity, and stomatal conductance was monitored at the le and plant scale for several weeks. Surprisingly, stomatal conductan of leaves on rewatered plants did not recover to control levels wh Ypd reached values lower than Y50 (that is, Yrecov; Fig. 4). Howev the Yrecov was similar across varieties [approximately −2.76 ± 0. and −2.61 ± 0.08 MPa (that is, approximately Y50) at the leaf an whole-plant scale, respectively]. Although some individuals produc new leaves that exhibited normal transpiration, stressed leaves d not recover, most likely because of an increase in leaf abscisic ac concentration (37) and/or petiole and leaf vein embolism. Limit Fig. 3. Hydraulic vulnerability to drought of different grapevine varieties, seasons, and techniques. (A to C) Percent stem loss of hydraulic conductivity de- pending on applied pressure (A and B) or minimum Ystem experienced by the plant (C). Vulnerability curves were obtained either during the growing season [n = 6 to 8; July (A)] or after growth cessation [n = 6 to 8; September (B)], using a dedicated 1-m- diameter Cavitron device. FC, flow centrifuge. Loss of hydraulic conductivity was also measured using the bench dehydration (BD) technique combined with the gravimetric method after relaxation of the tension in the xylem sap [n = 15 to 20 (C)]. Lines and colored areas represent modeled vulnerability curves and the confidence interval at 95% for each model. Fig. 4. Vineyards maintain a significant hydraulic safety margin Vineyards maintain a significant hydraulic safety margin Unrecoverable water potential [that is, the Y below which transpira- tion cannot be restored to normal values after rewatering (Yrecov)] varies between plant taxa and has been observed at Y50 in conifers (3) and at higher losses of conductivity in angiosperms [Y88 (35) or even Y99 (36)]. We hypothesized that Yrecov for grapevine would be similar to other angiosperms. Plants exposed to various levels of drought stress during the greenhouse experiment were rewatered to field capacity, and stomatal conductance was monitored at the leaf and plant scale for several weeks. Surprisingly, stomatal conductance of leaves on rewatered plants did not recover to control levels when Ypd reached values lower than Y50 (that is, Yrecov; Fig. 4). However, the Yrecov was similar across varieties [approximately −2.76 ± 0.07 and −2.61 ± 0.08 MPa (that is, approximately Y50) at the leaf and whole-plant scale, respectively]. Although some individuals produced new leaves that exhibited normal transpiration, stressed leaves did not recover, most likely because of an increase in leaf abscisic acid concentration (37) and/or petiole and leaf vein embolism. Limited on February 13, 2018 cemag.org/ Fig. 4. Leaf mortality (percent of whole leaves per plant) and recovery time (that is, inverse of the time to recover half of control gs) versus minimum Ypd experienced by different grapevine varieties. The intersection between linear regression and x axis gives the minimum Yrecov for each variety. Lines and colored areas represent modeled leaf mortality curves and the confidence interval at 95% level for each model (n = 21 to 24). The inset represents the re- covery time measured via Gs (that is, whole-plant stomatal conductance). Fig. 3. Hydraulic vulnerability to drought of different grapevine varieties, seasons, and techniques. (A to C) Percent stem loss of hydraulic conductivity de- pending on applied pressure (A and B) or minimum Ystem experienced by the plant (C). Vulnerability curves were obtained either during the growing season [n = 6 to 8; July (A)] or after growth cessation [n = 6 to 8; September (B)], using a dedicated 1-m- diameter Cavitron device. FC, flow centrifuge. Loss of hydraulic conductivity was also measured using the bench dehydration (BD) technique combined with the gravimetric method after relaxation of the tension in the xylem sap [n = 15 to 20 (C)]. S C I E N C E A D V A N C E S \| R E S E A R C H A R T I C L E Leaf mortality (percent of whole leaves per plant) and recovery tim (that is, inverse of the time to recover half of control gs) versus minimu Ypd experienced by different grapevine varieties. The intersection betwe linear regression and x axis gives the minimum Yrecov for each variety. Lin and colored areas represent modeled leaf mortality curves and the confiden interval at 95% level for each model (n = 21 to 24). The inset represents the covery time measured via Gs (that is, whole-plant stomatal conductance). S C I E N C E A D V A N C E S \| R E S E A R C H A R T I C L E vinifera (see Fig.1; colored by a blue-to-red gradient representing increasing water stress), and the box plots depict the distribution of Ystem in September observed during a decade in two regions of grapevine production (Saint-Emilion and Napa Valley). PLC, percentage loss of conductance. (B and C) The observed range of stem xylem pressure (that is, limited by the average of the three minimum Ystem observed under field conditions) in Saint- Emilion (B; over the 2003–2016 period in Cabernet Franc and Merlot) and in Napa Valley (C; over the 2010–2016 period in Cabernet Sauvignon, Cabernet Franc, Syrah, and Merlot) per month. In parallel, the dynamic patterns of Y12 (green squares; mean ± SE) and Y50 (blue circles; mean ± SE) along the growing season, based on results obtained using HRCT in May (18) and Cavi-1000 (July, September, and October on different Vitis varieties—V. vinifera cv. Cabernet Sauvignon, Grenache, Merlot, Regent, and Syrah). –1.5 –1.0 –0.5 0.0 1 mm 1 mm Predawn Midday Water potential (MPa) Fig. 6. Distribution of Ypd (blue) and Ymds (red) water potential observed during June and July in Napa Valley in V. vinifera cv. Syrah. The insets repre- sent transverse HRCT images of intact plants from the same variety at Y under this normal operating range: from predawn (Ypd = −0.15 MPa) to midday (Ymds = −1.3 MPa). Theoretical loss of hydraulic conductivity for each image is calculated from functional (gray) and air-filled (black) xylem vessels and indicated as PLC (%). White bars, 1 mm. transpiration, combined with the hydraulic vulnerability segmentation observed in grapevine petioles, appears to favor leaf shedding under strong and prolonged stress, buffering the perennial organs from reach- ing lower Y (18). Our greenhouse study demonstrate that when the minimum Y leads to 50% loss of stem hydraulic conductivity (that is, Y50), the vine sheds essentially all of its leaves (leaf mortality, 83.6%; Fig. 5A). Further- more, under the most extreme conditions observed over approximately a decade in two major cultivation areas, minimal Ystem never reached Y50 [that is, Saint-Emilion and Napa Valley; Fig. 5], although there was severe leaf drop. These data represent a broad range of field conditions and both nonirrigated (Saint-Emilion and Napa Valley) and irrigated (Napa Valley) vineyards. S C I E N C E A D V A N C E S \| R E S E A R C H A R T I C L E S C I E N C E A D V A N C E S \| R E S E A R C H A R T I C L E Fig. 5. Physiological thresholds for drought-induced mortality in stems and leaves versus long-term drought survey in Napa Valley and Saint-Emilion. (A) The upper panel depicts stomatal conductance (gs; green), leaf mortality(brown), and stemloss of hydraulic conductivity (red) depending on organ Y. Thethreecurves describe an average V. vinifera variety because no significant differences were observed across varieties in the responses of these physiological parameters to Y during the greenhouse experiment [namely, Y inducing 50% reduction in stomatal conductance (dotted line), leaf vitality (short-dashed line), and stem hydraulic conductivity (long-dashed line)]. The lower panel shows the standardized hydroscape for V. vinifera (see Fig.1; colored by a blue-to-red gradient representing increasing water stress), and the box plots depict the distribution of Ystem in September observed during a decade in two regions of grapevine production (Saint-Emilion and Napa Valley). PLC, percentage loss of conductance. (B and C) The observed range of stem xylem pressure (that is, limited by the average of the three minimum Ystem observed under field conditions) in Saint- Emilion (B; over the 2003–2016 period in Cabernet Franc and Merlot) and in Napa Valley (C; over the 2010–2016 period in Cabernet Sauvignon, Cabernet Franc, Syrah, and Merlot) per month. In parallel, the dynamic patterns of Y12 (green squares; mean ± SE) and Y50 (blue circles; mean ± SE) along the growing season, based on results obtained using HRCT in May (18) and Cavi-1000 (July, September, and October on different Vitis varieties—V. vinifera cv. Cabernet Sauvignon, Grenache, Merlot, Regent, and Syrah). Fig. 5. Physiological thresholds for drought-induced mortality in stems and leaves versus long-term drought survey in Napa Valley and Saint-Emilion. (A) The upper panel depicts stomatal conductance (gs; green), leaf mortality(brown), and stemloss of hydraulic conductivity (red) depending on organ Y. Thethreecurves describe an average V. vinifera variety because no significant differences were observed across varieties in the responses of these physiological parameters to Y during the greenhouse experiment [namely, Y inducing 50% reduction in stomatal conductance (dotted line), leaf vitality (short-dashed line), and stem hydraulic conductivity (long-dashed line)]. The lower panel shows the standardized hydroscape for V. S C I E N C E A D V A N C E S \| R E S E A R C H A R T I C L E Midsummer is the period with the smallest hy- draulic safety margin, and using the thresholds determined in this study, the most extreme drought events would have only generated an ~25% loss in stem hydraulic conductivity (Fig. 5B). Under the normal operating range of Y typically observed in Napa Valley in July (from Ypd = −0.36 ± 0.01 MPa to Ymds = −1.15 ± 0.03 MPa, mean ± SE), V. vinifera cv. Syrah would not present significant level of xylem embolism (Fig. 3A), and this is corroborated in intact plants using HRCT (Fig. 6). Predawn Midday Predawn Midday Fig. 6. Distribution of Ypd (blue) and Ymds (red) water potential observed during June and July in Napa Valley in V. vinifera cv. Syrah. The insets repre- sent transverse HRCT images of intact plants from the same variety at Y under this normal operating range: from predawn (Ypd = −0.15 MPa) to midday (Ymds = −1.3 MPa). Theoretical loss of hydraulic conductivity for each image is calculated from functional (gray) and air-filled (black) xylem vessels and indicated as PLC (%). White bars, 1 mm. Whereas the extent of drought stress can increase along growing sea- son, so does resistance to embolism. Furthermore, Y and gs regulation prevents Ystem from reaching these critical levels, and further protection is proved by petiole embolism and leaf mortality. Although extreme seasons surely result in significant leaf mortality and crop loss, more severe drought-induced embolism appears to be uncommon for grapevine. Nevertheless, the midsummer, withitssmaller safety margin, likely represents a critical period when considering management under extreme drought. viticultural sustainability and sound water management strategies. Stomatalregulation betweensupposedcontrasting varietieswasstrikingly similar and dynamic across the (an-)isohydric spectrum, representing an anisohydric to isohydric transition as stress increases. Vulnerability to embolism was almost identical between varieties and was not influ- enced by rootstock. Nevertheless, it is dynamic across the growing sea- son, decreasing as stress increases. When compared to the drought stress experienced over decades in two of the world’s premiere wine re- gions, severe drought-induced embolism appears to be uncommon for Vineyards maintain a significant hydraulic safety margin Lines and colored areas represent modeled vulnerability curves and the confidence interval at 95% for each model. Fig. 3. Hydraulic vulnerability to drought of different grapevine varieties, seasons, and techniques. (A to C) Percent stem loss of hydraulic conductivity de- pending on applied pressure (A and B) or minimum Ystem experienced by the plant (C). Vulnerability curves were obtained either during the growing season [n = 6 to 8; July (A)] or after growth cessation [n = 6 to 8; September (B)], using a dedicated 1-m- diameter Cavitron device. FC, flow centrifuge. Loss of hydraulic conductivity was also measured using the bench dehydration (BD) technique combined with the gravimetric method after relaxation of the tension in the xylem sap [n = 15 to 20 (C)]. Lines and colored areas represent modeled vulnerability curves and the confidence interval at 95% for each model. Fig. 4. Leaf mortality (percent of whole leaves per plant) and recovery time (that is, inverse of the time to recover half of control gs) versus minimum Ypd experienced by different grapevine varieties. The intersection between linear regression and x axis gives the minimum Yrecov for each variety. Lines and colored areas represent modeled leaf mortality curves and the confidence interval at 95% level for each model (n = 21 to 24). The inset represents the re- covery time measured via Gs (that is, whole-plant stomatal conductance). p linear regression and x axis gives the minimum Yrecov for each variety. Lines and colored areas represent modeled leaf mortality curves and the confidence interval at 95% level for each model (n = 21 to 24). The inset represents the re- covery time measured via Gs (that is, whole-plant stomatal conductance). 4 of 9 Charrier et al., Sci. Adv. 2018;4:eaao6969 31 January 2018 MATERIALS AND METHODS Greenhouse experiment Plant material The whole-plant stomatal conductance Gs (in mol m−2 s−1 MPa−1) can be calculated as follows (29) Gs ¼ KGðTÞ⋅ktot AL ⋅Ys Yg VPD ð2Þ ð2Þ where KG(T) is the conductance coefficient (in kPa·m3 kg−1) (39), ktot is the whole-plant hydraulic conductance (in mol s−1 MPa−1), Ys and Yg are the Y in soil and guard cells, respectively (in megapascals), and D is the vapor pressure deficit (in kilopascals, calculated from temperature and relative humidity data, as indicated in the infrared gas analyzer manual WALZ GFS-3000). where KG(T) is the conductance coefficient (in kPa·m3 kg−1) (39), ktot is the whole-plant hydraulic conductance (in mol s−1 MPa−1), Ys and Yg are the Y in soil and guard cells, respectively (in megapascals), and D is the vapor pressure deficit (in kilopascals, calculated from temperature and relative humidity data, as indicated in the infrared gas analyzer manual WALZ GFS-3000). g y Whole-plant conductance ktot was calculated as p p y Every 2 to 3 days, Ypd was measured on one basal leaf per plant before any light exposure (between 5:00 a.m. and 7:00 a.m.) on three to five plants per variety and treatment. Between 12:00 p.m. and 2:00 p.m., minimum Yleaf (Ymdl) were measured on the same plants, on fully expanded leaves on which gas exchange was measured (see below). For hydraulic conductivity measurements, Ymds values were measured between 12:00 p.m. and 2:00 p.m. on fully expanded leaves, which were baggedinplastic coveredwithaluminumbagsfrom7:00a.m. AllY values were measured using a Scholander pressure chamber (Precis 2000). ktot ¼ Dw Ys Yg ⋅MMw ð3Þ ð3Þ Finally, using Eqs. 1 to 3, Gs was subsequently calculated as Gs ¼ KGðTÞ⋅ E VPD ð4Þ ð4Þ On every sampled leaf, leaf area and length (using a leaf area meter; WinFOLIA 2007b, Regent Instrument) and dry weight (after oven drying for 1 week at 60°C) were measured (approximately 200 leaves per variety). Dead leaves (visual estimation) were counted on every sampled (hydraulic measurement) or under recovery plant. Balance data analysis y All 132 pots were weighed every 15 min for approximately 2 months on individual scales (CH15R11, OHAUS-type CHAMP). Transpiration per leaf area (E in mol m−2 s−1) was estimated as CONCLUSIONS Here, we define the dynamic hydraulic safety margin for grapevine acrossthegrowingseasonandestablishY thresholdscriticalinincreasing Charrier et al., Sci. Adv. 2018;4:eaao6969 31 January 2018 5 of 9 S C I E N C E A D V A N C E S \| R E S E A R C H A R T I C L E system including an infrared gas analyzer (GFS 3000, WALZ). Conditions in the cuvette were set steady to conditions that were sub- optimal but easily reproducible all along the experimental period [that is, photosynthetically active radiation (PAR), 1500 mmol m−2 s−1; tem- perature, 20°C, vapor pressure deficit (D), 1 kPa; and CO2, 400 parts per million). system including an infrared gas analyzer (GFS 3000, WALZ). Conditions in the cuvette were set steady to conditions that were sub- optimal but easily reproducible all along the experimental period [that is, photosynthetically active radiation (PAR), 1500 mmol m−2 s−1; tem- perature, 20°C, vapor pressure deficit (D), 1 kPa; and CO2, 400 parts per million). grapevine. Because of their perennial nature, vineyards are expected to be productive for decades and require a significant up-front investment. Extreme drought can result in vineyards being fallowed; this was the case for tens of thousands of acres during the recent drought in Califor- nia, with hugely negative economic consequences. The information elucidated here will aid growers in making more intelligent decisions regarding dry farming, fallowing, and irrigation management. MATERIALS AND METHODS Greenhouse experiment Plant material E ¼ Dw AL ⋅MMw ð1Þ ð1Þ Dynamic drydown of four varieties of grapevine [V. vinifera cv. Grenache; V. vinifera cv. Syrah; V. rupestris cv. Martin × berlandieri cv. Rességuier no. 2, also known as 110 Richter; and V. riparia cv. Gloire de Montpellier (38)] was measured in a mini-lysimeter greenhouse platform that mea- sures soil water availability and whole-plant transpiration for each in- dividual plant in real time. Temperature, light, and air humidity were monitored every 20 min in two positions of the greenhouse. Air tem- perature was maintained below approximately 25°C by the greenhouse cooling system, limiting D to approximately 2500 Pa. Cuttings of the year (n = 33 per variety), bearing one flushed bud, were planted into 7-liter pots filled with 1 kg of gravel and 5.5 kg of “Terre de Couhins” (20% clay, 18% silt, and 62% sand). Plants were placed in a greenhouse for 2 months before the experiment started and watered with nutritive solution [0.1 mM NH4H2PO4, 0.187 mM NH4NO3, 0.255 mM KNO3, 0.025 mM MgSO4, 0.002 mM Fe, and oligo-element (B, Zn, Mn, Cu, and Mo)] to prevent any deficiency during the experiment. Plants were not pruned or damaged during the course of the experiment to avoid air seeding in the xylem. On 16 August 2015, plants were wateredtofieldcapacity—wateredtosaturationattheendoftheafternoon and drained overnight. Drought stress was induced on 27 plants per variety by stopping irrigation at three different dates: 26 August on 12 plants per variety, 10 September on 12 plants per variety, and 22 September on 5 plants per variety. where Dw is the change in weight within the considered period (g s−1), AL is the leaf area (m2), and MMw is the molar mass of water (18 g mol−1). Unrealistic values of transpiration in the data set were filtered out. These values originated from plant watering (Dw > 0), gas exchange analysis, or other plant manipulations (Dw < −100 g hour−1). Low AL (<0.05 m2) and low Dw (<−2.5 g hour−1; close to balance weight reso- lution) led to overestimated transpiration rate and were therefore dis- carded. MATERIALS AND METHODS Greenhouse experiment Plant material measured through the sample, from an upper tank to a covered tank placed on a precision electronic balance (0.01-mg resolution; Sartorius). After steady flow was reached (after approximately 30 s to 1 min), mean flow rate was calculated as the average of 10 values measured at 10-s intervals. Flow rate was measured at four different water pres- sure gradients (ranging from 1 to 3 kPa), according to the procedure described by Torres‐Ruiz et al. (41). Hydraulic conductance of the sam- ple (ki) was calculated as the slope of the flow rate versus the applied pressure, after verification of the linearity of the relation (R2 > 0.99). Sample was then flushed at high pressure (140 kPa) for 1 min to re- move embolism, and conductance was remeasured (kmax). PLC was calculated as y In parallel, maximum and mean vessel lengths were measured by the air injection method. Compressed air was delivered to the basal end of a long stem (L > 3 m). Pressure was measured using a manom- eter and constantly set to 1.8 kPa, whereas air flow was measured using an air flow meter (F111B). The stem was cut shorter until air flowed through the samples (approximately 1 m) and every 10 cm (30- to 100-cm length) and 5 cm (5- to 30-cm length). Air flow was measured at each length after stabilization and computed as indicated by Pan et al. (43). PLC ¼ kmax ki kmax ð5Þ ð5Þ Long-term Y field monitoring Seasonal variations in Ypd, Ymdl, and Ymds water potentials were measured in V. vinifera plants growing near Bordeaux [Château Cheval Blanc, Saint-Emilion; Global Positioning System (GPS), 44.92°N, 0.19° W: Saint-Emilion], in California (Napa Valley; GPS, 38.43°N, 122.42° W: Napa Valley) and in Languedoc [Institut National de la Recherche Agronomique (INRA) Pech Rouge experimental unit (Gruissan); GPS, 43.08°N, 3.08°W: Pech Rouge]. In Saint-Emilion, two varieties (Cabernet Franc and Merlot) were monitored over the 2003–2016 period on three blocks with different soil compositions (clay, sand, and gravel). In Napa Valley, three varieties (Cabernet Franc, Cabernet Sauvignon, and Syr- ah) were monitored over the 2010–2015 period. In Pech Rouge (limestone soil), three varieties (Syrah, Grenache, and Marselan) were monitored in 2005. Y values were measured from June until harvest, every 2 weeks (Ypd in Saint-Emilion and Pech Rouge and Ymdl only in Pech Rouge) or every week (Ymds in all locations) according to the procedure detailed below. Ypd values were measured on one basal leaf per plant before any light exposure (between 3:00 a.m. and 5:00 a.m.) on 5 to 10 plants per variety and block. Between 1:00 p.m. and 3:00 p.m., Ymds values were measured on fully expanded leaves from the same plant, which were bagged in plastic covered with aluminum bags at least 1 hour before measurement. All Y values were measuredusing a Scholander pressure chamber (Saint-Emilion, Precis 2000; Napa Valley, PMS Model-1000; Pech Rouge, Soilmoisture Equipment Corp.) The theoretical specific hydraulic conductivity of a whole cross sec- tion (KH) was calculated from the Hagen-Poiseuille equation using the individual diameter of sap- and air-filled vessels as KH ¼ ∑ p⋅∅4 128⋅h⋅Axyl ð6Þ ð6Þ with KH being the specific theoretical hydraulic conductivity (in kg m−1 MPa−1 s−1), ∅being the mean Feret diameter of vessels (in meters), h being the viscosity of water (1.002 mPa·s at 20°C), and Axyl being the xylem area of the cross section (in square meters). with KH being the specific theoretical hydraulic conductivity (in kg m−1 MPa−1 s−1), ∅being the mean Feret diameter of vessels (in meters), h being the viscosity of water (1.002 mPa·s at 20°C), and Axyl being the xylem area of the cross section (in square meters). Determination of the minimum Yrecov The minimum Yrecov was determined as described by Brodribb and Co- chard (3). During the recovery phase, the time required for gs and Gs to reach50% of itsmaximumvalue (t1/2) wascalculated. Linearregressions were fitted to the inverse of t1/2 versus exposed Ypd. The Yrecov, corresponding to the Y inducing a failure of gs and Gs to recover, was assessed by determining the x intercept of the linear regression. High-resolution x-ray computed tomography Synchrotron-based computed microtomography was used to visualize air- and sap-filled vessels in the main stem of V. vinifera cv. Syrah. In spring 2015, six well-watered plants were brought to the HRCT beam- line [Pression Structure Imagerie par Contraste à Haute Énergie (PSICHE)] at the Source Optimisée de Lumière d’Energie Intermédiaire du LURE (SOLEIL) synchrotron facility and were scanned immediately and after 6 days of drydown. The Y was measured on one leaf, previously wrapped in a plastic bag for 3 hours to provide accurate Ystem values with a Scholander pressure chamber (Precis 2000). The middle part of the main stem was scanned using a high-flux (3 × 1011 photons mm−2), 25-keV monochromatic x-ray beam. The projections were recorded with a Hamamatsu ORCA-Flash sCMOS camera equipped with a 250-mm-thick LuAG scintillator. The complete tomographic scan in- cluded 1500 projections (50 ms each) for a 180° rotation. Tomographic reconstructions were performed using the PyHST2 software utilizing the Paganin method, resulting in 15363 32-bit volumetric images. The final spatial resolution was 33 mm3 per voxel. MATERIALS AND METHODS Greenhouse experiment Plant material Total plant leaf area (AL) was assessed (i) depending on the relation between leaf area and leaf length at the beginning of the experiment (every 2 weeks) and when any change in water regime happened and (ii) depending on the relation between leaf area and leaf dry weight (at the end of the exper- iment or when a plant was removed from the greenhouse, that is, for hydraulic conductivity measurement).AL was assumed to change linearly between two measurements, corrected by the area of sampled leaves. Gas exchange analysis On the basis of hourly change in weight (Dw) and relations be- tween RWC, Ypd, and Yleaf, whole-plant stomatal conductance Gs was compared to leaf level gs (R2 = 0.760; P < 0.001; fig. S6). Hydraulic conductivity y y When targeted Ymds values were reached, intact plants were brought to the laboratory for hydraulic conductivity measurements. The main stem was cut under water until a 7-cm-long piece was obtained, originating in the middle of the plant. Each cut was alternately per- formed at basal and apical end, with a 20-s delay between cuttings to relax sap tension within the xylem and prevent air bubble from ex- panding at the end of the sample (40). Both ends of the sample were refreshed with a clean razor blade and connected to a tubing system. Flow rate of a degassed KCl (10 mM) and CaCl2 (1 mM) solution was Leaf gas exchange measurements were conducted between 9:00 a.m. and 12:00 a.m. on mature, well-exposed leaves using a portable open- Charrier et al., Sci. Adv. 2018;4:eaao6969 31 January 2018 6 of 9 S C I E N C E A D V A N C E S \| R E S E A R C H A R T I C L E berlandieri, and SO4 V. berlandieri × V. riparia). The basal end of long stems (L > 3 m) was cut and shortened to 1-m length under water. The sample was flushed with air at low pressure (1 bar) to empty vessels open on both ends. Centrifugal force was used to establish neg- ative pressure in the xylem and to provoke drought stress–induced embolism, using a custom-built 1-m-diameter honeycomb rotor mounted on a high-speed centrifuge (DG MECA). The xylem pressure, ki, and PLC were determined at various speeds to obtain a vulnera- bility curve. REFERENCES AND NOTES 1. C. D. Allen, A. K. Macalady, H. Chenchouni, D. Bachelet, N. McDowell, M. Vennetier, 1. C. D. Allen, A. K. Macalady, H. Chenchouni, D. Bachelet, N. McDowell, M. Vennetier, T. Kitzberger, A. Rigling, D. D. Breshears, E. H. Hogg, P. Gonzalez, R. Fensham, Z. Zhang, J. Castro, N. Demidova, J.-H. Lim, G. Allard, S. W. Running, A. Semerci, N. Cobb, A global overview of drought and heat-induced tree mortality reveals emerging climate change risks for forests. For. Ecol. Manage. 259, 660–684 (2010). 1. C. D. Allen, A. K. Macalady, H. Chenchouni, D. Bachelet, N. McDowell, M. Vennetier, T. Kitzberger, A. Rigling, D. D. Breshears, E. H. Hogg, P. Gonzalez, R. Fensham, Z. Zhang, J. Castro, N. Demidova, J.-H. Lim, G. Allard, S. W. Running, A. Semerci, N. Cobb, A global overview of drought and heat-induced tree mortality reveals emerging climate change risks for forests. For. Ecol. Manage. 259, 660–684 (2010). where gsm corresponds to maximal stomatal conductance at Y = 0, slp is the sensitivity to decreasing Y, and gs50 is the Y inducing 50% stomatal closure. Loss of hydraulic conductivity, depending on minimal Ystem, was fit according to the sigmoid function 2. Intergovernmental Panel on Climate Change, Climate Change 2014—Impacts, Adaptation and Vulnerability: Regional Aspects (Cambridge Univ. Press, 2014) 3. T. J. Brodribb, H. Cochard, Hydraulic failure defines the recovery and point of death in water-stressed conifers. Plant Physiol. 149, 575–584 (2009). 3. T. J. Brodribb, H. Cochard, Hydraulic failure defines the recove water-stressed conifers. Plant Physiol. 149, 575–584 (2009). PLC ¼ 100 1 þ eslp⋅ðYY50Þ ð12Þ ð12Þ 4. T. J. Brodribb, N. M. Holbrook, Stomatal closure during leaf dehydration, correlation with other leaf physiological traits. Plant Physiol. 132, 2166–2173 (2003). other leaf physiological traits. Plant Physiol. 132, 2166–2173 (2003) 5. M. T. Tyree, J. S. Sperry, Do woody plants operate near the point of catastrophic xylem 5. M. T. Tyree, J. S. Sperry, Do woody plants operate near the point of catastrophic xylem dysfunction caused by dynamic water stress? Answers from a model. Plant Physiol. 88, 574–580 (1988). dysfunction caused by dynamic water stress? Answers from a model. Plant Physiol. 88, 574–580 (1988). where slp corresponds to the sensitivity to decreasing Y, and Y50 is the Y inducing 50% loss of conductivity. 6. H. G. Jones, R. A. Sutherland, Stomatal control of xylem embolism. Plant Cell Environ. 14, 607–612 (1991). SUPPLEMENTARY MATERIALS For Ypd < b 2⋅a ; Ymd ¼ minða⋅Ypd1 2 þ b⋅Ypd1 þ c; YpdÞ ð9Þ Supplementary material for this article is available at http://advances.sciencemag.org/cgi/ content/full/4/1/eaao6969/DC1 fig. S1. Ymdl depending on Ypd in different V.viniferavarietiesand under environmentalconditions. fig. S2. Correlations between Ypd and minimum Yleaf (Ymin; that is, average value of three lowest Ymdl per 0.1 MPa wide class of Ypd) in different V. vinifera varieties (Grenache and Syrah) and under environmental conditions (field and greenhouse). To extrapolate Y during the greenhouse experiment, Ypd was calculated from RWC through Campbell modified by van Genuchten equation (fig. S7) (44) fig. S3. Midmorning stomatal conductance measured on individual leaves, depending on predawn leafwater potentialYpdl in four grapevine varieties (V. vinifera cv.Grenache, blue; V.viniferacv.Syrah, red; 110 Richter, green; V. riparia, pink) during a drydown experiment in greenhouse. fig. S3. Midmorning stomatal conductance measured on individual leaves, depending on predawn leafwater potentialYpdl in four grapevine varieties (V. vinifera cv.Grenache, blue; V.viniferacv.Syrah, red; 110 Richter, green; V. riparia, pink) during a drydown experiment in greenhouse. Ypd ¼ a⋅ðRWCÞb þ Ye ð10Þ ð10Þ fig. S5. Percent stem loss of hydraulic conductivity depending on applied pressure in V. vinifera cv. Syrah and Grenache grafted on different rootstocks (V. riparia, SO4, and 110 Richter) after growth cessation (September), using a dedicated 1-m-diameter Cavitron device (Cavi-1000). fig. S6. Whole-plant stomatal conductance Gs depending on leaf-scale stomatal conductance gs, measured at the same moment (±1 hour). where Ye is the soil Y at the air entry point. where Ye is the soil Y at the air entry point. Stomatal conductance, at the leaf (gs) or plant scale (Gs), de- pending on Ymdl, was fit according to the sigmoid function fig. S7. Ypd depending on RWC in four grapevine varieties (V. vinifera cv. Grenache and Syrah, 110 Richter, and V. riparia) during a drydown experiment in a greenhouse. fig. S7. Ypd depending on RWC in four grapevine varieties (V. vinifera cv. Grenache and Syrah, 110 Richter, and V. riparia) during a drydown experiment in a greenhouse. gs ¼ gsm 1 þ eslp⋅ðYgs50Þ ð11Þ ð11Þ In situ flow centrifuge technique y The theoretical loss of hydraulic conductivity (PLC) was calculated as Xylem vulnerability to embolism was assessed on different varieties with the Cavitron technique, a centrifugation-based technique, as described by Cochard et al. (42). Four-year-old plants were not pruned or dam- aged during the course of the experiment to avoid air seeding in the xylem. Different varieties were used (on n = 5 to 6 replicates per variety), either fruit-oriented (V. vinifera Syrah, Grenache, Cabernet Sauvignon, Merlot and the interspecific hybrid Regent) or rootstock- oriented (V. riparia Gloire de Montpellier, 110 Richter V. rupestris × PLC ¼ 100⋅KHA KHmax ð7Þ ð7Þ with KHA and KHmax representing the theoretical hydraulic conductiv- ities of air-filled vessels in initial and cut cross sections, respectively. Charrier et al., Sci. Adv. 2018;4:eaao6969 31 January 2018 7 of 9 Statistical analyses and fit The slope sens and the intercept Gsref of the linear regression be- tween Gs and Ln(D) were calculated for each variety and level of water stress (Fig. 2). For a given variety, the slope sensx and the intercept Gsrefx were calculated across different ranges of Ypd. This method used a sliding window mechanism of Gs/D pair values ranked from 0 to the lowest observed Ypd value in which sensx and Gsrefx were estimated. The window was divided into a fixed number of equal-sized (500) by step increment of 1 (Fig. 2C). Statistical analyses and fit were performed using the R software (www.r-project.org). The slope s of the linear regression between Ymdl and Ypd was calculated for different varieties and growing conditions (Fig. 1 and fig. S1). For a given variety, the slope sx was calculated across different ranges of Ypd, that is, from 0 to x MPa (see top inset in Fig. 1 de- picting s in relation with x across the whole range). For each 0.1-MPaYpd class, the average of the three absolute Ymdl values was used to calculate the lower limit of the hydroscape, according to the procedure described by Meinzer et al. (10). Accounting forthe highvariability in Ymdl at high Ypd (>−0.5 MPa), nonlinear fit was also performed using a composite function Across varieties, comparisons in the parameters of different correla- tions (for example, Gsref, sens, and sx) were performed using t tests from estimate and SEs as proxy of mean and SEs. Different regressions (that is, linear and nonlinear) of the relation between Ymdl and Ypd were compared on the basis of the AIC. For Ypd > b 2⋅a ¼ Ypd1; Ymd ¼ a⋅Ypd 2 þ b⋅Ypd þ c ð8Þ For Ypd < b 2⋅a ; Ymd ¼ minða⋅Ypd1 2 þ b⋅Ypd1 þ c; YpdÞ ð9Þ For Ypd > b 2⋅a ¼ Ypd1; Ymd ¼ a⋅Ypd 2 þ b⋅Ypd þ c ð8Þ For Ypd > b 2⋅a ¼ Ypd1; Ymd ¼ a⋅Ypd 2 þ b⋅Ypd þ c ð8Þ b 2 ð8Þ S C I E N C E A D V A N C E S \| R E S E A R C H A R T I C L E Linder, Mean canopy stomatal conductance responses to water and nutrient availabilities in Picea abies and Pinus taeda. Tree Physiol. 21, 841–850 (2001). 17. C. Lovisolo, I. Perrone, A. Carra, A. Ferrandino, J. Flexas, H. Medrano, A. 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Funding: This study has been carried out with financial support from the Cluster of Excellence COTE (ANR-10-LABX-45, within Water Stress and Vivaldi projects) and AgreenSkills Fellowship program, which has received funding from the European Union’s Seventh Framework Program under grant agreement no. FP7 26719 (AgreenSkills contract 688). This work was also supported by the program “Investments for the Future” (ANR-10-EQPX-16, XYLOFOREST) from the French National Agency for Research and the program “Plan National Dépérissement du Vignoble” from the French Ministry of Agriculture (FranceAgriMer-CNIV) within the PHYSIOPATH project. This research was also supported by a grant from the French Research Agency (MARIS ANR-14-CE03-0007). Author contributions: G.C., S.D., J.-C.D., and G.A.G. designed all experiments. G.C., S.D., J.-C.D., L.Z., C.E.L.D., A.K., I.M., D.C., N.O., and G.A.G. carried out the experimentation. 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Adv. 2018;4:eaao6969 31 January 2018 drought observations in world's top wine regions Drought will not leave your glass empty: Low risk of hydraulic failure revealed by long-term drought observations in world's top wine regions Drought will not leave your glass empty: Low risk of hydraulic failure revealed by long-term drought observations in world's top wine regions Drought will not leave your glass empty: Low risk of hydraulic failure revealed by long-term Andrew King, Hernan Ojeda, Nathalie Ollat, Jorge A. Prieto, Thibaut Scholach, Paul Skinner, Cornelis van Leeuwen and Guillaume Charrier, Sylvain Delzon, Jean-Christophe Domec, Li Zhang, Chloe E. L. Delmas, Isabelle Merlin, Deborah Corso, Gregory A. Gambetta Andrew King, Hernan Ojeda, Nathalie Ollat, Jorge A. Prieto, Thibaut Scholach, Paul Skinner, Cornelis van Leeuwen and Guillaume Charrier, Sylvain Delzon, Jean-Christophe Domec, Li Zhang, Chloe E. L. Delmas, Isabelle Merlin, Deborah Corso, DOI: 10.1126/sciadv.aao6969 (1), eaao6969. 4 Sci Adv DOI: 10.1126/sciadv.aao6969 (1), eaao6969. 4 Sci Adv ARTICLE TOOLS http://advances.sciencemag.org/content/4/1/eaao6969 MATERIALS SUPPLEMENTARY http://advances.sciencemag.org/content/suppl/2018/01/29/4.1.eaao6969.DC1 REFERENCES http://advances.sciencemag.org/content/4/1/eaao6969#BIBL This article cites 43 articles, 7 of which you can access for free PERMISSIONS http://www.sciencemag.org/help/reprints-and-permissions http://advances.sciencemag.org/content/4/1/eaao6969 http://advances.sciencemag.org/content/suppl/2018/01/29/4.1.eaao6969.DC1 Terms of Service Use of this article is subject to the registered trademark of AAAS. is a Science Advances Association for the Advancement of Science. No claim to original U.S. Government Works. 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https://openalex.org/W3037872331	https://link.springer.com/content/pdf/10.1007/s10237-020-01356-x.pdf	English	null	Correction to: A two‑layer elasto‑visco‑plastic rheological model for the material parameter identification of bone tissue	Biomechanics and modeling in mechanobiology	2,020	cc-by	390	Correction to: A two‑layer elasto‑visco‑plastic rheological model for the material parameter identification of bone tissue Andreas G. Reisinger1,2 · Martin Frank2 · Philipp J. Thurner2 · Dieter H. Pahr1,2 Published online: 24 June 2020 © The Author(s) 2020 Published online: 24 June 2020 © The Author(s) 2020 Correction to: Biomechanics and Modeling in Mechanobiology https://doi.org/10.1007/s10237-020-01329-0 The original article has been corrected. The original article can be found online at https://doi.org/10.1007/ s10237-020-01329-0. CORRECTION CORRECTION Biomechanics and Modeling in Mechanobiology (2020) 19:1977 https://doi.org/10.1007/s10237-020-01356-x Biomechanics and Modeling in Mechanobiology (2020) 19:1977 https://doi.org/10.1007/s10237-020-01356-x CORRECTION Correction to: Biomechanics and Modeling in Mechanobiology https://doi.org/10.1007/s10237-020-01329-0 Open Access This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. In the original publication of the article, Eq. (29) was incor- rectly published as given below: 𝜎pr,i+1 = 𝜎trial pr,i+1 −𝛥𝛾Eprsign(𝜎trial pr,i+1), 𝜀p,i+1 = 𝜀trial p,i+1 + 𝛥𝛾sign(𝜎trial pr,i+1), 𝛼i+1 = 𝛼trial i+1 + 𝛥𝛾, fi+1 ≡0 ⎫ ⎪ ⎪ ⎬ ⎪ ⎪⎭ if f trial i+1 > 0 However, Eq. (29) should be as given below: Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 휎pr,i+1 = 휎trial pr,i+1, 휀p,i+1 = 휀trial p,i+1, 훼i+1 = 훼trial i+1 , fi+1 = f trial i+1 ⎫ ⎪ ⎪ ⎬ ⎪ ⎪⎭ if f trial i+1 ≤0 The original article can be found online at https://doi.org/10.1007/ s10237-020-01329-0. * Andreas G. Reisinger andreas.reisinger@kl.ac.at; andreas.reisinger@tuwien.ac.at * Andreas G. Reisinger andreas.reisinger@kl.ac.at; andreas.reisinger@tuwien.ac.at 1 Division Biomechanics, Department of Anatomy and Biomechanics, Karl Landsteiner University of Health Sciences, Krems an der Donau, Austria 2 Institute of Lightweight Design and Structural Biomechanics, Vienna University of Technology, Vienna, Austria .:(0123456789) 1 3
https://openalex.org/W2967730530	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0220817&type=printable	English	null	Action effect consistency and body ownership in the avatar-Simon task	PloS one	2,019	cc-by	9,456	Christian Bo¨ffelID, Jochen Mu¨sseler Christian Bo¨ffelID, Jochen Mu¨sseler Work and Cognitive Psychology, Institute of Psychology, RWTH Aachen University, Aachen, Germany a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 * boeffel@psych.rwth-aachen.de * boeffel@psych.rwth-aachen.de Editor: Jan De Houwer, Ghent University, BELGIUM Received: February 12, 2019 Accepted: July 23, 2019 Published: August 9, 2019 Copyright: © 2019 Bo¨ffel, Mu¨sseler. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. RESEARCH ARTICLE a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Abstract Putting oneself in the shoes of a digital alter ego becomes an increasingly relevant part of our everyday experience. In modern day psychology, these interactions can be examined within the frameworks of visual perspective taking and body ownership. Both target similar questions: What does it take, to become one with the avatar? When do we show the same behavior and make the same experiences, as if we were in its place? In this study, we want to address the role of action effect consistency for these concepts. We manipulated the participants’ sense of agency over a task-irrelevant avatar in a Simon task by providing either corresponding or random action effects. These effects could be either embodied and therefore linked to the avatar (Experiment 1) or independent of it (Experiment 2). We used stimulus-response compatibility effects from the avatar’s point of view as a measure for spontaneous visual perspective taking and a questionnaire to measure the perceived body ownership of the avatar. The results showed that corresponding action effects lead to increased spontaneous perspective taking of the avatar, regardless of whether the effect was linked to the avatar or not. Though the avatar compatibility effects were overall more pronounced in the embodied action effect condition. However, significant differences in per- ceived body ownership were only observed if the effects were linked to the avatar. The results might prove useful to further our understanding of subjective and objective measure- ments of interactions with avatars. OPEN ACCESS Citation: Bo¨ffel C, Mu¨sseler J (2019) Action effect consistency and body ownership in the avatar- Simon task. PLoS ONE 14(8): e0220817. https:// doi.org/10.1371/journal.pone.0220817 OPEN ACCESS Citation: Bo¨ffel C, Mu¨sseler J (2019) Action effect consistency and body ownership in the avatar- Simon task. PLoS ONE 14(8): e0220817. https:// doi.org/10.1371/journal.pone.0220817 OPEN ACCESS Citation: Bo¨ffel C, Mu¨sseler J (2019) Action effect consistency and body ownership in the avatar- Simon task. PLoS ONE 14(8): e0220817. https:// doi.org/10.1371/journal.pone.0220817 Action effect consistency and body ownership in the avatar-Simon task Christian Bo¨ffelID, Jochen Mu¨sseler Work and Cognitive Psychology, Institute of Psychology, RWTH Aachen University, Aachen, Germany Introduction Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. Avatars often serve as a means to interact with virtual worlds. Actions we perform with mice, keyboards or touchpads are transformed into movements of the avatar and such consequences of our actions are action effects. The anticipation of action effects plays a crucial role in guiding our actions and now represents a cornerstone of the cognitive psychology of action planning. The ideomotor principle for example [1] claims that we learn the relationship between our actions and the resulting action effects and that the subsequent anticipation of these action effects is a driving factor in action selection. If we experience, for example, that a certain switch turns the light on and if we want to illuminate the room, we can use this connection to select the appropriate action [2,3]. Even though this idea has caught a decent amount of criticism in the earlier days, (as was discussed by [4]) it has since been featured and expended on in promi- nent frameworks of action control such as the theory of event coding [5]. Funding: This study was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation: DFG MU 1298/11) awarded to Jochen Mu¨sseler and is associated with the DFG Priority Program "The Active Self" (DFG SPP 2134). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. 1 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0220817 August 9, 2019 Avatar action effects and body ownership One way to examine the influence of action effects on our behavior is to apply the concept of response-effect compatibility. It has been known for quite a while that certain combinations of stimuli and responses lead to a performance advantage over others, an idea known as stimu- lus-response compatibility (for an overview see: [6]). A frequent observation is that tasks with a spatial overlap between stimuli and responses lead to faster responses compared to tasks without such an overlap [7]. A typical approach to study these effects is the so-called Simon task, in which the participants respond to a certain stimulus feature, such as its color, while the task-irrelevant stimulus position is varied systematically. Introduction Even though the stimulus location is task-irrelevant, the conditions with corresponding stimulus and response locations lead to a performance advantage compared to the non-corresponding conditions [8]. A similar ap- proach can be applied to examine the relationship between responses and their action effects. In a series of experiments, Kunde [3] systematically manipulated this relationship. In the first experiment, he asked the participants to perform one of four key presses and each of the four keys was assigned a color. The task was to press the key with the same color as a stimulus pre- sented on a computer screen. After the participants gave their responses, a white box was dis- played on the screen as an action effect. In the congruent condition, the position of the action effect matched the position of the key. In the incongruent condition, the action effect was dis- played at a different position instead. The results revealed a significant reaction time advantage for the congruent condition over the incongruent one. Because the action effect followed the response, it is impossible that the action effect directly affects the response, if we only look at a single isolated trial. Instead, the results are only plausible if the participants did indeed learn the action-action effect relation and therefore anticipated the action effect in later trials. These correspondence advantages in response-effect pairings are not limited to spatial effect features and can for example also be observed in intensity [9], duration [10] or valence [11]. Using spatially neutral stimuli Ansorge [12] was able to demonstrate the importance of intention in effect compatibility. The participants were asked to move a centrally presented stimulus to the left or right by pressing left or right keys and Ansorge observed a compatibility effect with faster reaction times when the direction of the movement and the position of the key corresponded. However, this was only the case, when the participants were instructed to perform this movement and therefore had the intention to move the stimulus [12]. Following the reasoning of the theory of event coding [5], action-effect integration should take place if both, the action and its effect, overlap in time [13] and it has been shown that this integration is disrupted if the delay between action and effect is larger than 1 second [14]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0220817 August 9, 2019 Introduction In general, three major conditions have been identified that benefit action-action effect anticipa- tion: 1) A small delay between action and effect, 2) the effect only occurs rarely without the action and 3) the effect appears sufficiently often [14]. But how does action effect anticipation work, if the effect is linked to another agent? At first glance it seems difficult to anticipate an action effect that is performed by or through someone else. But we know that people interacting with avatars often show the same behavioral tenden- cies we would expect if they were actually in the avatar’s place [15,16]. If we present an avatar next to a vertical stimulus set in an orthogonal Simon task, we observe similar compatibility effects, as if the stimuli were regarded from the avatar’s perspective, overwriting the orthogonal Simon effect. A stimulus at the top position is compatible with a left response, when the avatar is on the left side but compatible to a right response, when the avatar is on the right. The usu- ally observed advantage of top to right and bottom to left mappings compared to the reverse is eliminated [15]. Such avatar-based compatibility effects can be interpreted within the frame- work of visual perspective taking [17], but also fall in line with predictions by the referential coding account [18,19]. In the current study, we will focus on the human-like aspect of the ava- tar and therefore think that the visual perspective taking framework is more appropriate, 2 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0220817 August 9, 2019 Avatar action effects and body ownership especially since broader definitions of visual perspective taking are not limited to real humans or animated objects as perspective taking targets [20]. Recent studies have shown that the per- spective information provided by the avatars is processed automatically, which differentiates avatars from simple objects with perspective information, such as arrows [21]. If these avatars also exhibit movements that correspond to the person’s own responses, this seems to further facilitate perspective taking of the avatar [15]. Pfister et al. [22] also investigated action effect compatibility between participants and a virtual agent and found that responses were facili- tated when the virtual agent imitated the duration of a key press performed by the participants compared to conditions in which a different duration was shown as an action effect. But the action effect itself is not the only important factor. Introduction It is also crucial how the effect is interpreted. This can have a significant influence on subjective measures of the interaction experience such as perceived body ownership of the avatar [23], the feeling that the avatar is a part of the person’s own body [24]. In a design context, for example in game design, it might be worthwhile to dilute the distinction between a person and her/his avatar in order to increase immersion [25] and create a more realistic and engaging experience. But not only subjective measures are affected by the interpretation of action effects. A simi- lar case can be made for objective measures of visuo-spatial perspective taking, for example measured as compatibility effects in reaction times from the avatar’s point of view. In a recent study [23], we influenced the participant’s interpretation of an action effect by giving two dif- ferent explanations of the exact same action effect. The participants performed a stimulus- response compatibility task from the avatar’s point of view and the avatar always moved the hand that corresponded to the response hand. However, the interpretation of this effect was manipulated in the instruction: One group was told that the action effect stems from the ava- tar’s own intention to move its hands and is only indirectly influenced by the participants. The other group was instructed that they directly control the avatar akin to a tool. Even though both conditions were identical on a purely physical level, the instruction differences led to dif- ferent results. We found that the direct control group showed stronger compatibility effects from the avatar’s point of view and reported higher levels of perceived body ownership of the avatar compared to the indirect control group [23]. These results are particularly interesting, because they seem to call the social aspect of visual perspective taking into question. The indi- rect control condition described an independent agent and was therefore closer to a real inter- action scenario than the direct control condition, which resembled a tool-use scenario. In the following experiments, we wanted to further examine the role of such action effects for perspective taking when performing a task with an avatar. In contrast to the top-down approach of [23], we aimed to employ a bottom-up approach by not explicitly stating the nature and role of the action effects. Introduction Instead, our goal was to systematically vary the correspon- dence between the response location and the effect location, as well as the relationship between effect and avatar. Experiment 1 The first experiment used avatar hand movements as action effects in a Simon task akin to the paradigm described in [15] (for a similar approach with hands instead of full avatars see [26]). The participants responded to the color of discs presented above or below the screen center with right or left key presses. Additionally, an avatar was presented either on the right or left side facing the stimuli. Based on the results of previous studies, we expected that the stimulus positions would be coded within the reference frame provided by the avatar on a horizontal axis. This was expected to cause spatial correspondence between the now left and right stimuli to the left and right responses. The resulting compatibility effects from the avatar’s point of PLOS ONE \| https://doi.org/10.1371/journal.pone.0220817 August 9, 2019 3 / 15 Avatar action effects and body ownership view were used as an objective measure for visual perspective taking. Following the partici- pant’s key press, the avatar moved either its right or left hand as an action effect and the partic- ipants performed the task in two different conditions: In the corresponding condition, the action effect was always performed by the avatar’s hand that corresponded to the participant’s response hand. Even though the connection between action and effect was not revealed in the instruction and the action effect was not defined as the action goal, we expected that the acqui- sition of the action-effect connection would be rather easy, because it followed the principle of anatomical correspondence and was shown instantaneously. We also predicted that this condi- tion would lead to relatively high values of perceived body ownership of the avatar, as the ava- tar and the action effect would both be included in the response code. Because a predictable action effect allows the participants to reliably interact with the scenario on the screen, the action effect and therefore the avatar are valuable to evaluate a performed action or to plan a future one. In contrast to this, we also used a random action effect condition, in which the action effect did not follow anatomical correspondence but was instead random, with an expected value of 50% corresponding and non-corresponding action effects. We expected that this condition would lead to difficulties connecting action and effect and make it less likely that the effect enters the response code necessary for action planning. Experiment 1 This should result in less perceived body ownership of the avatar. Because the avatar and the effect are not a reliable source of information for action planning, the influence of the avatar should be reduced and the compatibility effect from the avatar’s point of view diminished, indicating a decrease of spontaneous perspective taking. In Summary, we expected the following results: Firstly, we expected to replicate the overall performance advantage of conditions that are compatible from the avatar’s point of view, with faster response times and fewer errors, com- pared to incompatible conditions. Secondly, we predicted that this avatar-Simon effect is larger in the corresponding action effect condition, compared to the random action effect condition. Thirdly, we expected higher reported ownership and agency values with corresponding com- pared to random action effects. Method However, it is likely that the avatar effect in this study could be even larger if it is enhanced by the action effect manipulation. Apparatus and stimuli. The stimuli were presented on a 22” CRT monitor with a resolu- tion of 1024x768 pixels at a refresh-rate of 100 Hz. Matlab and the Psychtoolbox-3 Extension [29,30] were used to control the experiment. The participants were seated approximately 60 cm in front of the monitor and performed left or right key presses on a horizontally oriented keypad. Stimuli were dark and light blue discs (25 pixels in diameter) presented either above a below a fixation cross and in front of an avatar (240x190 pixels, Fig 1) that was facing the screen center, either from the left or right. The stimuli were presented against a gray background. Procedure. The participants were instructed to perform key presses based on the color of the disc. These discs were presented in the middle of the screen on two vertical positions, equi- distant above or below a central fixation cross. Additionally, an avatar was presented on the right or left of the stimulus set, facing the center of the screen and the stimuli. Consequently, the avatar’s hands were pointing towards both stimulus positions (Fig 1 top row). After a block of 192 trials, the avatar switched positions from left to right or from right to left depending on the starting position that was counterbalanced between participants. The discs’ color (light or dark blue) determined the required response. Half the participants were instructed to perform a left key press after a light blue disc, and a right key press after a dark blue disc. The other half used the opposite mapping. After the participants gave their response, the target disappeared and an action effect in the form of an avatar hand movement was displayed, as soon as possible on the next valid frame (Fig 1 bottom row). The delay was kept to a minimum to ensure that response and effect could be integrated into the same event file [14]. In the random condition, the movement was performed with a probability of 50% either with the left or right hand, while in the corresponding condition it was always performed with the corresponding hand of Fig 1. Avatars in Experiment 1. Avatars used in the experiment with the avatar on the left side. Method Participants. A total of 24 students (22 female) from RWTH Aachen University with a mean age of M = 20.9 (SD = 2.2) participated in this experiment for course credit. All partici- pants reported normal or corrected-to-normal vision and were naïve to the purpose of this study. All participants gave written and informed consent in accordance with the Declaration of Helsinki (2013) and participation was voluntary. Further, no undue physical or psychological stress by participating in this study was anticipated and the data obtained on individual partici- pants were not used to elucidate properties of the participant but to examine general laws of cognitive information processing. Furthermore, the study did not involve deception and had no risk of physical injury. Because the study is a basic reaction time experiment, it did not pose a risk of psychological, social or economic harm to the participants. As a result, no ethical con- cerns were identified in accordance to the ethics guidelines of the DFG (Deutsche Forschungs- gemeinschaft, [German Research Foundation], 2009 [27]) and therefore no further evaluation by an ethics committee was sought. The sample size was determined using GPower [28] and the predicted effect size is based on a rough estimate of effect sizes typically observed in similar paradigms. In a previous study [15, Experiment 1], we found an overall avatar compatibility effect of ηp 2 = .40 and a non-cen- trality parameter of λ = 14.8, with the same sample size of 24 participants. If we take this effect as a reference point, the current study is expected to have a power of approximately (1-β) = 4 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0220817 August 9, 2019 Avatar action effects and body ownership .96, with α = .05 with regards to the avatar-compatibility main effect in the within-subjects ANOVA. Since we had no data to estimate the size of the avatar-compatibilityaction effect interaction, we decided to base our sample size on the main effect instead. However, it is likely that the avatar effect in this study could be even larger if it is enhanced by the action effect manipulation. .96, with α = .05 with regards to the avatar-compatibility main effect in the within-subjects ANOVA. Since we had no data to estimate the size of the avatar-compatibilityaction effect interaction, we decided to base our sample size on the main effect instead. PLOS ONE \| https://doi.org/10.1371/journal.pone.0220817 August 9, 2019 Method The top row shows the initial setup before a response was given, the bottom row illustrates the action effects. https://doi org/10 1371/journal pone 0220817 g001 Fig 1. Avatars in Experiment 1. Avatars used in the experiment with the avatar on the left side. The top row shows the initial setup before a response was given, the bottom row illustrates the action effects. Fig 1. Avatars in Experiment 1. Avatars used in the experiment with the avatar on the left side. The top row shows the initial setup before a response was given, the bottom row illustrates the action effects. https://doi org/10 1371/journal pone 0220817 g001 https://doi.org/10.1371/journal.pone.0220817.g001 5 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0220817 August 9, 2019 Avatar action effects and body ownership the avatar. The following trial was initiated after a 1500 ms interval. False responses and responses faster than 100 ms or slower than 1500 ms led to an error sound to discourage time- outs and responses based on anticipations. The intertrial interval was prolonged for an addi- tional 1500 ms for each error sound. The participants performed two sets of blocks of either condition (random or correspond- ing), each of them with the avatar being on the left or right, followed by two blocks of the other condition. As a result, action effect correspondence switched from random to corresponding after half of the experiment was completed. After each quarter of the experiment, the avatar’s position changed from left to right or vice versa. The avatar’s starting position and the starting action effect correspondence were counterbalanced between participants. All other factors var- ied on a trial by trial basis and occurred equally often with randomized order in each block. Each block started out with 32 practice trials and was followed by a total of 160 trials with 40 repetitions of each condition. After each half of the experiment, the perceived ownership of the avatar and agency values were estimated using a questionnaire (Table 1). It contained 10 items [23] based on an earlier version of the body ownership questionnaire [31,32] that was adapted to fit the avatar scenario and in which the participants had to state their agreement to 10 items on a 7-point Likert scale. Items 1, 2 and 9 are targeting the ownership illusion and the remaining items can be regarded as filler items [33]. Method Additionally, one agency item was added as a manipulation check that asked the participants to estimate how often (in %) they felt responsible for the action effect location. The questionnaire was used in a German version and an English translation of the items is shown in Table 1. Design. The avatar’s position, the position of the stimuli and the response positions were collapsed into one factor: avatar compatibility. This factor describes stimulus-response com- patibility from the avatar’s perspective and key presses were compatible to stimuli presented on the same side, as seen from the avatar’s point of view. The second factor was action effect (corresponding or random) resulting in a 2x2 within-subjects design with repeated measures on both factors. Results The first 32 of all blocks were practice trials and not recorded. Outliers (5.5%) were identified using the Tukey criterion (1.5 times the interquartile range above the third or below the first quartile of the reaction time distribution, separately for each person and factor level) and removed from the reaction time analysis. Additionally, false responses (4.3% of all trials) were removed and analyzed separately. We used a 2x2 ANOVA with repeated measures on both Table 1. Items used in the ownership questionnaire. The items are based on the instruments developed by Botvinick and Cohen [31] and Ma and Hommel [32] . O1: It felt as if the avatar’s hands were part of my body. O2: It seemed that my hand was in the location where the hand of the avatar was. O3: I lost the feeling where my hands were located. O4: It seemed that my hands were no longer part of my body. O5: I had the feeling that I might have additional hands. O6: Sometimes I felt as if my hands were turning virtual. O7: The hands of the avatar began to resemble my hands in terms of shape or skin tone. O8: It appeared (visually) as if the hands of the avatar were drifting towards my hands. O9: It seemed like I could have moved the hand on the screen if I wanted, as if it were obeying my will. O10: It felt as if my hands took on the same size as the avatar’s hands. A: How often (in %) did you feel that your action controlled the location of the hand movement? https://doi.org/10.1371/journal.pone.0220817.t001 The first 32 of all blocks were practice trials and not recorded. Outliers (5.5%) were identified using the Tukey criterion (1.5 times the interquartile range above the third or below the first quartile of the reaction time distribution, separately for each person and factor level) and removed from the reaction time analysis. Additionally, false responses (4.3% of all trials) were removed and analyzed separately. We used a 2x2 ANOVA with repeated measures on both Table 1. Items used in the ownership questionnaire. The items are based on the instruments developed by Botvinick and Cohen [31] and Ma and Hommel [32] . Table 1. Items used in the ownership questionnaire. The items are based on the instruments developed by Botvinick and Cohen [31] and Ma and Hommel [32] . PLOS ONE \| https://doi.org/10.1371/journal.pone.0220817 August 9, 2019 Results Mean Reaction times (RT) in ms (left) and percentage errors (PE, right) as a function of avatar compatibility and action effect. Error bars represent 95% within-subject CIs [35]. https://doi.org/10.1371/journal.pone.0220817.g002 Fig 2. Results of Experiment 1. Mean Reaction times (RT) in ms (left) and percentage errors (PE, right) as a function of avatar compatibility and action effect. Error bars represent 95% within-subject CIs [35]. https://doi.org/10.1371/journal.pone.0220817.g002 factors (avatar compatibility and action effect) to analyze mean correct reaction times (RT) and percentage errors (PE). We report a ηp 2 as an effect size estimate that accounts for the correla- tion between repeated measures [34]. Reaction times. The results showed a significant main effect of avatar compatibility, F (1, 23) = 86.60, p < .001, ηp 2 = .790. Avatar-compatible conditions were associated with a reaction time advantage of 33 ms over avatar-incompatible conditions (Fig 2). We also observed a sig- nificant interaction of avatar compatibility and action effect, F (1, 23) = 14.93, p = .001, ηp 2 = .394. The avatar compatibility effect was more pronounced with corresponding action effects (Mcorresp. = 46 ms) compared to random action effects (Mrnd. = 21 ms). Post-hoc two-tailed t-tests showed, that the avatar-Simon effect was significant in both action effect conditions with t(23) = 4.97, p < 0.01 in the random and t(23) = 8.42, p < 0.01 in the corresponding action effect condition. Percentage errors. We observed a significant main effect of avatar compatibility of F(1, 23) = 12.70, p = .002, ηp 2 = .356 with a 2.1%-points avatar compatibility effect in favor of com- patible conditions. Furthermore, avatar compatibility and action effect interacted significantly with F (1, 23) = 5.02, p = .035, ηp 2 = .179 and the compatibility effect was larger in the corre- sponding conditions (Mcorresp. = 2.8%-points) compared to the random condition (Mrnd. = 1.4%-points). The avatar-Simon effect was significant in the corresponding action effect condi- tion with t(23) = 4.43, p < 0.01 but not in the random action effect condition t(23) = 1.90, p = 0.07. Questionnaire data. One answer to the agency question (Table 1: A) was missing and this person was therefore excluded from the analysis of this question. If a person answered the agency question by giving an interval estimate (e.g. “40–50%”), the central value of this interval was used as the answer in the following analysis. Results O1: It felt as if the avatar’s hands were part of my body. O2: It seemed that my hand was in the location where the hand of the avatar was. O3: I lost the feeling where my hands were located. O4: It seemed that my hands were no longer part of my body. O5: I had the feeling that I might have additional hands. O6: Sometimes I felt as if my hands were turning virtual. O7: The hands of the avatar began to resemble my hands in terms of shape or skin tone. O8: It appeared (visually) as if the hands of the avatar were drifting towards my hands. O9: It seemed like I could have moved the hand on the screen if I wanted, as if it were obeying my will. O10: It felt as if my hands took on the same size as the avatar’s hands. A: How often (in %) did you feel that your action controlled the location of the hand movement? https://doi.org/10.1371/journal.pone.0220817.t001 Table 1. Items used in the ownership questionnaire. The items are based on the instruments developed by Botvinick and Cohen [31] and Ma and Hommel [32] . O1: It felt as if the avatar’s hands were part of my body. O2: It seemed that my hand was in the location where the hand of the avatar was. O3: I lost the feeling where my hands were located. O4: It seemed that my hands were no longer part of my body. O5: I had the feeling that I might have additional hands. O6: Sometimes I felt as if my hands were turning virtual. O7: The hands of the avatar began to resemble my hands in terms of shape or skin tone. O8: It appeared (visually) as if the hands of the avatar were drifting towards my hands. O9: It seemed like I could have moved the hand on the screen if I wanted, as if it were obeying my will. O10: It felt as if my hands took on the same size as the avatar’s hands. A: How often (in %) did you feel that your action controlled the location of the hand movement? https://doi.org/10.1371/journal.pone.0220817.t001 6 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0220817 August 9, 2019 Avatar action effects and body ownership Fig 2. Results of Experiment 1. PLOS ONE \| https://doi.org/10.1371/journal.pone.0220817 August 9, 2019 Results To avoid effects of suggestibility, we calcu- lated a corrected ownership score for each participant, by subtracting the mean of the filler items (Q3-8, Q10) from the mean of the ownership items proper (Q1, Q2, Q9), as proposed by [36]. A significantly positive value indicates that ownership is experienced. The answers of the single agency question and the ownership scores were evaluated sepa- rately using one tailed t-tests for repeated measurements, because we predicted higher owner- ship scores and agency estimates in the corresponding condition. This way, we obtained a corrected ownership score of 0.83, (95% CI [0.49, 1.17]) in the corresponding action effect condition, and a score of 0.35 (95% CI [0.07, 0.64]) in the random action effect condition that are significantly different from one another t(23) = 3.17, p = .002, one tailed. The partici- pants also reported significantly different values of perceived agency, t(22) = 4.41, p < .001, one tailed, between both action effect conditions. The reported values were higher with 7 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0220817 August 9, 2019 Avatar action effects and body ownership corresponding action effects (Mcorresp. = 68.5%) compared to the random action effect condi- tion (Mrnd. = 35.0%). corresponding action effects (Mcorresp. = 68.5%) compared to the random action effect condi- tion (Mrnd. = 35.0%). Discussion The results confirm the expected importance of spatially corresponding and therefore predict- able and reliable action effects when interacting with an avatar. The participants reported higher perceived agency, which shows, that the action effect manipulation was successful and perceived consciously. The corrected ownership scores confirm that the ownership illusion was present in both action effect conditions and not only a result of suggestibility. Addition- ally, the participants had an easier time experiencing body ownership of the avatar in the cor- responding condition, which makes us believe that corresponding action effects help to destroy the perceptual barrier between oneself and the avatar. A process that can be described as self-other merging [37] and that leads to the person and the avatar forming a functional unit. A similar result was obtained with regards to the observed avatar-compatibility effects. Even though both, the corresponding and random action effect condition, revealed a compati- bility effect from the avatar’s point of view, the effect was significantly larger in the correspond- ing condition. This experiment therefore provides additional evidence that body ownership and perspective taking of an avatar are related [23]. This leads us to two conclusions: 1. Corresponding action effects aid spontaneous visual perspective taking and 2. random action effects are insufficient when it comes to eliminating the avatar-based compatibility effects. One possible reason for the second observation might be that the hand movement itself is such a natural and therefore easily anticipated effect, that it is not readily disrupted by action-effect inconsistencies. It therefore seems to be a plausible next step to eliminate the anatomical link between avatar and action effect in order to examine, if this leads to an elimination of the action effect influence. Experiment 2 The second experiment destroyed the anatomical connection between avatar and action effect by replacing the hand movement with the display of a red disc as an avatar-independent action effect. We expected that this change would disrupt the forming of an action-action effect con- nection related to the avatar because the effect was no longer linked to the avatar and it turned the task into a basic Simon task with an additional action effect manipulation, that just hap- pened to have an avatar nearby. Even though the participants’ responses were still followed by action effects, they had no control over the avatar itself, because it never moved. In contrast to past studies that used comparable action effect manipulations in a Simon task, e.g. [38], the effect in our study was not defined as the action goal. As a result, the influence of action effect correspondence might vanish as well (compare [39–41]). However, we still expected that stim- ulus-response compatibility would be determined by the avatar’s position, as is the case in sim- ilar tasks without action effects [15]. Method We used the same procedure as in Experiment 1 but changed the material slightly: Instead of an avatar hand movement, we now used the appearance of a red disc (25 pixels in diameter) as the action effect (Fig 3). We changed the agency question accordingly and asked participants how often (in %) they felt that their response determined the location of the red disc. A total of 24 students (21 female), with a mean age of M = 21.5 (SD = 3.2) participated in the second experiment. 8 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0220817 August 9, 2019 Avatar action effects and body ownership Fig 3. Avatars in Experiment 2. Avatars used in the second experiment with the avatar on the left side. The top row shows the initial setup before a response was given, the bottom row illustrates the two possible action effects. https://doi.org/10.1371/journal.pone.0220817.g003 Fig 3. Avatars in Experiment 2. Avatars used in the second experiment with the avatar on the left side. The top row shows the initial setup before a response was given, the bottom row illustrates the two possible action effects. Fig 3. Avatars in Experiment 2. Avatars used in the second experiment with the avatar on the left side. The top row shows the initial setup before a response was given, the bottom row illustrates the two possible action effects. https://doi.org/10.1371/journal.pone.0220817.g003 https://doi.org/10.1371/journal.pone.0220817.g003 https://doi.org/10.1371/journal.pone.0220817.g003 PLOS ONE \| https://doi.org/10.1371/journal.pone.0220817 August 9, 2019 Results We performed the same analysis as in Experiment 1 and excluded 2.8% errors and 4.7% outli- ers from the RT analysis, based on the same criteria. We performed the same analysis as in Experiment 1 and excluded 2.8% errors and 4.7% outli- ers from the RT analysis, based on the same criteria. Reaction times. The results revealed a significant main effect of avatar compatibility, F (1, 23) = 82.84, p < .001, ηp 2 = .783. Conditions that were compatible from the avatar’s point of view yielded a reaction time advantage of 23 ms over incompatible ones (Fig 4). We further observed a significant interaction of avatar compatibility and action effect, F (1, 23) = 26.30, p < .001, ηp 2 = .533. The avatar compatibility effect was again larger with corresponding action effects (Mcorresp. = 31 ms) compared to random action effects (Mrnd. = 15 ms). The post-hoc t- tests revealed, that the avatar-Simon effect was significant in both action effect conditions with t(23) = 4.94, p < 0.01 with random and t(23) = 10.79, p < 0.01 with corresponding action effects. Fig 4. Results of Experiment 2. Mean Reaction times (RT) in ms (left) and percentage errors (PE, right) as a function of avatar compatibility and action effect. Error bars represent 95% within-subject CIs [35]. https://doi.org/10.1371/journal.pone.0220817.g004 Fig 4. Results of Experiment 2. Mean Reaction times (RT) in ms (left) and percentage errors (PE, right) as a function of avatar compatibility and action effect. Error bars represent 95% within-subject CIs [35]. https://doi.org/10.1371/journal.pone.0220817.g004 PLOS ONE \| https://doi.org/10.1371/journal.pone.0220817 August 9, 2019 9 / 15 Avatar action effects and body ownership Percentage errors. The data revealed a significant main effect of avatar compatibility with F (1, 23) = 19.06, p < .001, ηp 2 = .453. Avatar-compatible conditions led to 2%-points fewer errors compared to incompatible conditions. A significant interaction effect was not observed in the error percentages. The avatar-Simon effect was significant in both action effects condi- tions with t(23) = 2.95, p = 0.07 in the random and t(23) = 4.81, p < 0.01 in the corresponding action effect condition. Questionnaire data. Two participants did not answer the agency question and were excluded from the analysis of this question. Discussion The results of the second experiment show several parallels to the first. For example, we repli- cated the avatar-based compatibility effect in both, corresponding and random action effects and again, the avatar-compatibility effect was larger with corresponding in comparison to ran- dom action effects. However, the overall avatar-compatibility effects were numerically smaller than in Experiment 1 and an additional analysis is needed to investigate if this difference is sta- tistically relevant (see below). Interestingly, even though the avatar itself did not move in Experiment 2, the unrelated action effect still affected the compatibility effects and therefore spontaneous perspective taking. This means that effects that are unrelated to the avatar can still increase its influence and even coincidental events could potentially influence avatar- based compatibility effects. Based on the questionnaire data, we can conclude that the action effect manipulation led to different questionnaire results compared to Experiment 1. The corrected ownership scores were significantly positive with either action effect type and did not differ between the two and there is likely no significant difference in perceived ownership of the avatar between corre- sponding and random action effects. This makes it plausible that the action effect has to be linked to the avatar to be relevant when determining perceived body ownership of the avatar. If the action effect is independent and therefore not directly linked to the avatar, it seems only logical that it is perceived differently. But it is important to note, that the results still indicate that the action effect is regarded within the reference frame provided by the avatar, or else we would not have been able to observe the interaction between avatar-compatibility and action effect. Overall, we observed a disparity between two measures of a person’s identification with an avatar. Objective measures of spontaneous perspective taking were influenced by the type of action effects, even when they were not linked to the avatar itself. The participants showed greater compatibility effects from the avatar’s perspective with corresponding action effects compared to random action effects, indicating improved perspective taking. However, the sub- jective measures of body ownership revealed that this action effect manipulation did not alter the participants’ subjective experience, even though the answers to the agency question showed that the manipulation itself was noticed. Results We observed a significant difference in perceived agency between random and corresponding action effects, as reported in the agency item, t(21) = 2.02, p = .028, (one tailed), with higher values in the corresponding action effect condi- tions (Mcorresp. = 46.1%) compared to the random action effect conditions (Mrnd. = 31.2%). Corrected ownership scores were calculated as in Experiment 1 and we obtained a score of 0.56, (95% CI [0.21, 0.90]) in the random condition and a score of .53 (95% CI [0.20, 0.87]) in the corresponding action effect condition. These corrected ownership scores were not signifi- cantly larger in the corresponding condition t(23) = 0.25, p = .403, one tailed. PLOS ONE \| https://doi.org/10.1371/journal.pone.0220817 August 9, 2019 Comparing Experiment 1 and 2 To examine whether the differences between both experiments are statistically significant, a combined ANOVA with the additional factor experiment was conducted. The results of the RT analysis revealed that the avatar compatibility effect was larger in the first experiment (ME1 = 33 ms) with anatomically linked action effects compared to the second experiment (ME2 = 23 ms) with independent action effects, resulting in a significant two-way interaction of experi- ment and avatar compatibility with F (1, 46) = 4.774, p = .034, ηp 2 = .094. However, the avatar compatibility effect was not differently influenced by the action effect correspondence between both experiments and the related three-way interaction was not significant F (1, 46) = 1.54 p = .221, ηp 2 = .032. p The analysis of percentage errors did not reveal any significant influence of the factor experiment. The analysis of the questionnaire data revealed that the two experiments did not signifi- cantly differ in overall agency ratings: F (1, 43) = 2.84 p = .099, ηp 2 = .062. Furthermore, the difference between random and corresponding action effects in perceived agency was compa- rable in both experiments with an interaction of experiment and action effect of F (1, 43) = 3.09, p = .086, ηp 2 = .067. However, while both experiments did result almost identical perceived ownership overall, F (1, 46) = .05, p = .82, ηp 2 = .001, the interaction effect of experiment and action effect was signif- icant with F (1, 46) = 6.47, p = .014, ηp 2 = .123 (Fig 5) and revealed larger differences between random and corresponding action effects in Experiment 1 compared to Experiment 2. Discussion While it has been shown in the past that body owner- ship and perspective taking of an avatar are related [23], the results of the present study show that they can be differently influenced by the same manipulation. It therefore seems likely that PLOS ONE \| https://doi.org/10.1371/journal.pone.0220817 August 9, 2019 10 / 15 Avatar action effects and body ownership Fig 5. Mean reported agency and corrected ownership scores of Experiment 1 (E1) and 2 (E2). Error bars represent 95% between-subject CIs. Fig 5. Mean reported agency and corrected ownership scores of Experiment 1 (E1) and 2 (E2). Error bars represent 95% between-subject CIs. https://doi.org/10.1371/journal.pone.0220817.g005 https://doi.org/10.1371/journal.pone.0220817.g005 a successful manipulation of body ownership might cause an increase in spontaneous perspec- tive taking, however, this is probably not a necessary condition, because differences in perspec- tive taking can be achieved by manipulations that do not influence perceived body ownership. a successful manipulation of body ownership might cause an increase in spontaneous perspec- tive taking, however, this is probably not a necessary condition, because differences in perspec- tive taking can be achieved by manipulations that do not influence perceived body ownership. General discussion The results of both experiments allow us to draw several conclusions: First, it has proven useful to implement corresponding action effects if the goal is to facilitate spontaneous visual per- spective taking of the avatar. With corresponding action effects, the avatar can be seen as a tool that transforms a key press (proximal effect) into a distal effect (changes on the screen) and this transformation might follow similar rules as the use of simpler tools [42]. This seems to be an overarching phenomenon as it is not limited to anatomically linked action effects and can 11 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0220817 August 9, 2019 Avatar action effects and body ownership instead also be observed for independent action effects. If we look at the compatibility differ- ences between both experiments, the effects are overall larger in the anatomically linked action effect scenario of Experiment 1. So, if we want to maximize spontaneous perspective taking, we should use a setup with reliable and linked action effects. But if our design space is limited to non-embodied action effects, we should also pay attention to action effect correspondence in order to maximize perspective taking, a result that seems surprising from the standpoint of embodiment. We can also think of several cases where a different design goal might be more important. For example, in an application that uses virtual agents in a teaching context or in a navigation tool, it could prove detrimental if the users spontaneously take this avatar’s perspec- tive over their own. In this case, actively avoiding any effect that could be interpreted as an action effect might be beneficial, especially, if the action effect is performed by the agent and always corresponds to the user’s input. However, the results of the subjective measurements might indicate that the situation is dif- ferent when it comes to perceived body ownership of the avatar. Here, the embodied nature of the action effect likely comes into play. While corresponding and anatomically linked action effects do produce the highest reported body ownership values, the case of independent action effects is not as clear-cut. In an independent action effect scenario, action effect correspon- dence seems to be of secondary importance as it does not seem to significantly increase per- ceived ownership compared to the random condition. General discussion When contrasting the ownership values of both experiments, the experiment—action effect interaction did not reach significance, even though it came very close. One possible reason for this is, that the subjective nature of the mea- surement is less reliable compared to the objective reaction time measurements. This is enhanced by the fact that the comparisons within each experiment were within-subject com- parisons, while the comparisons between the experiments are influenced by between-subjects variance. It is also possible that there are big interindividual differences when it comes to the interpretation of the action effects. It has been shown in the past that people can be categorized as either “embodiers” or “systemizers” when they complete similar tasks [43]. “Embodiers” usually show strong embodiment effects that are reduced or absent in “systemizers”. It is possi- ble that similar effects are at work in the current study, and that some people have an easier time experiencing an action effect as linked to the avatar or themselves compared to others. Overall this points us to the conclusion that our perception of the avatar and the observed compatibility effects from its point of view can be disjointed in certain set-ups. Whereas in more natural settings that use linked and therefore easily anticipated action effects, different mechanisms seem to be relevant when it comes to the perception of independent action effects. Whether these effects are social in nature is still unclear. However, the conditions with reliable and corresponding action effects share a lot of characteristics with tool-use scenarios. In this case, the avatars themselves are not social agents but means of interaction with a virtual envi- ronment. Additionally, if we expect that avatars can be fully integrated into a person’s self- representation, then the social aspect is again lost, because the avatar and the person form a functional unit. With this in mind, theoretical frameworks that do not rely on the social aspect of the human-avatar interaction—such as referential coding [19] or a broad definition of per- spective taking [20]—appear to suited best in order to explain the underlying mechanisms of avatar-based compatibility effects. Conclusion The goal of this experiment was to examine the role of action effects for spontaneous perspec- tive taking and perceived body ownership of task-irrelevant avatars. While past studies were able to demonstrate a relationship between both concepts using a top-down approach [23], 12 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0220817 August 9, 2019 Avatar action effects and body ownership this connection was only found in a bottom-up approach here, when the action effect was linked to the avatar. Even though we observed differences in avatar-compatibility between both experiments, the fundamental mechanism seems to hold true in both cases: The spatial coding of the scene seems to always occur with regards to the reference frame provided by the avatar and we can use stimulus-response compatibility to objectively measure these effects. In contrast to this, the subjective experience appears to rely more heavily on the nature of the action effect. Both questions regarding the action effect examined in this study seem to matter for perceived body ownership of the avatar: 1. Is the effect linked to the avatar? and 2. Is the effect predictable and reliable? This leads us to an interesting distinction between objective and subjective measures of the human-avatar interaction and different approaches should be used depending on the features of the scenario in question and the limitations of the design space in order to maximize the identification with the avatar or to prevent it. Acknowledgments The authors thank Marina Papke for her assistance in recruiting participants and data collection. Supporting information S1 File. Data. This dataset contains mean reaction times, error rates and questionnaire results per condition and participant. S2 File. Readme. This file contains brief explanations of the variables reported in the dataset. (TXT) Author Contributions Conceptualization: Christian Bo¨ffel, Jochen Mu¨sseler. Conceptualization: Christian Bo¨ffel, Jochen Mu¨sseler. Data curation: Christian Bo¨ffel. Data curation: Christian Bo¨ffel. Formal analysis: Christian Bo¨ffel. Investigation: Christian Bo¨ffel. Methodology: Christian Bo¨ffel. Project administration: Jochen Mu¨sseler. Project administration: Jochen Mu¨sseler. Resources: Jochen Mu¨sseler. Software: Christian Bo¨ffel. Supervision: Jochen Mu¨sseler. Validation: Christian Bo¨ffel. Visualization: Christian Bo¨ffel. Writing – original draft: Christian Bo¨ffel. Writing – review & editing: Jochen Mu¨sseler. 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https://openalex.org/W4289078052	https://zenodo.org/records/6945068/files/89-91.pdf	Tagalog	null	TALABALAR HAYOTIDA STRESSNI BOSHQARISHNING AHAMIYATI	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	645	ANNOTATSIYA Maqolada stressni boshqarishning talabalar hayotidagi ahamiyati, uning kelib chiqishiga sabab bo‘ladigan asosiy omillar haqida ma’lumot berilgani. Bundan tashqari, stressning talabalar sog‘lig‘iga va o‘qish jarayonlariga bo‘lgan salbiy ta’siri, stressni qanday boshqarish mumkinligi haqida ham qayd etilgan. Kalit so‘zlar: Stress, stressni boshqarish, talabalar, stressning talabalarga ta’siri. ABSTRACT The article provides information on the importance of stress management in students life and the main factors that cause it. In addition, the negative effects of stress on students’ health and learning processes, and how to manage stress are also noted. Keywords: Stress, stress management, students, effects of stress on students. VOLUME 1 \| ISSUE 4 \| 2022 VOLUME 1 \| ISSUE 4 \| 2022 ISSN: 2181-3191 RESEARCH AND EDUCATION KIRISH Stressni boshqarish keng spektrli texnikadir va psixoterapiya shaxsning darajasini boshqarishga qartilgan stress, ayniqsa surunkali stress, odatda, kundalik faoliyatni takomillashtirish maqsadi uchun. Stress har bir kishining vaziyat omillariga qarab o‘zgarib turadigan ko‘plab jismoniy va ruhiy alomatlar keltirib chiqaradi. Bularga 89 https://t.me/ResearchEdu_Journal Multidisciplinary Scientific Journal July, 2022 89 RESEARCH AND EDUCATION VOLUME 1 \| ISSUE 4 \| 2022 ISSN: 2181-3191 jismoniy sog‘liqning pasayishi ham kiradi. Stressni boshqarish jarayoni zamonaviy jamiyatda baxtli va muvaffaqiyatli hayotning kalitlaridan biri deb nomlangan. Garchi hayotni hal qilish qiyin bo‘lgan ko‘plab talablarni taqdim etsa-da, stressni boshqarish tashvishlarni boshqarish va umumiy farovonlikni saqlashning bir qancha usullarini taqdim etadi. Stress ko‘pincha sub’ektiv tajriba deb hisoblansa ham, stress darajasi har xil fiziologik testlardan foydalangan holda o‘xshashlik bilan aniqlanadi. Turli xil stressni boshqarish usullarining samaradorligini baholash qiyin bo‘lishi mumkin, chunki hozirda cheklangan tadqiqotlar mavjud. Binobarin, turli xil texnikalar uchun dalillarning miqdori va sifati juda farq qiladi. Ko‘pchilik talabalar o‘qish maqsadida o‘z uylaridan uzoqda yashay boshlaydi.Bu yerda ular notanish insonlar orasida bo‘ladi, darslar, imtihonlar va kurs ishlarini bajarishda qiyinchiliklarga duch keladi. Bu, o‘z navbatida, talabalardagi stress holatining kelib chiqishidagi asosiy omillardir. Stress talabalarning sog‘lig‘iga va o‘qish jarayonlariga salbiy ta’sir ko‘rsatishi mumkin. Shu sababli talabalar hayotida stressni boshqarish muhim ahamiyat kasb etadi. Talabalardagi stressning ularga bo‘lgan ta’siri, stressga sabab bo‘ladigan asosiy omillar va stressni boshqarishga yordam beradigan vositalar haqida shifokorlar,psixologlar va turli soha vakillari o‘z fikr va mulohazalarini bildirgan. RESEARCH AND EDUCATION ISSN: 2181-3191 4) Stimulyatorlarga tayanmaslik 4) Stimulyatorlarga tayanmaslik 5) Rejalarni aniq belgilash, kun tartibiga rioya qilish 6) Kechiktirishlardan saqlanish 7) Stressni yo‘qotishga harakat qilish. Elizabet Skottning ta’kidlashicha: “Ko‘pgina talabalar stressdan holi bo‘lish zarurligini biladi, ammo turli tadbirlar, darslarga bor vaqtini sarflaydi va stressni yo‘qotishga yordam beruvchi vositalarni sinab ko‘rishga vaqt topolmaydi. Natijada, stress uyqu tartibi, ovqatlanish kabi salomatlik bilan bog‘liq xatti - harakatlarga, shuningdek, talabalarning baholariga ham ta’sir ko‘rsatishi mumkin. Stressni boshqarish talabalar uchun muhim hisoblanadi. Shu tufayli ular stressni yo‘qotishda to‘g‘ri ovqatlanish, kun tartibiga rioya qilish, yetarlicha dam olish, sport bilan shug‘ullanish kabi masalalarga e’tibor qaratishi lozim. Aks holda, stress ularning ota- onalari bilan munosabatoga, ta’lim olish jarayonlariga va sog‘lig‘iga salbiy ta’sir ko‘rsatishi mumkin.” Kenton Levingsning aytishicha, stress nafaqat talabalarning hissiy va ruhiy salomatligiga, shuningdek, ularning ijtimoiy munosabatlariga, kerakli bilimlarni o‘zlashtirishiga va kelajakdagi muvaffaqiyatlariga ta’sir qilishi mumkin. Ko‘plab talabalar esa stressni boshqarish uchun zarur bo‘lgan bilim va ko‘nikmalarga ega emas.Stress talabalarda jiddiy kasalliklarni rivojlantirishi mumkin. Depressiya, tashvishlanish, yuqori qon bosimi va semizlik – bular duch kelishi mumkin bo‘lgan kasalliklardan bir nechtasi,xolos. Agar stress nazorat qilinmasa, u talabalarning salomatligiga va hayotiy faoliyatlariga jiddiy zarar yetkazishi mumkin. ADABIYOTLAR TAHLILI Tomas Broderikning fikriga ko‘ra:,,Agar stress e’tiborga olinmasa,u talabalarda tushkunlik va xavotirga olib kelishi mumkin. Natijada, talabalarning o‘qish natijalariga va oila, do‘stlar, tengdoshlar bilan shaxsiy munosabatlariga salbiy ta’sir ko‘rsatishi mumkin. "Shuningdek, Tomas Broderik talabalardagi stressni qanday boshqarish mumkinligi haqida maslahat va tavsiyalar berib o‘tadi: 1)Yetarlicha uxlash 2) Yaxshi ovqatlanish 3)Muntazam ravishda mashq qilish va sport bilan shug‘ullanish 90 https://t.me/ResearchEdu_Journal Multidisciplinary Scientific Journal July, 2022 90 https://t.me/ResearchEdu_Journal VOLUME 1 \| ISSUE 4 \| 2022 RESEARCH AND EDUCATION FOYDALANILGAN ADABIYOTLAR RO‘YXATI: FOYDALANILGAN ADABIYOTLAR RO‘YXATI: 1. Selye , H (1950) ‘‘Stress va umumiy moslashuv sindiromi’’ 2. https://www.bestcolleges.com 3. https://insightstobehavior.com 4. https://www.verywellmind.com 91 https://t.me/ResearchEdu_Journal Multidisciplinary Scientific Journal July, 2022 91 https://t.me/ResearchEdu_Journal Multidisciplinary Scientific Journal July, 2022 91
https://openalex.org/W4389109316	https://jgeb.springeropen.com/counter/pdf/10.1186/s43141-023-00597-4	English	null	Optimizing the generation of mature bone marrow-derived dendritic cells in vitro: a factorial study design	Journal of Genetic Engineering and Biotechnology /Journal of Genetic Engineering and Biotechnology	2,023	cc-by	8,263	Abstract Keywords Dendritic cells, Factorial design, Optimization, GM-CSF, IL4, DoE Correspondence: Najla Alotaibi najlabio@hotmail.com Full list of author information is available at the end of the article © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Alotaibi et al. Journal of Genetic Engineering and Biotechnology (2023) 21:144 https://doi.org/10.1186/s43141-023-00597-4 Alotaibi et al. Journal of Genetic Engineering and Biotechnology (2023) 21:144 https://doi.org/10.1186/s43141-023-00597-4 Journal of Genetic Engineering and Biotechnology Alotaibi et al. Open Access Open Access Optimizing the generation of mature bone marrow‑derived dendritic cells in vitro: a factorial study design Alotaibi1,2,3 , Alia Aldahlawi1,2, Kawther Zaher2, Fatemah Basingab1,2 and Jehan Alrahimi1,2 Abstract Background Factorial design is a simple, yet elegant method to investigate the effect of multiple factors and their interaction on a specific response simultaneously. Hence, this type of study design reaches the best optimization conditions of a process. Although the interaction between the variables is widely prevalent in cell culture procedures, factorial design per se is infrequently utilized in improving cell culture output. Therefore, we aim to optimize the exper- imental conditions for generating mature bone marrow-derived dendritic cells (BMDCs). Two different variables were investigated, including the concentrations of the inducing factors and the starting density of the bone marrow mononuclear cells. In the current study, we utilized the design of experiments (DoE), a statistical approach, to sys- tematically assess the impact of factors with varying levels on cell culture outcomes. Herein, we apply a two-factor, two-level (22) factorial experiment resulting in four conditions that are run in triplicate. The two variables investigated here are cytokines combinations with two levels, granulocyte–macrophage colony-stimulating factor (GM-CSF) alone or with interleukin-4 (IL4). The other parameter is cell density with two different concentrations, 2 × 106 and 4 × 106 cells/mL. Then, we measured cell viability using the trypan blue exclusion method, and a flow cytometer was used to detect the BMDCs expressing the markers FITC-CD80, CD86, CD83, and CD14. BMDC marker expression levels were calculated using arbitrary units (AU) of the mean fluorescence intensity (MFI). Results The current study showed that the highest total viable cells and cells yield obtained were in cell group seeded at 2 × 106 cells/mL and treated with GM-CSF and IL-4. Importantly, the expression of the co-stimulatory molecules CD83 and CD80/CD86 were statistically significant for cell density of 2 × 106 cells/mL (P < 0.01, two-way ANOVA). Bone marrow mononuclear cells seeded at 4 × 106 in the presence of the cytokine mix less efficiently dif- ferentiated and matured into BMDCs. Statistical analysis via two-way ANOVA revealed an interaction between cell density and cytokine combinations. Conclusion The analysis of this study indicates a substantial interaction between cytokines combinations and cell densities on BMDC maturation. However, higher cell density is not associated with optimizing DC maturation. Nota- bly, applying DoE in bioprocess designs increases experimental efficacy and reliability while minimizing experiments, time, and process costs. Background molecule. Plus, DC costimulatory molecules as signal 2, where the B7 family (CD80/CD86) is engaged to the CD28 receptor on the T cell surface [12, 17]. Engage- ment of CD28 on naïve T cells by either B7-1 or B7-2 ligands on APCs provides a potent costimulatory sig- nal which resulted in induction of IL-2 transcription, expression of CD25, and entry into the cell cycle. CD28 engagement also confers critical survival signals to acti- vated T cells [17]. g Dendritic cells (DCs) are immune cells that link the innate and adaptive immune systems. They are derived from bone marrow (BM) precursors and patrolling the blood. Functionally and morphologically, DCs consist of a heterogeneous population with different phenotypic subsets and locations [1]. They are abundant in epithe- lia and lymphoid organs, such as the skin [2], blood [3], lymph node [1], lungs [4], stomach [5], and intestines [6]. Crucially, DCs have a role in activating cytotoxic T cells and therefore have been extensively used in developing cancer immunotherapies [7]. DC maturation is triggered after immature DCs are exposed to pathogen-associated molecular patterns (PAMPs) [8, 9]. They have been rec- ognized for their essential role as professional antigen- presenting cells (APCs). This process is represented by recognizing the antigen and processing it into small peptides, then presenting it on their major histocompat- ibility molecule (MHC) to activate cytotoxic T cells [10, 11]. As a result, DCs will undergo morphological changes including migrating to lymphatic organs, expressing co- stimulatory molecules, and secreting cytokines [9]. Interestingly, there remains a noticeable gap in system- atic analyses investigating the influence of parameter lev- els and their interactions on culture performance. While the existing body of literature acknowledges the utility of factorial design in probing interactions, its applica- tion remains limited in cell culture research. We take a pioneering step by not only filling this gap but also con- tributing novel insights into the application of factorial design to optimize DC maturation, representing an inno- vative advancement in cell culture methodologies. Background This novel approach resonates with a similar mode of factorial study design that has emerged in recent works, wherein the application of cell-biomaterial full factorial design yielded valuable insights into optimizing cell density effi- ciency [18, 19], primary culture conditions, cytokine and serum doses on stem cells [20], solid lipid nanoparticles [21], cell suspension culture media for maximizing anti- body production [22], tissue engineering [18, 23], and various biotechnological studies [24, 25]. Our contribu- tion to this field is especially pertinent given the critical role of generating mature DCs in DC-based immuno- therapies. While optimization conditions for mature DCs in cell culture are underexplored, applying factorial design has proven effective in enhancing our understand- ing and knowledge to optimize cell maturation within this context. Given their potent ability to process and present anti- gens, DCs hold a promising treatment for multiple advanced diseases including cancer. Moreover, DC-based therapies are used for other medical purposes, such as preventing transplant rejection [10]. Furthermore, sev- eral studies have shed light on combining DC-based vac- cines with conventional therapies like chemotherapy to increase their efficacy [11–13], which helped the devel- opment of several DC-based therapeutic vaccines [14]. Due to the higher reachability and accessibility, BM became one of the primary sources of DC precursors. In in vitro studies, mature BMDCs are mainly generated by culturing BM monocyte progenitor with GM-CSF alone or combined with IL4 [8]. GM-CSF is essential and proven to maximize pure DCs [9]. Besides, cytokines like IL-4 enhance DC maturation from CD34+ and CD14+ precursors [8, 9]. The BM of BALB/c mice was used to obtain hematopoietic precursor cells, whereas early washings removed non-DCs [9, 10]. The aim of the present work is to use a DoE statisti- cal approach to investigate the main effects of culture variables and their interaction in the optimization of the optimization of generation of mature BMDCs. Thus, this statistical approach was applied to a two-factor, two- level (22) factorial experiment. The cytokine combina- tions are granulocyte–macrophage colony-stimulating factor (GM-CSF) alone and GM-CSF plus interleukin-4 (IL4). While the cell densities are 2 × 106 and 4 × 106 cells/ mL. Accordingly, BMDCs were generated from murine BM and differentiated using appropriate cytokines and seeded at specific cell densities according to the study design. Alotaibi et al. Journal of Genetic Engineering and Biotechnology (2023) 21:144 Alotaibi et al. Journal of Genetic Engineering and Biotechnology (2023) 21:144 Page 2 of 11 Factorial study design to optimize BMDC maturation ll f l l d ( k) Factorial study design to optimize BMDC maturation Full factorial experimental design (ik) is an experimental design that includes all possible combinations of k fac- tors at two or more levels [26, 27]. Herein, a combination of levels for each factor included in the study will be run separately and will result in ik set of experimental runs. In this work, optimal conditions of cytokine combina- tions and cell density to enhance BMDC maturation were determined using a factorial design (2 levels, 2 factors). The response to optimizing BMDC maturation (Y) was studied using independent variables of cytokine combi- nations (A) and cell density (B). The cytokine combina- tion was GM-CSF and GM-CSF plus IL4, while the cell density levels were 2 × 106 and 4 × 106 cells/mL. The experimental runs were determined according to the requirements of a full factorial design using the following equation [28, 29]: Table 1 The levels of two independent variables used in (22) factorial design GM-CSF granulocyte–macrophage colony-stimulating factor, IL-4 interleukin-4 a GM-CSF was 20ng/mL b IL4 was 10ng/mL Independent variables Model symbol Levels Low High Cytokine combination A GM-CSFa GM-CSFa & IL4b Cell density B 2 × 106 cells/mL 4 × 106 cells/mL Table 1 The levels of two independent variables used in (22) factorial design influence of desired factors and their interaction on cell culture. influence of desired factors and their interaction on cell culture. Generating mice bone marrow‑derived dendritic cells Generating of BMDCs was performed according to a paper described by Inaba, 1992, with a slight modifica- tion [30, 31]. Briefly, 6- to 8-week BALB/c mice (n = 2) were euthanized according to guidelines and regulations of local animal care by inhalational anesthetic isoflurane, and death was confirmed by cervical dislocation. The skin was removed, then the femurs were separated by cutting the connection point with a scissor. Muscles and tissues around the femur and tibia were removed under sterile conditions. The harvested bones were placed into a 100-mm non-tissue culture-treated plate filled with 70% ethanol for 2 min and then transferred to a culture- treated plate containing RPMI-1640 medium. After cut- ting both ends of the femur with scissors, a needle of a 1-mL syringe filled with complete cold RPMI-1640 was inserted into the bone cavity to flush out the marrow from the bone into the petri dish containing complete RPMI-1640. Then, the BM cell solution was pipetted to form a single cell suspension and transferred to a 15-mL conical tube by cell strainer to remove any residuals, then centrifuged at 300 × g for 10 min at 4 °C to pellet the cells. Briefly, the BM collected cell suspension was washed with PBS twice at 300 × g for 10 min at 4 °C. Cells were resuspended in 5–10 mL of ACK (ammonium-chloride- potassium) lysing buffer and were incubated for 3–5 min at room temperature to lyse red blood cells. Five to 10 mL of complete RPMI-1640 media was added, and cells were washed twice. Reagent and antibodies Roswell Park Memorial Institute medium (RPMI), phos- phate-buffered saline (PBS), heat-inactivated fetal bovine serum (FBS), Trypan blue exclusion assay, and lipopoly- saccharide (LPS) were purchased from Sigma Aldrich, MO, USA. Penicillin/Streptomycin 10,000 unit/mL and l-glutamine were purchased from Thermo Fisher Scien- tific, USA, Mouse recombinant IL4 and FITC-antibodies (CD80, CD86, CD83, and CD14) were purchased from BioLegend® (London, UK). Mouse recombinant GM-CSF was purchased from R&D systems® (MO, USA). Background The yield and viability of BMDCs were assessed by Trypan blue exclusion, and phenotypic characteriza- tion of mature BMDCs was evaluated by flow cytomet- ric analysis. By applying the statistical approach to the design of the experiment, we could contribute to improv- ing cell culture procedures by understanding the main Commonly, the maturation of DCs is evaluated by flow cytometric analysis of associated markers. The markers were expressed on the surface of the immune cells and each marker was investigated individually [15]. In DC flow cytometry analysis, the common markers include the monocyte surface marker CD14, which decreases when monocytes are differentiating into macrophages or DCs besides the common DC maturation marker CD83 and costimulatory molecules CD80/CD86 [16, 17]. For optimal T activation, DCs need to deliver two signals: signal 1, where DCs are effectively present- ing peptides to the T cell receptor (TCR) via the MHC Page 3 of 11 Alotaibi et al. Journal of Genetic Engineering and Biotechnology (2023) 21:144 Animal Female BALB/c (6 to 8 weeks, weight = 19.48 ± 0.21) were obtained from the Animal House and bred in specific pathogen-free conditions. The mice were maintained in the animal house with unrestricted access to water and a balanced diet. They were kept under laboratory condi- tions with a temperature of 22 °C (± 2), 40–60% humidity, and illuminations with a 12-h light–dark cycle in wire- bottomed cages. To apply factorial design, cells were counted using a hemocytometer and adjusted to two main densities equal to 2 × 106 cells/mL or 4 × 106 cells/mL. Cells were then seeded in 6-well plates in complete RPMI-1640 media (i.e., 3 mL/well) supplemented by cytokine combina- tions of recombinant murine GM-CSF and recombinant murine IL4 to a final concentration of 20 ng/mL and 10 ng/mL, respectively, while other cells were cultured in complete media supplemented by only a recombinant murine GM-CSF (Table 1). Cells were cultured at 37ºC in an incubator containing 5% CO2. On day 3 and day 5, half Generating and phenotyping of mature BMDCsh g p yp g The morphological changes of mature BMDCs on day 7 were observed by using an inverted microscope (Table 3) (Figs. 2 and 3). Mature BMDCs were characterized by surface markers of high expression of CD80, CD86, and CD83, and low or lack of expression of monocyte mark- ers CD14. The results showed that the lowest expression of CD14 was detected when both cytokines were used and with a cell density of 4 × 106 cells/mL. CD83 expres- sion was higher in the group of GM-CSF plus IL4 and with a density of 2 × 106 cells/mL. Both CD80 and CD86 markers were also highly expressed in the group of GM- CSF plus IL4 and with lower cell density (i.e., 2 × 106 cells/mL) compared to other sets of treatments (Table 3) (Figs. 1 and 4). Optimization of experimental designh The design of the experiment (DoE) of the two variable levels was coded in (Table 1). Response values and the matrix of the factorial design are represented in (Table 4). The highest MFI was observed when media were sup- plemented with both GM-CSF and IL4 regardless of cell density. The factors of interest have shown significant influence on different estimated responses. To illustrate, the MFI of CD14 ranged from 169.4 to 282.9 arbitrary units (AU), while the MFI of co-stimulatory CD80 and CD86 was 131.8 to 316.7 AU and 186.5 to 390.6 AU, respectively. While CD83 maturation markers expressed at levels from 76.1 to 157.6 AU compared to the other markers. Therefore, the effect of the independent fac- tors on marker expression (i.e., Y1, Y2 …) was illustrated by the factorial equation in (Table 5). Incorporating this information into the model, we can state that the calcu- lated adjusted R2 coefficients, which surpass 0.8 in all models, provide robust evidence of the excellent align- ment of the equations. This underscores that more than 80% of the variability in the response variable can be attributed to the fluctuations in the explanatory variables (A and B) within the model. Y = β0 + β1A + β2B + β12AB + e where Y is the estimated response, β0 is the intercept value, β1 and β2 are the linear coefficient, β12 is the fac- torial coefficient, A and B are the independent variables, and e is the residual error. Experiments number = LevelsFactors Full factorial experimental runs are constructed here by two-factor two-levels (22), resulting in a total of four con- ditions with triplicate; therefore, there will be 12 experi- mental runs. Alotaibi et al. Journal of Genetic Engineering and Biotechnology (2023) 21:144 Page 4 of 11 of the media was removed, and new media supplemented with appropriate cytokines was added. On day 6, BMDCs were activated by adding 1 μg/mL of LPS for 24 h before cells were collected [32]. Table 2 Comparison of dendritic cells yield and total viable cells GM-CSF granulocyte–macrophage colony-stimulating factor, IL-4 interleukin-4 * P value was based on two-way ANOVA GM-CSF & IL4 GM-CSF P 2 × 106 4 × 106 2 × 106 4 × 106 Yield (cells/ml) 6–6.5 × 107 4.5–5 × 106 4 × 106 5–6 × 106 0.465 Viable cells (%) 95.3 ± 1.4 91.5 ± 0.6 78.4 ± 3.4 83.1 ± 1.7 0.027 Table 2 Comparison of dendritic cells yield and total viable cells Statistical analysishff The effect of different levels of the two independent variables on the response was evaluated using analy- sis of variance (ANOVA). Regression models of param- eters (factorial equations) and their interactions and the coefficient of determination (R2) were estimated. All experiments were triplicated, and a P value of ≤ 0.05 was considered significant. Data analysis was performed using Rstudio (version 1.4.1106) (R Foundation for Sta- tistical Computing, Vienna, Austria). The graphics were made using “ggpubr” and “grid” packages. Yield and viability of BMDCsh The highest total viable cells obtained was 95.3% ± 1.4%, and cells yield were 6–6.5 × 107 cells/mL in BMDCs that were cultured in the presence of both cytokines and seeded at 2 × 106 cells/mL (Table 2) (Fig. 1). Phenotype analysis of mature bone marrow‑derived dendritic cell Phenotype properties of maturation status were inves- tigated by calculating the MFI of mature BMDC surface markers. BMDCs collected on day 7 were washed twice with flow cytometry staining buffer (FACS; PBS + 1% FBS) and incubated with the following antibodies; CD80, CD86, CD83, and CD14 for 30min at 4°C. Cells were washed twice with FACS to remove excessive antibodies [22, 33]. Stained and unstained cells were acquired using flow cytometry (Beckman Coulter Life Science, USA) using 5000 events. Data was analyzed using FCS Express 7 (De Novo software, CA, USA). A factorial equation was performed to estimate the response as follows [34]: Cells yield and viability At day 7, BMDCs were collected, and a Trypan blue exclusion assay was used to assess the yield and viabil- ity of BMDCs as described previously. Briefly, viable DCs were assessed by counting elongated, irregular with numerous and long cytoplasmic projection cells. The percentages of cells yield were estimated using the for- mula: yield (%) = BMDCs/total cells × 100. Main effects and interaction termf In terms of main effects for the CD83 marker, for instance, on average, one level increase in cytokine Alotaibi et al. Journal of Genetic Engineering and Biotechnology (2023) 21:144 Page 5 of 11 Fig. 1 Phenotypic analysis of dendritic cell markers using flow cytometry. Barplot presents mean fluorescence intensity (MFI) of the cell’s markers based on cell density and cytokine combinations Fig. 1 Phenotypic analysis of dendritic cell markers using flow cytometry. Barplot presents mean fluorescence intensity (MFI) of the cell’s markers based on cell density and cytokine combinations Table 3 Comparison of dendritic cell marker expression MFI mean fluorescence intensity, GM-CSF granulocyte–macrophage colony- stimulating factor, IL-4 interleukin-4 P value was based on two-way ANOVA GM-CSF & IL4 GM-CSF P 2 × 106 4 × 106 2 × 106 4 × 106 CD83 (MFI) 157.6 ± 6.7 135.0 ± 4.9 76.1 ± 14.0 105.5 ± 4.9 0.049 CD80 (MFI) 316.2 ± 3.5 230.9 ± 2.3 131.8 ± 1.5 135.7 ± 2.1 0.012 CD86 (MFI) 390.6 ± 13.3 266.2 ± 8.6 186.5 ± 2.9 214.9 ± 5.0 0.001 CD14 (MFI) 172.8 ± 15.3 169.4 ± 19.5 282.9 ± 9.3 195.9 ± 18.6 0.03 Table 3 Comparison of dendritic cell marker expression MFI mean fluorescence intensity, GM-CSF granulocyte–macrophage colony- stimulating factor, IL-4 interleukin-4 P value was based on two-way ANOVA GM-CSF & IL4 GM-CSF P 2 × 106 4 × 106 2 × 106 4 × 106 CD83 (MFI) 157.6 ± 6.7 135.0 ± 4.9 76.1 ± 14.0 105.5 ± 4.9 0.049 CD80 (MFI) 316.2 ± 3.5 230.9 ± 2.3 131.8 ± 1.5 135.7 ± 2.1 0.012 CD86 (MFI) 390.6 ± 13.3 266.2 ± 8.6 186.5 ± 2.9 214.9 ± 5.0 0.001 CD14 (MFI) 172.8 ± 15.3 169.4 ± 19.5 282.9 ± 9.3 195.9 ± 18.6 0.03 le 3 Comparison of dendritic cell marker expression elevation in CD86 MFI for the 4 × 106 cells/mL group over the 2 × 106 cells/mL group. These findings collec- tively underscore the differential impacts of cytokine combination and cell density on various markers, provid- ing a comprehensive understanding of their influence on the maturation of dendritic cells. The adjusted coefficient of R2 generated from the sta- tistical analysis of models was 0.999, 0.989, and 0.989 for CD83, CD80, and CD86, respectively. The two vari- ables and their interactions significantly contributed to the response of CD83 and CD86 (P < 0.001, two-way ANOVA). Main effects and interaction termf The maximum marker expression was asso- ciated with using GM-CSF and IL4 and lower cell den- sity, MFI = 157.6 AU and MFI = 390.6 AU, for CD83 and CD86, respectively. That could be due to the sufficient number of cytokines, 20ng/mL and 10ng/mL of GM- CSF and IL4 can boost the maturation of 2 × 106 cells/mL compared to 4 × 106 cells/mL. On the other hand, only cytokines combination significantly contributed to the response of CD80 (P < 0.001, two-way ANOVA). combination (i.e., GM-CSF and IL4) was associated with an 81.5 AU increase of CD83 MFI, keeping cell density constant (Table 5). The main effect of cell density was associated with only a 29.3 AU increase in CD83 AU for the group of 4 × 106 cells/mL than the group of 2 × 106 cells/mL. In a similar vein for CD14, using GM-CSF and IL4 cytokine combination corresponded to a decrease of 110.1 AU in MFI. Meanwhile, the main effect of cell den- sity led to a reduction of 86.9 AU in MFI for the group at 4 × 106 cells/mL compared to 2 × 106 cells/mL. Shift- ing our focus to CD80, the main effect of cytokine com- bination was associated with a substantial increase of 184.3 AU in MFI, regardless of cell density. Conversely, the impact of cell density resulted in a modest 3.8 AU increase in CD80 MFI for the 4 × 106 cells/mL group compared to the 2 × 106 cells/mL group. Similarly, inves- tigating CD86 revealed that the main effect of cytokine combination led to a noteworthy 204.1 AU increase in CD86 MFI, irrespective of cell density. On the other hand, cell density’s main effect contributed to a 28.4 AU combination (i.e., GM-CSF and IL4) was associated with an 81.5 AU increase of CD83 MFI, keeping cell density constant (Table 5). The main effect of cell density was associated with only a 29.3 AU increase in CD83 AU for the group of 4 × 106 cells/mL than the group of 2 × 106 cells/mL. In a similar vein for CD14, using GM-CSF and IL4 cytokine combination corresponded to a decrease of 110.1 AU in MFI. Meanwhile, the main effect of cell den- sity led to a reduction of 86.9 AU in MFI for the group at 4 × 106 cells/mL compared to 2 × 106 cells/mL. Main effects and interaction termf Shift- ing our focus to CD80, the main effect of cytokine com- bination was associated with a substantial increase of 184.3 AU in MFI, regardless of cell density. Conversely, the impact of cell density resulted in a modest 3.8 AU increase in CD80 MFI for the 4 × 106 cells/mL group compared to the 2 × 106 cells/mL group. Similarly, inves- tigating CD86 revealed that the main effect of cytokine combination led to a noteworthy 204.1 AU increase in CD86 MFI, irrespective of cell density. On the other hand, cell density’s main effect contributed to a 28.4 AU Interestingly, the significant interaction between cytokine combination and cell densities across all four markers reveals the complex dynamics governing den- dritic cell maturation (Table 5). The interaction plots pro- vide a comprehensive picture of how these factors jointly influence the observed marker responses, underscoring the importance of considering their combined effects when optimizing dendritic cell culture conditions (Fig. 5). The magnitude of the positive interaction between cytokines combination and cell density is shown in Fig. 5 by non-parallel lines. That is, the effect of cell density of BMDC marker expression is different for different levels Alotaibi et al. Journal of Genetic Engineering and Biotechnology (2023) 21:144 Page 6 of 11 Fig. 2 Morphological changes of mature BMDCs. A, B Unstimulated DCs (controls) were cultured for 7 days without cytokines at cell densities of 4 × 106 and 2 × 106 cells/mL, respectively, and appeared rounded in shape with no cytoplasmic projections. C DCs were cultured in the presence of GM-CSF and IL4 and seeded with a density of 2 × 106 cells/mL. D DCs were cultured in the presence of GM-CSF and IL4 and seeded with a density of 4 × 106 cells/mL while in E DCs were cultured in the lack of IL4 and with a density of 2 × 106 cells/mL (the weakest cell appearance among other combination) and lastly in F DCs were also supplemented with only GM-CSF and lack of IL4 with a density of 4 × 106 cells/mL. Pictures were taken with an inverted microscope (magnifications were 40 ×); arrows indicate dendrons of DCs Fig. 2 Morphological changes of mature BMDCs. A, B Unstimulated DCs (controls) were cultured for 7 days without cytokines at cell densities of 4 × 106 and 2 × 106 cells/mL, respectively, and appeared rounded in shape with no cytoplasmic projections. Main effects and interaction termf C DCs were cultured in the presence of GM-CSF and IL4 and seeded with a density of 2 × 106 cells/mL. D DCs were cultured in the presence of GM-CSF and IL4 and seeded with a density of 4 × 106 cells/mL while in E DCs were cultured in the lack of IL4 and with a density of 2 × 106 cells/mL (the weakest cell appearance among other combination) and lastly in F DCs were also supplemented with only GM-CSF and lack of IL4 with a density of 4 × 106 cells/mL. Pictures were taken with an inverted microscope (magnifications were 40 ×); arrows indicate dendrons of DCs of cytokine combinations. For example, provided that low level of cytokine combination (i.e., GM-CSF), one level increase in cell density was associated with a 29.4 AU increase in CD83 marker expression compared to 22.5 AU when cells supplemented with GM-CSF and IL4. Indicating that higher cell density contributed to increas- ing the expression of BMDC markers if only GM-CSF was used. That might be due to the spontaneous matura- tion of using more than 2 × 106 cells per mL. Fig. 3 BMDC of optimized conditions. BMDCs supplemented GM-CSF and IL4 and seeded with a cell density of 2 × 106 cells/mL. The picture was taken with an inverted microscope Discussion Although factorial design has been recognized for its usefulness and importance in investigating interac- tions, several published protocols suggest a lack of sys- tematic analysis into the effect of parameter levels and their interaction in culture performance. However, only a few experimental studies have used this design, such as cell-biomaterial [18, 19], optimization of culture con- ditions [20–22], tissue engineering [18, 23], and other Fig. 3 BMDC of optimized conditions. BMDCs supplemented GM-CSF and IL4 and seeded with a cell density of 2 × 106 cells/mL. The picture was taken with an inverted microscope Alotaibi et al. Journal of Genetic Engineering and Biotechnology (2023) 21:144 Page 7 of 11 Fig. 4 Representative histograms of BMDC surface marker expression. The histogram illustrates the role of combinations of two levels of the two variables individually based on the DC expression of surface markers. The highest fluorescence intensities of the mature BMDC markers (CD83, CD80, and CD86) were repeatedly detected in the cell group seeded at 2 × 106 in the presence of GM-CSF and IL-4, followed by the cell group seeded at the higher density and treated with the cytokine mix Fig. 4 Representative histograms of BMDC surface marker expression. The histogram illustrates the role of combinations of two levels of the two variables individually based on the DC expression of surface markers. The highest fluorescence intensities of the mature BMDC markers (CD83, CD80, and CD86) were repeatedly detected in the cell group seeded at 2 × 106 in the presence of GM-CSF and IL-4, followed by the cell group seeded at the higher density and treated with the cytokine mix according to [30–32]. Together, GM-CSF and IL-4 play complementary roles in the differentiation and activa- tion of dendritic cells. Therefore, in the current study, the bone marrow of female BALB/c mice was used to obtain hematopoietic precursor cells, whereas non-DCs were removed by early washings [30, 31]. Morphological observations of unstimulated BMDCs showed a rounded shape with short or no projections (Fig. 2A, B). This biotechnological studies [24, 25]. Herein, applying facto- rial design in cell culture settings [26] has provided suffi- cient knowledge on optimizing the cell’s maturation. In the current study, we systematically isolate and gen- erate mature DCs from mice bone marrow under the aegis of GM-CSF and IL-4. Discussion For the generation of BMDCs, GM-CSF is essential to differentiate and the survival of DC progenitors and has proven to maximize pure DCs, Alotaibi et al. Journal of Genetic Engineering and Biotechnology (2023) 21:144 Page 8 of 11 Table 4 The matrix of the experimental values and response values of BMDC marker expression conditions using (22) factorial design MFI mean fluorescence intensity Run Factor 1 (A) Factor 2 (B) Response 1 Response 2 Response 3 Response 4 Cytokine combinations (ng/ mL) Cell density (cells/mL) CD14 expression (MFI AU) CD83 expression (MFI AU) CD80 expression (MFI AU) CD86 expression (MFI AU) 1 GM-CSF 2 × 106 272.53 75.63 133.14 186.48 2 GM-CSF 2 × 106 290.32 90.32 130.2 189.35 3 GM-CSF 2 × 106 285.89 62.32 132.18 183.65 4 GM-CSF + IL4 2 × 106 170.2 159.05 315.52 395.62 5 GM-CSF + IL4 2 × 106 158.97 150.32 320.02 400.7 6 GM-CSF + IL4 2 × 106 189.32 163.52 312.96 375.5 7 GM-CSF 4 × 106 194.23 105.75 137.89 214.01 8 GM-CSF 4 × 106 215.32 100.35 133.85 210.36 9 GM-CSF 4 × 106 178.24 110.25 135.23 220.32 10 GM-CSF + IL4 4 × 106 168.17 134.82 230.95 265.31 11 GM-CSF + IL4 4 × 106 150.52 130.25 228.52 275.2 12 GM-CSF + IL4 4 × 106 189.52 139.96 233.12 257.98 MFI mean fluorescence intensity markers such as DCs and macrophages [8, 17]. Therefore, we used four markers to identify DCs, including CD80, CD86, CD83, and CD14. Functional DCs activate T cells through two major signals: interaction between TCR and MHC complex and co-stimulatory signaling [2, 30, 31]. Discussion Unlike macrophages, mature DCs are characterized by high expression of co-stimulatory molecules, CD80, CD86, and membrane-bound CD83, and low expression or lack of monocyte markers CD14 [3].h Table 5 The used linear regression equations to estimate the markers’ expression response Adj.R2 adjusted R-squared P value was based on two-way ANOVA Response Linear regression equation R2 Adj.R2 *P CD14 282.9 – 110.1 (A) – 86.9 (B) + 83.6 (AB) 0.924 0.895 < 0.001 CD83 76.1 + 81.5 (A) + 29.4 (B) – 51.9 (AB) 0.951 0.999 < 0.01 CD80 131.8 + 184.3 (A) + 3.8 (B) – 89.1 (AB) 0.999 0.989 < 0.001 CD86 186.5 + 204.1 (A) + 28.4 (B) – 152.8 (AB) 0.992 0.989 < 0.001 Table 5 The used linear regression equations to estimate the markers’ expression response The results of this work indicated that the cell yield, viability, and phenotypic markers’ expression are corre- lated with higher cytokine levels. Also, the effect of the maturation conditions and their interaction significantly contributed to the CD14 expression level (Y1) (P < 0.001, two-way ANOVA) (Table 4). Both cytokine combination and cell density were negatively correlated with CD14 expression level, the higher the level (i.e., two cytokines and higher cell density) the lower the monocyte CD14 expression (MFI = 169.4 AU) that indicates a success- ful differentiation of monocytes into DCs as reported by previous studies [3, 9]. Importantly, CD14 is a monocyte marker that occasionally decreases while monocytes dif- ferentiate into DCs [9]. observation aligns with the expectation that cytokine stimulation is necessary for inducing dendritic cell differ- entiation and maturation. However, upon the addition of GM-CSF and IL4 at a cell density of 2 × 10^6 cells/mL, the DCs exhibited dis- tinct morphological features (Figs. 2C and 3). This sug- gests that the combination of these cytokines at this particular density promoted dendritic cell maturation and the development of dendritic projections, which are essential for their immune-presenting function [10–12]. The distinct appearances of the mature BMDCs under different conditions shed light on the influence of cytokines and cell density on their morphology and mat- uration status (Fig. 2C–F). Besides, the expression of the co-stimulatory mol- ecules was significantly higher in DCs that were supple- mented with both cytokines and seeded at 2 × 106 cells/ mL. Discussion However, CD86 was expressed at higher levels than CD80, as shown in (Table 2) that may be because CD86 expression on the APC surface is rapidly upregulated upon stimulation; CD80, on the other hand, requires stimulation to be expressed [33, 35]. Additionally, the membrane-bound CD83 is increased on the surface of BMDCs’ phenotypic characteristics were further investigated using flow cytometric analysis. This analy- sis is performed for different purposes; the most impor- tant reason is to accurately identify the desired subset of cells, especially when expressing the same surface Alotaibi et al. Journal of Genetic Engineering and Biotechnology (2023) 21:144 Page 9 of 11 Fig. 5 Two-factor interaction plot of the independent variables. There were significant interaction effects between cytokine combination and cell density on all DC markers (P < 0.001, two-way ANOVA) Fig. 5 Two-factor interaction plot of the independent variables. There were significant interaction effects between cytokine combination and cell density on all DC markers (P < 0.001, two-way ANOVA) activated DCs and is considered a major marker to dis- tinguish between mature and immature DCs [36–38]. In general, the results of the expression of co-stimula- tory molecules and mature DC markers were correlated with the low cell density. Conclusion To our knowledge, the work presented here is one of a few investigations into the interaction effects of cell- culture variables using the DoE statistical approach. The maturation of DCs is implemented by culturing cells with the presence of cytokines. Then, they seeded at the desired number of cells per mL. Notably, because of the significant role of IL4 in DC maturation. Also, to avoid seeding more than 2 million cells per mL to eliminate spontaneous maturation. The current work has success- fully optimized the DC maturation. In addition, it high- lighted the importance of factorial experimental design in minimizing experiments, time, and process costs while maintaining high-quality responses. y Cytokine combinations have a positive effect on CD80 expression levels. The higher the level of cytokine combination, the higher the CD80 expres- sion (MFI = 316.2 AU). Cell density has a difference of only 4.2 AU between DCs that are seeded in lower cell density compared to higher cell density. However, the higher MFI was correlated with a cell density of 2 × 106 cells/mL and both cytokine combinations. To sum up, a cell density level of 2 × 106 was positively correlated with BMDC marker expression when cells were sup- plemented with GM-CSF and IL4. Using only GM- CSF increased BMDC maturation with a cell density of 4 × 106 cells/mL compared to IL4. Remarkably, the role of cytokines is strongly associated with BMDC maturation, even though all factors have significantly influenced BMDC maturation. Following the facto- rial approach, our optimization conditions ultimately resulted in an overall maturation for cells supplemented with GM-CSF and IL4 and seeded at a cell density of 2 × 106 cells/mL. Availability of data and materials 12. Calmeiro J, Carrascal MA, Tavares AR, Ferreira DA, Gomes C, Falcão A et al (2020) Dendritic cell vaccines for cancer immunotherapy: the role of human conventional type 1 dendritic cells. Pharmaceutics 12(2):158 12. Calmeiro J, Carrascal MA, Tavares AR, Ferreira DA, Gomes C, Falcão A et al (2020) Dendritic cell vaccines for cancer immunotherapy: the role of human conventional type 1 dendritic cells. Pharmaceutics 12(2):158 The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. 13. Ogasawara M, Miyashita M, Yamagishi Y, Ota S (2021) Dendritic cell vac- cination combined with a conventional chemotherapy for patients with relapsed or advanced pancreatic ductal adenocarcinoma: a single-center phase I/II trial. Ther Apher Dial 25(4):415–424 Conclusion Abbreviations DC Dendritic cell BMDC Bone-marrow dendritic cells DoE Design of experiment MHC Major histocompatibility molecule PAMP Pathogen-associated molecular patterns FACS Flow cytometry staining buffer FITC Fluorescein isothiocyanate GM-CSF Granulocyte-macrophage colony-stimulating factor IL4 Interleukin-4 AU Arbitrary units MFI Mean fluorescence intensity APC Antigen-presenting cell Abbreviations DC Dendritic cell BMDC Bone-marrow dendritic cells DoE Design of experiment MHC Major histocompatibility molecule PAMP Pathogen-associated molecular patterns FACS Flow cytometry staining buffer FITC Fluorescein isothiocyanate GM-CSF Granulocyte-macrophage colony-stimulating factor IL4 Interleukin-4 AU Arbitrary units MFI Mean fluorescence intensity APC Antigen-presenting cell Abbreviations DC Dendritic cell BMDC Bone-marrow dendritic cells DoE Design of experiment MHC Major histocompatibility molecule PAMP Pathogen-associated molecular patterns FACS Flow cytometry staining buffer FITC Fluorescein isothiocyanate GM-CSF Granulocyte-macrophage colony-stimulating factor IL4 Interleukin-4 AU Arbitrary units MFI Mean fluorescence intensity APC Antigen-presenting cell Page 10 of 11 Alotaibi et al. 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https://openalex.org/W4220668206	https://repositorium.sdum.uminho.pt/bitstream/1822/78408/1/Materials_2022_2.pdf	English	null	Physical Properties of Eco-Sustainable Form-Stable Phase Change Materials Included in Mortars Suitable for Buildings Located in Different Continental Regions	Materials	2,022	cc-by	11,111	Citation: Sarcinella, A.; de Aguiar, J.L.B.; Frigione, M. Physical Properties of Eco-Sustainable Form-Stable Phase Change Materials Included in Mortars Suitable for Buildings Located in Different Continental Regions. Materials 2022, 15, 2497. https://doi.org/10.3390/ ma15072497 Keywords: Phase Change Materials (PCMs); Thermal Energy Storage (TES); sustainable mortars; Circular Economy (CE); Polyethylene Glycol (PEG) Keywords: Phase Change Materials (PCMs); Thermal Energy Storage (TES); sustainable mortars; Circular Economy (CE); Polyethylene Glycol (PEG) Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Antonella Sarcinella 1 , José Luís Barroso de Aguiar 2,* and Mariaenrica Frigione 1 1 Department of Innovation Engineering, University of Salento, 73100 Lecce, Italy; antonella.sarcinella@unisalento.it (A.S.); mariaenrica.frigione@unisalento.it (M.F.) 2 Department of Civil Engineering, University of Minho, 4800-058 Guimaraes, Portugal * Correspondence: aguiar@civil.uminho.pt p g g y g * Correspondence: aguiar@civil.uminho.pt Abstract: Starting from two low-cost, low-environmental-impact polymers belonging to the Polyethylene Glycol (PEG) family, i.e., PEG 800 and PEG 1000, two form-stable phase change materials were produced. The two PEGs differ in molecular weight and, as a consequence, the melting and crystallization range of temperatures. The PCMs were obtained, including the PEG, in a liquid state, inside the pores of Lecce Stone ﬂakes, obtained as waste pieces from its processing. A simple and inexpensive impregnation process was selected to produce the PCMs, thus adopting low-environmental-impact materials and cheap processes, and respecting circular economy principles. The two PCMs, the ﬁrst composed of PEG 800, namely LS/PEG800, and the second composed of a 50/50%wt. mix of the different LS/PEGs, i.e., LS/PEG800_LS/PEG1000, were added as aggregates to four types of mortars, based on aerial and hydraulic lime, gypsum, and cement. The obtained mortars were characterized in their fresh state to assess their workability, and in a solid state after a proper cure to determine their characteristic Latent Heat Thermal Energy Storage (LHTES) properties and mechanical properties in both ﬂexural and compressive modes, taking the mortars not containing any PCM as the reference. The results revealed that, with the proper selection of mortar formulations, it was possible to achieve suitable workability and adequate mechanical characteristics. The selection of a PEG with a low range of phase change temperatures, such as PEG 800, allows one to obtain mortars characterized by a melting/crystallization range that can be considered appropriate in applications characterized by cold climates. The production of a mixed PCM, composed of both PEGs, led to mortars displaying a large interval of melting/crystallization temperatures, which could be suitable in both warm and cold climates. materials materials Keywords: Phase Change Materials (PCMs); Thermal Energy Storage (TES); sustainable mortars; Circular Economy (CE); Polyethylene Glycol (PEG) 1. Introduction In recent years, many different PCMs have been proposed and investigated, and this represents a great advantage since it is possible to select the most appropriate system according to the application in which it must be implemented [7,8]. When the objective to be pursued is the energy efﬁciency of buildings, the selected PCM can be directly incorporated into mortars [9,10]. Mortars, in fact, can be adapted to many applications, due to their versatility [11]. In addition, they are particularly efﬁcient due to their large heat exchange surfaces [12,13]. So far, according to the literature, the most widely used PCMs are the so-called “microencapsulated PCMs” that can be easily adapted to many different applications [14–16]. Lately, a novel way to produce PCMs, i.e., the form-stable method, has been proving to be more advantageous and equally effective. q y According to the “form-stable” method, which is widely employed to produce PCMs to include in mortars, the active component of the PCM is absorbed in inert support, which, in turn, will be added as aggregate in the mortar. This procedure is able to prevent polymer leakage, improving the thermal durability of the PCM [17]. In this context, the idea of the project “Development and analysis of mortars, based on different binders, containing original sustainable Phase Change Materials (PCMs) able to improve the energy efﬁciency of the buildings located in different area” was conceived. The eco-sustainable polymer chosen for the realization of new form-stable PCMs is Polyethylene Glycol (PEG) selected for its favorable characteristics, i.e.,: low toxicity coupled with limited ﬂammability achievable at low costs, all features particularly important in the construction ﬁeld. Furthermore, PEGs of different molecular weights (MWs) are available. Since MW greatly affects the melting/crystallization temperature range of a polymer, on the basis of the typical outdoor temperatures of a location, it is possible to select the most appropriate PEG, i.e., that possessing a more favorable range of melting/crystallization temperatures to produce the PCM-mortar required in a speciﬁc climate condition. As inert support for the form-stable PCM, waste pieces from the processing of a very porous stone are proposed. A local stone has been selected, i.e., Lecce stone, widely employed in many (civil and religious) buildings typical of the Salento region, for the production of friezes and other constructive elements. 1. Introduction It is well known that the production of the energy required to regulate the internal temperature of buildings, thus ensuring the optimal comfort of the inhabitants irrespective of the external climate, represents the largest sector of energy consumption. To meet this (ever-growing) global energy demand, large quantities of fossil fuels are needed. Referring to the countries belonging to the European Union, almost 40% of the consumed energy is used for the heating/cooling of buildings [1]; this huge amount is directly related to CO2 emissions, causing alarming climate change. The resulting global warming, on the other hand, is rapidly increasing the need for energy to reduce internal temperatures in homes and ofﬁces. In this regard, the International Energy Agency (IEA) reports an increase in global energy consumption in the construction sector by 30% in 2060 if the current rate of growth is maintained [2–4]. Achieving high levels of energy efﬁciency in buildings is among the most pressing priorities of governments globally, as this would Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/materials Materials 2022, 15, 2497. https://doi.org/10.3390/ma15072497 Materials 2022, 15, 2497 2 of 15 contribute to reducing energy consumption and the environmental issues associated with CO2 emissions. The high concern of the international community toward these issues is proven by the released EU codes and the numerous calls published periodically by the European Commission to support research in this ﬁeld (such as Green Deal or the new Agenda 2030 for sustainable development goal 7 “Affordable and clean Energy”). To address this need, Thermal Energy Storage (TES) systems seem to be the most suitable tool because they can store thermal energy and release it later when the reduction in the outdoor temperature can negatively affect the internal comfort of inhabitants [5]. In fact, this role is performed by a phase change material (PCM), which can, in fact, change its physical status according to the environmental temperature. Thus, a PCM can store thermal energy during its phase change from solid to liquid states and release the same thermal energy when it returns to a solid form [6]. 1. Introduction The utilization of waste materials from other processes, cutting down on costs for their disposal, represents an added value of our choices and respects the circular economy principles. Still in line with the adoption of sustainable and low-cost processes, the inclusion of the polymer in the porous stone granules can be achieved through a simple, cheap procedure, employing a hot plate and a vacuum pump. g p p p p y g p p p PEG polymers, thanks to their adaptability [18], have been proposed to realize different efﬁcient form-stable PCMs [19,20]. They are typically incorporated into porous, organic, or more frequently inorganic, matrices [21,22]. Several studies on the thermal performance of form-stable PEG-based PCMs added to mortars demonstrated that the selection of this polymer leads to materials able to adequately regulate the temperature in a room, thus enhancing the thermal comfort of humans [23,24]. In our previous studies [25,26], a form-stable PCM based on PEG 1000 was successfully included in mortars produced by different binders. It was found that mortars, composed of hydraulic lime or cement, containing the PEG 1000-based PCM, were able to reduce the Materials 2022, 15, 2497 3 of 15 com- ble to 3 of 15 com- bl t energetic heating/cooling needs if applied in buildings located in warm Mediterranean regions [24]. As a continuation of this project, a PEG of a different grade, i.e., PEG 800, was selected on the basis of its melting/crystallization range of temperatures, which shifted towards lower values with respect to PEG 1000. The purpose of this selection was to evaluate whether a PEG800-based PCM, to be included in different mortars, will act as an effective PCM if applied in buildings located in colder climates. In the ﬁrst paper [27], PEG 800 was thermally characterized, alone or when included in the Lecce Stone granules to form the PCM, in order to assess the thermal characteristics and latent heats. In Figure 1, the calorimetric curves measured by DSC (Differential Scanning Calorimetry) of the PEG-based PCMs produced so far, i.e., LS/PEG800 and LS/PEG1000, are shown. terranean regions [24]. As a continuation of this project, a PEG of a different grade, i.e., PEG 800, was selected on the basis of its melting/crystallization range of temperatures, which shifted towards lower values with respect to PEG 1000. 1. Introduction The purpose of this selec- tion was to evaluate whether a PEG800-based PCM, to be included in different mortars, will act as an effective PCM if applied in buildings located in colder climates. In the first paper [27], PEG 800 was thermally characterized, alone or when included in the Lecce Stone granules to form the PCM, in order to assess the thermal characteristics and latent heats. In Figure 1, the calorimetric curves measured by DSC (Differential Scanning Calo- rimetry) of the PEG-based PCMs produced so far, i.e., LS/PEG800 and LS/PEG1000, are shown. Figure 1. DSC curves of LS/PEG800 and LS/PEG1000 PCM composites, with the indication of the peak temperatures. Data from [25,27]. Figure 1. DSC curves of LS/PEG800 and LS/PEG1000 PCM composites, with the indication of the peak temperatures. Data from [25,27]. Figure 1. DSC curves of LS/PEG800 and LS/PEG1000 PCM composites, with the indication of the peak temperatures. Data from [25,27]. Figure 1. DSC curves of LS/PEG800 and LS/PEG1000 PCM composites, with the indication of the peak temperatures. Data from [25,27]. The DSC data confirmed that, with the selection of a PEG with a lower molecular weight (i.e., PEG 800), it is possible to shift both the melting and crystallization ranges of temperatures of the PEG-based PCM composite to lower values with respect to the LS/PEG1000 PCM. This observation is supported by the peak temperatures of melting and crystallization processes indicated on the curves in Figure 1 for both PEG-based PCMs. The previous observations suggest that LS/PEG800 is a PCM potentially suitable for mor- tars to be applied in buildings located in cold climates The DSC data conﬁrmed that, with the selection of a PEG with a lower molecular weight (i.e., PEG 800), it is possible to shift both the melting and crystallization ranges of temperatures of the PEG-based PCM composite to lower values with respect to the LS/PEG1000 PCM. This observation is supported by the peak temperatures of melting and crystallization processes indicated on the curves in Figure 1 for both PEG-based PCMs. The previous observations suggest that LS/PEG800 is a PCM potentially suitable for mortars to be applied in buildings located in cold climates. tars to be applied in buildings located in cold climates. 2. Materials and Methods 2.1. Materials As a continuation of the research, the PCMs based on LS/PEG800 and on the mix of LS/PEG800 with LS/PEG1000 (50:50 in weight) were added to mortars based on the same binders (aerial and hydraulic limes, gypsum, and cement mortars), even though greater mortar contents were employed in the present study (1000 kg for 1 m3 of each mortar) in order to enhance their mechanical properties. Several phys- l f h d d d I h h d Two different grades of Poly-Ethylene Glycol (PEG), i.e., PEG 800 and PEG 1000, were selected as the active components of the PCMs. They were both supplied in solid form: PEG 800 was produced by Wuhan Fortuna Chemical Co. (Wuhan, China) and PEG 1000 by Sigma—Aldrich Company (Darmstadt, Germany), respectively. PEG is a thermoplastic semi-crystalline polymer; it is commercially available in different molecular weights, corresponding to different ranges of melting/crystallization temperatures [18,28]. In a previous phase of the research [25,26,29], a PEG possessing a melting temperature in the range 37–40 ◦C was selected (i.e., PEG 1000), corresponding to a molecular weight of 1000. Our aim, in that case, was, in fact, the production of a sustainable In a previous phase of the research [25,26], the PCM produced with LS/PEG1000 was added to mortar formulations based on different binders (i.e., aerial lime, hydraulic lime, gypsum, and cement) analyzing the properties of the obtained mortars in the fresh state, i e their workability as well as after their curing determining mechanical properties and Two different grades of Poly-Ethylene Glycol (PEG), i.e., PEG 800 and PEG 1000, were selected as the active components of the PCMs. They were both supplied in solid form: PEG 800 was produced by Wuhan Fortuna Chemical Co. (Wuhan, China) and PEG 1000 by Sigma—Aldrich Company (Darmstadt, Germany), respectively. i.e., their workability, as well as after their curing, determining mechanical properties and latent heat characteristics. As a continuation of the research, the PCMs based on LS/PEG800 and on the mix of LS/PEG800 with LS/PEG1000 (50:50 in weight) were added to mortars based on the same binders (aerial and hydraulic limes, gypsum, and cement mortars), even though greater mortar contents were employed in the present study (1000 kg for 1 m3 of each mortar) in order to enhance their mechanical properties. 1. Introduction The investigation proceeded further with the aim of producing a PEG-based PCM appropriate for a wider range of outdoor temperatures, including both high and low tem- peratures that can be recorded during the winter and summer seasons, respectively. To address this request, a mixed PEG-based PCM was produced, i.e., a PCM composed of 50 %wt LS/PEG800 and 50 %wt LS/PEG1000 The investigation proceeded further with the aim of producing a PEG-based PCM appropriate for a wider range of outdoor temperatures, including both high and low temperatures that can be recorded during the winter and summer seasons, respectively. To address this request, a mixed PEG-based PCM was produced, i.e., a PCM composed of 50 %wt LS/PEG800 and 50 %wt LS/PEG1000. %wt LS/PEG800 and 50 %wt LS/PEG1000. In this article, the two original PEG-based PCMs, i.e., LS/PEG800 and LS/PEG800_LS/PEG1000, were included in mortars based on different binders, namely aerial and hydraulic lime, cement, and gypsum. The properties of these mortars in fresh and solid states, if compared to the same mortars not containing the two PCMs, were In this article, the two original PEG-based PCMs, i.e., LS/PEG800 and LS/PEG800_LS/ PEG1000, were included in mortars based on different binders, namely aerial and hydraulic lime, cement, and gypsum. The properties of these mortars in fresh and solid states, if compared to the same mortars not containing the two PCMs, were evaluated with the aim to assess if it is possible to produce mortars including these new PCMs, suitable for even demanding applications. The thermal characteristics and latent heats of these mortars were also evaluated with the DSC technique, in order to analyze the phase change temperature intervals and thermal capacity of the mortars and to conﬁrm that the selection of PEG 800 to produce the new PCMs was correct. 4 of 15 Materials 2022, 15, 2497 2. Materials and Methods 2.1. Materials The eco-sustainable, form-stable PCMs were easily and cheaply produced by a combination of a porous stone and a family of low-cost polymers known to display low toxicity and ﬂammability. y y Lecce Stone (LS) is a unique, easily malleable, calcareous limestone extracted in the Salento area (located in the south-east area in Italy). The stone quarries are located close to the city of Lecce, hence the name “Lecce Stone”. During its extraction and workmanship, a large quantity of LS is wasted, posing the problem of its disposal. Starting from the opportunity to valorize a discarded material, waste Lecce Stone fragments were used as the inert support matrix for the form-stable PCMs. LS can, in fact, accomplish this task because it is a very porous material, as our earlier works have demonstrated [25,26]. In particular, characterization of the LS porosity, performed through mercury intrusion porosimetry (MIP), was previously performed and reported in [25]. These measurements revealed that 61% of pore size displays a radius in the range 0.25–2 µm, with an average pore radius of 0.054 ± 0.036 µm, and an open porosity equal to 30.33 ± 0.99%. Flakes of Lecce Stone were sourced from a quarry located in the Salento area (Cursi, Lecce, Italy). The as-received stone fragments were then milled and sieved, and small LS granules were ﬁnally obtained (observable in Figure 2a), with a granulometry of 1.6–2.0 mm. The LS particles, impregnated or not with a PEG polymer, were employed as aggregates of the different mortars investigated in the present study. VIEW 5 of 16 Figure 2. (a) Granules of Lecce Stone before the impregnation process; (b) granules of Lecce Stone impregnated with PEG800 (i.e., LS/PEG800). Figure 2. (a) Granules of Lecce Stone before the impregnation process; (b) granules of Lecce Stone impregnated with PEG800 (i.e., LS/PEG800). Figure 2. (a) Granules of Lecce Stone before the impregnation process; (b) granules of Lecce Stone impregnated with PEG800 (i.e., LS/PEG800). Figure 2. (a) Granules of Lecce Stone before the impregnation process; (b) granules of Lecce Stone impregnated with PEG800 (i.e., LS/PEG800). In a previous phase of the research [25,26], the PCM produced with LS/PEG1000 was added to mortar formulations based on different binders (i.e., aerial lime, hydraulic lime, gypsum, and cement) analyzing the properties of the obtained mortars in the fresh state, i.e., their workability, as well as after their curing, determining mechanical properties and latent heat characteristics. 2. Materials and Methods 2.1. Materials Several phys- PEG is a thermoplastic semi-crystalline polymer; it is commercially available in different molecular weights, corresponding to different ranges of melting/crystallization temperatures [18,28]. In a previous phase of the research [25,26,29], a PEG possessing a melting temperature in the range 37–40 ◦C was selected (i.e., PEG 1000), corresponding to a molecular weight of 1000. Our aim, in that case, was, in fact, the production of a sustainable Materials 2022, 15, 2497 5 of 15 5 of 15 PCM that could carry out its action, that is to change state from solid to liquid, at the temperatures characteristic of a Mediterranean warm climate; this ability was conﬁrmed by PEG 1000, as illustrated in [24]. Starting with the illustrated results, a PEG with a lower molecular weight, i.e., PEG 800, and a melting range of temperatures around 26–32 ◦C was analyzed in [27]. The aim was, in fact, the production of a PCM to be added to mortars suitable for cold climates, as in those climates characteristic of countries of Northern Europe. In the continuation of the project, it was established to also manufacture a mixed PCM, composed of both PEG 800 and PEG 1000 polymers. The mixed PCM is expected to be efﬁcient, if included in a mortar, in reducing the indoor temperature ﬂuctuations in a wider range of outdoor temperatures. Therefore, appropriately sized granules of Lecce Stone were impregnated by the PEG 800 polymer, and other LS granules by PEG 1000; in this way, two form-stable PCM composites were obtained, indicated as LS/PEG800 and LS/PEG1000, respectively. LS/PEG800 is visible in Figure 2b. The impregnation procedure, reported in detail in [25], allows one to introduce, under vacuum, the PEGs, brought into the liquid state at appropriate temperatures, into LS granules. p g In a previous phase of the research [25,26], the PCM produced with LS/PEG1000 was added to mortar formulations based on different binders (i.e., aerial lime, hydraulic lime, gypsum, and cement) analyzing the properties of the obtained mortars in the fresh state, i.e., their workability, as well as after their curing, determining mechanical properties and latent heat characteristics. 2. Materials and Methods 2.1. Materials Compositions of the produced and analyzed mortars (reported as kg/m3 of produced mortar). Mortars Binder a/ Content Aggregates SP Water Saturation b Water Water/ Binder LS PEG800 Content PEG1000 Content AL_LS AL/1000 668 0 0 20 168 347 0.35 AL_LS/PEG800 979 225 0 20 0 310 0.31 _LS/PEG800_LS/PEG1000 979 113 113 20 0 310 0.31 HL_LS HL/1000 682 0 0 20 171 380 0.38 HL_LS/PEG800 1082 249 0 20 0 320 0.32 _LS/PEG800_LS/PEG1000 1082 124 124 20 0 320 0.32 G_LS G/1000 763 0 0 20 192 385 0.39 G_LS/PEG800 1129 260 0 20 0 336 0.34 LS/PEG800_LS/PEG1000 1129 130 130 20 0 340 0.34 C_LS C/1000 772 0 0 20 194 390 0.39 C_LS/PEG800 1307 301 0 20 0 300 0.30 LS/PEG800_LS/PEG1000 1307 150 150 20 0 300 0.30 a Each binder is indicated with an acronym: AL represents Aerial Lime; HL represents Hydraulic Lime; G represents Gypsum; and C represents Cement. b The “water saturation” label is the amount of water used to saturate the LS aggregates in order to avoid them to absorb water required to manufacture the mortars. When an LS/PEG composite is used, no additional water was required, since the stone pores were almost completely saturated by PEG 800 or PEG 1000. A total of twelve compositions were developed, whose specimens can be observed in Figure 3. s 2022, 15, x FOR PEER REVIEW 7 of 16 Figure 3. Mortar specimens with indication of the binder and the aggregate used. 2.2. Methods The properties of the manufactured mortars, whose compositions are reported in Ta- ble 1, were evaluated in a fresh state (i.e., workability) as well as in a hardened state, de- termining their thermal properties and mechanical characteristics. The workability test, performed according to the EN 1015-3 standard [33], was per- formed on fresh mortars employing the flow table method (Figure 4a). Th h i i ld i d d di i d d Figure 3. Mortar specimens with indication of the binder and the aggregate used. 2.2. Methods The properties of the manufactured mortars, whose compositions are reported in Table 1, were evaluated in a fresh state (i.e., workability) as well as in a hardened state, determining their thermal properties and mechanical characteristics. Table 1. Compositions of the produced and analyzed mortars (reported as kg/m3 of produced mortar). 2. Materials and Methods 2.1. Materials Mortars Binder a/ Content Aggregates SP Water Saturation b Water Water/ Binder LS PEG800 Content PEG1000 Content AL_LS AL/1000 668 0 0 20 168 347 0.35 AL_LS/PEG800 979 225 0 20 0 310 0.31 AL_LS/PEG800_LS/PEG1000 979 113 113 20 0 310 0.31 HL_LS HL/1000 682 0 0 20 171 380 0.38 HL_LS/PEG800 1082 249 0 20 0 320 0.32 HL_LS/PEG800_LS/PEG1000 1082 124 124 20 0 320 0.32 G_LS G/1000 763 0 0 20 192 385 0.39 G_LS/PEG800 1129 260 0 20 0 336 0.34 G_LS/PEG800_LS/PEG1000 1129 130 130 20 0 340 0.34 C_LS C/1000 772 0 0 20 194 390 0.39 C_LS/PEG800 1307 301 0 20 0 300 0.30 C_LS/PEG800_LS/PEG1000 1307 150 150 20 0 300 0.30 a Each binder is indicated with an acronym: AL represents Aerial Lime; HL represents Hydraulic Lime; G represents Gypsum; and C represents Cement. b The “water saturation” label is the amount of water used to saturate the LS aggregates in order to avoid them to absorb water required to manufacture the mortars. When an LS/PEG composite is used, no additional water was required, since the stone pores were almost completely saturated by PEG 800 or PEG 1000. O EE E IE f a Each binder is indicated with an acronym: AL represents Aerial Lime; HL represents Hydraulic Lime; G represents Gypsum; and C represents Cement. b The “water saturation” label is the amount of water used to saturate the LS aggregates in order to avoid them to absorb water required to manufacture the mortars. When an LS/PEG composite is used, no additional water was required, since the stone pores were almost completely saturated by PEG 800 or PEG 1000. f a Each binder is indicated with an acronym: AL represents Aerial Lime; HL represents Hydraulic Lime; G represents Gypsum; and C represents Cement. b The “water saturation” label is the amount of water used to saturate the LS aggregates in order to avoid them to absorb water required to manufacture the mortars. When an LS/PEG composite is used, no additional water was required, since the stone pores were almost completely saturated by PEG 800 or PEG 1000. A total of twelve compositions were developed, whose specimens can be observed in Figure 3. A total of twelve compositions were developed, whose specimens can be observed in Figure 3. A total of twelve compositions were developed, whose specimens can be observed in Figure 3. 2. Materials and Methods 2.1. Materials As a continuation of the research, the PCMs based on LS/PEG800 and on the mix of LS/PEG800 with LS/PEG1000 (50:50 in weight) were added to mortars based on the same binders (aerial and hydraulic limes, gypsum, and cement mortars), even though greater mortar contents were employed in the present study (1000 kg for 1 m3 of each mortar) in order to enhance their mechanical properties. Several physical properties of the produced mortars were assessed. In [27], the characteristics determined on the aerial lime mortar containing LS/PEG800 only were reported. However, for comparison purposes, they are also reported in the present manuscript. The binders analyzed in this study were supplied by the following: Hydraulic lime (NHL) with a density of 2700 kg/m3 was obtained from the CIMPOR company (Lisbon, Portugal); LHOIST (Valverde, Alcanede, Portugal) provided aerial lime, possessing a density of 2450 kg/m3; CEM I 42.5 R cement (density of 3030 kg/m3) was supplied by SECIL (Lisbon, Portugal); conventional gypsum, possessing high ﬁneness and a density of 2960 kg/m3, was ﬁnally obtained by SIVAL (Souto da Carpalhosa, Leira, Portugal). All the compositions employed to produce the mortars were based on a binder content of 1000 kg/m3, and in order to achieve good mechanical properties in the developed mortars, a superplasticizer (SP) was employed, able to allow a substantial reduction of the mixing water. A superplasticizer, SP, (density of 1050 kg/m3) based on polyacrylate commercialized by the BASF company with the trade name Master Glenium SKY 627 (BASF, Porto, Portugal), was employed. The amount of superplasticizer was set at 20 kg/m3, in order to appreciably reduce the water content and, thus, improve the (expected) mechanical properties. It has been reported, in fact, that the addition of a PCM to mortars leads to severe reductions in its mechanical characteristics [9,30,31]. For each formulation containing LS/PEG800 or LS/PEG800_LS/PEG1000 PCMs, a reference mortar containing the same amount of non-impregnated Lecce Stone was manufactured for comparison purposes. All the mortar mixtures, of which the compositions are summarized in Table 1, were manufactured according to the European standard EN 998-1 [32]. For each binder, three mortar formulations were manufactured: The ﬁrst containing the composite material LS/PEG800; another one containing 50%wt. of LS/PEG800 and 50%wt. of LS/PEG1000; and the last, as a control, prepared by introducing pristine LS granules as aggregates. Materials 2022, 15, 2497 6 of 15 Table 1. 2. Materials and Methods 2.1. Materials Results and Discussion The physical properties of the mortars reported in Table 1, those containing the two The mortars were then cast in iron molds possessing standard dimensions and cured for 28 days in an environment maintained at standard levels of temperature (25 ◦C) and humidity (50%). mode. 3. Results and Discussion The physical properties of the mortars reported in Table 1, those containing the two The mortars were then cast in iron molds possessing standard dimensions and cured for 28 days in an environment maintained at standard levels of temperature (25 ◦C) and humidity (50%). The physical properties of the mortars reported in Table 1, those containing the two PCM composites (i.e., LS/PEG800 and LS/PEG800_LS/PEG1000) and the control formula- tions, were analyzed in both fresh and solid states. The flow table test was first performed on the produced mortars, in order to evaluate their workability and, consequently, their application adequacy. It is well known, in fact, that the workability values of mortar formulations must be comprised in the range of 160– 180 mm [35,36]. As it can be observed in Table 2, all the produced mortars show an ade- quate workability value, confirming the suitability of the selected formulations. Table 2. Workability values of the produced mortars (their compositions are reported in Table 1). Mortars Workability (mm) AL_LS 178 ± 2.0 The mortars, with or without the different PCMs, were thermally characterized with the aid of a Differential Scanning Calorimeter (DSC1 Stare System, Mettler Toledo, Columbus, OH, USA). The melting and crystallization processes of the polymeric phase contained in the PCM-based mortars were analyzed, monitoring the phase change of the LS/PEG composing any PCM. During the DSC analysis, the specimens of the mortars, containing or not one of the two PCMs, were subjected to a heating step from −10 ◦C to 80 ◦C and a subsequent cooling stage from 80 ◦C to −10 ◦C. The experiments were always carried out under an inert atmosphere employing nitrogen gas with a ﬂow rate of 60 mL/min, and at a heating/cooling rate of 10 ◦C/min rate was used. This rate was employed in order to compare the calorimetric data found in the present research with those previously measured on both LS/PEG PCMs. Three samples for each mortar (with a weight between 10 and 20 mg) were analyzed, averaging the obtained results. 2. Materials and Methods 2.1. Materials A total of twelve compositions were developed, whose specimens can be observed i Figure 3. Figure 3. Mortar specimens with indication of the binder and the aggregate used. 2.2. Methods Figure 3. Mortar specimens with indication of the binder and the aggregate used. 2.2. Methods igure 3. Mortar specimens with indication of the binder and the aggregate used. 2. Methods Figure 3. Mortar specimens with indication of the binder and the aggregate used. 2.2. Methods e o The properties of the manufactured mortars, whose compositions are reported in Ta- ble 1, were evaluated in a fresh state (i.e., workability) as well as in a hardened state, de- ermining their thermal properties and mechanical characteristics. The properties of the manufactured mortars, whose compositions are reported in Table 1, were evaluated in a fresh state (i.e., workability) as well as in a hardened state, determining their thermal properties and mechanical characteristics. Materials 2022, 15, 2497 7 of 15 7 of 15 The workability test, performed according to the EN 1015-3 standard [33], was performed on fresh mortars employing the ﬂow table method (Figure 4a). IEW 8 of 16 The workability test, performed according to the EN 1015-3 standard [33], was performed on fresh mortars employing the ﬂow table method (Figure 4a). IEW 8 of 16 The workability test, performed according to the EN 1015-3 standard [33], was performed on fresh mortars employing the ﬂow table method (Figure 4a). VIEW 8 of 16 Figure 4. Characterization tests performed on mortars in fresh and hardened states. (a) Determina- tion of workability with flow table test; (b) measurements of mechanical properties in compressive mode. 3 Results and Discussion Figure 4. Characterization tests performed on mortars in fresh and hardened states. (a) Determination of workability with ﬂow table test; (b) measurements of mechanical properties in compressive mode. The mortars were then cast in iron molds possessing standard dimensions and cured for 28 days in an environment maintained at standard levels of temperature (25 ◦C) and Figure 4. Characterization tests performed on mortars in fresh and hardened states. (a) Determina- tion of workability with flow table test; (b) measurements of mechanical properties in compressive d Figure 4. Characterization tests performed on mortars in fresh and hardened states. (a) Determination of workability with ﬂow table test; (b) measurements of mechanical properties in compressive mode. mode. 3. 2. Materials and Methods 2.1. Materials AL_LS/PEG800 160 ± 3.0 AL_LS/PEG800_LS/PEG1000 175 ± 2.0 HL_LS 175 ± 1.0 HL_LS/PEG800 170 ± 3.0 HL_LS/PEG800_LS/PEG1000 163 ± 2.0 G LS 170 ± 4 0 The mechanical characteristics of the cured mortars were, finally, determined (Figure 4b). The mechanical properties were measured in ﬂexural and compression mode, following the EN 1015-11 standard [34]. For each of the mortar formulations reported in Table 1, 3 prismatic specimens (40 × 40 × 160 mm3) were tested. The ﬂexural and compression tests were performed employing a Lloyd dynamometer instrument (LR50K Plus by Ametek Company, Berwyn, PA, USA) at speeds of 6 µm/s and 12 µm/s, respectively. G_LS/PEG G LS/PEG800 LS 3. Results and Discussion The presence of a high amount of superplasticizer in the fresh mixtures reduced the amount of water required by each mix (as shown in Figure 5), the latter being the prerequisite for obtaining mortars with high mechanical characteristics. VIEW 9 of 16 Figure 5. Water content for each mortar composition. 0 50 100 150 200 250 300 350 400 AL HL G C Water Content (L/m³) Mortars LS LS/PEG800 LS/PEG800_LS/PEG1000 Figure 5. Water content for each mortar composition. Figure 5. Water content for each mortar composition. 0 50 100 150 200 250 300 350 400 AL HL G C Water Content (L/m³) Mortars LS LS/PEG800 LS/PEG800_LS/PEG1000 Figure 5. Water content for each mortar composition. Figure 5. Water content for each mortar composition. Figure 5. Water content for each mortar composition. As can be seen in Figure 5, the mortar formulations based on the same binder and containing one of the two PCMs, i.e., LS/PEG800 or LS/PEG800_ LS/PEG1000, were man- ufactured with the same water content. In this way, it was possible to assess the effect of the employed PCM system on the properties of mortars in the fresh state, as well as in the hardened state. Upon the addition of both PCMs, a decrease in the water content needed to achieve suitable values of workability was recorded with respect to the control mortars. The greatest reduction in water was observed for the mortar based on cement. This be- havior could be attributed to small portions of the PEG800 polymer possessing a low vis- cosity at room temperature [27], with portions lying on the surface of the Lecce Stone grains that can contribute to improving the workability of the mortar mix with replace- As can be seen in Figure 5, the mortar formulations based on the same binder and containing one of the two PCMs, i.e., LS/PEG800 or LS/PEG800_ LS/PEG1000, were manufactured with the same water content. In this way, it was possible to assess the effect of the employed PCM system on the properties of mortars in the fresh state, as well as in the hardened state. Upon the addition of both PCMs, a decrease in the water content needed to achieve suitable values of workability was recorded with respect to the control mortars. The greatest reduction in water was observed for the mortar based on cement. G_LS/PEG G LS/PEG800 LS 3. Results and Discussion G_LS/PEG800_LS/PEG1000 163 ± 1.0 C_LS 180 ± 0.5 C_LS/PEG800 170 ± 1.0 The physical properties of the mortars reported in Table 1, those containing the two PCM composites (i.e., LS/PEG800 and LS/PEG800_LS/PEG1000) and the control formulations, were analyzed in both fresh and solid states. C_LS/PEG800_LS/PEG1000 170 ± 4.0 The presence of a high amount of superplasticizer in the fresh mixtures reduced the t f t i d b h i ( h i Fi 5) th l tt b i th The ﬂow table test was ﬁrst performed on the produced mortars, in order to evaluate their workability and, consequently, their application adequacy. It is well known, in fact, that the workability values of mortar formulations must be comprised in the range of Materials 2022, 15, 2497 8 of 15 160–180 mm [35,36]. As it can be observed in Table 2, all the produced mortars show an adequate workability value, conﬁrming the suitability of the selected formulations. adequate workability value, conﬁrming the suitability of the selected formulations. Table 2. Workability values of the produced mortars (their compositions are reported in Table 1). Mortars Workability (mm) AL_LS 178 ± 2.0 AL_LS/PEG800 160 ± 3.0 AL_LS/PEG800_LS/PEG1000 175 ± 2.0 HL_LS 175 ± 1.0 HL_LS/PEG800 170 ± 3.0 HL_LS/PEG800_LS/PEG1000 163 ± 2.0 G_LS 170 ± 4.0 G_LS/PEG800 165 ± 3.0 G_LS/PEG800_LS/PEG1000 163 ± 1.0 C_LS 180 ± 0.5 C_LS/PEG800 170 ± 1.0 C_LS/PEG800_LS/PEG1000 170 ± 4.0 The presence of a high amount of superplasticizer in the fresh mixtures reduced the amount of water required by each mix (as shown in Figure 5), the latter being the prerequisite for obtaining mortars with high mechanical characteristics. VIEW 9 of 16 Figure 5 Water content for each mortar composition 0 50 100 150 200 250 300 350 400 AL HL G C Water Content (L/m³) Mortars LS LS/PEG800 LS/PEG800_LS/PEG1000 Figure 5 Water content for each mortar composition Table 2. Workability values of the produced mortars (their compositions are reported in Table 1). Mortars Workability (mm) AL_LS 178 ± 2.0 AL_LS/PEG800 160 ± 3.0 AL_LS/PEG800_LS/PEG1000 175 ± 2.0 HL_LS 175 ± 1.0 HL_LS/PEG800 170 ± 3.0 HL_LS/PEG800_LS/PEG1000 163 ± 2.0 G_LS 170 ± 4.0 G_LS/PEG800 165 ± 3.0 G_LS/PEG800_LS/PEG1000 163 ± 1.0 C_LS 180 ± 0.5 C_LS/PEG800 170 ± 1.0 C_LS/PEG800_LS/PEG1000 170 ± 4.0 Table 2. Workability values of the produced mortars (their compositions are reported in Table 1). G_LS/PEG G LS/PEG800 LS 3. Results and Discussion This behavior could be attributed to small portions of the PEG800 polymer possessing a low viscosity at room temperature [27], with portions lying on the surface of the Lecce Stone grains that can contribute to improving the workability of the mortar mix with Materials 2022, 15, 2497 9 of 15 9 of 15 replacement of a limited amount of the required water. This feature can, nevertheless, represent an important advantage, potentially counteracting the detrimental effects on mechanical properties brought about by the addition of a PCM in the mortars, as previously reported [26,37–39]. The experimental campaign was then focused on determining the properties of the mortars, alone or containing the two PCMs, in the solid state, i.e., after adequate curing. The measurement of the thermal properties of the mortars allowed us to assess if the selection of PEG 800, and the production of two PCMs (i.e., LS/PEG800 and LS/PEG800_LS/PEG1000) based on this polymer, achieved the expected results in terms of suitable melting/crystallization temperature ranges. Determination of the mechanical properties helped to identify the best-performing mortars, i.e., those exhibiting suitable values of compressive and ﬂexural strengths. In Figure 6, the DSC curves of the mortars produced in this study are presented. For comparison purposes, the thermograms relative to the aerial lime mortar, containing the LS/PEG800 composite or not and previously determined [27], are also reported in Figure 6a. From the analysis of the DSC curves, the values shown in Table 3 were obtained. In particu- lar, the characteristic temperatures (initial, peak, and ﬁnal temperatures) monitored during the melting and the crystallization processes, and the relative latent heats measured on the produced mortars, containing LS/PEG800 or LS/PEG800_ LS/PEG1000 PCMs or not, are summarized. To facilitate evaluations and comparisons of data, the thermal characteristics previously determined on the mortar AL_LS/PEG800 are shown in Table 3 [27]. Table 3. Start, peak, and end temperatures and enthalpy measured during melting (heating stage) and subsequent crystallization (cooling stage) on the mortars based on different binders and containing one of the two LS/PEG PCM composites produced in this study. Table 3. Start, peak, and end temperatures and enthalpy measured during melting (heating stage) and subsequent crystallization (cooling stage) on the mortars based on different binders and containing one of the two LS/PEG PCM composites produced in this study. Table 3. G_LS/PEG G LS/PEG800 LS 3. Results and Discussion Start, peak, and end temperatures and enthalpy measured during melting (heating stage) d subsequent crystallization (cooling stage) on the mortars based on different binders and con- ning one of the two LS/PEG PCM composites produced in this study. T (°C) T (°C) T (°C) ΔH (J/ ) Observation of the calorimetric curves reported in Figure 6 allows us to conclude that the mortars not including any PCM based on a polymeric phase do not exhibit any melting/crystallization processes, irrespective of the kind of binder. No peaks are, in fact, noted in the range of temperatures analyzed. ystem Tstart (°C) Tm (°C) Tend (°C) ΔHm (J/g) S/PEG800 3.3 ± 1.9 15.0 ± 1.0 24.1 ± 1.2 11.8 ± 0.4 800_LS/PEG1000 4.3 ± 0.9 32.3 ± 0.8 45.2 ± 0.5 9.7 ± 2.1 S/PEG800 −2.3 ± 0.8 14.0 ± 0.8 21.2 ± 0.5 9.1 ± 0.9 800_LS/PEG1000 6.9 ± 3.1 32.4 ± 2.6 38.4 ± 0.8 9.1 ± 1.2 S/PEG800 2.5 ± 1.1 16.4 ± 0.8 24.6 ± 2.0 7.8 ± 0.6 00_LS/PEG1000 3.2 ± 0.9 30.8 ± 1.6 38.9 ± 2.7 8.1 ± 0.4 S/PEG800 −2.0 ± 0.8 17.3 ± 0.2 25.8 ± 1.8 9.5 ± 0.5 00_LS/PEG1000 3.5 ± 1.8 33.5 ± 0.2 42.3 ± 0.4 9.7 ± 0.9 ystem Tstart (°C) Tc (°C) Tend (°C) ΔHc (J/g) S/PEG800 17.7 ± 1.5 13.1 ± 1.1 −6.4 ± 0.9 12.5 ± 1.0 800_LS/PEG1000 29.0 ± 0.9 18.6 ± 1.6 −0.6 ± 1.2 10.8 ± 1.4 S/PEG800 14.6 ± 1.4 12.4 ± 3.0 −3.3 ± 0.6 10.3 ± 1.2 800_LS/PEG1000 25.1 ± 0.7 15.2 ± 0.5 −1.4 ± 2.2 9.2 ± 3.5 S/PEG800 15.8 ± 0.2 11.0 ± 0.3 −1.5 ± 0.9 9.2 ± 1.1 00_LS/PEG1000 25.7 ± 1.9 17.5 ± 2.3 −1.8 ± 3.1 9.2 ± 1.8 Conversely, peaks related to the melting of the PCM polymeric component in the heating stage and its crystallization in the cooling stage, are always present in the DSC thermograms recorded on the mortars containing a PCM system. G_LS/PEG G LS/PEG800 LS 3. Results and Discussion Start, peak, and end temperatures and enthalpy measured during melting (heating stage) and subsequent crystallization (cooling stage) on the mortars based on different binders and containing one of the two LS/PEG PCM composites produced in this study. System Tstart (◦C) Tm (◦C) Tend (◦C) ∆Hm (J/g) Heating Stage AL_LS/PEG800 3.3 ± 1.9 15.0 ± 1.0 24.1 ± 1.2 11.8 ± 0.4 AL_LS/PEG800_LS/PEG1000 4.3 ± 0.9 32.3 ± 0.8 45.2 ± 0.5 9.7 ± 2.1 HL_LS/PEG800 −2.3 ± 0.8 14.0 ± 0.8 21.2 ± 0.5 9.1 ± 0.9 HL_LS/PEG800_LS/PEG1000 6.9 ± 3.1 32.4 ± 2.6 38.4 ± 0.8 9.1 ± 1.2 G_LS/PEG800 2.5 ± 1.1 16.4 ± 0.8 24.6 ± 2.0 7.8 ± 0.6 G_LS/PEG800_LS/PEG1000 3.2 ± 0.9 30.8 ± 1.6 38.9 ± 2.7 8.1 ± 0.4 C_LS/PEG800 −2.0 ± 0.8 17.3 ± 0.2 25.8 ± 1.8 9.5 ± 0.5 C_LS/PEG800_LS/PEG1000 3.5 ± 1.8 33.5 ± 0.2 42.3 ± 0.4 9.7 ± 0.9 System Tstart (◦C) Tc (◦C) Tend (◦C) ∆Hc (J/g) Cooling Stage AL_LS/PEG800 17.7 ± 1.5 13.1 ± 1.1 −6.4 ± 0.9 12.5 ± 1.0 AL_LS/PEG800_LS/PEG1000 29.0 ± 0.9 18.6 ± 1.6 −0.6 ± 1.2 10.8 ± 1.4 HL_LS/PEG800 14.6 ± 1.4 12.4 ± 3.0 −3.3 ± 0.6 10.3 ± 1.2 HL_LS/PEG800_LS/PEG1000 25.1 ± 0.7 15.2 ± 0.5 −1.4 ± 2.2 9.2 ± 3.5 G_LS/PEG800 15.8 ± 0.2 11.0 ± 0.3 −1.5 ± 0.9 9.2 ± 1.1 G_LS/PEG800_LS/PEG1000 25.7 ± 1.9 17.5 ± 2.3 −1.8 ± 3.1 9.2 ± 1.8 C_LS/PEG800 19.1 ± 0.6 13.0 ± 1.3 −0.4 ± 0.2 10.5 ± 1.0 C_LS/PEG800_LS/PEG1000 25.9 ± 0.5 19.5 ± 2.4 0.3 ± 0.2 11.3 ± 3.4 10 of 15 en- Materials 2022, 15, 2497 Figure 6. DSC curves recorded for mortars, containing LS/PEG800 and LS/PEG800_ LS/PEG1000 PCM composites or not, based on (a) aerial lime; (b) hydraulic lime; (c) gypsum; (d) cement. Figure 6. DSC curves recorded for mortars, containing LS/PEG800 and LS/PEG800_ LS/PEG1000 PCM composites or not, based on (a) aerial lime; (b) hydraulic lime; (c) gypsum; (d) cement. ure 6. DSC curves recorded for mortars, containing LS/PEG800 and LS/PEG800_ LS/PEG1000 M composites or not, based on (a) aerial lime; (b) hydraulic lime; (c) gypsum; (d) cement. Figure 6. DSC curves recorded for mortars, containing LS/PEG800 and LS/PEG800_ LS/PEG1000 PCM composites or not, based on (a) aerial lime; (b) hydraulic lime; (c) gypsum; (d) cement. ble 3. G_LS/PEG G LS/PEG800 LS 3. Results and Discussion Comparing the melting/crystallization initial and ﬁnal temperatures, as well as the relative peaks, reported in Table 3, the following conclusions can be drawn: The peak temperatures for both melting and crystallization processes depend on the speciﬁc PCM added to each mortar; both the melting and crystallization processes are shifted to lower temperatures in the case of mortars containing the LS/PEG800 PCM with respect to those containing the LS/PEG800_ LS/PEG1000 composite. In addition, the melting/crystallization ranges of temperature are larger when the mortars include the LS/PEG800_ LS/PEG1000 PCM; for such mortars, the melting process is completed at approximately 39–45 ◦C. In the case of mortars including LS/PEG800 PCM, the melting process ends before 26 ◦C. The initial melting temperature, on the other hand, ranges between −2 and 6 ◦C, with no clear trend with respect to the kind of mortar or the added PCM; it essentially depends on the initial melting temperature of the polymer possessing the lower melting temperature, in all cases being PEG 800. It can be concluded, therefore, that the kind of mortar has no effect on melting/crystallization ranges of temperature, nor on the peak temperatures. Similar considerations can be made by analyzing the calorimetric data relating to the crystallization process, with the initial crystallization (solidiﬁcation) temperatures depending on the kind of PCM and the ﬁnal value being similar for all the mortars, basically depending on the presence of PEG 800 in both PCMs. Materials 2022, 15, 2497 11 of 15 11 of 15 The previous observations conﬁrm the hypothesis that led to the selection of the PEG 800 to produce a PCM displaying a phase change range of temperatures shifted to lower values with respect to PEG 1000, thus efﬁcient in colder climates. As already mentioned, in fact, a form-stable PCM composite based on PEG 1000, i.e., LS/PEG1000, was demonstrated to be able to reduce the energetic heating/cooling needs when added to mortars used in buildings located in warm climates [24]. On the other hand, the realization of a PCM based on two different polymers characterized by different molecular weights (i.e., PEG 800 and PEG 1000), thus exhibiting different melting/crystallization ranges, is a good omen for obtaining mortars containing PCMs that are effective at both low and high external temperatures. p As observed in Table 3, the latent heats for melting and crystallization processes for all the mortars containing a PCM range between 8 and 12 J/g. G_LS/PEG G LS/PEG800 LS 3. Results and Discussion These values appear independent of both the type of mortar and the added PCM. As already underlined, in the range of temperatures analyzed, the thermal characteristics of the mortars containing a PCM depend on the thermal characteristics of the polymeric phase of the speciﬁc PCM. It is expected, therefore, that the kind of mortar cannot affect the melting/crystallization enthalpies. Furthermore, since very similar melting/crystallization enthalpies were measured on the two LS/PEG PCMs (LS/PEG800: ∆Hm = 28.3 J/g, ∆Hc = 28.1 J/g; LS/PEG1000: ∆Hm = 27.7 J/g, ∆Hc = 26.2 J/g) [27], this explains why the type of composite PCM, i.e., LS/PEG800 or LS/PEG800_ LS/PEG1000, has a negligible inﬂuence on the measured latent heats, provided that the amount (% in weight) of the PEG in each PCM composite is the same in the produced mortars, as in the present case (the amount of both PEG 800 and PEG 1000 absorbed in the porous Lecce Stone granules is, in fact, similar, and equal to 23% [25,27]). The values of melting/crystallization enthalpies found in the present study are roughly in line with those previously reported for mortars containing the LS/PEG1000 PCM, mortars based on the same binders even if in a different composition [26]; it was demonstrated that the latent heats of these latter mortars were sufﬁcient to inﬂuence the internal temperature of an environment [24]. This observation suggests, once again, that the mortars containing one of the two PCMs produced in this study, i.e., LS/PEG800 or LS/PEG800_ LS/PEG1000, will also be able to act as an energy storage system when enclosed in mortars applied in buildings. The mechanical properties (tested in ﬂexural and compressive modes) of the mortar formulations reported in Table 1 were, ﬁnally, assessed after 28 days of aging. Table 4 reports the measured values of ﬂexural and compressive strengths recorded for each mortar, with the indication of the classiﬁcation according to the standard EN 998-1 [32]. Table 4. Mechanical properties measured in ﬂexural and compressive mode with the classiﬁcation, according to the standard EN 998-1:2010. according to the standard EN 998-1:2010. G_LS/PEG G LS/PEG800 LS 3. Results and Discussion (a) Values of flexural strength recorded for the mortars containing the different PEG-based PCMs; (b) values of compressive strength recorded for the mortars containing the different PEG- based PCMs. Figure 7. (a) Values of ﬂexural strength recorded for the mortars containing the different PEG- based PCMs; (b) values of compressive strength recorded for the mortars containing the different PEG-based PCMs. Keeping in mind the type of applications in which the PCM-based mortars can be proposed, the compressive strength of the mortars under analysis must at least fall within the CSII class. In this regard, mortars with the experimented PCMs based on hydraulic lime, gypsum, and cement fall within the requirements of the CSII class while those based on aerial lime do not meet the qualification. This result, as already evidenced in our pre- vious work for an aerial lime mortar based on LS/PEG800 [27], was also confirmed in the present study when the LS/PEG800_LS/PEG1000 compound is added to the same binder. It can be concluded, therefore, that the addition of a PCM based on LS/PEG to an aerial lime mortar does not allow it to achieve suitable mechanical characteristics. Keeping in mind the type of applications in which the PCM-based mortars can be proposed, the compressive strength of the mortars under analysis must at least fall within the CSII class. In this regard, mortars with the experimented PCMs based on hydraulic lime, gypsum, and cement fall within the requirements of the CSII class while those based on aerial lime do not meet the qualiﬁcation. This result, as already evidenced in our previous work for an aerial lime mortar based on LS/PEG800 [27], was also conﬁrmed in the present study when the LS/PEG800_LS/PEG1000 compound is added to the same binder. It can be concluded, therefore, that the addition of a PCM based on LS/PEG to an aerial lime mortar does not allow it to achieve suitable mechanical characteristics. lime mortar does not allow it to achieve suitable mechanical characteristics. The analysis of the flexural and compressive strength data, reported in Figure 7a,b, respectively, for the different mortars containing the experimented PCMs, allows us to conclude that the mortars offering satisfactory (flexural and compressive) mechanical characteristics are those based on hydraulic lime and cement, irrespective of the kind of PCM added. G_LS/PEG G LS/PEG800 LS 3. Results and Discussion Sample Flexural Strength (N/mm2) Compressive Strength (N/mm2) Classiﬁcation EN 998-1:2010 AL_LS 0.63 ± 0.19 1.47 ± 0.16 CSI AL_LS/PEG800 0.28 ± 0.12 0.44 ± 0.01 AL_LS/PEG800_LS/PEG1000 0.40 ± 0.01 0.61 ± 0.08 HL_LS 5.20 ± 1.53 11.66 ± 0.47 CSIV HL_LS/PEG800 2.32 ± 0.37 3.50 ± 0.15 CSII-CSIII HL_LS/PEG800_LS/PEG1000 2.13 ± 0.52 3.61 ± 0.54 G_LS 9.28 ± 1.26 22.34 ± 0.21 CSIV G_LS/PEG800 1.69 ± 0.04 4.33 ± 1.74 CSII-CSIII G_LS/PEG800_LS/PEG1000 2.34 ± 0.02 6.17 ± 0.51 C_LS 11.76 ± 1.11 65.55 ± 6.09 CSIV C_LS/PEG800 2.11 ± 0.11 3.86 ± 1.21 CSII-CSIII C_LS/PEG800_LS/PEG1000 2.03 ± 0.18 4.42 ± 0.72 Sample Flexural Strength (N/mm2) Compressive Strength (N/mm2) Materials 2022, 15, 2497 12 of 15 12 of 15 As expected, an important decrease in mechanical properties is generally observed as a consequence of the introduction of a PCM, irrespective to the kind of PEG composing the PCM. This behavior has been observed in several other studies; with the inclusion of PCM, the mechanical properties of mortars can signiﬁcantly decrease [23,38,39]. Several reasons may explain this occurrence: (i) An increase in the porosity of the mortar containing the PCM; (ii) a delay of the hydration reaction of the mortar; and (iii) in the case of a form-stable PCM, as in the present study, the polymeric component that constitutes the PCM, a part of which can be located on the surface of the inert support, can take part in the mixing of the mortar, reducing its mechanical properties. g p p No speciﬁc trend can be attributed to the PCM composition when measuring the ﬂexural strength. Referring to the compressive strength, slightly greater values of this property are achieved when adding the mixed PCM, i.e., that produced with the 50/50 % wt LS/PEG800_LS/PEG1000 composite, particularly in the case of the mortars based on gypsum and cement. To better appreciate the mechanical strength values recorded upon the addition of the two PCMs, the results of the mechanical characterization are additionally illustrated in Figure 7. VIEW 13 of 16 Figure 7. (a) Values of flexural strength recorded for the mortars containing the different PEG-based PCMs; (b) values of compressive strength recorded for the mortars containing the different PEG- based PCMs. Figure 7. (a) Values of ﬂexural strength recorded for the mortars containing the different PEG- based PCMs; (b) values of compressive strength recorded for the mortars containing the different PEG-based PCMs. Figure 7. G_LS/PEG G LS/PEG800 LS 3. Results and Discussion Mortars based on gypsum, on the other hand, displayed a lower value of flexural strength when LS/PEG800 was employed The analysis of the ﬂexural and compressive strength data, reported in Figure 7a,b, respectively, for the different mortars containing the experimented PCMs, allows us to conclude that the mortars offering satisfactory (ﬂexural and compressive) mechanical characteristics are those based on hydraulic lime and cement, irrespective of the kind of PCM added. Mortars based on gypsum, on the other hand, displayed a lower value of ﬂexural strength when LS/PEG800 was employed. flexural strength when LS/PEG800 was employed. Thermal experimental tests are, consequently, in progress on the mortars based on hydraulic lime and cement binders to assess the effective efficiency of LS/PEG-based com- posites as phase change materials in different climatic environments. The results of these tests, performed in a climatic chamber able to simulate different outdoor climatic condi- tions while recording the temperature inside a small room covered with the mortar under analysis will be the subject of a forthcoming manuscript Thermal experimental tests are, consequently, in progress on the mortars based on hydraulic lime and cement binders to assess the effective efﬁciency of LS/PEG-based composites as phase change materials in different climatic environments. The results of these tests, performed in a climatic chamber able to simulate different outdoor climatic conditions while recording the temperature inside a small room covered with the mortar under analysis, will be the subject of a forthcoming manuscript. Materials 2022, 15, 2497 13 of 15 2. Dulac, J.; Petrichenko, K.; Graham, P. Towards Zero-Emission Efﬁcient and Resilient Buildings. 2016. Available online: https: //backend.orbit.dtu.dk/ws/portalﬁles/portal/127199228/GABC_Global_Status_Report_V09_november.pdf (accessed on 23 February 2022). y 3. Delmastro, C. Thibaut Abergel IEA (2020). In Building Envelopes; IEA: Paris, France, 2020. 1. Tyagi, V.V.; Chopra, K.; Kalidasan, B.; Chauhan, A.; Stritih, U.; Anand, S.; Pandey, A.K.; Sarı, A.; Kothari, R. Phase Change Material Based Advance Solar Thermal Energy Storage Systems for Building Heating and Cooling Applications: A Prospective Research Approach. Sustain. Energy Technol. Assess. 2021, 47, 101318. [CrossRef] 4. REN21. Renewables 2021 Global Status Report; REN21 Secretariat: Paris, France, 2021; ISBN 978-3-94 Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The study does not report any data, please exclude the statement. Acknowledgments: The authors are grateful to the CRUI Foundation, which supported one of the authors of this research (Antonella Sarcinella) in the framework of the “GO FOR IT” project. Moreover, the authors wish to thank the L’essenza della Pietra Company (Taviano, Lecce, Italy) for supplying the Lecce stone pieces and the Eurostrade Company (Melissano, Lecce, Italy) for grinding the Lecce stone. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. g g p Renewables 2021 Global Status Report; REN21 Secretariat: Paris, France, 2021; ISBN 978-3-948393-03-8. g g p 4. REN21. Renewables 2021 Global Status Report; REN21 Secretariat: Paris, France, 2021; ISBN 978-3-948393-03-8. 4. Conclusions Institutional Review Board Statement: Not applicable. 4. Conclusions In this work, the main results of the physical analyses conducted on different mortar formulations containing two form-stable PCMs, are presented. The two phase change materials were realized by impregnating very porous Lecce Stone granules with Polyethylene Glycol polymers possessing different molecular weights and, in turn, different melting/crystallization ranges of temperatures, i.e., PEG 800 and PEG 1000. The obtained PCMs, LS/PEG800, and LS/PEG800_LS/PEG1000 were included as aggregates in aerial lime, hydraulic lime, gypsum, and cement mortar mixtures. The effect of the incorporation of each PCM on the workability of the fresh mortar mixtures and on the mechanical and thermal properties of the same cured mortars was evaluated, to establish the suitability and the efﬁcacy of the mortar mixtures produced with the PCMs. Although the addition of both PCMs in mortar led to modiﬁcations in their characteristics, in both fresh and solid states, with the proper selection of the formulation, it was possible to obtain mixtures possessing suitable workability and still adequate mechanical properties. In particular, it was possible to obtain mortars, containing one of the PEG-based PCMs, with a compressive strength classiﬁed within CSII/CSIII classes, except for the systems based on an aerial lime binder. Referring to the LHTES properties, measured on the mortars with the aid of calorimetric analysis, the selection of a PEG characterized by lower melting/crystallization temperatures allows one to obtain mortars with phase change temperatures characteristic of continental climates, as expected. Furthermore, the mortars containing the mixed PCM, i.e., that composed by LS/PEG800:LS/PEG1000 50:50%wt., showed a large interval of melting/crystallization temperatures, suggesting that this mortar could be suitable in both warm and cold climates. Thermal experimental tests are in progress in a climatic chamber able to simulate different climatic conditions on some of the best-performing mortars, i.e., those based on hydraulic lime and cement binders, to assess the effective efﬁciency of LS/PEG-based- composites as phase change materials in different weather conditions. Author Contributions: Conceptualization, M.F. and J.L.B.d.A.; experimentation, A.S.; validation, M.F. and J.L.B.d.A.; formal analysis, A.S.; data curation, A.S.; writing—original draft preparation, A.S.; writing—review and editing, M.F. and J.L.B.d.A.; project administration, M.F.; funding acquisition, M.F. All authors have read and agreed to the published version of the manuscript. Funding: This research was partly funded by the CRUI Foundation (Fondazione CRUI, Conferenza dei Rettori delle Università Italiane, Rome, Italy), in the framework of the GO FOR IT Project, which supported one of the authors. 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https://openalex.org/W3159623420	https://scholarworks.utrgv.edu/cgi/viewcontent.cgi?article=1178&context=mss_fac	English	null	Local Zeta Functions and Koba-Nielsen String Amplitudes	arXiv (Cornell University)	2,021	cc-by	32,628	Miriam Bocardo-Gaspar Hugo García-Compeán Edgar Y. López Wilson A. Zúñiga-Galindo Miriam Bocardo-Gaspar Hugo García-Compeán Edgar Y. López Wilson A. Zúñiga-Galindo Follow this and additional works at: https://scholarworks.utrgv.edu/mss_fac Part of the Mathematics Commons University of Texas Rio Grande Valley University of Texas Rio Grande Valley ScholarWorks @ UTRGV ScholarWorks @ UTRGV School of Mathematical and Statistical Sciences Faculty Publications and Presentations College of Scienc 5-29-2021 Local Zeta Functions and Koba–Nielsen String Amplitudes Local Zeta Functions and Koba–Nielsen String Amplitudes Miriam Bocardo-Gaspar Hugo García-Compeán Edgar Y. López Wilson A. Zúñiga-Galindo University of Texas Rio Grande Valley University of Texas Rio Grande Valley ScholarWorks @ UTRGV ScholarWorks @ UTRGV School of Mathematical and Statistical Sciences Faculty Publications and Presentations College of Sciences Miriam Bocardo-Gaspar 1, Hugo García-Compeán 2 , Edgar Y. López 2 and Wilson A. Zúñiga-Galindo 3,4,* Miriam Bocardo-Gaspar 1, Hugo García-Compeán 2 , Edgar Y. López 2 and Wilson A. Zúñiga-Galindo 3,4,* 1 Departamento de Matemáticas, Universidad de Guadalajara, Cucei, Blvd. Marcelino García Barragán #1421, 44430 Guadalajara, Mexico; miriam.bocardo@academicos.udg.mx j g 2 Centro de Investigación y de Estudios Avanzados del I.P.N., Departamento de Física, Av. Instituto Politécnico Nacional 2508, Col. San Pedro Zacatenco, 07360 Ciudad de México, Mexico; compean@ﬁs.cinvestav.mx (H.G.-C.); elopez@ﬁs.cinvestav.mx (E.Y.L.) 2 Centro de Investigación y de Estudios Avanzados del I.P.N., Departamento de Física, Av. Instituto Politécnico Nacional 2508, Col. San Pedro Zacatenco, 07360 Ciudad de México, Mexico; compean@ﬁs.cinvestav.mx (H.G.-C.); elopez@ﬁs.cinvestav.mx (E.Y.L.) p p 3 University of Texas Rio Grande Valley, School of Mathematical & Statistical Sciences, One West University Blvd, Brownsville, TX 78520, USA ne West University Blvd, Brownsville, TX 78520, USA y 4 Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional Departamento de Matemáticas, Unidad Querétaro Libramiento Norponiente #2000, Fracc, Real de Juriquilla, 76230 Santiago de Querétaro Mexico 76230 Santiago de Querétaro, Mexico 76230 Santiago de Querétaro, Mexico * Correspondence: wilson.zunigagalindo@utrgv.edu or wazuniga@math.cinvestav.edu.mx 76230 Santiago de Querétaro, Mexico * Correspondence: wilson.zunigagalindo@utrgv.edu or wazuniga@math.cinvestav.edu.mx * Correspondence: wilson.zunigagalindo@utrgv.edu or wazuniga@math.cinvestav.edu Abstract: This article is a survey of our recent work on the connections between Koba–Nielsen amplitudes and local zeta functions (in the sense of Gel’fand, Weil, Igusa, Sato, Bernstein, Denef, Loeser, etc.). Our research program is motivated by the fact that the p-adic strings seem to be related in some interesting ways with ordinary strings. p-Adic string amplitudes share desired characteristics with their Archimedean counterparts, such as crossing symmetry and invariance under Möbius transformations. A direct connection between p-adic amplitudes and the Archimedean ones is through the limit p →1. Gerasimov and Shatashvili studied the limit p →1 of the p-adic effective action introduced by Brekke, Freund, Olson and Witten. They showed that this limit gives rise to a boundary string ﬁeld theory, which was previously proposed by Witten in the context of background independent string theory. Explicit computations in the cases of 4 and 5 points show that the Feynman amplitudes at the tree level of the Gerasimov–Shatashvili Lagrangian are related to the limit p →1 of the p-adic Koba–Nielsen amplitudes. At a mathematical level, this phenomenon is deeply connected with the topological zeta functions introduced by Denef and Loeser. Academic Editor: Vladimir Al. Osipov Received: 1 May 2021 Accepted: 24 May 2021 Published: 29 May 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Keywords: string amplitudes; Koba–Nielsen amplitudes; Virasoro–Shapiro amplitudes; regulariza- tion; p-adic string amplitudes; local zeta functions; resolution of singularities Citation: Bocardo-Gaspar, M.; García-Compeán, H.; López, E.; Zúñiga-Galindo, W.A. Local Zeta Functions and Koba–Nielsen String Amplitudes. Symmetry 2021, 13, 967. https://doi.org/10.3390/ sym13060967 Miriam Bocardo-Gaspar 1, Hugo García-Compeán 2 , Edgar Y. López 2 and Wilson A. Zúñiga-Galindo 3,4,* A Koba– Nielsen amplitude is just a new type of local zeta function, which can be studied using embedded resolution of singularities. In this way, one shows the existence of a meromorphic continuations for the Koba–Nielsen amplitudes as functions of the kinematic parameters. The Koba–Nielsen local zeta functions are algebraic-geometric integrals that can be deﬁned over arbitrary local ﬁelds (for instance R, C, Qp, Fp((T))), and it is completely natural to expect connections between these objects. The limit p tends to one of the Koba–Nielsen amplitudes give rise to new amplitudes which we have called Denef–Loeser amplitudes. Throughout the article, we have emphasized the explicit calculations in the cases of 4 and 5 points. Recommended Citation Recommended Citation Bocardo-Gaspar, M.; García-Compeán, H.; López, E.; Zúñiga-Galindo, W.A. Local Zeta Functions and Koba–Nielsen String Amplitudes. Symmetry 2021, 13, 967. https://doi.org/10.3390/ sym13060967 Bocardo-Gaspar, M.; García-Compeán, H.; López, E.; Zúñiga-Galindo, W.A. Local Zeta Functions and Koba–Nielsen String Amplitudes. Symmetry 2021, 13, 967. https://doi.org/10.3390/ sym13060967 This Article is brought to you for free and open access by the College of Sciences at ScholarWorks @ UTRGV. It has been accepted for inclusion in School of Mathematical and Statistical Sciences Faculty Publications and Presentations by an authorized administrator of ScholarWorks @ UTRGV. For more information, please contact justin.white@utrgv.edu, william.flores01@utrgv.edu. symmetry S S symmetry S S Review 1. Introduction In the recent years, the connections between string amplitudes and arithmetic geome- try, p-adic analysis, combinatorics, etc. have been studied extensively, see e.g., [1–9] and the references therein. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). The string amplitudes were introduced by Veneziano in the 60s, In [10], further generalizations were obtained by Virasoro [11], Koba and Nielsen [12], among others. In the 80s, Freund, Witten and Volovich, among others, studied string amplitudes at the tree level over different number ﬁelds, and suggested the existence of connections between these amplitudes, see e.g., [4,13,14]. In this framework the connection with local zeta https://www.mdpi.com/journal/symmetry Symmetry 2021, 13, 967. https://doi.org/10.3390/sym13060967 Symmetry 2021, 13, 967 2 of 53 functions appears naturally. This article is intended as a survey of our recent work on the connections between Koba–Nielsen amplitudes and local zeta functions [6–9] in the sense of Gel’fand, Weil, Igusa, Sato, Bernstein, Denef, Loeser, etc. g The p-adic string theories have been studied over the time with some periodic fluctu- ations in their interest (for some reviews, see [3–5,15]). Recently aconsiderable amount of work has been performed on this topic in the context of the AdS/CFT correspondence [16–19]. String theory with a p-adic worldsheet was proposed and studied for the first time in [20]. Later this theory was formally known as p-adic string theory. The adelic scattering amplitudes which are related to the Archimedean ones were studied in [21]. The tree-level string ampli- tudes were explicitly computed in the case of p-adic string worldsheet in [14,22]. One can obtain these amplitude, in a formal way, from a suitable action using general principles [23]. In [24], it was established that the tree-level string amplitudes may be obtained starting with a discrete field theory on a Bruhat–Tits tree. Determining the convergence of the amplitudes in momentum space is a difficult task, both in the standard and p-adic case; however, for the latter, this was precisely done for the N-point tree amplitudes in [8]. 1. Introduction In this article we show (in a rigorous mathematical way) that the p-adic open string N-point tree amplitudes are bona fide integrals that admit meromorphic continuations as rational functions, this is done by associating to them multivariate local zeta functions (also called multivariate Igusa local zeta functions) [25–28]. In [6] we establish in a rigorous mathematical way that Koba–Nielsen amplitudes defined on any local field of characteristic zero (for instance R, C, Qp) are bona fide integrals that admit meromorphic continuations in the kinematic parameters. In the regularization process we use techniques of local zeta functions and embedded resolution of singularities. g The p-adic strings are related to ordinary strings at least in two different ways. First, connections through the adelic relations [21] and second, through the limit when p →1 [23,29]. In [29] was showed that the limit p →1 of the effective action gives rise to a boundary string field theory, this was previously proposed by Witten in the context of background independent string theory [30,31]. The limit p →1 in the effective theory can be performed without any problem, since one can consider p as a real parameter and take formally the limit p →1. The resulting theory is related to a field theory describing an open string tachyon [32]. In the limit p →1 also there are exact noncommutative solitons, some of these solutions were found in [33]. In [34] a very interesting physical interpretation of this limit was given in terms of a lattice discretization of ordinary string worldsheet. In the worldsheet theory we cannot forget the nature of p as a prime number, thus the analysis of the limit is more subtle. The correct way of taking the limit p →1 involves the introduction of finite extensions of the p-adic field Qp. The totally ramified extensions give rise to a finer discretization of the worldsheet following the rules of the renormalization group [34]. In [35] Denef and Loeser showed that the limit p approaches to one of a local zeta function give rise a new object called a topological zeta function. By using Denef–Loeser’s theory of topological zeta functions, in [9], we show that the limit p →1 of tree-level p-adic string amplitudes give rise to new amplitudes, that we have named Denef–Loeser string amplitudes. 1. Introduction Gerasimov and Shatashvili showed that in the limit p →1 the well-known non-local effective Lagrangian (reproducing the tree-level p-adic string amplitudes) gives rise to a simple Lagrangian with a logarithmic potential [29]. In [9], we conjecture that the Feynman amplitudes of this last Lagrangian are related to the Denef–Loser amplitudes. In [7] we establish rigorously the regularization of the p-adic open string amplitudes, with Chan–Paton rules and a constant B-ﬁeld. These amplitudes were originally introduced by Ghoshal and Kawano. To study these amplitudes, we introduce new multivariate local zeta functions depending on multiplicative characters (Chan–Paton factors) and a phase factor (the B-ﬁeld) which involves an antisymmetric bilinear form. We show that these integrals admit meromorphic continuations in the kinematic parameters, this result allows us to regularize the Ghoshal–Kawano amplitudes, the regularized amplitudes do not have ultraviolet divergencies. The theory is only valid for p congruent to 3 mod 4, this is to preserve a certain symmetry. The limit p →1 is also considered for the noncommutative Symmetry 2021, 13, 967 3 of 53 effective ﬁeld theory and the Ghoshal–Kawano amplitudes. We show that in the case of four points, the limit p →1 of the regularized Ghoshal–Kawano amplitudes coincides with the Feynman amplitudes attached to the limit p →1 of the noncommutative Gerasimov– Shatashvili Lagrangian. We denote by K a local ﬁeld of characteristic zero (for instance R, C, Qp), and set f := ( f1, . . . , fm) and s := (s1, . . . , sm) ∈Cm, where the fi(x) are non-constant polynomials in the variables x := (x1, . . . , xn) with coefﬁcients in K. The multivariate local zeta function attached to ( f, Θ), where Θ is a test function, is deﬁned as ZΘ( f, s) = Z Kn Θ(x) m ∏ i=1 \| fi(x)\|si K n ∏ i=1 dxi, when Re(si) > 0 for all i, when Re(si) > 0 for all i, and where ∏n i=1dxi is the normalized Haar measure of (Kn, +). These integrals admit meromorphic continuations to the whole Cm, [25,26,36], see also [37,38]. The study of Archimedean and non-Archimedean local zeta functions was started by Weil in the 60 s, in connection to the Poisson-Siegel formula. In the 70 s, Igusa developed a uniform theory for local zeta functions in characteristic zero [25,26], see also [36,39,40]. 1. Introduction In the p-adic setting, the local zeta functions are connected with the number of solutions of polynomial congruences mod pl and with exponential sums mod pl [28]. Recently, Denef and Loeser introduced the motivic zeta functions which constitute a vast generalization of p-adic local zeta functions [41,42]. In the 50 s, Gel’fand and Shilov studied the local zeta functions attached to real polyno- mials in connection with fundamental solutions, indeed, the meromorphic continuation of Archimedean local zeta functions implies the existence of fundamental solutions (i.e., Green functions) for differential operators with constant coefﬁcients. This fact was established, independently, by Atiyah [43] and Bernstein [44]. It is relevant to mention here that in the p-adic framework, the existence of fundamental solutions for pseudodifferential operators is also a consequence of the fact that the Igusa local zeta functions admit a meromorphic continuation, see ([45], Chapter 5), ([46], Chapter 10). This analogy turns out to be very important in the rigorous construction of quantum scalar ﬁelds in the p-adic setting, see [47] and the references therein. Take N ≥4, and complex variables s1j and s(N−1)j for 2 ≤j ≤N −2 and sij for 2 ≤i < j ≤N −2. Put s := sij ∈CD, where D = N(N−3) 2 denotes the total number of indices ij. In [6] we introduce the multivariate local zeta function Z(N) K (s) := Z KN−3 N−2 ∏ i=2 xj s1j K 1 −xj s(N−1)j K ∏ 2≤i<j≤N−2 xi −xj sij K N−2 ∏ i=2 dxi, (1) (1) where ∏N−2 i=2 dxi is the normalized Haar measure on KN−3. We called these integrals Koba- Nielsen local zeta functions. In ([6], Theorems 4.1 and 6.1), we show that these functions are bona ﬁde integrals, which are holomorphic in an open part of CD, containing the set given by 2 2 −2 N −2 < Re(sij) < −2 N for all ij. (2) (2) Furthermore, they admit meromorphic continuations to the whole CD. Furthermore, they admit meromorphic continuations to the whole CD. The Koba–Nielsen open string amplitudes for N-points over K are formally deﬁned as The Koba–Nielsen open string amplitudes for N-points over K are formally deﬁned as A(N) K (k) := Z KN−3 N−2 ∏ i=2 xj k1kj K 1 −xj kN−1kj K ∏ 2≤i<j≤N−2 xi −xj kikj K N−2 ∏ i=2 dxi, where k = (k1,. . .,kN), ki = (k0,i,. . 1. Introduction .,kl,i) ∈Rl+1, for i = 1,. . ., N (N ≥4), is the mo- mentum vector of the i-th tachyon (with Minkowski product kikj = −k0,ik0,j + k1,ik1,j + · · · + kl,ikl,j), obeying 4 of 53 Symmetry 2021, 13, 967 N ∑ i=1 ki = 0, kiki = 2 for i = 1, . . . , N. The parameter l is an arbitrary positive integer. Typically, l is taken to be 25 for bosonic strings. However, we do not require using the critical dimension. We choose units such that the tachyon mass is m2 = −2. In the real case, A(N) R (k) is (up to multiplication by a positive constant) the open Koba–Nielsen amplitude of N-points, see ([4], Section 8), ([48], Section 2). If N = 4, A(4) R (k) is the Veneziano amplitude [10]. In the complex case, by using the results of ([48], Section 2), in [49], the N-point, closed string amplitude at the tree level is the product of A(N) C (k) times a polynomial function in the momenta k. This fact implies that the techniques and results presented in [6] are applicable to classical closed string amplitudes at the tree level. We use the integrals Z(N) K (s) as regularizations of the amplitudes A(N) K (k). More precisely, we redeﬁne A(N) K (k) = Z(N) K (s) \|sij=kikj, (3) (3) where Z(N) K (s) now denotes the meromorphic continuation of (1) to the whole CD, see ([6], Theorem 6.1). Furthermore, A(N) K (k) extends to a meromorphic function to the whole CN(l+1), see ([6], Theorem 7.1). The articles [6,8] deal with the meromorphic continuation of local zeta functions of type Z(N) K (s), but their results are complementary. The main result of [8] is the existence of a meromorphic continuation for Z(N) K (s) in the case in which K is a non-Archimedean local ﬁeld of arbitrary characteristic. For instance Qp or Fq((T)), the ﬁeld of formal Laurent series with coefﬁcients in a ﬁnite ﬁeld Fq. The methods used are based on the stationary phase formula, which allows the explicit calculation of Z(N) K (s). On the other hand, the results presented in [6] depend completely on Hironaka’s resolution of singularities theorem [50], which is available only for ﬁelds of characteristic zero. 1. Introduction which in turn implies the existence of new type of ‘motivic string amplitude’ at the three level A(N) C((T))(L) which is a generalization of the p-adic Koba–Nielsen and Denef–Loeser amplitudes. Problem 1. Does the motivic string amplitudes A(N) C((T))(L) have a physical meaning? Is there an effective action such that the Feynman integrals at the three level are exactly the A(N) C((T))(L)? Problem 1. Does the motivic string amplitudes A(N) C((T))(L) have a physical meaning? Is there an effective action such that the Feynman integrals at the three level are exactly the A(N) C((T))(L)? In [9], we veriﬁed that the four and ﬁve-point Feynman amplitudes of the Gerasimov-Shatashvili Lagrangian are related by limp→1 A(N) Qp (s). Then we propose the following conjecture: Conjecture 2. The Feynman amplitudes at the three level of the Gerasimov–Shatashvili Lagrangian are by related limp→1 A(N) Qp (s). Conjecture 2. The Feynman amplitudes at the three level of the Gerasimov–Shatashvili Lagrangian are by related limp→1 A(N) Qp (s). A precise formulation of this conjecture is given at the end of Section 6. This survey is organized as follows. In Section 2, we review brieﬂy basic aspects of string amplitudes. Section 3 aims to provide an introduction to p-adic analysis. Section 4 is dedicated to the p-adic Koba–Nielsen amplitudes. We compute explicitly the amplitudes for 4 and 5 points. At the end of the section we provide an outline of the proof that the p-adic Koba–Nielsen amplitudes admit meromorphic continuations as rational functions in p−kikj. The Koba–Nielsen amplitudes can be deﬁned over any local ﬁeld (i.e., R, C, Qp, Fp((T)), etc.). Section 5 is dedicated to amplitudes deﬁned on local ﬁelds such as Qp, Fp((T)). A central idea is that the limit p tends to one of the p-adic Koba–Nielsen amplitudes gives a new amplitude, the Denef–Loeser amplitude. We compute the Denef– Loeser amplitudes for 4 and 5 points. Section 6 deals with the physical interpretation of the limit p →1 of p-adic string amplitudes. In this section we show, in the case of 4 and 5 particles, that the Feynman integrals at the tree level of the Gerasimov–Shatashvili Lagrangian are related Denef–Loeser amplitudes. We also give a precise formulation of Conjecture 2. Section 7 deals with the N-point p-adic string amplitudes, with Chan–Paton rules and a constant B-ﬁeld. We emphasize the explicit calculations of these amplitudes in the cases N = 4, 5. 1. Introduction In Section 8, we brieﬂy review some basic deﬁnitions and some central results about analytic manifolds on local ﬁelds of characteristic zero, Hironaka’s resolution of singularities theorem and multivariate Igusa zeta functions. In Section 10, we give an outline of the proof of the fact that Koba–Nielsen amplitudes deﬁned on local ﬁelds of characteristic zero admit meromorphic continuations in kikj ∈C. In the cases N = 4, 5, we show explicitly the existence of a meromorphic continuations for amplitudes by outlining the construction of certain embedded resolution of singularities. 1. Introduction In this framework, the results of [6] show that for any ﬁeld of characteristic zero K, all the zeta functions Z(N) K (s) converge on a common domain, see (2), and that the possible poles can be described in a geometric form. We propose the following conjecture: Conjecture 1. There is a meromorphic function Γ(s) in CD such that Z(N) R (s) = Γ(s) ∏ p<∞ Z(N) Qp (s) for any s ∈CD. This conjecture and ([6], Theorem 6.1) imply that ∏p<∞Z(N) Qp (s) admits a meromorphic continuation to the whole CD. By using (3), this conjecture implies that This conjecture and ([6], Theorem 6.1) imply that ∏p<∞Z(N) Qp (s) admits a meromorphic continuation to the whole CD. By using (3), this conjecture implies that A(N) R (s) = Γ(s) ∏ p<∞ A(N) Qp (s) is meromorphic function in CD. Freund and Witten established (formally) that the ordinary Veneziano and Virasoro–Shapiro four-particle scattering amplitudes can be factored in terms of an inﬁnite product of non-Archimedean string amplitudes [21]. More precisely, they established that the product of the p-adic string amplitudes multiplied by the crossing- symmetric Veneziano amplitude is equal to one. Later Aref’eva, Dragovich and Volovich noted that the inﬁnite product used by Freund and Witten is divergent, and established that the product of the p-adic string amplitudes multiplied by the crossing-symmetric Veneziano amplitude is equal to an expression containing the Riemann zeta function [51]. Symmetry 2021, 13, 967 5 of 53 We already mentioned that the Denef–Loeser amplitudes A(N) top (s) are the limit of the Koba–Nielsen amplitudes A(N) Qp (s) when p tends to one. This is a consequence of the fact that Z(N) top (s) = limp→1 Z(N) Qp (s), which in turn is a consequence of the motivic integration theory, see e.g., [41,52]. Motivic integration is a generalization of the p-adic integration and the integration with respect to the Euler characteristic. By using this theory one can construct a function Z(N) C((T))(L) such that Z(N) Qp (s) = Z(N) C((T))(L) \|L=p and Z(N) top (s) = Z(N) C((T))(L) \|L=1, which in turn implies the existence of new type of ‘motivic string amplitude’ at the three level A(N) C((T))(L) which is a generalization of the p-adic Koba–Nielsen and Denef–Loeser amplitudes. 2. Generalities of String Amplitudes String theory is the theory of tiny one-dimensional extended objects propagating in an underlying Riemannian or pseudo-Riemannian spacetime manifold M. The theory is deﬁned as a theory for a dynamical embedding map X : Σg,b,N →M, where Σg,b,N is a compact and oriented Riemann surface with a possible nonempty boundary (∂Σ ̸= 0) Symmetry 2021, 13, 967 6 of 53 characterized by the genus g, their number of boundaries b and N marked points. Σg,b,N has local coordinates σ = (σ0, σ1) (σa with a = 0, 1). Under a Wick rotation σ1 →σ1 and σ2 = iσ0, the metric written in terms of coordinates (σ1, σ2) becomes a metric with Euclidean signature. In addition to X there is a non-dynamical ﬁled hab on Σg,N, which is the intrinsic metric. The string may oscillate in the target space M and it has an inﬁnite number of quantum modes of oscillation carrying a representation of the Poincaré group of the target space ( In the present review will be enough to consider M to be the ﬂat Minkowskian spacetime) and characterized by a mass and a spin. In the bosonic string the spectrum consists of a tachyonic mode with negative square mass. There are a ﬁnite number of massless modes described by massless ﬁelds on M. These modes are the target space metric gµν with Lorentzian signature (−, +, . . . , +), the antisymmetric ﬁeld Bµν, and the dilaton ﬁeld Φ, where µ, ν = 0, . . . , D −1. M is the target space which is a Riemannian manifold of 26 dimensions in the case of the bosonic string and 10 dimensions for the superstring [53–55]. The Euclidean worldsheet action for the bosonic open string is given by I = S + S′, (4) (4) where where S = T 2 Z Σ d2σ √ h hab∂aXµ∂bXνgµν(X) −2πiα′Bµνϵab∂aXµ∂bXν = T 2 Z Σ d2σ √ hhab∂aXµ∂bXνgµν(X) −i 2 Z ∂Σ dlBµνXµ∂tXν, (5) (5) where T = 1 2πα′ is the string tension, here α′ = ℓ2 s and ℓs stands for the string length. In the above action the physical degrees of freedom are Xµ and hab, ∂t is the tangential derivative along ∂Σ and Bµν is an antisymmetric ﬁeld. 2. Generalities of String Amplitudes The perturbative scattering amplitudes of N particles are deﬁned as the correlation function of N vertex operators VΛi(ki). Furthermore, these amplitudes admit an expansion over the genus g of χ(Σ) = 2 −2g −b as follows: A(Λ1, k1, . . . , ΛN, kN) = ∞ ∑ g=0 λ2g−2+b s Ag(Λ1, k1, . . . , ΛN, kN), where λs := eΦ0 is the coupling constant of string and Ag is the Feynman functional integral where λs := eΦ0 is the coupling constant of string and Ag is the Feynman functional integral Ag(Λ1, k1, . . . , ΛN, kN) = 1 Vol Z Mg,b,N DXDh exp(−S) N ∏ i=1 VΛi(ki), here Vol stands for the volume of the symmetry groups leaving invariant the action (4). Here the integration is carried out over Mg,b,N the moduli space of Riemann surfaces of genus g, b boundaries and N marked points inserted on the boundary. The difﬁculty of integration increases with higher values of g, b and N. Fortunately it can be carried out in an explicit form for a few simple cases with lower values of g, b and N. Thus, for instance, the case for closed strings with genus g = 0 and N = 4, or open strings with g = 0, b = 1 and N = 4, are explicitly performed. Vertex operators VΛi(ki) are functionals of the embedding ﬁelds X and their derivatives. They are given by here Vol stands for the volume of the symmetry groups leaving invariant the action (4). Here the integration is carried out over Mg,b,N the moduli space of Riemann surfaces of genus g, b boundaries and N marked points inserted on the boundary. The difﬁculty of integration increases with higher values of g, b and N. Fortunately it can be carried out in an explicit form for a few simple cases with lower values of g, b and N. Thus, for instance, the case for closed strings with genus g = 0 and N = 4, or open strings with g = 0, b = 1 and N = 4, are explicitly performed. Vertex operators VΛi(ki) are functionals of the embedding ﬁelds X and their derivatives. They are given by VΛ(k) = Z d2σ √ hWΛ(σ)eikX(σ), where WΛ(σ) represents a functional of X and its derivatives associated with the species of ﬁeld in the string spectrum, X(σ) = (X0, . . 2. Generalities of String Amplitudes Moreover, the action S′ is given by S′ = Φ0χ(Σ), S′ = Φ0χ(Σ), where χ(Σ) = 1 4π R d2σ √ hR + BT is the Euler characteristic of Σ and BT is the Gibbons- Hawking boundary term that we encode in χ(Σ) with the number of boundaries b. Notice that the vacuum expectation value of the dilaton Φ0 can also be incorporated in the action through this topological term. It is also worth noting that this is the most general action that has worldsheet reparametrization invariance, in particular Weyl invariance (hab →Λ(σ)hab), and is renormalizable by power counting (this implies that there must be two worldsheet derivatives in each term). The second term is analogous to placing an electromagnetic ﬁeld in the background with which the string interacts; it is a gauge ﬁeld in that sense. Mathematically it is the integration of the pullback of the B-ﬁeld to the string worldsheet. If we deﬁne Aµ := BµνXν, the second term of the second line in (5) may be written as i i 2 Z ∂Σ Aµ∂tXµ, (6) (6) which is exactly the term considered in [56] where the partition function of the action was obtained explicitly at tree level. Strings propagating in non-trivial backgrounds have been considered in other works [57]. We now consider ﬂat space as well as a constant B-ﬁeld. The open string action (5) determines the Neumann boundary conditions gµν∂σXν + 2πiα′Bµν∂tXν\|∂Σ = 0, where ∂σ is the normal derivative to ∂Σ. We should note that these conditions are in general complex, because we have taken a Wick rotation to a Euclidean signature. We will be concerned about the case when the string worldsheet Σ is a disk, which corresponds to the open string in the classical limit. It is known that the disk can be transformed into the Symmetry 2021, 13, 967 7 of 53 upper half plane via a conformal transformation, whose boundary is the real line. In these variables the Neumann boundary conditions are [58] upper half plane via a conformal transformation, whose boundary is the real line. In these variables the Neumann boundary conditions are [58] gµν(∂−¯∂)Xν + 2πα′Bµν(∂+ ¯∂)Xν\|z=¯z = 0, where ∂= ∂/∂z and ¯∂= ∂/∂¯z, with Im z ≥0. where ∂= ∂/∂z and ¯∂= ∂/∂¯z, with Im z ≥0. where ∂= ∂/∂z and ¯∂= ∂/∂¯z, with Im z ≥0. 2. Generalities of String Amplitudes . , X25), k = (k0, . . . , k25) are the position and momentum vectors in target space M and kX(σ) := 25 ∑ µ,ν=0 kµηµνXν(σ) is the Minkowskian inner product. For instance for tachyons Λ = t, Wt(σ) = 1. For gauge ﬁelds Λ = A and WA(σ) = εµ∂tXµ, where εµ is the polarization vector. The graviton is WG(σ) = εµνhab∂aXµ∂bXν, where εµν is the polarization tensor. For bosonic closed strings, the tachyon is the lowest state and it has a k2 = 4 α′ , where k2 := kk is the Minkowskian norm. The open string tachyon has k2 = 1 α′ . In units where the Regge slope α′ = 1 2 or where the string tension T = 1 π we have that for the closed string tachyon k2 = 8 and for the open string k2 = 2, which will be used in the following sections of the present survey. The massless open string gauge ﬁeld and the closed string graviton have k2 = 0. For open strings one can see that in the functional integral with action (5) the expansion in Taylor’s series of exponential of the second term of (5), leads to a superposition of amplitudes with different powers of the vertex operator of the gauge ﬁeld (6) inserted in the boundary ∂Σ. From now on, we restrict ourselves to the case of bosonic closed strings with g = 0 and bosonic open strings with g = 0 and b = 1, i.e., the 2-sphere and the disk. For closed strings, by worldsheet diffeomorphisms and Weyl transformations of Σ, allow us to ﬁx the worldsheet metric hab to the Euclidean ﬂat metric δab. This is the called the conformal gauge. In the absence of a B-ﬁeld, there is a remnant SL(2, C) symmetry which cannot be ﬁxed by the local Weyl symmetry. This is called the conformal Killing symmetry. This symmetry does act on the complex worldsheet coordinates (z, z) of the 2-sphere separately. In the holomorphic sector (for the anti-holomorphic sector a similar argument is applied) Symmetry 2021, 13, 967 8 of 53 z 7→az + b cz + d, z 7→az + b cz + d, where ad −bc = 1 and a, b, c, d ∈R. This symmetry allows the choice of three points on the sphere which could be at 0, 1 and ∞. 2. Generalities of String Amplitudes For open strings the remnant symmetry preserves the boundary is SL(2, R). In the open string case the vertex operators are inserted on the boundary of the disk and this symmetry can be used to ﬁx three points on the boundary that also may be 0, 1 and ∞. As with the case of closed strings, for open strings the presence of a B-ﬁeld spoils the SL(2, R) symmetry avoiding the possibility to ﬁx three points of the positions of the vertex operators on the boundary ∂Σ. p p y The two-point function with these boundary conditions is given by ⟨Xµ(z)Xν(z′)⟩= −α′h gµν log \|z −z′\| −gµν log \|z −¯z′\| + Gµν log \|z −¯z′\|2 + 1 2πα′ θµν log z −¯z′ ¯z −z′ + Dµνi (7) (7) with with Gµν = 1 g + 2πα′B g 1 g −2πα′B µν , (8) θµν = −(2πα′)2 1 g + 2πα′B B 1 g −2πα′B µν , (8) and where Dµν are quantities independent on z. Basically, what we want are the N-tachyon scattering amplitudes at tree level for the action (5). This is done by inserting the following N tachyon vertex operators V(k, z) = eikX(z) at different points of the boundary of the open string worldsheet ∂Σ and obtaining the correlation functions of these operators. To obtain it we must realize that the correlators in the path integral formalism can be computed as Gaussian integrals [54]. Consider the following integral Z DX exp Z d2z(X∆X + iJX) ∼exp 1 2 Z d2zd2z′J(z)K(z, z′)J(z′) , (9) (9) where ∆is a differential operator. The symbol ∼has been used to indicate that there are some proportional factors not relevant to the analysis. Here K is the inverse operator, or Green’s function, that satisﬁes ∆K(z, z′) = δ(z −z′). ∆K(z, z′) = δ(z −z′). We can see that we can use this result to obtain the scattering amplitudes by choosing the appropriate source J(z). ⟨V(k1, z1)V(k2, z2) . . . V(kN, zN)⟩ ∼ Z DX exp{−S + i(k1X1(σ) + · · · + kNXN(σ))}. ⟨V(k1, z1)V(k2, z2) . . . V(kN, zN)⟩ ∼ Z DX exp{−S + i(k1X1(σ) + · · · + kNXN(σ))}. Since we are interested in the vertex operators inserted at the boundary, we should restrict the propagator to it. 2. Generalities of String Amplitudes By taking z = τ and z′ = τ′ for real τ, τ′ in (7) we get Since we are interested in the vertex operators inserted at the boundary, we should restrict the propagator to it. By taking z = τ and z′ = τ′ for real τ, τ′ in (7) we get ⟨Xµ(τ)Xν(τ′)⟩= −α′Gµν log τ −τ′ + i 2θµνsgn(τ −τ′), (10) (10) where sgn is the sign function. Now we can see that it sufﬁces to choose the current as where sgn is the sign function. Now we can see that it sufﬁces to choose the current as J(τ) = ∑ l δ(τ −τl)kl Symmetry 2021, 13, 967 9 of 53 9 of 53 in (9). From this we can obtain the scattering amplitudes by finally integrating the expected values of the vertex operators over the entire boundary of the worldsheet, which now is just integrating over the real variable τ. The result is in (9). From this we can obtain the scattering amplitudes by finally integrating the expected values of the vertex operators over the entire boundary of the worldsheet, which now is just integrating over the real variable τ. The result is Z dNτ⟨V(k1, τ1)V(k2, τ2) · · · V(kN, τN)⟩∼ Z dNτ exp −i 2 ∑ l>m klθkmsgn(τl −τm) ! × exp α′ ∑ l,m klGkm log\|τl −τm\| ! = Z dNτ exp −i 2 ∑ l>m klθkmsgn(τl −τm) ! ∏ l,m \|τl −τm\|α′klGkm, (11) Z dNτ⟨V(k1, τ1)V(k2, τ2) · · · V(kN, τN)⟩∼ Z dNτ exp −i 2 ∑ l>m klθkmsgn(τl −τm) ! × exp α′ ∑ l,m klGkm log\|τl −τm\| ! (11) (11) = Z dNτ exp −i 2 ∑ l>m klθkmsgn(τl −τm) ! ∏ l,m \|τl −τm\|α′klGkm, where klθkm := kl µθµνkm ν and klGkm := kl µGµνkm ν . These amplitudes are of the Koba– Nielsen type. The factor involving the sign function in the last line is actually more general, because it only depends on the variables through the sign functions, and any derivatives of it are 0. This allows us to write * N ∏ l=1 Pl(∂X(τl), ∂2X(τl) . . . )eiklX(τl) + G,θ = e i 2 ∑l>m klµθµνkmν sgn(τl−τm) * N ∏ l=1 Pl(∂X(τl), ∂2X(τl) . . . )eiklX(τl) + G,θ=0 . 2. Generalities of String Amplitudes The symbol ⟨· · · ⟩G,θ = 0 means that the expectation value taken on the second line is using the propagator (10) without the second term. This is achieved by writing the polynomial as an exponential and keeping only the corresponding linear terms. For example [48]: The symbol ⟨· · · ⟩G,θ = 0 means that the expectation value taken on the second line is using the propagator (10) without the second term. This is achieved by writing the polynomial as an exponential and keeping only the corresponding linear terms. For example [48]: V(ζ, k, z) = iζµ∂XµeikX(z) = [exp(ikX + iζµ∂Xµ)]linear, where [· · · ]linear indicates to only keep linear terms in ζ. So, we can see two direct effects of the B-ﬁeld on the action (5). The ﬁrst is that the effective metric on spacetime becomes Gµν given in (8). The second is that at tree level (Remember, this came from the fact that we are in the full upper half plane with the simplest zero genus topology) the scattering amplitudes acquire a phase factor that depends on the cyclic ordering of the momenta kµ and the matrix θ. But the overall form of the amplitudes is the same as without the B-ﬁeld. In the case of open strings for N points with vanishing B-ﬁeld and with ﬂat metric Gµν = ηµν and units with α′ = 1 and for a ﬁxed ordering of the external momenta ki, from (11) we obtain the Koba–Nielsen amplitude, with the three ﬁxed points in x1 = 0, xN−1 = 1 and xN = ∞, eA(N) R (k) = Z 0<x2<x3<···<xN−2<1 dx2dx3 · · · dxN−2 N−2 ∏ j=2 \|xj\|k1kj\|1 −xj\|kN−1kj × ∏ 2≤i<j≤N−2 \|xi −xj\|kikj, eA(N) R (k) = Z 0<x2<x3<···<xN−2<1 dx2dx3 · · · dxN−2 N−2 ∏ j=2 \|xj\|k1kj\|1 −xj\|kN−1kj where we have changed the variables τ to x’s variables in (11), In [48,53–55]. The four-point amplitude is Veneziano’s amplitude where we have changed the variables τ to x’s variables in (11), In [48,53–55]. The four-point amplitude is Veneziano’s amplitude eA(4) R (k) = Z 1 0 dx\|x\|k1k2\|1 −x\|k2k3. 2. Generalities of String Amplitudes (12) (12) It is known that this deﬁnition of Veneziano’s amplitude describes only one of the three decay channels s, t and u corresponding to the different inequivalent permutations of It is known that this deﬁnition of Veneziano’s amplitude describes only one of the three decay channels s, t and u corresponding to the different inequivalent permutations of Symmetry 2021, 13, 967 10 of 53 10 of 53 the momenta. If we consider the sum over the three channels and performing a suitable change of variables [59] is possible to write the total amplitude as the momenta. If we consider the sum over the three channels and performing a suitable change of variables [59] is possible to write the total amplitude as A(4) R (k) = Z R dx\|x\|k1k2\|1 −x\|k2k3. In terms of the gamma function the above amplitude writes A(4) R (k) = Γ(−1 −α′s)Γ(−1 −α′t) Γ(−2 −α′(s + t)) + Γ(−1 −α′s)Γ(−1 −α′u) Γ(−2 −α′(s + u)) + Γ(−1 −α′t)Γ(−1 −α′u) Γ(−2 −α′(t + u)) where s, t and u are the Mandelstam variables and they are deﬁned as s := −(p1 + p2)2, t := −(p1 + p3)2 and u := −(p1 + p4)2 and they satisfy s + t + u = −∑i k2 i = ∑i M2 i , and Γ(u) = Z ∞ 0 dt tu−1e−t is the gamma function. Thus, the 4-point amplitudes whose integration is R it already includes the three channels. Moreover, this procedure also works for N point amplitudes [4,48] and it yields Moreover, this procedure also works for N point amplitudes [4,48] and it yields A(N) R (k) = Z RN−3 dx2dx3 · · · dxN−2 N−2 ∏ j=2 \|xj\|k1kj\|1 −xj\|kN−1kj × ∏ 2≤i<j≤N−2 \|xi −xj\|kikj. For completeness we write the form of closed strings in absence of B-ﬁeld the N-point amplitude is given by A(N) C (k) = Z CN−2 ∏d2zi ∏ i<j \|zi −zj\|kikj/2. The four-point amplitude is the Virasoro–Shapiro amplitude The four-point amplitude is the Virasoro–Shapiro amplitude A(4) C (k) = Z C2 d2z\|z\|k1k4/2\|1 −z\|k2k4/2 = Γ(−1 −α′s 4 )Γ(−1 −α′t 4 )Γ(−1 −α′u 4 ) Γ(2 + α′s 4 )Γ(2 + α′t 4 )Γ(2 + α′u 4 ) . 3. Essential Ideas of p-Adic Analysis In this section, we review some ideas and results on p-adic analysis that we will use throughout this article. For an in-depth exposition, the reader may consult [3,61,62]. 3.1. The Field of p-Adic Numbers Throughout this article p will denote a prime number. The ﬁeld of p-adic numbers Qp is deﬁned as the completion of the ﬁeld of rational numbers Q with respect to the p-adic norm \| · \|p, which is deﬁned as \|x\|p =    0 if x = 0 p−γ if x = pγ a b, \|x\|p =    0 if x = 0 p−γ if x = pγ a b, where a and b are integers coprime with p. The integer γ := ord(x), with ord(0) := +∞, is called the p-adic order of x. We extend the p-adic norm to Qn p by taking where a and b are integers coprime with p. The integer γ := ord(x), with ord(0) := +∞, is called the p-adic order of x. We extend the p-adic norm to Qn p by taking \|\|x\|\|p := max 1≤i≤n \|xi\|p, for x = (x1, . . . , xn) ∈Qn p. We deﬁne ord(x) = min1≤i≤n{ord(xi)}, then \|\|x\|\|p = p−ord(x). The metric space Qn p, \|\| · \|\|p is a complete ultrametric space. As a topological space Qp is homeomorphic to a Cantor-like subset of the real line, see e.g., [3,61]. Any p-adic number x ̸= 0 has a unique expansion of the form x = pord(x) ∞ ∑ i=0 xipi, where xi ∈{0, 1, 2, . . . , p −1} and x0 ̸= 0. For r ∈Z, denote by Bn r (a) = {x ∈Qn p; \|\|x −a\|\|p ≤pr} the ball of radius pr with center at a = (a1, . . . , an) ∈Qn p, and take Bn r (0) := Bn r . Please note that Bn r (a) = Br(a1) × · · · × Br(an), where Br(ai) := {x ∈Qp; \|xi −ai\|p ≤pr} is the one-dimensional ball of radius pr with center at ai ∈Qp. The ball Bn 0 equals the product of n copies of B0 = Zp, the ring of p-adic integers. In addition, Bn r (a) = a + p−rZp n. We also denote by Sn r (a) = {x ∈Qn p; \|\|x −a\|\|p = pr} the sphere of radius pr with center at a ∈Qn p, and take Sn r (0) := Sn r . We notice that S1 0 = Z× p (the group of units of Zp), but Z× p n ⊊Sn 0. 2. Generalities of String Amplitudes It is worth noting that the Veneziano four-point amplitude (12), describing only one channel, can be written also as an integral over the whole R if we introduce some multiplicative characters as sgn(x): eA(4) R (k) = 1 2 Z R dx\|x\|k1k2\|1 −x\|k2k3 + 1 2 Z R dx\|x\|k1k2\|1 −x\|k2k3sgn(x) sgn(1 −x), where the function sign(x) is +1 of x > 0 and is −1 if x < 0 [14]. g ( ) The generalization for N-point amplitudes is given by ˜A(4) R (k) = Z RN−3 dx2dx3 · · · dxN−2 N−2 ∏ j=2 \|xj\|k1kj\|1 −xj\|kN−1kj 1 −sgn −xj(1 −xj) 2 11 of 53 Symmetry 2021, 13, 967 × ∏ 2≤i<j≤N−2 \|xi −xj\|kikj 1 2 1 −sgn (−1)(xi −xj) , In [14]. The open string amplitude (11) with a non-vanishing B-ﬁeld can be carried out in this form using the multiplicative character sign(x). This form is of particular interest in connection to the Ghoshal–Kawano amplitudes [60] studied later in this survey. Remark 1. There is a natural map, called the reduction mod p and denoted as ·, from Zp onto Fp, the ﬁnite ﬁeld with p elements. More precisely, if x = ∑∞ j=0 xjpj ∈Zp, then x = x0 ∈Fp = {0, 1, . . . , p −1}. If a = (a1, . . . , an) ∈Zn p, then a = (a1, . . . , an). 3.1. The Field of p-Adic Numbers The balls and spheres are both open and closed subsets in Qn p. In addition, two balls in Qn p are either disjoint or one is contained in the other. As a topological space Qn p, \|\| · \|\|p is totally disconnected, i.e., the only connected subsets of Qn p are the empty set and the points. A subset of Qn p is compact if and only if it is closed and bounded in Qn p, see e.g., ([3], Section 1.3), or ([61], Section 1.8). The balls and spheres are compact subsets. Thus, Qn p, \|\| · \|\|p is a locally compact topological space. Remark 1. There is a natural map, called the reduction mod p and denoted as ·, from Zp onto Fp, the ﬁnite ﬁeld with p elements. More precisely, if x = ∑∞ j=0 xjpj ∈Zp, then x = x0 ∈Fp = {0, 1, . . . , p −1}. If a = (a1, . . . , an) ∈Zn p, then a = (a1, . . . , an). Remark 1. There is a natural map, called the reduction mod p and denoted as ·, from Zp onto Fp, the ﬁnite ﬁeld with p elements. More precisely, if x = ∑∞ j=0 xjpj ∈Zp, then x = x0 ∈Fp = {0, 1, . . . , p −1}. If a = (a1, . . . , an) ∈Zn p, then a = (a1, . . . , an). 12 of 53 Symmetry 2021, 13, 967 12 of 53 3.2. Integration on Qn p Deﬁnition 1. A function ϕ ∈L1 loc is said to be integrable in Qn p if lim m→+∞ Z Bnm ϕ(x)dnx = lim m→+∞ m ∑ j=−∞ Z Sn j ϕ(x)dnx exists. If the limit exists, it is denoted as R Qnp ϕ(x)dnx, and we say that the (improper) integral exists. 3.2. Integration on Qn p Since (Qp, +) is a locally compact topological group, there exists a Borel measure dx, called the Haar measure of (Qp, +), unique up to multiplication by a positive constant, such that R U dx > 0 for every nonempty Borel open set U ⊂Qp, and satisfying R E+z dx = R E dx for every Borel set E ⊂Qp, see e.g., ([63], Chapter XI). If we normalize this measure by the condition R Zp dx = 1, then dx is unique. From now on we denote by dx the normalized Haar measure of (Qp, +) and by dnx the product measure on (Qn p, +). (Qp ) y p (Qp ) A function ϕ : Qn p →C is said to be locally constant if for every x ∈Qn p there exists an open compact subset U, x ∈U, such that ϕ(x) = ϕ(u) for all u ∈U. Any locally constant function ϕ : Qn p →C can be expressed as a linear combination of characteristic functions of the form ϕ(x) = ∑∞ k=1 ck1Uk(x), where ck ∈C and 1Uk(x) is the characteristic function of Uk, an open compact subset of Qn p, for every k. If ϕ has compact support, then ϕ(x) = ∑L k=1 ck1Uk(x) and in this case ϕ(x) = ∑L k=1 ck1Uk(x) and in this case Z Qnp ϕ(x)dnx = c1 Z U1 dnx + . . . + cL Z UL dnx. A locally constant function with compact support is called a Bruhat-Schwartz function. These functions form a C-vector space denoted as D Qn p . By using the fact that D Qn p is a dense subspace of Cc Qn p , the C-space of continuous functions on Qn p with compact support, with the topology of the uniform convergence, the functional ϕ →R Qnp ϕ(x)dnx, ϕ ∈D Qn p has a unique continuous extension to Cc Qn p , as an unbounded linear functional. For integrating more general functions, say locally integrable functions, the following notion of improper integral will be used. Deﬁnition 1. A function ϕ ∈L1 loc is said to be integrable in Qn p if 3.3. Analytic Change of Variables 3.3. Analytic Change of Variables A function h : U →Qp is said to be analytic on an open subset U ⊂Qn p, if for every b ∈U there exists an open subset eU ⊂U, with b ∈eU, and a convergent power series ∑i ai(x −b)i for x ∈eU, such that h(x) = ∑i∈Nn ai(x −b)i for x ∈eU, with xi = xi1 1 · · · xinn , i = (i1, . . . , in). In this case, ∂ ∂xl h(x) = ∑i∈Nn ai ∂ ∂xl (x −b)i is a convergent power series. Let U, V be open subsets of Qn p. A mapping h : U →V, h = (h1, . . . , hn) is called analytic if each hi is analytic. y Let ϕ : V →C be a continuous function with compact support, and let h : U →V be an analytic mapping. Then R V ϕ(y)dny = R U ϕ(h(x))\|Jac(h(x))\|pdnx, (13) (13) where Jac(h(z)) := det h ∂hi ∂xj (z) i 1≤i≤n 1≤j≤n , see e.g., ([64], Section 10.1.2). Example 1. In dimension one, the change of variables Formula (13) implies tha Example 1. In dimension one, the change of variables Formula (13) implies that Z aU dx = \|a\|p Z U dy, Symmetry 2021, 13, 967 13 of 53 Symmetry 2021, 13, 967 13 of 53 13 of 53 for any a ∈Q× p and any Borel set U ⊆Qp. For example, Z Br dx = Z p−rZp dx = \|p−r\|p Z Zp dy = pr Z Zp dy = pr, x = p−ry (14) and Z Sr dx = Z Br dx − Z Br−1 dx = pr −pr−1 = pr(1 −p−1). (15) Z Br dx = Z p−rZp dx = \|p−r\|p Z Zp dy = pr Z Zp dy = pr, x = p−ry (14) (14) and Z Sr dx = Z Br dx − Z Br−1 dx = pr −pr−1 = pr(1 −p−1). (15) (15) Example 2. We now compute the following integral Z(s) = Z Zp \|x\|s pdx, s ∈C, with Re(s) > 0 ∈C. We use the partition Zp = pZp ⊔Z× p , where Z× p is the group of units of Zp: Z× p := {x ∈Zp; \|x\|p = 1} = ( ∞ ∑ i=0 xipi ∈Zp; x0 ̸= 0 ) . 3.3. Analytic Change of Variables Using (15), Using (15), Z(s) = Z pZp \|x\|s pdx + Z Z×p \|x\|s pdx = p−1−s Z Zp \|x\|s pdx + Z Z×p dx = p−1−s Z Zp \|x\|s pdx + (1 −p−1) = p−1−sZ(s) + (1 −p−1). Therefore, Therefore, Z(s) = 1 −p−1 1 −p−1−s for Re(s) > −1. (16) (16) Example 3. We now consider the integral Example 3. We now consider the integral Z(s) := Z Z×p \|1 −x\|s pdx, s ∈C, with Re(s) > 0 ∈C. Assuming that Z× p = ⊔a0∈F×p a0 + pZp , where F× p = {1, 2, ..., p −1}, and (16) we have Z(s) = Z 1+pZp \|1 −x\|s pdx + p−1 ∑ a0=2 Z a0+pZp \|1 −x\|s pdx =p−1−s Z Zp \|y\|s pdy +p−1 p−1 ∑ a0=2 Z Zp \|1 −(a0 + py)\|s pdy = p−1−s 1 −p−1 1 −p−1−s + p−1(p −2). +p−1 p−1 ∑ a0=2 Z Zp \|1 −(a0 + py)\|s pdy = p−1−s 1 −p−1 1 −p−1−s + p−1(p −2). 3.4. Some p-Adic Integrals In this section, we compute some integrals that we will use later on. In this section, we compute some integrals that we will use later on. Example 4. We consider the following integral: Example 4. We consider the following integral: Z(s) := Z Zp×Zp \|x −y\|s pdxdy, s ∈C, with Re(s) > 0 ∈C. Symmetry 2021, 13, 967 14 of 53 14 of 53 Using the partition Z2 p = (pZp)2 ⊔S2 0, with S2 0 = pZp × Z× p ⊔Z× p × pZp ⊔Z× p × Z× p , we have Using the partition Z2 p = (pZp)2 ⊔S2 0, with S2 0 = pZp × Z× p ⊔Z× p × pZp ⊔Z× p × Z× p , we have we have Z(s) = Z (pZp)2 \|x −y\|s pdxdy + Z pZp×Z×p \|x −y\|s pdxdy + Z Z×p ×pZp \|x −y\|s pdxdy + Z Z×p ×Z×p \|x −y\|s pdxdy = p−2−sZ(s) + p−1 Z Zp×Z×p dxdy + p−1 Z Z×p ×Zp dxdy + Z Z×p ×Z×p \|x −y\|s pdxdy. 3.3. Analytic Change of Variables To compute the last integral, we use the partition (Z× p )2 = F a0,a1∈F×p a0 + pZp × a1 + pZp as follows: Z Z×p ×Z×p \|x −y\|s pdxdy = ∑ a0,a1∈F×p Z (a0+pZp)×(a1+pZp) \|x −y\|s pdxdy = p−2 ∑ a0,a1∈F×p a0̸=a1 Z Zp×Zp \|a0 + px −a1 −py\|s pdxdy + p−2−s ∑ a0,a1∈F×p a0=a1 Z Zp×Zp \|x −y\|s pdxdy = p−2(p −1)(p −2) + p−2−s(p −1)Z(s). = p−2(p −1)(p −2) + p−2−s(p −1)Z(s). Hence, Z(s) = 1 −p−1 1 −p−1−s . (17) e, Z(s) = 1 −p−1 1 −p−1−s . (17) (17) Example 5. We now show that Example 5. We now show that Z(s) := Z Z×p ×Z×p \|x −y\|s pdxdy = p−2(p −1)(p −2) + p−2−s(p −1) 1 −p−1 1 −p−1−s . (18) (18) Indeed, by changing variables as x = uv, y = v, Indeed, by changing variables as x = uv, y = v, Z(s) = Z Z×p ×Z×p \|u −1\|s pdudv = 1 −p−1Z Z×p \|u −1\|s pdu. Now the result follows from Example 3. Now the result follows from Example 3. Now the result follows from Example 3. Example 6. We now compute the following integral Example 6. We now compute the following integral Z(s1, s2, s3) := Z Z×p ×Z×p \|1 −x\|s1p \|1 −y\|s2p \|x −y\|s3p dxdy, where s1, s2, s3 ∈C satisfying Re(s1), Re(s2), Re(s3) > 0. By using the partition where s1, s2, s3 ∈C satisfying Re(s1), Re(s2), Re(s3) > 0. By using the partition (Z× p )2 = F a0,a1∈F×p a0 + pZp × a1 + pZp , where F× p = {1, 2, ..., p −1}, Symmetry 2021, 13, 967 15 of 53 Z(s1, s2, s3) = p−1 ∑ a0=1 p−1 ∑ a1=1 Z (a0+pZp)×(a1+pZp) \|1 −x\|s1p \|1 −y\|s2p \|x −y\|s3p dxdy =: p−1 ∑ a0=1 p−1 ∑ a1=1 Ja0,a1(s1, s2, s3). ) \|1 −x\|s1p \|1 −y\|s2p \|x −y\|s3p dxdy =: p−1 ∑ a0=1 p−1 ∑ a1=1 Ja0,a1(s1, s2, s3). By changing variables as x = a0 + pu, y = a1 + pv we have By changing variables as x = a0 + pu, y = a1 + pv we have a0,a1(s1, s2, s3) = p−2 Z Z2p \|1 −(a0 + pu)\|s1p \|1 −(a1 + pv)\|s2p \|(a0 + pu) −(a1 + pv)\|s3p dudv. 3.3. Analytic Change of Variables Ja0,a1(s1, s2, s3) = p−2 Z Z2p \|1 −(a0 + pu)\|s1p \|1 −(a1 + pv)\|s2p \|(a0 + pu) −(a1 + pv)\|s3p dudv. Now, we compute the integrals Ja0,a1. This calculation involves several cases. Now, we compute the integrals Ja0,a1. This calculation involves several cases. Case 1. If a0̸=1, a1̸=1 a0 ̸= a1, then \|1 −(a0 + pu)\|p = 1, \|1 −(a1 + pv)\|p = 1, and \|(a0 + pu) −(a1 + pv)\|p = 1. and Ja0,a1(s1, s2, s3) = p−2 Z Z2p dudv = p−2. There are (p −2)(p −3) integrals of this type. Case 2. If a0 ̸= 1, a1 ̸= 1 a0 = a1, then, \|1 −(a0 + pu)\|p = 1, \|1 −(a1 + pv)\|p = 1. By Example (17), f By Example (17), Ja0,a1(s1, s2, s3) = p−2−s3 Z Zp×Zp \|u −v\|s3p dudv = p−2−s3(1 −p−1) 1 −p−1−s3 , for Re(s3) > −1. There are p −2 integrals of this type. C 3 If 1 ̸ 1 h \|1 ( ) for Re(s3) > −1. There are p −2 integrals of this type. Case 3. If a0 = 1, a1 ̸= 1, then \|1 −(a1 + pv)\|p = 1, \|(a0 + pu) −(a1 + pv)\|p = 1. By using (16) f ( ) p g f yp Case 3. If a0 = 1, a1 ̸= 1, then \|1 −(a1 + pv)\|p = 1, \|(a0 + pu) −(a1 + pv)\|p = 1. By using (16) By using (16) Ja0,a1(s1, s2, s3) = p−2−s1 Z Z2p \|u\|s1p dudv = p−2−s1(1 −p−1) 1 −p−1−s1 , for Re(s1) > −1. There are p −2 integrals of this type. Case 4. If a0 ̸= 1, a1 = 1, this case is similar to Case 3, for Re(s1) > −1. There are p −2 integrals of this type. Case 4. If a0 ̸= 1, a1 = 1, this case is similar to Case 3, Ja0,a1(s1, s2, s3) = p−2−s2 Z Zp \|x3\|s2p dx3 = p−2−s2(1 −p−1) 1 −p−1−s2 for Re(s2) > −1. Therefore Z(s1, s2, s3) = p−2(p −2)(p −3) + (p −2) p−2−s3(1 −p−1) 1 −p−1−s3 + (p −2) p−2−s1(1 −p−1) 1 −p−1−s1 + (p −2) p−2−s2(1 −p−1) 1 −p−1−s2 . on the region of C3 deﬁned by Re(s1) > −1, Re(s2) > −1, and Re(s3) > −1. Symmetry 2021, 13, 967 16 of 53 4. p-Adic Open String Amplitudes 4. p-Adic Open String Amplitudes In [14], Brekke, Freund, Olson and Witten work out the N-point amplitudes in explicit form and investigate how these can be obtained from an effective Lagrangian. The p-adic open string N-point tree amplitudes are deﬁned as A(N)(k) = Z QN−3 p N−2 ∏ i=2 \|xi\|k1ki p \|1 −xi\|kN−1ki p ∏ 2≤i<j≤N−2 xi −xj kikj p N−2 ∏ i=2 dxi, (19) (19) where ∏N−2 i=2 dxi is the normalized Haar measure of QN−3 p , k = (k1, . . . , kN), ki = (k0,i, . . . , kl,i), i = 1, . . . , N, N ≥4, is the momentum vector of the i-th tachyon (with Minkowski product kikj = −k0,ik0,j + k1,ik1,j + · · · + kl,ikl,j) obeying N ∑ i=1 ki = 0, kiki = 2 for i = 1, . . . , N. The parameter l is an arbitrary positive integer. Typically, l is taken to be 25. However, we do not require using the critical dimension. In [8], we show that the p-adic open string amplitudes (19) are bona ﬁde integrals and that they have meromorphic continuations as rational functions in the kinematic parameters. We attach to these amplitudes a mul- tivariate Igusa-type zeta function. Fix an integer N ≥4 and set T := {2, . . . , N −2}, D = (N−3)(N−4) 2 + 2(N −3) and CD as    sij ∈C; i ∈{1, N −1}, j ∈T if N = 4 sij ∈C; i ∈{1, N −1}, j ∈T or i, j ∈T with i < j if N ≥5. if N = 4 We now put s = sij ∈CD and kikj = sij ∈C satisfying sij = sji for 1 ≤i < j ≤N −1. 4. p-Adic Open String Amplitudes For the second integral, we use that \|x\|s0 p is locally integrable, and thus 1 p \|x\|s0 p is locally integrable, and thus J1(s0) = ∞ ∑ j=1 Z p−jZ×p \|x\|s0 p dx = ∞ ∑ j=1 pj+js0 Z Z×p dx = 1 −p−1 ∞ ∑ j=1 pj(1+s0) < ∞ and only if s0 < −1. Then, integral J(s) does not exist for any complex value s. if and only if s0 < −1. Then, integral J(s) does not exist for any complex value s. Theorem 1 in [8] establishes that the p-adic open string N-point zeta function is a holomorphic function in a certain domain of CD and that it admits an analytic continuation to CD (denoted as Z(N)(s)) as a rational function in the variables p−sij, i, j ∈{1, . . . , N −1}. Hence, in the theory of local zeta functions, the convergence and the existence of a mero- morphic continuation as a rational function in the variables p−sij, i, j ∈{1, . . . , N −1} of integrals of type (20) is a new and remarkable result. The p-adic open string N-point zeta functions Z(N)(s) are regularizations of the amplitudes A(N)(k). More precisely, we deﬁne A(N)(k) = Z(N)(s) \|sij=kikj with i ∈{1, . . . , N −1}, j ∈T or i, j ∈T, where T = {2, . . . , N −2}. By Theorem 1 in [8], A(N)(k) are well-deﬁned rational func- tions of the variables p−kikj, i, j ∈{1, . . . , N −1}, which agree with integrals (87) when they converge. where T = {2, . . . , N −2}. By Theorem 1 in [8], A(N)(k) are well-deﬁned rational func- tions of the variables p−kikj, i, j ∈{1, . . . , N −1}, which agree with integrals (87) when they converge. where T = {2, . . . , N −2}. By Theorem 1 in [8], A(N)(k) are well-deﬁned rational func- tions of the variables p−kikj, i, j ∈{1, . . . , N −1}, which agree with integrals (87) when they converge. y g In the following two sections, using the methods given in [8], we compute the p-adic open string amplitudes in the cases N = 4, 5. 4.1. p-Adic Open String 4-Point Amplitude The 4-point string amplitude is given by 4.1. 4. p-Adic Open String Amplitudes The p-adic open string N-point zeta function is defined as We now put s = sij ∈CD and kikj = sij ∈C satisfying sij = sji for 1 ≤i < j ≤N −1 The p-adic open string N-point zeta function is defined as Z(N)(s) = Z QN−3 p ∖Λ N−2 ∏ i=2 \|xi\|s1i p \|1 −xi\| s(N−1)i p ∏ 2≤i<j≤N−2 xi −xj sij p N−2 ∏ i=2 dxi, (20) (20) where s = sij ∈CD, ∏N−2 i=2 dxi is the normalized Haar measure of QN−3 p , and where s = sij ∈CD, ∏N−2 i=2 dxi is the normalized Haar measure of QN−3 p , and Λ := ( (x2, . . . , xN−2) ∈QN−3 p ; N−2 ∏ i=2 xi(1 −xi) ∏ 2≤i<j≤N−2 xi −xj = 0 ) . In the deﬁnition of integral (20) we remove the set Λ from the domain of integration to use the formula as = es ln a for a > 0 and s ∈C. The reason for using the name ‘zeta function’ is that the integral (20) is a ﬁnite sum of multivariate local zeta functions. g Take φ(x2, . . . , xN−2) a locally constant function with compact support, then Z(N) φ (s) = Z QN−3 p ∖Λ φ(x2, . . . , xN−2) N−2 ∏ i=2 \|xi\|s1i p \|1 −xi\| s(N−1)i p ∏ 2≤i<j≤N−2 xi −xj sij p N−2 ∏ i=2 dxi, for Re(sij) > 0 for any ij, is a multivariate Igusa local zeta function. In characteristic zero, a general theory for this type of local zeta functions was elaborated by Loeser in [36]. In particular, these local zeta functions admit analytic continuations as rational functions of the variables p−sij. We want to highlight that the convergence of the multivariate local zeta functions depends on the fact that the locally constant function φ has compact support. For example, we consider the following integral: 17 of 53 Symmetry 2021, 13, 967 17 of 53 J(s) = Z Qp \|x\|s pdx, s ∈C. J(s) = Z Qp \|x\|s pdx, s ∈C. If the integral J(s0) exists for some s0 ∈R, then the integrals J0(s0) = Z Zp \|x\|s0 p dx and J1(s0) = Z Qp∖Zp \|x\|s0 p dx exist. The ﬁrst integral J0(s0) = 1−p−1 1−p−1−s0 for s0 > −1. 4. p-Adic Open String Amplitudes p-Adic Open String 4-Point Amplitude The 4-point string amplitude is given by 4.1. p-Adic Open String 4-Point Amplitude The 4-point string amplitude is given by A(4)(k) = Z Qp \|x2\|k1k2 p \|1 −x2\|k3k2 p dx2. We attach to this amplitude the local zeta function: We attach to this amplitude the local zeta function: We attach to this amplitude the local zeta function: We attach to this amplitude the local zeta function: Z(4)(s) = Z Qp \|x2\|s12 p \|1 −x2\|s32 p dx2. We now divide the integration domain (Qp) into sectors. Given I ⫅T = {2}, the at- tached sector is deﬁned as Sect(I) = xi ∈Qp; \|xi\|p ≤1 ⇐⇒i ∈I . Sect(I) = xi ∈Qp; \|xi\|p ≤1 ⇐⇒i ∈I . Then, we have two sectors: I T⧹I Sect(I) {2} ∅ Zp ∅ {2} Qp\Zp, 18 of 53 Symmetry 2021, 13, 967 d and Z(4)(s) = Z(4)(s, ∅) + Z(4)(s, {2}), where Z(4)(s; {2}) := Z Zp \|x2\|s12 p \|1 −x2\|s32 p dx2 and Z(4)(s; ∅) := Z Q Z \|x2\|s12 p \|1 −x2\|s32 p dx2. and Z(4)(s) = Z(4)(s, ∅) + Z(4)(s, {2}), where (4) Z and and Z(4)(s) = Z(4)(s, ∅) + Z(4)(s, {2}), h Z(4)(s) = Z(4)(s, ∅) + Z(4)(s, {2}), Z(4)(s) = Z(4)(s, ∅) + Z(4)(s, {2}), Z(4)(s; {2}) := Z Zp \|x2\|s12 p \|1 −x2\|s32 p dx2 Z(4)(s; {2}) := Z Zp \|x2\|s12 p \|1 −x2\|s32 p dx2 and Z(4)(s; ∅) := Z Qp∖Zp \|x2\|s12 p \|1 −x2\|s32 p dx2. We now compute Z(4)(s; {2}). By using the Examples 2 and 3: We now compute Z(4)(s; {2}). By using the Examples 2 and 3: Z(4)(s; {2}) = Z Zp \|x2\|s12 p dx2 + Z Z×p \|1 −x2\|s42 p dx2 (21) = 1 −2p−1 + 1 −p−1 p−1−s12 1 −p−1−s12 + 1 −p−1 p−1−s32 1 −p−1−s32 , (21) for Re(s12) > −1 and Re(s32) > −1. (4) for Re(s12) > −1 and Re(s32) > −1. (4) for Re(s12) > −1 and Re(s32) > −1. We now consider integral Z(4)(s;∅). By using the ultrametric inequality \|1−x2\|p = \|x2\|p for x2 ∈Qp ∖Zp, ( ) ( ) We now consider integral Z(4)(s;∅). By using the ultrametric inequality \|1−x2\|p = \|x2\|p for x2 ∈Qp ∖Zp, Z(4)(s; ∅) = Z Qp∖Zp \|x2\|s12+s32 p dx2. To compute this last integral we proceed as follows. 4. p-Adic Open String Amplitudes For l ∈N\{0}, we set Qp ∖Zp −l := x2 ∈ Qp ∖Zp ; −l ≤ord(x2) ≤−1 , pZp l := x2 ∈ pZp ; 1 ≤ord(x2) ≤l , Qp ∖Zp −l := x2 ∈ Qp ∖Zp ; −l ≤ord(x2) ≤−1 , pZp l := x2 ∈ pZp ; 1 ≤ord(x2) ≤l , and I−l(s) := Z (Qp∖Zp)−l \|x2\|s12+s32 p dx2. Notice that Qp ∖Zp −l, pZp l are compact sets and that Notice that Qp ∖Zp −l, pZp l are compact sets and that Qp ∖Zp −l → pZp l x2 → 1 y2 , Qp ∖Zp −l → pZp l x2 → 1 y2 , Qp ∖Zp −l → pZp l x2 → 1 y2 , is an analytic change of variables satisfying dx2 = dy2 \|y2\|2 p , then using this change of variables, we have I−l(s) = Z (pZp)l dy2 \|y2\|s12+s32+2 p . Now, by using that R pZp 1 \|y\|s p dy converges for Re(s) < 1 and the dominated conver- gence theorem, liml→∞I−l(s) = Z(4)(s; ∅), i.e. 19 of 53 19 of 53 Symmetry 2021, 13, 967 Z(4)(s; {∅}) = Z pZp dy2 \|y2\|s12+s32+2 p = (1 −p−1)p1+s12+s32 1 −p1+s12+s32 , on Re(s12) + Re(s32) < −1. Therefore Z(4)(s) = p −2 p + 1 −p−1 p−1−s12 1 −p−1−s12 + p−1−s32 1 −p(−1−s32) + p1+s12+s32 1 −p1+s12+s32 , this function is holomorphic in Re(s12) > −1, Re(s32) > −1 and Re(s12) + Re(s32) < −1, which is a nonempty open subset in C2 because it contains the open set deﬁned by the conditions: which is a nonempty open subset in C2 because it contains the open set deﬁned by the conditions: −2 3 < Re(s12) < −1 2 and −2 3 < Re(s32) < −1 2. We denote the meromorphic continuation of integral Z(4)(s) also by Z(4)(s). Now, we regularize the p-adic open string 4-point amplitude using Z(4)(s): A(4)(k) := Z(4)(s) \|s12=k1k2,s32=k3k2 = p −2 p + 1 −p−1( p−1−k1k2 1 −p−1−k1k2 + p−1−k2k3 1 −p−1−k2k3 + p1+k1k2+k2k3 1 −p1+k1k2+k2k3 ) = p −2 p + 1 −p−1( p−1−k1k2 1 −p−1−k1k2 + p−1−k2k3 1 −p−1−k2k3 + p−1−k2k4 1 −p−1−k2k4 ) . 4. p-Adic Open String Amplitudes (22) This amplitude can be rewritten as This amplitude can be rewritten as This amplitude can be rewritten as A(4)(k) = K4 + 1 2 ∑ i<j xij, with 1 k k A(4)(k) = K4 + 1 2 ∑ i<j xij, with xij := p −1 p p−1−kikj 1 −p−1−kikj , xij := p −1 p p−1−kikj 1 −p−1−kikj , where ∑i<j means the summation over all pairs of compatible channels ij. Then A(4)(k) = K4 + 1 2{x12 + x13 + x14 + x23 + x24 + x34}. Indeed, by using that ∑4 i=1 ki = 0, k2 i = 2, we obtain A(4)(k) = K4 + 1 2{x12 + x13 + x14 + x23 + x24 + x34}. using that ∑4 i ki = 0 k2 i = 2 we obtain A(4)(k) = K4 + 1 2{x12 + x13 + x14 + x23 + x24 + x34}. Indeed, by using that ∑4 i=1 ki = 0, k2 i = 2, we obtain k1k3 + k2k3 + k3k4 + 2 = 0, (23) k1k2 + k1k3 + k1k4 + 2 = 0, (24) k1k2 + k2k3 + k2k4 + 2 = 0, (25) k1k4 + k2k4 + k3k4 + 2 = 0. (26) k1k3 + k2k3 + k3k4 + 2 = 0, (23) k1k2 + k1k3 + k1k4 + 2 = 0, (24) k1k2 + k2k3 + k2k4 + 2 = 0, (25) k1k4 + k2k4 + k3k4 + 2 = 0. (26) (23) (26) From (23)–(26), we obtain From (23)–(26), we obtain From (23)–(26), we obtain k1k2 = k3k4, k1k3 = k2k4, k1k4 = k2k3. k1k2 = k3k4, k1k3 = k2k4, k1k4 = k2k3. k1k2 = k3k4, k1k3 = k2k4, k1k4 = k2k3. k1k2 = k3k4, k1k3 = k2k4, k1k4 = k2k3. Then A(4)(k) = K4 + x12 + x23 + x24, (27) Then Then Then A(4)(k) = K4 + x12 + x23 + x24, (27) A(4)(k) = K4 + x12 + x23 + x24, (27) (27) A(4)(k) = K4 + x12 + x23 + x24, (27) 20 of 53 Symmetry 2021, 13, 967 20 of 53 which agrees with (22) which agrees with (22). 4.2. p-Adic Open String 5-Point Amplitude 4.2. p-Adic Open String 5-Point Amplitude 4.2. 4. p-Adic Open String Amplitudes The integral Z(5)(s; {∅}) is holomorphic in the solution set of the following conditions: The integral Z(5)(s; {∅}) is holomorphic in the solution set of the following conditions: Symmetry 2021, 13, 967 21 of 53 Re(s12) + Re(s42) + Re(s23) < −1, (29) Re(s13) + Re(s43) + Re(s23) < −1, Re(s23) > −1, (29) Re(s23) > −1, Re(s12) + Re(s42) + Re(s13) + Re(s43) + Re(s23) < −2. Re(s12) + Re(s42) + Re(s13) + Re(s43) + Re(s23) < −2. Case 2. Sect({2}) = Zp × Qp\Zp . Case 2. Sect({2}) = Zp × Qp\Zp . In this case, \|x2\|p ≤1 and \|x3\|p > 1, now by ultrametric property \|1 −x3\|p = \|x3\|p and \|x2 −x3\|p = \|x3\|p, then Z(5)(s; {2}) = Z Zp×(Qp\Zp) \|x2\|s12 p \|x3\|s13 p \|1 −x2\|s42 p \|1 −x3\|s43 p \|x2 −x3\|s23 p dx2d = Z Zp×(Qp\Zp) \|x2\|s12 p \|x3\|s13+s43+s23 p \|1 −x2\|s42 p dx2dx3 = Z Zp \|x2\|s12 p \|1 −x2\|s42 p dx2 Z (Qp\Zp) \|x3\|s13+s43+s23 p dx3 =: Z(5) 0 (s; {2})Z(5) 1 (s; {2}). =: Z(5) 0 (s; {2})Z(5) 1 (s; {2}). The calculation of Z(5)(s; {2}, 0) is similar to the case of integral (21): The calculation of Z(5)(s; {2}, 0) is similar to the case of integral (21): Z(5)(s; {2}, 0) = p−1(p −2) + (1 −p−1)p−1−s12 1 −p−1−s12 + (1 −p−1)p−1−s42 1 −p−1−s42 . The integral Z(5)(s; {2}, 0) is holomorphic in the domain Re(s12) > −1 and Re(s42) > −1. (30) Re(s12) > −1 and Re(s42) > −1. (30) The calculation of Z(5)(s; {2}, 1) is similar to the calculation of Z(4)(s; {∅}) given in the Subsection of 4-point zeta function. By changing of variables as x3 = 1 y3 , dx3 = dy3 \|y3\|2p , we obtain Z(5)(s; {2}, 1) = Z Qp\Zp \|x3\|s13+s43+s23 p dx3 = Z pZp dy3 \|y3\|2+s13+s43+s23 p = (1 −p−1)p1+s13+s43+s23 1 −p1+s13+s43+s23 . Z(5)(s; {2}, 1) = Z Qp\Zp \|x3\|s13+s43+s23 p dx3 = Z pZp dy3 \|y3\|2+s13+s43+s23 p = (1 −p−1)p1+s13+s43+s23 1 −p1+s13+s43+s23 . = (1 −p−1)p1+s13+s43+s23 1 −p1+s13+s43+s23 . The integral Z(5)(s; {2}, 1) is holomorphic in The integral Z(5)(s; {2}, 1) is holomorphic in Re(s13) + Re(s43) + Re(s23) < −1. (31) (31) Case 3. Sect({3}) = Qp\Zp × Zp. Case 3. Sect({3}) = Qp\Zp × Zp. Case 3. Sect({3}) = Qp\Zp × Zp. 4. p-Adic Open String Amplitudes p-Adic Open String 5-Point Amplitude p p g p The p-adic open string 5-point amplitude is given by he p-adic open string 5-point amplitude is given by The p-adic open string 5-point amplitude is given by The p-adic open string 5-point amplitude is given by A(5)(k) = Z Q2p \|x2\|k1k2 p \|x3\|k1k3 p \|1 −x2\|k4k2 p \|1 −x3\|k4k3 p \|x2 −x3\|k2k3 p dx2dx, and the 5-point local zeta function is given by Z(5)(s) = Z Q2p \|x2\|s12 p \|x3\|s13 p \|1 −x2\|s42 p \|1 −x3\|s43 p \|x2 −x3\|s23 p dx2dx3, where sij ∈C. We divide the integration domain Q2 p in sectors. Given I ⫅T = {2, 3} the attached sector is deﬁned as Sect(I) = n (x1, x2) ∈Q2 p; \|xi\|p ≤1 ⇐⇒i ∈I o . Table 1 shows all the sectors for the computation of the 5-point amplitude. Table 1. Sectors of the 5-point amplitude. I T⧹I Sect(I) ∅ {2, 3} Qp\Zp × Qp\Zp {2} {3} Zp × Qp\Zp {3} {2} Qp\Zp × Zp {2, 3} ∅ Zp × Zp. Now, by using the notation Now, by using the notation Z(5)(s; I) = Z Sect(I) \|x2\|s12 p \|x3\|s13 p \|1 −x2\|s42 p \|1 −x3\|s43 p \|x2 −x3\|s23 p dx2dx3, we have Z(5)(s) = Z(5)(s; ∅) + Z(5)(s; {2}) + Z(5)(s; {3}) + Z(5)(s; {2, 3}). Case 1. Sect({∅}) = Qp\Zp × Qp\Zp . By changing of variables as x2 →1 u, x3 →1 v and using Lemma 7 of [8], we have Z(5)(s; {∅}) = Z (Qp\Zp)2 \|x2\|s12+s42 p \|x3\|s13+s43 p \|x2 −x3\|s23 p dx2dx3. = Z (pZp)2 \|u\|−s12−s42−2 p \|v\|−s13−s43−2 p \| 1 u −1 v\|s23 p dudv \| \|s23 +s12+s42+s23+2+s13+s43+s23+2−s23 Z Z2p \|v −u\|s23 p \|u\|s12+s42+s23+2 p \|v\|s13+s43+s23+2 p dudv (28) = p2+s12+s42+s23+s13+s43(1 −p−1) 1 −p2+s12+s42+s23+s13+s43 (1 −p−1)p1+s12+s42+s23 1 −p1+s12+s42+s23 + (1 −p−1)p1+s13+s43+s23 1 −p1+s13+s43+s23 + (1 −p−1)p−1−s23 1 −p−1−s23 + (p −2)p−1 . (28) Z Z2p \|u\|s12+s42+s23+2 p \|v\|s13+s43+s23+2 p = p2+s12+s42+s23+s13+s43(1 −p−1) 1 −p2+s12+s42+s23+s13+s43 (1 −p−1)p1+s12+s42+s23 1 −p1+s12+s42+s23 + (1 −p−1)p1+s13+s43+s23 1 −p1+s13+s43+s23 + (1 −p−1)p−1−s23 1 −p−1−s23 + (p −2)p−1 . 4. p-Adic Open String Amplitudes In this case, \|x2\|p > 1, by the ultrametric inequality \|1 −x2\|p = \|x2\|p and \|x2 −x3\|p = \|x2\|p, then 22 of 53 Symmetry 2021, 13, 967 Z(5)(s; {3}) = Z (Qp\Zp)×Zp \|x2\|s12 p \|x3\|s13 p \|1 −x2\|s42 p \|1 −x3\|s43 p \|x2 −x3\|s23 p dx2dx3 = Z Qp\Zp×Zp \|x2\|s12+s42+s23 p \|x3\|s13 p \|1 −x3\|s43 p dx2dx3 = Z Zp \|x3\|s13 p \|1 −x3\|s43 p dx3 Z Qp\Zp \|x2\|s12+s42+s23 p dx2 =: Z(N) 0 (s; {3})Z(N) 1 (s; {3}). These integrals are similar to the ones obtained in the case of Sect({2}): These integrals are similar to the ones obtained in the case of Sect({2}): Z(5)(s; {3}, 0) = p−1(p −2) + (1 −p−1)p−1−s13 1 −p−1−s13 + (1 −p−1)p−1−s43 1 −p−1−s43 , (32) (32) the integral Z(5)(s; {3}, 0) is holomorphic in Re(s13) > −1 and Re(s43) > −1. (33) Re(s13) > −1 and Re(s43) > −1. (33) And Z(5)(s; {3}, 1) = (1 −p−1)p1+s12+s42+s23 1 −p1+s12+s42+s23 , the integral Z(5)(s; {3}, 1) is holomorphic in the integral Z(5)(s; {3}, 1) is holomorphic in Re(s12) + Re(s42) + Re(s23) < −1. (34) Re(s12) + Re(s42) + Re(s23) < −1. (34) Case 4. Sect({2, 3}) = Zp × Zp. Case 4. Sect({2, 3}) = Zp × Zp. Using the partition Z2 p = (pZp)2 ⊔S2 0, with S2 0 = pZp × Z× p ⊔Z× p × pZp ⊔Z× p × Z× p , we have Using the partition Z2 p = (pZp)2 ⊔S2 0, with S2 0 = pZp × Z× p ⊔Z× p × pZp ⊔Z× p × Z× p , we have Z(5)(s; {2, 3}) = Z Zp×Zp \|x2\|s12 p \|x3\|s13 p \|1 −x2\|s42 p \|1 −x3\|s43 p \|x2 −x3\|s23 p dx2dx3 =: I31(s) + I32(s) + I33(s) + I34(s), where I31(s) := R (pZp) 2 \|x2\|s12 p \|x3\|s13 p \|x2 −x3\|s23 p dx2dx3, I32(s) := R pZp×Z×p \|x2\|s12 p \|1 −x3\|s43 p dx2dx3, I33(s) := R Z×p ×pZp \|x3\|s13 p \|1 −x2\|s42 p dx2dx3, I34(s) := R Z×p ×Z×p \|1 −x2\|s42 p \|1 −x3\|s43 p \|x2 −x3\|s23 p dx2dx3. Using Lemma 4 of [8], we have Using Lemma 4 of [8], we have Using Lemma 4 of [8], we have I31(s) = p−2−s12−s13−s23 1 −p−2−s12−s13−s23 p−1−s12(1 −p−1)2 1 −p−1−s12 + p−1−s13(1 −p−1)2 1 −p−1−s13 + p−1−s23(1 −p−1)2 1 −p−1−s23 + (p −1)(p −2)p−2 . 4. p-Adic Open String Amplitudes Symmetry 2021, 13, 967 23 of 53 The integral I31(s) is holomorphic in The integral I31(s) is holomorphic in The integral I31(s) is holomorphic in The integral I31(s) is holomorphic in Re(s12) > −1, Re(s13) > −1, Re(s23) > −1, and Re(s12) + Re(s13) + Re(s23) > −2. (35) (35) Using Examples 2 and 3, Using Examples 2 and 3, I32(s) = (1 −p−1)p−1−s12 1 −p−1−s12 (1 −p−1)p−1−s43 1 −p−1−s43 + p−1(p −2) , and I33(s) = (1 −p−1)p−1−s13 1 −p−1−s13 (1 −p−1)p−1−s42 1 −p−1−s42 + p−1(p −2) . These integrals are holomorphic in Re(s12) > −1, Re(s13) > −1, Re(s42) > −1, and Re(s43) > −1. (36) (36) Finally, by Example 6, Finally, by Example 6, I34(s) = p−2(p −2)(p −3) + (p −2) p−2−s23(1 −p−1) 1 −p−1−s23 (37) + (p −2) p−2−s42(1 −p−1) 1 −p−1−s42 + (p −2) p−2−s43(1 −p−1) 1 −p−1−s43 . (37) This integral is holomorphic in This integral is holomorphic in This integral is holomorphic in Re(s42) > −1, Re(s43) > −1, and Re(s23) > −1. (38) (38) In conclusion, the 5-point local zeta functions is holomorphic on the region of C5 deﬁned by In conclusion, the 5-point local zeta functions is holomorphic on the region of C5 deﬁned by Re(s12) > −1, Re(s13) > −1, Re(s42) > −1, and Re(s43) > −1, Re(s12) + Re(s42) + Re(s23) < −1, Re(s13) + Re(s43) + Re(s23) < −1, Re(s12) + Re(s13) + Re(s23) > −2. Re(s12) + Re(s42) + Re(s13) + Re(s43) + Re(s23) < −2. which is a nonempty subset, since it contains the open set which is a nonempty subset, since it contains the open set −2 3 < Re(sij) < 0 , −2 3 < Re(s1i) < −1 2, −2 3 < Re(s(N−1)i) < −1 2. We regularize the p-adic open string 5-point amplitude using the meromorphic continuation of Z(5)(s) by setting We regularize the p-adic open string 5-point amplitude using the meromorphic continuation of Z(5)(s) by setting A(5)(k) := Z(5)(s) \|s12=k1k2,s13=k1k3,s42=k4k2,s43=k4k3,s23=k2k3 . The amplitude A(5)(k) agrees with the one computed in [14] using the Feynman rules of the effective Lagrangian: A(5)(k) = K5 + K4 ∑ i<j xij + ∑ {i,j,k,l}⊂{1,2,3,4,5} i<j,k<l xijxkl, (39) (39) Symmetry 2021, 13, 967 24 of 53 where K5 = (p−2)(p−3) p2 , K4 = p−2 p , and ij and kl are summed over all pairs of compatible channels. 4. p-Adic Open String Amplitudes By a pair of compatible channels ij, kl we mean that i, j, k and l are different, and that i < j, k < l. where K5 = (p−2)(p−3) p2 , K4 = p−2 p , and ij and kl are summed over all pairs of compatible channels. By a pair of compatible channels ij, kl we mean that i, j, k and l are different, and that i < j, k < l. 4.3. p-Adic Open String N-Point Amplitudes 4.3. p-Adic Open String N-Point Amplitudes We ﬁx an integer N ≥4 and consider the general N-point zeta function: 4.3. p-Adic Open String N-Point Amplitudes We ﬁx an integer N ≥4 and consider the general N-point zeta function: We ﬁx an integer N ≥4 and consider the general N-point zeta function: Z(N)(s) = Z QN−3 p N−2 ∏ i=2 \|xi\|s1i p \|1 −xi\| s(N−1)i p ∏ 2≤i<j≤N−2 xi −xj sij p N−2 ∏ i=2 dxi. (40) (40) We divide the domain of integration QN−3 p into sectors. Given I ⊆T = {2, 3, . . . , N −2} the attached sector is defined as Sect(I) = n (x2, . . . , xN−2) ∈QN−3 p ; \|xi\|p ≤1 ⇔i ∈I o . Hence, the N-point zeta function (40) can be written as Hence, the N-point zeta function (40) can be written as Z(N)(s) = ∑ I⊆T Z(N)(s; I), where Z(N)(s; I) := Z Sect(I) F(s, x; N) N−2 ∏ i=2 dxi, Z(N)(s; I) := Z Sect(I) F(s, x; N) N−2 ∏ i=2 dxi, with with F(s, x; N) := N−2 ∏ i=2 \|xi\|s1i p \|1 −xi\| s(N−1)i p ∏ 2≤i<j≤N−2 xi −xj sij p and x = (x2, . . . , xN−2) ∈QN−3 p . 4. p-Adic Open String Amplitudes In [8] we show that Z(N)(s) has an analytic continuation to the whole CD as a rational function in the variables p−sij by showing that all functions that appear on the right-hand side of (41) are holomorphic in a region H(C) in CD (Deﬁnition 3 and Remarks 9 and 10 in [8]) deﬁned by \|J\| + ∑ i∈J (Re(s1i) + Re s(N−1)i ) + ∑ 2≤i<j≤N−2 i∈J Re sij (42) + ∑ 2≤i<j≤N−2 i∈T∖J,j∈J Re sij < 0 for J ∈F1; \|K\| −1 + ∑ 2≤i<j≤N 2 Re(sij) > 0 for K ∈F2; (43) \|J\| + ∑ i∈J (Re(s1i) + Re s(N−1)i ) + ∑ 2≤i<j≤N−2 i∈J Re sij (42) (42) + ∑ 2≤i<j≤N−2 i∈T∖J,j∈J Re sij < 0 for J ∈F1; \|K\| −1 + ∑ 2≤i<j≤N−2 i,j∈K Re(sij) > 0 for K ∈F2; (43) (43) 1 + Re(sij) > 0 for ij ∈G ⊆{ij; 2 ≤i < j ≤N −2}, (44) 1 + Re(sij) > 0 for ij ∈G ⊆{ij; 2 ≤i < j ≤N −2}, (44) 1 + Re(sij) > 0 for ij ∈G ⊆{ij; 2 ≤i < j ≤N −2}, (44) (44) where F1, F2 are families of nonempty subsets of T, and G is a nonempty subset of {ij; 2 ≤i < j ≤N −2, i, j ∈T}. where F1, F2 are families of nonempty subsets of T, and G is a nonempty subset of {ij; 2 ≤i < j ≤N −2, i, j ∈T}. \|J\| + ∑ i∈S Re(sti) + ∑2≤i<j≤N−2, i,j∈J Re(sij) > 0 for J × S ∈F3, (45) (45) with S ⊆J, t ∈{1, N −1},and F3 a family of nonempty subsets of I × I; with S ⊆J, t ∈{1, N −1},and F3 a family of nonempty subsets of I × I; \|K\| −1 + ∑ 2≤i<j≤N−2 i,j∈K Re(sij) > 0 for K ∈F4, (46) (46) where F4’s a family of nonempty subsets of I; where F4’s a family of nonempty subsets of I; 1 + Re(sij) > 0 for ij ∈GT, (47) (47) where GT is a nonempty subset of {2 ≤i < j ≤N −2, i, j ∈J} with (N −1)i, 1i ∈GT. 4. p-Adic Open String Amplitudes By Lemma 2 in [8], Z(N)(s; I) = pM(s)        Z Z\|I\| p ∏ i∈I \|xi\|s1i p \|1 −xi\| s(N−1)i p ∏ 2≤i<j≤N−2 i,j∈I xi −xj sij p ∏ i∈I dxi        ×              Z Z\|T∖I\| p ∏ 2≤i<j≤N−2 i,j∈T∖I xi −xj sij p ∏ i∈T∖I \|xi\| 2+s1i+s(N−1)i+∑2≤j≤N−2,j̸=i sij p ∏ i∈T∖I dxi              =: pM(s)Z(N) 0 (s; I)Z(N) 1 (s; T ∖I), Z(N)(s; I) = pM(s)        Z Z\|I\| p ∏ i∈I \|xi\|s1i p \|1 −xi\| s(N−1)i p ∏ 2≤i<j≤N−2 i,j∈I xi −xj sij p ∏ i∈I dxi        ×              Z Z\|T∖I\| p ∏ 2≤i<j≤N−2 i,j∈T∖I xi −xj sij p ∏ i∈T∖I \|xi\| 2+s1i+s(N−1)i+∑2≤j≤N−2,j̸=i sij p ∏ i∈T∖I dxi              =: pM(s)Z(N) 0 (s; I)Z(N) 1 (s; T ∖I), where M(s) := \|T ∖I\| + ∑ i∈T∖I (s1i + s(N−1)i) + ∑ 2≤i<j≤N−2 i∈T∖I,j∈T sij + ∑ 2≤i<j≤N−2 i∈I,j∈T∖I sij. I dditi Z(N)( I) Z(N)( T I) lti i t l l t f ti d where where where M(s) := \|T ∖I\| + ∑ i∈T∖I (s1i + s(N−1)i) + ∑ 2≤i<j≤N−2 i∈T∖I,j∈T sij + ∑ 2≤i<j≤N−2 i∈I,j∈T∖I sij. In addition Z(N) 0 (s; I), Z(N) 1 (s; T ∖I) are multivariate local zeta functions and In addition Z(N) 0 (s; I), Z(N) 1 (s; T ∖I) are multivariate local zeta functions and Z(N)(s) = ∑ I⊆T pM(s)Z(N) 0 (s; I)Z(N) 1 (s; T ∖I), (41) (41) Symmetry 2021, 13, 967 25 of 53 with the convention that Z(N) i (s; ∅) = 1 for i = 0, 1. with the convention that Z(N) i (s; ∅) = 1 for i = 0, 1. 4. p-Adic Open String Amplitudes In Lemma 9 of [8], we show that region H(C) contains and open and connected subset of CD deﬁned by the conditions −2 3N1 < Re(sij) < 0 , (48) −2 3 < Re(s1i) < −1 2, (49) −2 3 < Re(s(N−1)i) < −1 2, (50) (48) (49) (50) for N ⩾5, N1 = (N−4)(N−3) 2 , i, j ∈{2, ..., N −2}. For the case N = 4 we only consider conditions (49) and (50). Like in the cases N = 4 and N = 5, the key point is to reduce the in- tegrals Z(N) 0 (s; I), Z(N) 1 (s; T ∖I) to certain simple integrals, for which admit meromorphic continuations to the whole CD as rational functions in the variables p−sij. We now state the meromorphic continuation of the open string N-point zeta function. Theorem 1 (Theorem 1 in [8]). (1) The p-adic open string N-point zeta function, Z(N)(s), gives rise to a holomorphic function on H(C), which contains an open and connected subset of CD. Furthermore, Z(N)(s) admits an analytic continuation to CD, denoted also as Z(N)(s), as a rational function in the variables p−sij, i, j ∈{1, . . . , N −1}. The real parts of the poles of Z(N)(s) belong to a ﬁnite union of hyperplanes, the equations of these hyperplanes have the form (42)–(47) with the symbols ‘<’, ‘>’ replaced by ‘=’. (2) If s = sij ∈CD, with Re(sij) ≥0 for i, j ∈{1, . . . , N −1}, then the integral Z(N)(s) diverges to +∞. Symmetry 2021, 13, 967 26 of 53 5.1. Non-Archimedean Local Fields 5.1. Non-Archimedean Local Fields A non-Archimedean local ﬁeld K is a locally compact topological ﬁeld with respect to a non-discrete topology, which comes from a norm \|·\|K satisfying \|x + y\|K ≤max{\|x\|K, \|y\|K}, for x, y ∈K. A such norm is called an ultranorm or non-Archimedean. Any non-Archimedean local ﬁeld K of characteristic zero is isomorphic (as a topological ﬁeld) to a ﬁnite extension of Qp. The ﬁeld Qp is the basic example of non-Archimedean local ﬁeld of characteristic zero. In the case of positive characteristic, K is isomorphic to a ﬁnite extension of the ﬁeld of formal Laurent series Fq((T)) over a ﬁnite ﬁeld Fq, where q is a power of a prime number p. The ring of integers of K is deﬁned as RK = {x ∈K; \|x\|K ≤1}. Geometrically RK is the unit ball of the normed space (K, \|·\|K). This ring is a domain of principal ideals with a unique maximal ideal, which is given by PK = {x ∈K; \|x\|K < 1}. We ﬁx a generator π of PK i.e., PK = πRK. A such generator is also called a local uniformizing parameter of K, and it plays the same role as p in Qp. The group of units of RK is deﬁned as R× K = {x ∈RK; \|x\|K = 1}. The natural map RK →RK/PK ∼= Fq is called the reduction mod PK. The quotient RK/PK ∼= Fq, q = p f , is called the residue ﬁeld of K. Every nonzero element x of K can be written uniquely as x = πord(x)u, u ∈R× K. We set ord(0) = ∞. The normalized valuation of K is the mapping K → Z ∪{∞} x → ord(x). Then \|x\|K = q−ord(x) and \|π\|K = q−1. We ﬁx S ⊂RK a set of representatives of Fq in RK, i.e., S is a set which is mapped bijectively onto Fq by the reduction mod PK. We assume that 0 ∈S. Any nonzero element x of K can be written as x = πord(x) ∞ ∑ i=0 xiπi, where xi ∈S and x0 ̸= 0. This series converges in the norm \|·\|K. We extend the norm \|·\|K to Kn by taking where xi ∈S and x0 ̸= 0. This series converges in the norm \|·\|K. We extend the norm \|·\|K to Kn by taking where xi ∈S and x0 ̸= 0. 5.1. Non-Archimedean Local Fields This series converges in the norm \|·\|K. We extend the norm \|·\|K to Kn by taking We extend the norm \|·\|K to Kn by taking \|\|x\|\|K := max 1≤i≤n \|xi\|K, for x = (x1, . . . , xn) ∈Kn.We deﬁne ord(x) = min1≤i≤n{ord(xi)}, then \|\|x\|\|K = q−ord(x). The metric space (Kn, \|\| · \|\|K) is a complete ultrametric space. for x = (x1, . . . , xn) ∈Kn.We deﬁne ord(x) = min1≤i≤n{ord(xi)}, then \|\|x\|\|K = q−ord(x) The metric space (Kn, \|\| · \|\|K) is a complete ultrametric space. As we mentioned before, any ﬁnite extension K of Qp is a non-Archimedean local ﬁeld. Then pRK = πmRK, m ∈N. Symmetry 2021, 13, 967 27 of 53 27 of 53 If m = 1 we say that K is a unramiﬁed extension of Qp. In other case, we say that K is a ramiﬁed extension. It is well known that for every positive integer e there exists a unique unramiﬁed extension Ke of Qp of degree e, which means that Ke is a Qp-vector space of dimension e. From now on, π denotes a local uniformizing parameter of Ke, thus pRKe = πRKe, RKe/PKe ∼= Fpe and \|π\|Ke = p−e. For an in-depth exposition of non-Archimedean local ﬁelds, the reader may consult [62,65], see also [3,61]. 5.2. Open String Amplitudes over Non-Archimedean Local Fields 5.2. Open String Amplitudes over Non-Archimedean Local Fields The open string amplitudes can be deﬁned over any local ﬁeld. In this section, we consider Koba–Nielsen string amplitudes on Ke, the unique unramiﬁed extension of Qp of degree e for all e ∈N\{0}. We recall that if Ke is the unramiﬁed extension of degree e of Qp, then pRKe = πRKe, RKe/PKe ∼= Fpe and \|π\|Ke = p−e. Thus, π in Ke plays the role of p in Qp. Qp The Koba–Nielsen amplitudes on Ke are deﬁned as Qp The Koba–Nielsen amplitudes on Ke are deﬁned as A(N) Ke (k) = Z KN−3 e N−2 ∏ i=2 \|xi\|k1ki Ke \|1 −xi\|kN−1ki Ke ∏ 2≤i<j≤N−2 xi −xj kikj Ke N−2 ∏ i=2 dxi, (51) (51) (51) where ∏N−2 i=2 dxi is the Haar measure of KN−3 e , + normalized so that the measure of RN−3 Ke is 1 where ∏N−2 i=2 dxi is the Haar measure of KN−3 e , + normalized so that the measure of RN−3 Ke is 1. where ∏N−2 i=2 dxi is the Haar measure of KN−3 e , + normalized so that the measure of RN−3 Ke is 1 The procedure used to regularize the p-adic amplitudes extends to amplitudes of the form (51). In this case, the open string N-point zeta function is deﬁned as Z(N) Ke (s) := Z KN−3 e F(s, x; N, Ke) N−2 ∏ i=2 dxi, (52) (52) where where F(s, x; N, Ke) = N−2 ∏ i=2 \|xi\|s1i Ke\|1 −xi\| s(N−1)i Ke ∏ 2≤i<j≤N−2 xi −xj sij Ke. where s = sij ∈CD, with D = (N−3)(N−4) 2 + 2(N −3). Let T = {2, 3, . . . 5.3. The Limit p Tends to One The functions 5.3. The Limit p Tends to One The functions The functions Z(N) Ke (s; I, 0), Z(N) Ke (s; T ∖I, 1) are multivariate local zeta functions. Thus, to make mathematical sense of the limit of Z(N) Qp (s) as p →1 we use the work of Denef and Loeser, see [35,66], and compute the limit of Z(N) Ke (s) as e →0 instead of the limit of Z(N) Qp (s) as p →1. In order to compute the limit e →0 is necessary to have an explicit formula for Z(N) Ke (s), so in [9] we determined the explicit formula by ﬁnding explicit formulae for integrals Z(N) Ke (s; I, 0) and Z(N) Ke (s; T ∖I, 1), see Theorem B in [9]. After that, we deﬁne are multivariate local zeta functions. Thus, to make mathematical sense of the limit of Z(N) Qp (s) as p →1 we use the work of Denef and Loeser, see [35,66], and compute the limit of Z(N) Ke (s) as e →0 instead of the limit of Z(N) Qp (s) as p →1. In order to compute the limit e →0 is necessary to have an explicit formula for Z(N) Ke (s), so in [9] we determined the explicit formula by ﬁnding explicit formulae for integrals Z(N) Ke (s; I, 0) and Z(N) Ke (s; T ∖I, 1), see Theorem B in [9]. After that, we deﬁne Z(N) top (s; I, 0) = lim e→0 Z(N) Ke (s; I, 0) Z(N) top (s; I, 0) = lim e→0 Z(N) Ke (s; I, 0) and and Z(N) top (s; T ∖I, 1) = lim e→0 Z(N) Ke (s; T ∖I, 1), which are elements of Q sij, i, j ∈{1, . . . , N −1} , the ﬁeld of rational functions in the variables sij, i, j ∈{1, . . . , N −1} with coefﬁcients in Q. Then, by using (53), we deﬁned the open string N-point topological zeta function as Z(N) top (s) = ∑ I⊆T Z(N) top (s; I, 0)Z(N) top (s; T ∖I, 1). The open string N-point topological zeta function Z(N) top (s) is a rational function of Q sij, i, j ∈{1, . . . , N −1} . We now deﬁne the Denef–Loeser open string N-point amplitudes at the tree level as A(N) top (k) = Z(N) top (s) \|sij=ki·kj, with i ∈{1, . 5.2. Open String Amplitudes over Non-Archimedean Local Fields , N −2}, then Z(N) Ke (s) = ∑ I⊆T peM(s)Z(N) Ke (s; I, 0) Z(N) Ke (s; T ∖I, 1), (53) Z(N) Ke (s) = ∑ I⊆T peM(s)Z(N) Ke (s; I, 0) Z(N) Ke (s; T ∖I, 1), (53) (53) where M(s) := \|T ∖I\| + ∑ i∈T∖I (s1i + s(N−1)i) + ∑ 2≤i<j≤N−2 i∈T∖I, j∈T sij + ∑ 2≤i<j≤N−2 i∈I,j∈T∖I sij Z(N) Ke (s; I, 0) = Z R\|I\| Ke ∏ i∈I \|xi\|s1i Ke\|1 −xi\| s(N−1)i Ke ∏ 2≤i<j≤N−2 i,j∈I xi −xj sij Ke ∏ i∈I dxi, Z(N) Ke (s; I, 0) = Z R\|I\| Ke ∏ i∈I \|xi\|s1i Ke\|1 −xi\| s(N−1)i Ke ∏ 2≤i<j≤N−2 i,j∈I xi −xj sij Ke ∏ i∈I dxi, and Z(N) Ke (s; T ∖I, 1, ) = Z R\|T∖I\| Ke F1(s, x; N, Ke) ∏ i∈T∖I dxi, where Symmetry 2021, 13, 967 28 of 53 28 of 53 F1(s, x; N, Ke) := ∏ 2≤i<j≤N−2 i,j∈T∖I xi −xj sij Ke ∏ i∈T∖I \|xi\| 2+s1i+s(N−1)i+∑2≤j≤N−2,j̸=i sij Ke . By convention Z(N) Ke (s; ∅, 0) = 1, Z(N) Ke (s; ∅, 1) = 1. Ke Ke All the zeta functions appearing in the right-hand side of Formula (53) admit analytic continuations to the whole CD as rational functions in the variables p−esij and they are holomorphic on a common domain in CD. Therefore Z(N) Ke (s) is a holomorphic function in a certain domain of CD admitting a meromorphic continuation to the whole CD as a rational function in the variables p−esij, see Theorem 1 of [8]. ( ) We use Z(N) Ke (s) as regularizations of Koba–Nielsen amplitudes A(N)(k, Ke), more precisely, we deﬁne A(N) Ke (k) = Z(N) Ke (s) \|sij=ki·kj . Then A(N) Ke (k) is a well-deﬁned rational function in the variables p−eki·kj, which agree with the integral (51) when it converges. 5.3. The Limit p Tends to One The functions 5.3. The Limit p Tends to One The functions . . , N −1}, j ∈T or i, j ∈T, where T = {2, . . . , N −2}. Thus, the Denef–Loeser amplitudes are rational functions of the variables ki · kj, i, j ∈{1, . . . , N}. Symmetry 2021, 13, 967 29 of 53 29 of 53 5.4. Feynman Rules, Explicit Formulae and Denef–Loeser Amplitudes Using the Feynman rules of the effective Lagrangian, an ‘explicit formula’ of the type 5.4. Feynman Rules, Explicit Formulae and Denef–Loeser Amplitudes Using the Feynman rules of the effective Lagrangian, an ‘explicit formula’ of the type 5.4. Feynman Rules, Explicit Formulae and Denef–Loeser Amplitudes Using the Feynman rules of the effective Lagrangian, an ‘explicit formula’ of the type A(N)(k) = KN + N−3 ∑ l=2 Ki1...il ∑ 1≤a<b≤l p −1 p p−kia kib−1 1 −p−kia kib−1 ! was given in ([14]), where the constants KN, Ki1...il ∈Q(p). Of course, a rigorous demon- stration of a such formula is an open problem. Taking formally the limit p tends to one, we get A(N) top (k) = Ktop N + N−3 ∑ l=2 Ktop i1...il ∑ 1≤a<b≤l 1 kiakib + 1. 5.5. Denef–Loeser Open String 4-Point Amplitudes The open string 4-point zeta function on Ke is deﬁned as 5.5. Denef–Loeser Open String 4-Point Amplitudes The open string 4-point zeta function on Ke is deﬁned as Z(4) Ke (s) = Z Ke \|x2\|s12 Ke \|1 −x2\|s32 Ke dx2. We divide the integration domain in sectors We divide the integration domain in sectors Sect(I) = {xi ∈Q : \|xi\|Ke ≤1 ⇐⇒i ∈I}, I ⫅T = {2, 3}, I T⧹I Sect(I) {2} ∅ RKe ∅ {2} Ke\RKe. In this way, we obtain that In this way, we obtain that In this way, we obtain that Z(4) Ke (s) = Z(4) Ke (s; {2}, 0) + pe(1+s12+s32)Z(4) Ke (s{2}, 1) = Z RKe \|x2\|s12 Ke \|1 −x2\|s32 Ke dx2 + pe(1+s12+s32) Z RKe \|x2\|−2−s12−s32 Ke dx2, with Z(4) Ke (s; {2}, 0) = 1 −2p−e + (1 −p−e)pe(−1−s12) 1 −pe(−1−s12) + (1 −p−e)pe(−1−s32) 1 −pe(−1−s32) , and Z(4) Ke (s; {2}, 1) = (1 −p−e)pe(1+s12+s32) 1 −pe(1+s12+s32) . Taking the limit e approaches to zero, Z(4) top(s; {2}, 0) = −1 + 1 s12 + 1 + 1 s32 + 1 and Z(4) top(s; {2}, 1) = − 1 s12 + s32 + 1. 5.3. The Limit p Tends to One The functions Denef–Loeser Open String 5-Point Amplitudes The open string 5-point zeta function on Ke is given by The open string 5-point zeta function on Ke is given by The open string 5-point zeta function on Ke is given by Z(5) Ke (s) = Z K2e \|x2\|s12 Ke \|x3\|s13 Ke \|1 −x2\|s42 Ke \|1 −x3\|s43 Ke \|x2 −x3\|s23 Ke dx2dx3. By dividing the integration domain in sectors as in Section 4.2, we obtain the results presented in Table 2. Table 2. Sectors of the 5-point Denef-Loeser amplitude. Table 2. Sectors of the 5-point Denef-Loeser amplitude. I Ic Sect(I) {2} {3} RKe × Ke\RKe {3} {2} Ke\RKe × RKe {2, 3} ∅ RKe × RKe ∅ {2, 3} Ke\RKe × Ke\RKe. The open string 5-point topological zeta function is deﬁned as Z(5) top(s) = ∑ I⊆T Z(5) top(s; I, 0)Z(5) top(s; T ∖I, 1). Table 3 contains explicit formulae for all the integrals Z(5) top(s; I, 0) and Z(5) top(s; T ∖I, 1). Table 3 contains explicit formulae for all the integrals Z(5) top(s; I, 0) and Z(5) top(s; T ∖I, 1). Table 3. Explicit calculation of Z(5) top(s; I, 0), Z(5) top(s; T ∖I, 1). Table 3. Explicit calculation of Z(5) top(s; I, 0), Z(5) top(s; T ∖I, 1). I Z(5) top(s; I, 0) Z(5) top(s; T ∖I, 1) {2} −1 + 1 1+s12 + 1 1+s42 − 1 1+s13+s43+s23 {3} −1 + 1 1+s13 + 1 1+s43 − 1 1+s12+s42+s23 {2, 3} h 1 1+s12 + 1 1+s13 + 1 1+s23 −1 i 1 2+s12+s13+s23 + 1 1+s12 h 1 1+s43 −1 i + 1 1+s13 h 1 1+s42 −1 i +2 − 1 1+s23 − 1 1+s42 − 1 1+s43 + 1 2+s42+s43+s23 h 1 1+s42 + 1 1+s43 + 1 1+s23 −1 i 1 {∅} 1 − 1 2+s52+s53+s23 × " 1 1+s12+s42+s23 + 1 1+s13+s43+s23 + 1 1+s23 −1 # Table 3. Explicit calculation of Z(5) top(s; I, 0), Z(5) top(s; T ∖I, 1). 5.3. The Limit p Tends to One The functions Consequently Z(4) top(s) = −1 + 1 s12 + 1 + 1 s32 + 1 − 1 s12 + s32 + 1. Z(4) Ke (s) = Z(4) Ke (s; {2}, 0) + pe(1+s12+s32)Z(4) Ke (s{2}, 1) = Z RKe \|x2\|s12 Ke \|1 −x2\|s32 Ke dx2 + pe(1+s12+s32) Z RKe \|x2\|−2−s12−s32 Ke dx2, with Z(4) Ke (s; {2}, 0) = 1 −2p−e + (1 −p−e)pe(−1−s12) 1 −pe(−1−s12) + (1 −p−e)pe(−1−s32) 1 −pe(−1−s32) , and Z(4) Ke (s; {2}, 1) = (1 −p−e)pe(1+s12+s32) 1 −pe(1+s12+s32) . Z(4) Ke (s) = Z(4) Ke (s; {2}, 0) + pe(1+s12+s32)Z(4) Ke (s{2}, 1) = Z RKe \|x2\|s12 Ke \|1 −x2\|s32 Ke dx2 + pe(1+s12+s32) Z RKe \|x2\|−2−s12−s32 Ke dx2, h Z(4) Ke (s; {2}, 0) = 1 −2p−e + (1 −p−e)pe(−1−s12) 1 −pe(−1−s12) + (1 −p−e)pe(−1−s32) 1 −pe(−1−s32) , Z(4) Ke (s) = Z(4) Ke (s; {2}, 0) + pe(1+s12+s32)Z(4) Ke (s{2}, 1) = Z RKe \|x2\|s12 Ke \|1 −x2\|s32 Ke dx2 + pe(1+s12+s32) Z RKe \|x2\|−2−s12−s32 Ke dx2, with Z(4) Ke (s; {2}, 0) = 1 −2p−e + (1 −p−e)pe(−1−s12) 1 −pe(−1−s12) + (1 −p−e)pe(−1−s32) 1 −pe(−1−s32) , and Z(4) Ke (s; {2}, 1) = (1 −p−e)pe(1+s12+s32) 1 −pe(1+s12+s32) . Z(4) Ke (s; {2}, 1) = (1 −p−e)pe(1+s12+s32) 1 −pe(1+s12+s32) . Taking the limit e approaches to zero, Taking the limit e approaches to zero, Z(4) top(s; {2}, 0) = −1 + 1 s12 + 1 + 1 s32 + 1 and Z(4) top(s; {2}, 1) = − 1 s12 + s32 + 1. Consequently Z(4) top(s) = −1 + 1 s12 + 1 + 1 s32 + 1 − 1 s12 + s32 + 1. Z(4) top(s; {2}, 0) = −1 + 1 s12 + 1 + 1 s32 + 1 and Symmetry 2021, 13, 967 30 of 53 30 of 53 Using the kinematic relations k1 + k2 + k3 + k4 = 0 and k2 i = 2 we obtain k1k2 + k3k2 + 1 = −1 −k2k4, then the Denef–Loeser string 4-point amplitude is given by A(4) top(k) = −1 + 1 k1k2 + 1 + 1 k3k2 + 1 + 1 k2k4 + 1. (54) (54) 5.6. Denef–Loeser Open String 5-Point Amplitudes 5.6. 5.3. The Limit p Tends to One The functions I Z(5) top(s; I, 0) Z(5) top(s; T ∖I, 1) {2} −1 + 1 1+s12 + 1 1+s42 − 1 1+s13+s43+s23 {3} −1 + 1 1+s13 + 1 1+s43 − 1 1+s12+s42+s23 {2, 3} h 1 1+s12 + 1 1+s13 + 1 1+s23 −1 i 1 2+s12+s13+s23 + 1 1+s12 h 1 1+s43 −1 i + 1 1+s13 h 1 1+s42 −1 i +2 − 1 1+s23 − 1 1+s42 − 1 1+s43 + 1 2+s42+s43+s23 h 1 1+s42 + 1 1+s43 + 1 1+s23 −1 i 1 {∅} 1 − 1 2+s52+s53+s23 × " 1 1+s12+s42+s23 + 1 1+s13+s43+s23 + 1 1+s23 −1 # 1 − 1 2+s52+s53+s23 × " 1 1+s12+s42+s23 + 1 1+s13+s43+s23 + 1 1+s23 −1 # {∅} Therefore, the Denef–Loeser open string 5-point amplitude is given by A(5) top(k) = 2 −∑ i<j 1 1 + kikj + ∑ {i,j,k,l}⊂{1,2,3,4,5} i<j,k<l 1 1 + kikj 1 1 + kkkl , 31 of 53 Symmetry 2021, 13, 967 where where ∑ {i,j,k,l}⊂{1,2,3,4,5} i<j,k<l 1 1 + kikj 1 1 + kkkl = 1 1 + k1k2 1 1 + k3k4 + 1 1 + k1k2 1 1 + k3k5 + 1 1 + k1k2 1 1 + k4k5 + 1 1 + k1k3 1 1 + k2k4 + 1 1 + k1k3 1 1 + k2k5 + 1 1 + k1k3 1 1 + k4k5 + 1 1 + k1k4 1 1 + k2k3 + 1 1 + k1k4 1 1 + k2k5 + 1 1 + k1k4 1 1 + k3k5 + 1 1 + k1k5 1 1 + k2k3 + 1 1 + k1k5 1 1 + k2k4 + 1 1 + k1k5 1 1 + k3k4 + 1 1 + k2k3 1 1 + k4k5 + 1 1 + k3k5 1 1 + k2k4 + 1 1 + k2k5 1 1 + k3k4 This amplitude agrees with the calculation A(5) top(k) = lim p→1 A(5)(k) (55) =Ktop 5 + Ktop 4 ∑ i<j 1 kikj + 1 + ∑{i,j,k,l}⊂{1,2,3,4,5} i<j,k<l 1 kikj + 1 1 kkkl + 1 one using the explicit formula for given by the Feynman rules given in [14]. Here top 5 = −2, Ktop 4 = −1. 5.3. The Limit p Tends to One The functions here ∑ {i,j,k,l}⊂{1,2,3,4,5} i<j,k<l 1 1 + kikj 1 1 + kkkl = 1 1 + k1k2 1 1 + k3k4 + 1 1 + k1k2 1 1 + k3k5 + 1 1 + k1k2 1 1 + k4k5 + 1 1 + k1k3 1 1 + k2k4 + 1 1 + k1k3 1 1 + k2k5 + 1 1 + k1k3 1 1 + k4k5 + 1 1 + k1k4 1 1 + k2k3 + 1 1 + k1k4 1 1 + k2k5 + 1 1 + k1k4 1 1 + k3k5 + 1 1 + k1k5 1 1 + k2k3 + 1 1 + k1k5 1 1 + k2k4 + 1 1 + k1k5 1 1 + k3k4 + 1 1 + k2k3 1 1 + k4k5 + 1 1 + k3k5 1 1 + k2k4 + 1 1 + k2k5 1 1 + k3k4 This amplitude agrees with the calculation A(5) top(k) = lim p→1 A(5)(k) (55) =Ktop 5 + Ktop 4 ∑ i<j 1 kikj + 1 + ∑{i,j,k,l}⊂{1,2,3,4,5} i<j,k<l 1 kikj + 1 1 kkkl + 1 (55) done using the explicit formula for given by the Feynman rules given in [14]. Here Ktop 5 = −2, Ktop 4 = −1. done using the explicit formula for given by the Feynman rules given in [14]. Here Ktop 5 = −2, Ktop 4 = −1. 6. A Physical View of the Limit p →1 on p-Adic String Amplitudes The problem of ﬁnding a relation between the physical string amplitudes and the p-adic one has been present since the initial proposal. One of the ﬁrst attempts is that of the Adelic amplitudes which can be written as an inﬁnite product of physical non-Archimedean amplitudes [14,21]. There are other possibilities to look for a link between Archimedean and Non-Archimedean amplitudes. One of them is to consider the limit p →1. This limit is very intriguing and there are different interpretations of it [23,29,34]. For instance, in [23] it was argued that the analytic continuation of p to the complex numbers can be reflected in the Lagrangian and equations of motion by turning the non-local equations of motion into a local and linear dy- namical description with a logarithmic potential. This is an ordinary theory with real physical amplitudes. In [29] it was argued that as similarly found in [23], the expansion of the effective action around p →1 leads to a linear theory with logarithmic potential. Besides that, this theory is deeply related to the boundary string field theory proposed by Witten [30] in the context of the developing of a background independence string theory. The limit p →1 also can be interpreted in terms of some scaling transformations of the renormalization group for the Bruhat–Tits tree [34]. As we have seen in this survey, the non-Archimedean nature is encoded in the world- sheet theory. In this context we must recall that p as a prime number, thus the analysis necessarily needs to be carry out in a rigorous way. The right way of taking the limit p →1 involves the introduction of unramiﬁed ﬁnite extensions of the p-adic ﬁeld Qp. In [9] the limit p →1 was discussed for tree-level string amplitudes, by using the topological zeta functions introduced by Denef and Loeser [35,66]. We found that the limit p →1 of p-adic string amplitudes leads to certain string amplitudes (which are rational functions) that we termed the Denef–Loeser open string amplitudes. These Denef–Loeser amplitudes for N = 4, 5 points were computed in Sections 5.5 and 5.6, for more details see [9]. On the other hand for N = 4, 5, in [9], we computed the limit p →1 of the effective ﬁeld theory of p-adic amplitudes, i.e., the Gerasimov and Shatashvili Lagrangian (61) involving a logarithmic potential. 5.7. Non-Archimedean Closed Strings In Archimedean string theory, it is known that closed strings can be produced from the scattering of open strings [55]. Therefore, for p-adic string theory, it is desirable to construct a p-adic closed string theory. This was ﬁrst studied in [20], see also [4,67]. The usual Archimedean closed string is described by two coordinates (τ, σ) with the periodical condition σ′ = σ + 2π. That is, the string worldsheet is a cylinder, which can be conformally mapped to the whole complex plane C. In order to obtain tree-level scattering amplitudes we need to insert vertex operators on C. Using the SL(2, C) symmetry, we can ﬁx three insertions points. As with the open string, it is conventional to ﬁx three points to 0, 1 and ∞. The simplest non-trivial example is the 4-point tachyon scattering amplitude for closed strings, also known as the Virasoro–Shapiro amplitude, is A(4) C (k) = Z C \|z\|k1k2/4 C \|1 −z\|k1k3/4 C dz = ΓC(−α(s)/2)ΓC(−α(t)/2) ΓC(−α(s)/2 −α(t)/2) , where ki ∈CD and kikj = −k0,ik0,j + · · · kl,ikl,j is the Minkowski product; \|z\|C := z¯z is the square complex norm, α(x) = x/4 + 2, and ΓC(s) := Z C exp[2πi(z + ¯z)]\|z\|s−1 C dz is the Gelfand–Graev gamma function over C [4]. For the closed strings case, the momenta vectors satisfy is the Gelfand–Graev gamma function over C [4]. For the closed strings case, the momenta vectors satisfy N k2 i = 8, N ∑ i=1 ki = 0. (56) (56) Symmetry 2021, 13, 967 32 of 53 32 of 53 This explains the factor of 1/4 in the exponents relative to the open strings case. The generalization of these amplitudes to N-points is given by A(N) C (k) = Z CN−3 N−2 ∏ i=2 \|zi\|k1ki/4 C \|1 −zi\|kN−1ki/4 C ∏ 2≤i<j≤N−2 \|zi −zj\| kikj/4 C N−2 ∏ i=2 dzi, (57) (57) where k = (k1, . . . , kN). As we can see, these amplitudes are very similar to the Koba– Nielsen amplitudes, except for the fact that they are being integrated over complex variables and the replacement ki →1 2ki. 2 To construct p-adic versions of Virasoro–Shapiro amplitudes, one can consider Qp as the analog of R, and a quadratic extension of Qp as the analog of C. However, this quadratic extension is not unique, and is not an algebraically closed ﬁeld. 5.7. Non-Archimedean Closed Strings This naive approach is followed in [4,20]. This approach is not very useful here, because we work with open string amplitudes over any ﬁnite extension of Qp. Deﬁnition 2. Given an open string N-point amplitude A(N) K (k) deﬁned over a non-Archimedean local ﬁeld K, we attach to it a Virasoro–Shapiro amplitude deﬁned as A(N) K2 (k) = A(N) K2 ( 1 2k), where K2 is the unique unramiﬁed extension of K of degree 2. Then, by the results of [6], A(N) K2 (k) admits a meromorphic continuation as a rational function in the variables q−2sij. 6. A Physical View of the Limit p →1 on p-Adic String Amplitudes By computing the interacting generating functional at the tree level, we veriﬁed that the corresponding amplitudes coincide exactly with the Denef–Loeser Symmetry 2021, 13, 967 33 of 53 33 of 53 amplitudes. Based on this fact we have formulated a conjecture, which was already stated in the introduction of this survey. In what follows we review the evidence of the mentioned conjecture in the cases N = 4, 5 following [9]. First, we review some basic results from [29] starting with the effective action which is a ﬁeld theory whose perturbative analysis leads to the p-adic scattering amplitudes [14], it can be written as S(φ) = 1 g2 p2 p −1 Z dDx −1 2φp−1 2 ∆φ + 1 p + 1φp+1 , (58) (58) where ∆is the Laplacian on M and g is the coupling constant. The corresponding Euler- Lagrange equation is 1 p−1 2 ∆φ = φp. (59) (59) In the source space (and consequently in the amplitudes) p is a prime number; how- ever, in the target space (and consequently in the Lagrangian and in the equation of motion) p is a real parameter. Thus, one can formally proceed to approach p to one and perform a Taylor expansion at (p −1) of the expression exp(−1 2∆log p) and exp(p log φ). Then the resulting equation of motion is given by ∆φ = −2φ log φ. (60) (60) This is the motion equation of the Gerasimov–Shatashvili action S(φ) = Z dDx (∂φ)2 −V(φ) , (61) (61) where (∂φ)2 = ηµν∂µφ · ∂νφ and V(φ) is the potential where (∂φ)2 = ηµν∂µφ · ∂νφ and V(φ) is the potential V(φ) = φ2 log φ2 e . We consider that action (61) is the limit p tends to one of the effective action (58). The action corresponding to the free theory with a source is written as We consider that action (61) is the limit p tends to one of the effective acti The action corresponding to the free theory with a source is written as S0(φ) = Z dDx (∂φ)2 + φ2(x) + J(x)φ(x) . Now, the action (61) can be conveniently rewritten as Now, the action (61) can be conveniently rewritten as S(φ) = Z dDx (∂φ)2 + m2 −U(φ) , (62) (62) where U(φ) = 2φ2 log φ. 6. A Physical View of the Limit p →1 on p-Adic String Amplitudes Then the potential U(φ) can be expanded in Taylor series around the origin in the form U(φ) = Aφ2 + Bφ3 + Cφ4 + Dφ5 + · · · , (63) (63) where A, B, C and D are constants and they are real numbers. where A, B, C and D are constants and they are real numbers. y Now, we brieﬂy review the form of obtaining the four-point and ﬁve-point amplitudes from the Lagrangian (61) [29]. In quantum ﬁeld theory N-point correlation functions of N local operators bφ in N different points x1, x2, . . . , xN of M, can be written as ⟨T(bφ(x1)bφ(x2) · · · bφ(xN))⟩= (−i¯h)N Z[J] δnZ[J] δJ(x1)δJ(x2) · · · δJ(xN) J=0 , Symmetry 2021, 13, 967 34 of 53 34 of 53 where Z[J] is the generating functional constructed using interacting Lagrangian (62). The functional can be computed as where Z[J] is the generating functional constructed using interacting Lagrangian (62). The functional can be computed as Z[J] = exp −iB ¯h Z dDx −i¯h δ δJ(x) 3 −iC ¯h Z dDx −i¯h δ δJ(x) 4 −iD ¯h Z dDx −i¯h δ δJ(x) 5 + · · · Z0[J], where the generating functional of the correlation function for the free theory with sources is given by Z0[J] = N [det(∆−1)]−1/2 exp −i 4¯h Z dDx Z dDx′J(x)GF(x −x′)J(x′) . Moreover, GF(x −x′) is the Green-Feynman function of the differential operator (∆−1). For the computation of the interacting 4-point amplitudes can be obtained through the generating functional Z[J] = · · · −iC¯h3 Z dDx δ δJ(x) 4 Z0[J] + · · · . In terms of Z[J], 4-point amplitudes implies the computation of In terms of Z[J], 4-point amplitudes implies the computation of δ4Z[J] δJ(x1)δJ(x2)δJ(x3)δJ(x4) J=0 (64) = −4!iC¯h3 Z dDx −i 2¯h GF(x −x1) −i 2¯h GF(x −x2) × −i 2¯h GF(x −x3) −i 2¯h GF(x −x4) = −3iC 2¯h Z dDx GF(x −x1) GF(x −x2) GF(x −x3) GF(x −x4). (64) Here GF(x −y) is the Green-Feynman propagator. This amplitude corresponds to the Feynman diagram with only one vertex and four external legs. following the notation of [14], we write for it the symbol K4. The contribution to the 4-point amplitudes of Bφ3 in (63) has a contribution at the second order of the expansion. 6. A Physical View of the Limit p →1 on p-Adic String Amplitudes The corresponding Feynman diagrams have two vertices at points x and y with two external legs, which are connected by a propagator GF(x −y). Thus, we have Z[J] = · · · + B2¯h4 2 Z dDx Z dDy δ δJ(x) 3 δ δJ(y) 3 Z0[J] + · · · . (65) (65) Moreover, the contribution to the 4-point amplitudes from the term Cφ3 arises to second order. Thus, it yields Moreover, the contribution to the 4-point amplitudes from the term Cφ3 arises to second order. Thus, it yields δ4Z[J] δJ(x1)δJ(x2)δJ(x3)δJ(x4) J=0 = 18B2¯h4 Z dDx Z dDy −i 2¯h GF(x −y) × −i 2¯h GF(x −x4) −i 2¯h GF(x −x3) −i 2¯h GF(y −x2) −i 2¯h GF(y −x1) Symmetry 2021, 13, 967 35 of 53 + −i 2¯h GF(x −x4) −i 2¯h GF(y −x3) −i 2¯h GF(x −x2) −i 2¯h GF(y −x1) + −i 2¯h GF(x −x4) −i 2¯h GF(y −x3) −i 2¯h GF(y −x2) −i 2¯h GF(x −x1) + −i 2¯h GF(y −x4) −i 2¯h GF(y −x3) −i 2¯h GF(x −x2) −i 2¯h GF(x −x1) + −i 2¯h GF(y −x4) −i 2¯h GF(x −x3) −i 2¯h GF(y −x2) −i 2¯h GF(x −x1) + −i 2¯h GF(y −x4) −i 2¯h GF(x −x3) −i 2¯h GF(x −x2) −i 2¯h GF(y −x1) . (66) (66) The sum of expressions (64) and (66) constitutes the total amplitude which contains the sum of the three channels s, t and u. They are expected to be the p-adic 4-point amplitudes arising in the limit p →1. Thus, in the Fourier space it is expected to be written schematically as A(4) GS = KGS 4 + ∑ i<j xGS ij , A(4) GS = KGS 4 + ∑ i<j xGS ij , where xGS ij is the propagator xGS ij = 1 kikj+1 in momentum space. Then A(4) GS agrees with A(4) top(k) = −1 + ∑i<j xGS ij up to the constant KGS 4 , see (54). where xGS ij is the propagator xGS ij = 1 kikj+1 in momentum space. Then A(4) GS agrees with A(4) top(k) = −1 + ∑i<j xGS ij up to the constant KGS 4 , see (54). 6. A Physical View of the Limit p →1 on p-Adic String Amplitudes j In the case of amplitudes of 5-points, the contribution of the term Dφ5 in the La- grangian is written as Z[J] = · · · −D¯h4 Z dDx δ δJ(x) 5 Z0[J] + · · · . Thus, the corresponding 5-point amplitude is Thus, the corresponding 5-point amplitude is Thus, the corresponding 5-point amplitude is δ5Z[J] δJ(x1)δJ(x2)δJ(x3)δJ(x4)δJ(x5) J=0 = −5!D¯h4 Z dDx −i 2¯h GF(x −x1) × −i 2¯h GF(x −x2) −i 2¯h GF(x −x3) −i 2¯h GF(x −x4) −i 2¯h GF(x −x5) . (67) (67) The contribution will be encoded in the symbol KGS 5 . 3 4 5 Another contribution come from the term Bφ3 × Cφ4. In the Fourier space the corre- sponding diagrams have precisely 2-vertices, 5 external legs attached to these vertices and one internal leg between the two vertices (see, Section 3 of [14]). This latter contribution to generating functional is written as Z[J] = · · · −iBC¯h5 Z dDx Z dDy δ δJ(x) 3 δ δJ(y) 4 Z0[J] + · · · . (68) (68) The 5-point amplitude yields δ5Z[J] δJ(x1)δJ(x2)δJ(x3)δJ(x4)δJ(x5) J=0 = −iBC(12)2¯h5 Z dDx Z dDy −i 2¯h GF(x −y) −i 2¯h GF(x −x5) −i 2¯h GF(x −x4) × −i 2¯h GF(y −x3) −i 2¯h GF(y −x2) −i 2¯h GF(y −x1) + · · · δ5Z[J] δJ(x1)δJ(x2)δJ(x3)δJ(x4)δJ(x5) J=0 = −iBC(12)2¯h5 Z dDx Z dDy −i 2¯h GF(x −y) −i 2¯h GF(x −x5) −i 2¯h GF(x −x4) × −i 2¯h GF(y −x3) −i 2¯h GF(y −x2) −i 2¯h GF(y −x1) + · · · Symmetry 2021, 13, 967 36 of 53 36 of 53 + −i 2¯h GF(y −x5) −i 2¯h GF(x −x4) −i 2¯h GF(x −x3) × −i 2¯h GF(y −x2) −i 2¯h GF(y −x1) + · · · . (69) + −i 2¯h GF(y −x5) −i 2¯h GF(x −x4) −i 2¯h GF(x −x3) × −i 2¯h GF(y −x2) −i 2¯h GF(y −x1) + · · · . (69) (69) Moreover, in the computation of the 5-point amplitudes, the last contribution comes from the term Moreover, in the computation of the 5-point amplitudes, the last contribution comes from the term Z[J] = · · · + B3¯h6 3! 6. A Physical View of the Limit p →1 on p-Adic String Amplitudes Z dDx Z dDy Z dDz δ δJ(x) 3 · δ δJ(y) 3 · δ δJ(z) 3 Z0[J] + · · · . (70) (70) The diagrams associated with this expansion are diagrams with three vertices. Two of them have two external legs and the other leg is internal. The remaining leg has one external line and two internal lines attached. The diagrams associated with this expansion are diagrams with three vertices. Two of them have two external legs and the other leg is internal. The remaining leg has one external line and two internal lines attached. Then the total contribution is Then the total contribution is δ5Z[J] δJ(x1)δJ(x2)δJ(x3)δJ(x4)δJ(x5) J=0 = B3¯h6a 3! Z dDx Z dDy Z dDz −i 2¯h GF(x −y) × −i 2¯h GF(y −z) −i 2¯h GF(y −x5) −i 2¯h GF(z −x4) −i 2¯h GF(z −x3) × −i 2¯h GF(x −x2) −i 2¯h GF(x −x1) + · · · , (71) × −i 2¯h GF(y −z) −i 2¯h GF(y −x5) −i 2¯h GF(z −x4) −i 2¯h GF(z −x3) 2h 2h 2h 2h × −i 2¯h GF(x −x2) −i 2¯h GF(x −x1) + · · · , (71) (71) where a is a constant. where a is a constant. Summarizing the 5 point amplitudes A obtained from Lagrangian (61) can be written where a is a constant. Summarizing, the 5-point amplitudes A5 obtained from Lagrangian (61) can be written Summarizing, the 5-point amplitudes A5 obtained from Lagrangian (61) can be written schematically (in notation from [14]) by Summarizing, the 5-point amplitudes A5 obtained from Lagrangian (61) can be written schematically (in notation from [14]) by A(5) GS = KGS 5 + ∑ KGS 4 i<j xGS ij + ∑ {i,j,k,l}⊂{1,2,3,4,5} i<j, k<l xGS ij xGS kl , where the three terms in the sum corresponds to the contributions of Equations (67), (69) and (71) respectively. Notice that A(5) GS agrees with where the three terms in the sum corresponds to the contributions of Equations (67), (69) and (71) respectively. Notice that A(5) GS agrees with A(5) top(k) = 2 −∑ i<j xGS ij + ∑ {i,j,k,l}⊂{1,2,3,4,5} i<j, k<l xGS ij xGS kl up to the constants KGS 5 , KGS 4 , see (55). 6. A Physical View of the Limit p →1 on p-Adic String Amplitudes up to the constants KGS 5 , KGS 4 , see (55). up to the constants KGS 5 , KGS 4 , see (55). up to the constants KGS 5 , KGS 4 , see (55). We now give a precise formulation of Conjecture 2 announced in the Introduction. By using the Feynman rules for the effective Lagrangian given in [14], p 5 4 ( ) We now give a precise formulation of Conjecture 2 announced in the Introduction. By using the Feynman rules for the effective Lagrangian given in [14], p 5 4 ( ) We now give a precise formulation of Conjecture 2 announced in the Introduction. By using the Feynman rules for the effective Lagrangian given in [14], A(N) Qp = K + ∑N−3 l=2 Ki1...il ∑ 1≤a<b≤l p −1 p p−kia kib−1 1 −p−kia kib−1 ! , where the constants K, Ki1...il are rational functions in p with rational coefﬁcients. Further- more, if N ≤p + 1, the constant term K is not zero. Formally, we have A(N) top = lim p→1 A(N) Qp = Ktop + ∑N−3 l=2 Ktop i1...il ∑ 1≤a<b≤l 1 kiakib + 1 . We denote by A(N) GS the Feynman amplitude of Lagrangian (61). We conjecture that A(N) GS = KGS + ∑N−3 l=2 KGS i1...il ∑ 1≤a<b≤l xGS iaib , Symmetry 2021, 13, 967 37 of 53 where xGS iaib = 1 kia kib+1 = c GF kia −kib . Notice that we are not asserting the existence of any relation between the constants Ktop, Ktop i1...il and the constants KGS, KGS i1...il. 7.1. The p-Adic Sign Function A p-adic sign function is a multiplicative character of Q× p that takes values in {±1}. We set h Q× p i2 as the multiplicative subgroup of squares in Q× p , i.e. We set h Q× p i2 as the multiplicative subgroup of squares in Q× p , i.e. h Q× p i2 = {a ∈Qp; a = b2 for some b ∈Q× p }. Let ε ∈{1, . . . , p −1} such that ( ε p) = −1, where ( · ·) is the Legendre symbol (see for instance the Appendix of [7]). For p ̸= 2 we have Let ε ∈{1, . . . , p −1} such that ( ε p) = −1, where ( · ·) is the Legendre symbol (see for instance the Appendix of [7]). For p ̸= 2 we have Q× p / h Q× p i2 = {1, ε, p, εp}, Q× p / h Q× p i2 = {1, ε, p, εp}, this means that any nonzero p-adic number can be written uniquely as x = τa2, with a ∈Q× p and τ ∈Q× p / h Q× p i2 . For a ﬁxed τ ∈{ε, p, εp}, and x ∈Q× p , we deﬁne the p-adic sign function sgnτ(x) := ( 1 if x = a2 −τb2 for a, b ∈Qp −1 otherwise. (72) (72) The following is the list of all the possible p-adic sign functions: p ≡1 mod 4 p ≡3 mod 4 sgnε(x) = (−1)ord(x) sgnε(x) = (−1)ord(x) sgnp(x) = x0 p sgnp(x) = (−1)ord(x) x0 p sgnεp(x) = (−1)ord(x) x0 p sgnεp(x) = x0 p , (73) p ≡1 mod 4 p ≡3 mod 4 sgnε(x) = (−1)ord(x) sgnε(x) = (−1)ord(x) sgnp(x) = x0 p sgnp(x) = (−1)ord(x) x0 p sgnεp(x) = (−1)ord(x) x0 p sgnεp(x) = x0 p , (73) (73) see [68]. The function sgnτ is a multiplicative character, which means that sgnτ(xy) = sgnτ(x)sgnτ(y). sgnτ(xy) = sgnτ(x)sgnτ(y). sgnτ(xy) = sgnτ(x)sgnτ(y). Additionally sgnτ is a locally constant function in Q× p , which means that sgnτ(x −y) = sgnτ(x) if \|y\|p < \|x\|p. Additionally sgnτ is a locally constant function in Q× p , which means that sgnτ(x −y) = sgnτ(x) if \|y\|p < \|x\|p. g τ( ) \|y\|p \| \|p We need the symmetry sgnτ(−y) = −sgnτ(y) (i.e. 7. p-Adic Open String Amplitudes Coupled to a B-Field with Chan–Paton Factors Strings propagating in a background with gauge ﬁelds was discussed many years ago [57]. In particular, incorporating a Neveu–Schwarz B ﬁeld in the target space leads to a noncommutative effective gauge theory on the world-volume of D-branes [58]. In [7], the N-point p-adic string amplitudes, with Chan–Paton rules and a constant B-ﬁeld (the Ghoshal–Kawano amplitudes) were studied, these amplitudes were introduced in [60]. The study was done by attaching twisted local multivariate zeta functions to the Ghoshal– Kawano amplitudes. In this section we discuss the meromorphic continuation of the Ghoshal–Kawano amplitudes, we also compute the 4 and 5 points amplitudes. Due to the need for a particular symmetry, in this section, we take p ≡3 mod 4. 7.1. The p-Adic Sign Function sgnτ(−1) = −1), this requires p ≡3 mod 4 and τ ∈{p, εp}. Finally, for any x ∈Q× p , we deﬁne the p-adic Heaviside step function as p p We need the symmetry sgnτ(−y) = −sgnτ(y) (i.e. sgnτ(−1) = −1), this requires p ≡3 mod 4 and τ ∈{p, εp}. Finally, for any x ∈Q× p , we deﬁne the p-adic Heaviside step function as We need the symmetry sgnτ(−y) = −sgnτ(y) (i.e. sgnτ(−1) = −1), this requires p ≡3 mod 4 and τ ∈{p, εp}. Finally, for any x ∈Q× p , we deﬁne the p-adic Heaviside step function as Symmetry 2021, 13, 967 38 of 53 Hτ(x) = 1 2(1 + sgnτ(x)) =    1 if sgnτ(x) = 1 0 if sgnτ(x) = −1. 7.2. A Class of Twisted Multivariate Local Zeta Functions 7.2. A Class of Twisted Multivariate Local Zeta Functions 7.2. A Class of Twisted Multivariate Local Zeta Functions Let f := ( f1, . . . , fm), χ := (χ1, . . . , χm) be vectors of non-constant polynomials and multiplicative characters, respectively. And s := (s1, . . . , sm) ∈Cm. The twisted multivariate local zeta functions have the form: ZΘ(s, χ, f) = Z Qnp∖∪m i=1 f −1(0) Θ(x) m ∏ i=1 χi(ac( fi(x)))\| fi(x)\|si p n ∏ i=1 dxi, (74) (74) with Re(si) > 0 for all i, ac stands for the angular component and Θ(x) is a test function. Integrals of type (74) are holomorphic functions in s, which admit meromorphic continua- tions as rational functions in the variables p−s1, . . . , p−sm to the whole Cm, Théorème 1.1.4. of [36], see also [26]. The case when χi is the trivial character has been studied previously, see e.g., Lemma 8.2.1 of [26]. The case when χi = sgnτ is new. τ Using Hironaka’s resolution of singularities theorem [50], in [7] we show that ZΘ(s, χ, f) admits a meromorphic continuation as a rational function in the variables p−s1, . . . , p−sm. More precisely, ZΘ(s, χ, f) = LΘ,χ(s) ∏ j∈T 1 −p−∑m i=1 Ni,jsj−vj , (75) (75) where LΘ,χ(s) is a polynomial in the variables p−s1, . . . , p−sm, and the real parts of its poles belong to the ﬁnite union of hyperplanes where LΘ,χ(s) is a polynomial in the variables p−s1, . . . 7.1. The p-Adic Sign Function , p−sm, and the real parts of its poles belong to the ﬁnite union of hyperplanes m ∑ i=1 Ni,j Re(si) + vj = 0, for j ∈T . m ∑ i=1 Ni,j Re(si) + vj = 0, for j ∈T . This result is a variation of Théorème 1.1.4. in [36]. This result is a variation of Théorème 1.1.4. in [36]. This result is a variation of Théorème 1.1.4. in [36]. 7.3. The Ghoshal–Kawano Local Zeta Function 7.3. The Ghoshal–Kawano Local Zeta Function In [60] Ghoshal and Kawano proposed the following amplitude (for the N-point tree-level, p-adic open string amplitude, with Chan–Paton rules in a constant B-ﬁeld): A(N)(k, θ, τ; x1, xN−1) := Z QN−3 p ∖Λ N−2 ∏ i=2 \|xi\|k1ki p \|1 −xi\|kN−1ki p Hτ(xi)Hτ(1 −xi) × ∏ 2≤i<j≤N−2 xi −xj kikj p Hτ xi −xj × exp ( − √−1 2 ∑ 1≤i<j≤N−1 (kiθkj)sgnτ(xi −xj) !) N−2 ∏ i=2 dxi, (76 × exp ( − √−1 2 ∑ 1≤i<j≤N−1 (kiθkj)sgnτ(xi −xj) !) N−2 ∏ i=2 dxi, (76) (76) where N ≥4, k = (k1, . . . , kN), ki = (k0,i, . . . , kl,i), i = 1, . . . , N, is the momentum vector of the i-th tachyon (with Minkowski product kikj = −k0,ik0,j + k1,ik1,j + · · · + kl,ikl,j), θ is a N 2 where N ≥4, k = (k1, . . . , kN), ki = (k0,i, . . . , kl,i), i = 1, . . . , N, is the momentum vector of the i-th tachyon (with Minkowski product kikj = −k0,ik0,j + k1,ik1,j + · · · + kl,ikl,j), θ is a N 2 where N ≥4, k = (k1, . . . , kN), ki = (k0,i, . . . , kl,i), i = 1, . . . , N, is the momentum vector of the i-th tachyon (with Minkowski product kikj = −k0,ik0,j + k1,ik1,j + · · · + kl,ikl,j), θ is a ﬁxed antisymmetric bilinear form that is built with the inverse of the B-ﬁeld; ∏N−2 i=2 dxi is h li d H f QN 3 ﬁxed antisymmetric bilinear form that is built with the inverse of the B-ﬁeld; ∏N−2 i=2 dxi is the normalized Haar measure of QN−3 p , Symmetry 2021, 13, 967 39 of 53 39 of 53 Λ := ( (x2, . . . 7.1. The p-Adic Sign Function , xN−2) ∈QN−3 p ; N−2 ∏ i=2 xi(1 −xi) ∏ 2≤i<j≤N−2 xi −xj =0 ) , and the momentum vectors obey N ∑ i=1 ki = 0, kiki = 2 for i = 1, . . . , N. (77) (77) To preserve the symmetry under the exchange of external momentum vectors, we require that sgnτ(xi −xj) = −sgnτ(xj −xi), or equivalently sgnτ(−1) = −1. Then for the rest of this section we assume that τ ∈{p, εp}. {p p} To simplify the notation, we introduce the variables sij ∈C, and ˜sij ∈R for 1 ≤i < j ≤N −1. We also set F(x, s, τ) := N−2 ∏ i=2 \|xi\|s1i p \|1 −xi\| s(N−1)i p Hτ(xi)Hτ(1 −xi) × ∏ 2≤i<j≤N−2 \|xi −xj\| sij p Hτ(xi −xj), F(x, s, τ) := N−2 ∏ i=2 \|xi\|s1i p \|1 −xi\| s(N−1)i p Hτ(xi)Hτ(1 −xi) × ∏ 2≤i<j≤N−2 \|xi −xj\| sij p Hτ(xi −xj), and E(x,es, τ; x1, xN−1) := exp ( −√−1 2 ∑ 1≤i<j≤N−1 esijsgnτ(x1 −xj) !) . (78) (78) Now, we deﬁne the Ghoshal–Kawano local zeta function as Z(N)(s,es, τ; x1, xN−1) = Z QN−3 p ∖Λ F(x, s, τ)E(x,es, τ; x1, xN−1) N−2 ∏ i=2 dxi. (79) (79) For the sake of simplicity, we use QN−3 p as domain of integration in (79) from now on. We now consider the convergence of the amplitudes (76). By using that \|E(x,es, τ; x1, xN−1)\| = 1, \|Hτ(xi)\| ≤1, \|Hτ(1 −xi)\| ≤1, for any i, and that Hτ(xi −xj) ≤1, for any i, j, we have Z(N)(s,es, τ; x1, xN−1) ≤ Z QN−3 p N−2 ∏ i=2 \|xi\|Re(s1i) p \|1 −xi\| Re(s(N−1)i) p ∏ 2≤i<j≤N−2 xi −xj Re(sij) p N−2 ∏ i=2 dxi = Z(N)(Re(s)), where Z(N)(s) is the Koba–Nielsen string amplitude studied in [6,8]. By applying the results of [6,8], integral Z(N)(Re(s)) is holomorphic in an open set K ⊂CD, therefore Z(N)(s,es, τ) is holomorphic in s ∈K for any es, τ, x1, xN−1. Z(N)(s,es, τ) is holomorphic in s ∈K for any es, τ, x1, xN−1. It is important to notice that if any of the integration variables is in Qp ∖Zp, then due to the local constancy of sgnτ, the factor Hτ(x)Hτ(−x) appears in F(x, s, τ), and Hτ(x)Hτ(−x) = 0. For this reason, we redeﬁne the Ghoshal–Kawano local zeta function as Z(N)(s,es, τ; x1, xN−1) := Z ZN−3 p F(x, s, τ)E(x,es, τ; x1, xN−1) N−2 ∏ i=2 dxi. (80) (80) Symmetry 2021, 13, 967 40 of 53 40 of 53 7.4. Ghoshal–Kawano Amplitude as a Local Zeta Function For ˜s ∈R we have 7.4. Ghoshal–Kawano Amplitude as a Local Zeta Function p For ˜s ∈R we have exp ( −√−1es 2 sgnτ(x) ) = cos es 2 − √ −1 sgnτ(x) sin es 2 . (81) (81) Using this identity and the convention x1 = 0, xN−1 = 1, we have Using this identity and the convention x1 = 0, xN−1 = 1, we have E(x,es, τ) = ∑ I,J,K EI,J,K(es) ∏ j∈I sgnτ(xj) ∏ j∈J sgnτ(1 −xj) ∏ i,j∈K sgnτ(xi −xj). (82) (82) In a similar way, we obtain that In a similar way, we obtain that N−2 ∏ i=2 Hτ(xi)Hτ(1 −xi) ∏ 2≤i<j≤N−2 Hτ(xi −xj) = ∑ I,J,K eI,J,K∏ j∈I sgnτ(xj) ∏ j∈J sgnτ(1 −xj) ∏ i,j∈K sgnτ(xi −xj), (83) (83) Using Formulae (82) and (83), and assuming x1 = 0, xN−1 = 1, xN = ∞, we can write the Ghoshal–Kawano zeta function as a ﬁnite sum of integrals of type Using Formulae (82) and (83), and assuming x1 = 0, xN−1 = 1, xN = ∞, we can write the Ghoshal–Kawano zeta function as a ﬁnite sum of integrals of type C(es) R ZN−3 p N−2 ∏ i=2 \|xi\|s1i p \|1 −xi\| s(N−1)i p ∏ 2≤i<j≤N−2 \|xi −xj\| sij p ∏ j∈I χτ(xj)∏ j∈J χτ(1 −xj) × ∏ i,j∈K χτ(xi −xj) N−2 ∏ i=2 dxi, where C(es) is an R-analytic function and χτ is a trivial character or sgnτ. This expression is a local zeta functions of the type (74), which implies that amplitudes (76) are equal to a ﬁnite sum of local zeta functions and consequently they admit a meromorphic continuation in the kinematic parameters. Z(N)(s,es, τ) is holomorphic in s ∈K for any es, τ, x1, xN−1. Finally, we point out that the meromorphic continuation of the Ghoshal–Kawano local zeta function (80) is also valid without taking the normalization x1 = 0, xN−1 = 1, xN = ∞, see Section V-C of [7]. 7.5. Explicit Computation of Z(4)(s,es, τ) We ﬁrst notice that the exponential factor E(4)(˜s, τ) can be taken outside the integral, i.e., the four-point Ghoshal–Kawano zeta function is Z(4)(s,es, τ) = exp i 2(es13 + es12 + es23) × R Zp \|x2\|s12 p \|1 −x2\|s32 p Hτ(x2)Hτ(1 −x2)dx2. Z(4)(s,es, τ) = exp i 2(es13 + es12 + es23) × R Zp \|x2\|s12 p \|1 −x2\|s32 p Hτ(x2)Hτ(1 −x2)dx2. The computation of this integral is based on the calculation of the following integrals: assume that S ⊂Zp ∖{0} satisﬁes −S = S, then R S \|x2\|s12 p sgnτ(x2)dx2 = 0, for τ ∈{p, εp}, and and I(s, τ) = R Zp \|x2\|s12 p \|1 −x2\|s32 p Hτ(x2)Hτ(1 −x2)dx2 = p −3 4p + p−1−s121 −p−1 2(1 −p−1−s12) + p−1−s321 −p−1 2(1 −p−1−s32) . Symmetry 2021, 13, 967 41 of 53 41 of 53 For further details, the reader may consult [7]. The explicit expression for the 4-point amplitude, also reported in [60], is Z(4)(s,es, τ) = exp i 2(es13 + es12 + es23) × p −3 4p + p−1−s121 −p−1 2(1 −p−1−s12) + p−1−s321 −p−1 2(1 −p−1−s32) ! which is holomorphic in which is holomorphic in Re(s12) > −1 and Re(s32) > −1. 7.6. Explicit Computation of Z(5)(s,es, τ) 7.6. Explicit Computation of Z(5)(s,es, τ) After some simple considerations involving sign functions, After some simple considerations involving sign functions, Z(5)(s,es, τ) = E(5)(es)L(s, τ) := E(5)(es) Z Z2p F(5)(x2, x3, s, τ)dx2dx3. where where F(5)(x2, x3, s, τ) = \|x2\|s12 p \|x3\|s13 p \|1 −x2\|s42 p \|1 −x3\|s43 p \|x2 −x3\|s23 p × Hτ(x2)Hτ(x3)Hτ(1 −x2)Hτ(1 −x3)Hτ(x2 −x3). and E(5)(es) = exp (√−1 2 (es14 + es12 + es13 + es24 + ˜s34 + ˜s32) ) , see [7] for further details. To compute the integral L(s, τ), we subdivide the domain of integration as see [7] for further details. To compute the integral L(s, τ), we subdivide the domain of integration as Z2 p = p−1 G i,j=0 i + pZp × j + pZp , then L(s, τ) becomes L(s, τ) = p−1 ∑ i,j=0 Lij(s, τ) =: p−1 ∑ i,j=0 Z i+pZp×j+pZp F(5)(x2, x3, s, τ)dx2dx3. The computation of the integrals Lij(s, τ) is a simple but technical calculation, see [7] for further details. The explicit expression for the 5-point Ghoshal–Kawano zeta function is Z(5)(s,es) = E(5)(es) (p −3)(p −7) 32p2 + p −3 8p p−1−s23(1 −p−1) (1 −p−1−s23) + p−1−s13(1 −p−1) (1 −p−1−s13) + p−1−s42(1 −p−1) (1 −p−1−s42) + 1 −p−12 4 p−2−s13−s42 (1 −p−1−s13)(1 −p−1−s42) +1 4 p−s12−s13−s23−21 −p−1 1 −p−s12−s13−s23−2 " 1 2 −3 2p + p−1−s231 −p−1 1 −p−1−s23 + p−1−s131 −p−1 1 −p−1−s13 # +1 4 p−s42−s43−s23−21 −p−1 1 −p−s42−s43−s23−2 " 1 2 −3 2p + p−1−s231 −p−1 1 −p−1−s23 + p−1−s421 −p−1 1 −p−1−s42 #) . Symmetry 2021, 13, 967 42 of 53 This function is holomorphic in This function is holomorphic in This function is holomorphic in Re(s13) > −1; Re(s23) > −1; Re(s42) > −1; Re(s12 + s13 + s23) > −2; Re(s42 + s43 + s23) > −2. 8.1. Local Fields We take K to be a non-discrete locally compact ﬁeld of characteristic zero. Then K is R, C, or a ﬁnite extension of Qp, the ﬁeld of p-adic numbers. If K is R or C, we say that K is an R-ﬁeld, otherwise we say that K is a p-ﬁeld. For a ∈K, we deﬁne the modulus \|a\|K of a by For a ∈K, we deﬁne the modulus \|a\|K of a by \|a\|K =        the rate of change of the Haar measure in (K, +) under x →ax for a ̸= 0, 0 for a = 0. \|a\|K =        the rate of cha for a ̸= 0, 0 for a = 0. is well-known that if K is an R-ﬁeld, then \|a\|R = \|a\| and \|a\|C = \|a\|2, where \|·\| denotes the usual absolute value in R or C, and, if K is a p-ﬁeld, then \|·\|K is the normalized absolute value in K. is well-known that if K is an R-ﬁeld, then \|a\|R = \|a\| and \|a\|C = \|a\|2, where \|·\| denotes the usual absolute value in R or C, and, if K is a p-ﬁeld, then \|·\|K is the normalized absolute value in K. 8. Resolution of Singularities and Multivariate Igusa Zeta Functions 8.1. Local Fields 8.2. K-Analytic Manifolds and Resolution of Singularities 8.2. K-Analytic Manifolds and Resolution of Singularities 8.2. K-Analytic Manifolds and Resolution of Singularities We review the basic deﬁnitions of K-analytic manifolds following Igusa’s book [26]. y g g [ ] Let K be a local ﬁeld of characteristic zero, and let V be a nonempty open subset of Kn and let f : V →K be a function. We say that f is a K-analytic function on V, if for every point a = (a1, . . . , an) ∈V, there exists an element fa(x) of K⟨⟨x −a⟩⟩= K⟨⟨x1 − a1, . . . , xn −an⟩⟩(the ring of convergent power series around a) such that f (x) = fa(x) for any point x near a. A map g = (g1, . . . , gm) : V →Km is called K- analytic mapping on V if each gi is an analytic function on V. Let X Hausdorff space and let n be a ﬁxed non- negative. Let U be a nonempty open subset of X, if φU : U →φU(U) is a homeomorphism, where φU(U) is a nonempty open subset of Kn, then we say that the pair (U, φU(U)) is a chart. For a variable point x ∈U, φ(x) = (x1, . . . , xn) are called the local coordinates of x. A collection of charts {U, φU} is called an atlas of X if the union of all open sets V is X and for every U, U′ such that U ∩U′ ̸= ∅the map φU′ ◦φ−1 U : φU(U ∩U′) →φU′(U ∩U′) is K- analytic. There is an equivalence relation over the set of atlases on X. Two atlases are equivalent if their union is also an atlas. Thus, any equivalence class is called an n-dimensional K-analytic structure on X. Hence, we say that X is a K-analytic manifold and we write dim(X) = n. Suppose that X and Y are two K- analytical manifolds defined by the atlases {(U, φU(U))} and {(V, ψV(V))}, respectively, and f : X →Y a map. If for every U, V such that U ∩f −1(V) ̸= ∅, the map ψV ◦f ◦φ−1 U : φU(U ∩f −1(V)) →Kdim(Y) is K- analytic, then f is called a K-analytic map. If Y = K we say that f is a K-analytic function on X. This deﬁnition does not depend on the choice of atlases into the equiva- lence class. is K- analytic, then f is called a K-analytic map. 8.2. K-Analytic Manifolds and Resolution of Singularities If Y = K we say that f is a K-analytic function on X. This deﬁnition does not depend on the choice of atlases into the equiva- lence class. Let X be a K-analytic manifold deﬁned by an atlas {(U, φU)} and let Y be a nonempty open set of X. If U′ = U ∩Y ̸= ∅we put φU′ = φU′ = φU\|U′. Then {(U′, φU′)} is an atlas for Y. Hence, Y is an open K- analytic submanifold of X. Furthermore, dim(Y) = dim(X). Symmetry 2021, 13, 967 43 of 53 Now let Y be a nonempty closed subset of a K-analytic manifold X of dimension n. Suppose that X is defined by an atlas {(U, φU)} with the following property: If φU(x) = (x1, . . . , xn) and U′ = Y ∩U ̸= ∅, there exist F1, . . . , Fm K-analytic functions on U with 0 < m ≤n such that U′ becomes the set of all x in U such that F1(x) = . . . = Fm(x) = 0 and det " ∂Fi ∂xj # 1≤i≤m 1≤j≤m (a) ̸= 0, for every a ∈U′. By the implicit function theorem, the mapping for every a ∈U′. By the implicit function theorem, the mapping x 7→(F1(x), . . . , Fm(x), xm+1, . . . , xn) gives a K-bianalytic map from a neighborhood of a in U to its image in Kn. If we denote by V the intersection of such neighborhood and Y, and put ψV(x) = (xm+1, . . . , xn) for every x ∈V. Then (V, ψV) for all V and for each U as above, gives an atlas on Y. Thus, Y becomes a K-analytic closed submanifold of X of dimension n −m. Assume that U and V are nonempty open sets that contain a point a of X, and let f, g be two K- analytic functions respectively on U, V such that f \|W = g\|W for some nonempty open set W such that a ∈W ⊆U ∩V. Then we say that f and g are equivalent. An equivalence class is called a germ of analytic function at a. The set of such equivalence classes becomes a local ring OX,a with maximal ideal mX,a = { f ∈OX,a : f (a) = 0}. In addition, K ⊆OX,a. 9. Multivariate Igusa Zeta Functions Let K be a local ﬁeld. If K is a p-ﬁeld, resp. an R-ﬁeld, we denote by D(Kn) the C-vector space consisting of all C-valued locally constant functions, resp. all smooth functions, on Kn, with compact support. An element of D(Kn) is called a test function. p pp ( ) f Let fi(x) ∈K[x1, . . . , xn] be a non-constant polynomial for i = 1, . . . , m. We set f = ( f1, . . . , fm) and s = (s1, . . . , sm) ∈Cm. Let Φ be a test function. The multivariate local zeta function attached to ( f, Φ) is deﬁned as ZΦ( f, s) = Z Kn∖DK Φ(x) m ∏ i=1 \| fi(x)\|si K n ∏ i=1 dxi, when Re(si) > 0 for all i. (84) (84) where DK := ∪m i=1 f −1 i (0) is the divisor attached to fi(x), i = 1, . . . , m. In the multivariate case i.e., for m ≥1, the local zeta functions over local ﬁelds of zero characteristic were studied by Loeser [36]. In the case of zero characteristic, the main tool to show the existence of a meromorphic continuation of the multivariate local function is the Hironaka’s resolu- tion of singularities theorem. By applying this theorem to the divisor DK, the mutivariate local zeta function is reduced to the case of monomial integrals [25,26,36]. Currently, the methods used by Igusa are not available in positive characteristic, so the problem of the meromorphic continuations in this setting it is still an open problem. Theorem 2 (Hironaka, [50]). Let K be a local ﬁeld of characteristic zero. There exists an embedded resolution σ : X →Kn of DK, i.e., Theorem 2 (Hironaka, [50]). Let K be a local ﬁeld of characteristic zero. There exists an embedded resolution σ : X →Kn of DK, i.e., f ﬁ of σ−1(DK); 1 Theorem 2 (Hironaka, [50]). Let K be a local ﬁeld of characteristic zero. There exists an embedded resolution σ : X →Kn of DK, i.e., (i) X is an n-dimensional K-analytic manifold, σ is a proper K-analytic map which is a composition of a ﬁnite number of blow-ups at closed submanifolds, and which is an isomorphism outside of σ−1(DK); (ii) σ−1(DK) is a normal crossings divisor, meaning that σ−1(DK) = ∪i∈TEi, where the Ei are closed submanifolds of X of codimension one, each equipped with an m-tuple of non-negative integers Nf1,i, . . . , Nfm,i and a positive integer vi, satisfying the following. At every point b of X Symmetry 2021, 13, 967 44 of 53 44 of 53 there exist local coordinates (y1, . . . , yn) on X around b such that if E1, . . . , Er are the Ei containing b, we have on some open neighborhood V of b that Ei is given by yi = 0 for i ∈{1, . . . , r}, there exist local coordinates (y1, . . . , yn) on X around b such that if E1, . . . , Er are the Ei containing b, we have on some open neighborhood V of b that Ei is given by yi = 0 for i ∈{1, . . . , r}, σ∗(dx1 ∧. . . ∧dxn) = η(y) r ∏ i=1 yvi−1 i ! dy1 ∧. . . ∧dyn, (85) (85) and and f ∗ j (y) := fj ◦σ (y) = ε fj(y) r ∏ i=1 y Nfj,i i , for j = 1, . . . , m, (86) (86) where η(y) and the ε fj(y) belong to O× X,b, the group of units of the local ring of X at b. The Hironaka resolution theorem allows expressing a multivariate local zeta function as a linear combination of monomial integrals, through a ﬁnite sequence of changes of variables. We have the following theorem on multivariate local zeta functions over local ﬁelds of zero characteristic. Theorem 3. (Lemma 6.4, Remark 2 of [6]) Let f1(x), . . . , fm(x) ∈K[x1, . . . , xn] be non- constant polynomials and Φ : Kn →C a smooth function with compact support, to which we associate the multivariate local zeta function ZΦ( f, s). Fix an embedded resolution σ : X →K of DK = ∪m i=1 f −1 i (0) as in Theorem 2 . Theorem 2 (Hironaka, [50]). Let K be a local ﬁeld of characteristic zero. There exists an embedded resolution σ : X →Kn of DK, i.e., Then (i) ZΦ( f, s) is convergent and deﬁnes a holomorphic function in the region m ∑ j=1 Nfj,i Re(sj) + vi > 0, for i ∈T; (ii) ZΦ( f, s) admits a meromorphic continuation to the whole Cm, with poles belonging to (ii) ZΦ( f, s) admits a meromorphic continuation to the whole Cm, with poles belonging to [ i∈T [ t∈N ( m ∑ j=1 Nfj,isj + vi + t = 0 ) with t ∈N if K = R and t = 1 2N if K = C, and with poles belonging to [ 1≤i≤r ( m ∑ j=1 aj,isj + bi = 0 ) , in the p-ﬁeld case. In addition, in the p-adic case the multivariate local zeta function has a meromorphic continuation as a rational function ZΦ( f, s) = PΦ(s) ∏ i∈T    1 −q −   m ∑ j=1 Nfj,isj+vi       in q−s1, . . . , q−sm, where PΦ(s) is a polynomial in the variables q−si. in q−s1, . . . , q−sm, where PΦ(s) is a polynomial in the variables q−si. 10. Meromorphic Continuation of Koba–Nielsen Amplitudes Deﬁned on Local Fields of Characteristic Zero In the case N = 4 and K = R, the amplitude (87) is the Veneziano amplitude, see [10]. In Section 2 of [48] and [49], it was established that the N-point closed string amplitude at the tree level is the product of A(N) C (k) times a polynomial function in the momenta k. Hence, the results established in [6] are still valid for closed string amplitudes at tree level. If we take the Minkowski products of the kinematic parameters as follows kikj = sij ∈C, the string amplitude (87) becomes to the following integral which is a type of multivariate local zeta function Z(N) K (s) := Z KN−3∖DN N−2 ∏ i=2 xj s1j K 1 −xj s(N−1)j K ∏ 2≤i<j≤N−2 xi −xj sij K N−2 ∏ i=2 dxi, (89) (89) where ∏N−2 i=2 dxi is the normalized Haar measure on KN−3, s := sij ∈CD, with D = N(N−3) 2 denotes the total number of indices ij, and DN := ( x ∈KN−3; N−2 ∏ i=2 xi N−2 ∏ i=2 (1 −xi) ∏ 2≤i<j≤N−2 xi −xj = 0 ) . We have called integrals of type (89) Koba–Nielsen local zeta functions. For simplicity of notation, we put KN−3 instead of KN−3 ∖DN in (89). We have called integrals of type (89) Koba–Nielsen local zeta functions. For simplicity of notation, we put KN−3 instead of KN−3 ∖DN in (89). In [6], we establish, in a rigorous mathematical way that the Koba–Nielsen string amplitudes deﬁned on any local ﬁeld of characteristic zero are bona ﬁde integrals and that they can be extended to meromorphic functions in the kinematic parameters. In order to prove the meromorphic continuation of (89), we express it as linear combinations of local zeta functions. These computations were ﬁrst made in the case of K = R, but they can be easily extended to other local ﬁelds of characteristic zero. Thus, we only review the real case. We consider RN−3 as an R-analytic manifold, with N ≥4, and use {x2, . . . , xN−2} as a coordinate system. In order to regularize the integral (89), we use a partition of RN−3 constructed using a smooth function χ : R →R satisfying χ(x) =    1 if x ∈[−2, 2] 0 if x ∈(−∞, −2 −ϵ] ∪[2 + ϵ, +∞), for some ﬁxed positive ϵ sufﬁciently small. 10. Meromorphic Continuation of Koba–Nielsen Amplitudes Deﬁned on Local Fields of Characteristic Zero The Koba–Nielsen open string amplitudes for N-points over a local ﬁeld K of charac- teristic zero are deﬁned as A(N) K (k) := Z KN−3 N−2 ∏ i=2 xj k1kj K 1 −xj kN−1kj K ∏ 2≤i<j≤N−2 xi −xj kikj K N−2 ∏ i=2 dxi, (87) (87) Symmetry 2021, 13, 967 45 of 53 where k = (k1, . . . , kN), ki = (k0,i, . . . , kl,i) ∈Cl+1, for i = 1, . . . , N, is the momentum vector of the i-th tachyon (with Minkowski product kikj = −k0,ik0,j + k1,ik1,j + · · · + kl,ikl,j), obeying where k = (k1, . . . , kN), ki = (k0,i, . . . , kl,i) ∈Cl+1, for i = 1, . . . , N, is the momentum vector of the i-th tachyon (with Minkowski product kikj = −k0,ik0,j + k1,ik1,j + · · · + kl,ikl,j), obeying where k = (k1, . . . , kN), ki = (k0,i, . . . , kl,i) ∈Cl+1, for i = 1, . . . , N, is the momentum vector of the i-th tachyon (with Minkowski product kikj = −k0,ik0,j + k1,ik1,j + · · · + kl,ikl,j), obeying N ∑ i=1 ki = 0, kiki = 2 for i = 1, . . . , N. (88) (88) The parameter l is typically taken to be 25. However, we do not require using the critical dimension, thus l can be any positive number. These amplitudes were introduced by Brekke, Freund, Olson and Witten, among others in their works about string amplitudes, see e.g., Section 8 of [4]. In the case N = 4 and K = R, the amplitude (87) is the Veneziano amplitude, see [10]. In Section 2 of [48] and [49], it was established that the N-point closed string amplitude at the tree level is the product of A(N) C (k) times a polynomial function in the momenta k. Hence, the results established in [6] are still valid for closed string amplitudes at tree level. The parameter l is typically taken to be 25. However, we do not require using the critical dimension, thus l can be any positive number. These amplitudes were introduced by Brekke, Freund, Olson and Witten, among others in their works about string amplitudes, see e.g., Section 8 of [4]. 10. Meromorphic Continuation of Koba–Nielsen Amplitudes Deﬁned on Local Fields of Characteristic Zero This function is well-known, see e.g., Section 1.4 of [69], Section 5.2 of [26]. The number 2 was chosen in an arbitrary form, the key point is that the interval [0, 1] is included in the locus where χ ≡1. Now, we can write Z(N)(s) = ∑ I Z(N) I (s) (90) (90) with Z(N) I (s) := Z RN−3 ϕI(x) N−2 ∏ j=2 xj s1j N−2 ∏ j=2 1 −xj s(N−1)j ∏ 2≤i<j≤N−2 xi −xj sij N−2 ∏ i=2 dxi, (91) (91) Symmetry 2021, 13, 967 46 of 53 where the functions {ϕI} are deﬁned as where the functions {ϕI} are deﬁned as ϕI : RN−3 →R; x 7→∏ i∈I χ(xi)∏ i/∈I (1 −χ(xi)), (92) (92) for I ⊆{2, . . . , N −2}, including the empty set, with the convention that ∏i∈∅· ≡1. Notice that ϕI ∈C∞RN−3 and ∑I ϕI(x) ≡1, for x ∈RN−3, i.e., the functions {ϕI} form a partition of the unity. In the case I = {2, . . . , N −2}, Z(N) I (s) is a classical multivariate Igusa local zeta function (since ϕI(x) has compact support). It is well known that these integrals are holo- morphic functions in a region including Re sij > 0 for all ij, and they admit meromorphic continuations to the whole CD, see ([6], Theorem 3.2). 1 In the case I ̸= {2, . . . , N −2}, by changing variables in (91) as xi → 1 xi for i ̸∈I, and xi →xi for i ∈I, we have ∏N−2 i=2 dxi →∏i̸∈I 1 \|xi\|2 ∏N−2 i=2 dxi, and by setting eχ(xi) := 1 1 f i ̸∈I i 1 −χ 1 xi for i ̸∈I, i.e., 1 −χ 1 xi for i ̸∈I, i.e., eχ(xi) =    1 if \|xi\| ≤ 1 2+ϵ 0 if \|xi\| ≥1 2, we have that supp eχ ⊆ h −1 2, 1 2 i and eχ ∈C∞(R). Now setting eϕI(x) := ∏i̸∈I eχ(xi) ∏i∈I χ(xi), and we have that supp eχ ⊆ h −1 2, 1 2 i and eχ ∈C∞(R). 10. Meromorphic Continuation of Koba–Nielsen Amplitudes Deﬁned on Local Fields of Characteristic Zero Now setting eϕI(x) := ∏i̸∈I eχ(xi) ∏i∈I χ(xi), and ∏i∈I χ(xi), and FI(x, s) := ∏ j∈I xj s1j N−2 ∏ j=2 1 −xj s(N−1)j ∏ 2≤i<j≤N−2 i, j∈I xi −xj sij × ∏ 2≤i<j≤N−2 i, j̸∈I xi −xj sij ∏ 2≤i<j≤N−2 i̸∈I, j∈I 1 −xixj sij ∏ 2≤i<j≤N−2 i∈I, j̸∈I 1 −xixj sij, we have Z(N) I (s) = Z RN−3∖DI eϕI(x)FI(x, s) ∏ i̸∈I \|xi\| s1i+s(N−1)i+∑2≤j≤N−2 j̸=i sij+2 N−2 ∏ i=2 dxi, (93) (93) where DI is the divisor deﬁned by the polynomial where DI is the divisor deﬁned by the polynomial where DI is the divisor deﬁned by the polynomial N−2 ∏ i=2 xi N−2 ∏ i=2 (1 −xi) ∏ 2≤i<j≤N−2 i, j∈I xi −xj ∏ 2≤i<j≤N−2 i, j/∈I xi −xj × ∏ 2≤i<j≤N−2 i/∈I, j∈I 1 −xixj ∏ 2≤i<j≤N−2 i∈I, j/∈I 1 −xixj . N−2 I xi −xj ∏ 2≤i<j≤N−2 i, j/∈I xi −xj × ∏ 2≤i<j≤N−2 i/∈I, j∈I 1 −xixj ∏ 2≤i<j≤N−2 i∈I, j/∈I 1 −xixj . The integrals Z(N) I (s), with I ̸= {2, . . . , N −2}, are not classical multivariate local zeta functions thus we do not apply the theory of local zeta functions. Thus, in [6] we show that they deﬁne holomorphic functions on some nonempty open in CD, and admit meromorphic continuations to the whole CD. Lemma 1. (Lemma 4.1 of [6]) For any I ⊆{2, . . . , N −2}, the function Z(N) I (s) is holomorphic in s on the solution set H(I) of a system of inequalities of the form H(I) :=   sij ∈CD; ∑ ij∈M(I) Nij,k(I) Re sij + γk(I) > 0, for k ∈T(I)   ̸= ∅, (94) (94) Symmetry 2021, 13, 967 47 of 53 where Nij,k(I), γk(I) ∈Z, and M(I), T(I) are ﬁnite sets. More precisely, for each k, either all numbers Nij,k(I) are equal to 0 or 1 and γk(I) > 0, or all numbers Nij,k(I) are equal to 0 or −1 and γk(I) < 0. where Nij,k(I), γk(I) ∈Z, and M(I), T(I) are ﬁnite sets. More precisely, for each k, either all numbers Nij,k(I) are equal to 0 or 1 and γk(I) > 0, or all numbers Nij,k(I) are equal to 0 or −1 and γk(I) < 0. 10. Meromorphic Continuation of Koba–Nielsen Amplitudes Deﬁned on Local Fields of Characteristic Zero In addition, Z(N) I (s) admits an analytic continuation to the whole CD, as a meromorphic function with poles belonging to P(I) := [ t∈N [ k∈T(I)   sij ∈CD; ∑ ij∈M(I) Nij,k(I)sij + γk(I) + t = 0   . (95) (95) To show the existence of the meromorphic continuation of Z(N)(s) it is necessary that all the integrals Z(N) I (s) be holomorphic in a common domain, and then Formula (90) allows us to construct a meromorphic continuation of Z(N)(s). We show that ∩IH(I) contains a nonempty open subset of CD by studying the possible poles of integrals Z(N) I (s) := Z(N) I (s) \|sij=s for any I, and proving that Z(N)(s) := Z(N)(s) \|sij=s is a holomorphic function in the region − 2 N −2 < Re(s) < −2 N , − 2 N −2 < Re(s) < −2 N , This fact was proved in Theorem 4.1 in [6]. Furthermore, the Koba–Nielsen local zeta function Z(N)(s) is convergent and holo- morphic in the region determined by the following inequalities: Re(sij) > −1 for all N(N−3) 2 variables sij, for all N(N−3) 2 variables sij, ∑ j∈J Re(s1j) + ∑ i,j∈J i<j Re(sij) > −#J for all subsets J ⊂{2, . . . , N −2} with #J ≥2, ∑ j∈J Re(s(N−1)j) + ∑ i,j∈J i<j Re(sij) > −#J ∑ j∈J Re(s1j) + ∑ i,j∈J i<j Re(sij) > −#J j for all subsets J ⊂{2, . . . , N −2} with #J ≥2, ∑ j∈J Re(s(N−1)j) + ∑ i,j∈J i<j Re(sij) > −#J ∑ j∈J Re(s(N−1)j) + ∑ i,j∈J i<j Re(sij) > −#J for all subsets J ⊂{2, . . . , N −2} with #J ≥2, ∑ i,j∈J i<j Re(sij) > −#J + 1 ∑ i j∈J Re(sij) > −#J + 1 for all subsets J ⊂{2, . . . , N −2} with #J ≥3, for all subsets J ⊂{2, . . . , N −2} with #J ≥3, ∑ j∈J Re(s1j) + ∑ j∈J Re(s(N−1)j) + ∑ i∈{2,...,N−2}\J j∈J Re(sij) + ∑ i,j∈J i<j Re(sij) < −#J for all subsets J ⊂{2, . . . , N −2} with #J ≥3, for all subsets J ⊂{2, . . . , N −2} with #J ≥3, ubsets J ⊂{2, . . . 10. Meromorphic Continuation of Koba–Nielsen Amplitudes Deﬁned on Local Fields of Characteristic Zero , N −2} with #J ≥3, ∑ j∈J Re(s1j) + ∑ j∈J Re(s(N−1)j) + ∑ i∈{2,...,N−2}\J j∈J Re(sij) + ∑ i,j∈J i<j Re(sij) < −#J for all subsets J ⊂{2, . . . , N −2} with ♯J ≥1. This region contains the open subset defined by for all subsets J ⊂{2, . . . , N −2} with ♯J ≥1. This region contains the open subset defined by − 2 N −2 < Re(sij) < −2 N , f ll i di ij for all subsets J ⊂{2, . . . , N −2} with ♯J ≥1. This region contains the open subset defined by 2 < Re(s ) < 2 − 2 N −2 < Re(sij) < −2 N , − 2 N −2 < Re(sij) < −2 N , for all indices ij. for all indices ij. j As in the p-adic case, we use the meromorphic continuation of (89) to the whole CD, which is denoted by Z(N) K (s), as regularizations of the amplitudes A(N) K (k) by redeﬁning Symmetry 2021, 13, 967 48 of 53 A(N) K (k) = Z(N) K (s) \|sij=kikj, A(N) K (k) = Z(N) K (s) \|sij=kikj, see Theorem 6.1 of [6]. It is important to mention here that in the regularization of A(N) K (k), we do not use the kinematic restrictions (88). ( ) Furthermore, in [6] we show that A(N) K (k) converges on some open subset of C(N−1)(l+1) by showing that this open is mapped into the domain of convergency of Z(N) K (s) by k →sij = kikj. In addition, we prove that A(N) K (k) extends to a meromorphic func- tion to the whole C(N−1)(l+1), and that its polar set is contained in the inverse image of the polar set of Z(N) K (s) under that mapping, where the possible poles are described in terms of numerical data of suitable resolutions of singularities: Theorem 4. (Theorem 7.1 of [6]) Let K be a local ﬁeld of characteristic zero. The integral A(N) K (k) converges and is holomorphic in the open set U ⊂C(N−1)(l+1). It extends to a meromorphic function in k on the whole C(N−1)(l+1). 10. Meromorphic Continuation of Koba–Nielsen Amplitudes Deﬁned on Local Fields of Characteristic Zero Hence, Symmetry 2021, 13, 967 49 of 53 Z(4)(s) = Z(4) {2}(s) + Z(4) ∅(s), Z(4) {2}(s) = Z R χ(x2)\|x2\|s12\|1 −x2\|s32dx2 (98) (98) and Z(4) ∅(s) = Z R (1 −χ(x2))\|x2\|s12\|1 −x2\|s32dx2. (99) (99) The integral (98) is a multivariate local zeta function since χ(x2) has compact support. By a classical result of local zeta functions Z(4) {2}(s) converges when Re(s12) > −1 and Re(s32) > −1. Furthermore, it admits a meromorphic continuation to the whole C2, see Theorem 3.2 of [6]. The second integral (99) is not a multivariate local zeta function but it can be trans- formed in one by changing of variables as x2 → 1 x2 , then dx2 → 1 \|x2\|2 dx2, and by setting The second integral (99) is not a multivariate local zeta function but it can be trans- formed in one by changing of variables as x2 → 1 x2 , then dx2 → 1 \|x2\|2 dx2, and by setting eχ(x2) := 1 −χ 1 x2 , we have that eχ(x2) := 1 −χ 1 x2 , we have that eχ(x2) := 1 −χ 1 x2 , we have that eχ(x2) =    1 if \|x2\| ≤ 1 2+ϵ 0 if \|x2\| ≥1 2, . Notice that the support of eχ is contained in h −1 2, 1 2 i and eχ ∈C∞(R). Thus, integral (99) becomes Notice that the support of eχ is contained in h −1 2, 1 2 i and eχ ∈C∞(R). Thus, integral (99) becomes Notice that the support of eχ is contained in h −1 2, 1 2 i and eχ ∈C∞(R). Thus, integral (99) becomes Z(4) ∅(s) = Z R eχ(x2)\|x2\|−s12−s32−2\|1 −x2\|s32dx2, which is analytic when −Re(s12) −Re(s32) −1 > 0 and Re(s32) + 1 > 0. We concluded that Z(4)(s) is analytic in the region which is analytic when −Re(s12) −Re(s32) −1 > 0 and Re(s32) + 1 > 0. We concluded that Z(4)(s) is analytic in the region Re(s12) > −1, Re(s32) > −1, Re(s12) + Re(s32) < −1. Which contains the open set −1 < Re(s12) < −1 2 and −1 < Re(s32) < −1 2. 10.2. Example: 5-Point Koba–Nielsen String Amplitude 10.2. Example: 5-Point Koba–Nielsen String Amplitude Fix N = 5. By using the function χ(x), see (97), we deﬁne the following partition of the unity: p g p Fix N = 5. 10. Meromorphic Continuation of Koba–Nielsen Amplitudes Deﬁned on Local Fields of Characteristic Zero If K is an R-ﬁeld, then the possible poles of A(N) K (k) belong to If K is an R-ﬁeld, then the possible poles of A(N) K (k) belong to [ I⊆{2,...,N−2} [ t∈N [ r∈T(I)   k ∈C(N−1)(l+1); ∑ ij∈M(I) Nij,r(I)kikj + γr(I) + t [K : R] = 0   , (96) (96) where Nij,r(I), γr(I) ∈Z, and M(I), T(I) are ﬁnite sets, and [K : R] = 1 if K = R, and [K : R] = 2 if K = C. If K is a p-adic ﬁeld, then A(N) K (k) is a rational function in the variables q−kikj, and its possible poles belong to where Nij,r(I), γr(I) ∈Z, and M(I), T(I) are ﬁnite sets, and [K : R] = 1 if K = R, and [K : R] = 2 if K = C. If K is a p-adic ﬁeld, then A(N) K (k) is a rational function in the variables q−kikj, and its possible poles belong to [ I⊆{2,...,N−2} [ r∈T(I)   k ∈C(N−1)(l+1); ∑ ij∈M(I) Nij,r(I) Re(kikj) + γr(I) = 0   . More precisely, for each r, either all numbers Nij,r(I) are equal to 0 or 1 and γr(I) > 0, or all numbers Nij,r(I) are equal to 0 or −1 and γr(I) < 0. We now show explicitly the existence of a meromorphic continuation for Z(N) R (s) in the cases N = 4, 5, by using Hironaka’s resolution of singularities theorem. 10.1. Example: 4-Point Koba–Nielsen String Amplitude The 4-point Koba–Nielsen open string amplitude is deﬁned as The 4-point Koba–Nielsen open string amplitude is deﬁned as The 4-point Koba–Nielsen open string amplitude is deﬁned as Z(4)(s) = Z R \|x2\|s12\|1 −x2\|s32dx2. Using the function χ(x2) =    1 if x2 ∈[−2, 2] 0 if x2 ∈(−∞, −2 −ϵ] ∪[2 + ϵ, +∞), (97) (97) where ϵ > 0 is sufﬁciently small, we construct the following partition of the unity: where ϵ > 0 is sufﬁciently small, we construct the following partition of the unity: ϕ{2} : R → R; x2 7→χ(x2) ϕ∅: R → R; x2 7→1 −χ(x2) . Notice that ϕ∅, ϕ{2} ∈C∞(R) and ϕ{2}(x) + ϕ∅(x) ≡1, for x ∈R. Hence, Notice that ϕ∅, ϕ{2} ∈C∞(R) and ϕ{2}(x) + ϕ∅(x) ≡1, for x ∈R. 10. Meromorphic Continuation of Koba–Nielsen Amplitudes Deﬁned on Local Fields of Characteristic Zero By using the function χ(x), see (97), we deﬁne the following partition of the unity: ϕ{2,3} : R2 → R; (x2, x3) 7→χ(x2)χ(x3), ϕ{2} : R2 → R; (x2, x3) 7→χ(x2)(1 −χ(x3)), ϕ{3} : R2 → R; (x2, x3) 7→χ(x3)(1 −χ(x2)), ϕ∅: R2 → R; (x2, x3) 7→(1 −χ(x2))(1 −χ(x3)). Then Z(5)(s) = Z(5) {2,3}(s) + Z(5) {3}(s) + Z(5) {2}(s) + Z(5) ∅(s). We consider the ﬁrst integral Z(5) {2,3}(s) = Z R2 χ(x2)χ(x3)\|x2\|s12\|x3\|s13\|1 −x2\|s42\|1 −x3\|s43\|x2 −x3\|s23dx2dx3. (100) (100) Since ϕ{2,3} has compact support, then integral (100) is a local zeta function. Thus, we use resolution of singularities of the divisor D5 defined by x2x3(1−x2)(1−x3)(x2 −x3) = 0. This arrangement is not locally monomial only at the points (0, 0) and (1, 1). Hence, we pick a partition of the unity, ∑2 i=0 Ωi(x2, x3) = 1, and we write Z(5) {2,3}(s) = 2 ∑ j=0 Z(5) Ωj (s), Z(5) {2,3}(s) = 2 ∑ j=0 Z(5) Ωj (s), 50 of 53 Symmetry 2021, 13, 967 where where Z(5) Ωj (s) = Z R2 Ωj(x2, x3)\|x2\|s12\|x3\|s13\|1 −x2\|s42\|1 −x3\|s43\|x2 −x3\|s23dx2dx3. We assume that Ω0 and Ω1 are smooth functions with support in a small neighborhood of (0, 0) and (1, 1), respectively. So, we only compute the integrals Z(5) Ω0 (s) and Z(5) Ω1 (s). In terms of convergence and holomorphy, around (0, 0), the factor \|1 −x2\|s42\|1 −x3\|s43 b l t d h l d b dd d l ti f ( ) 0 hi h We assume that Ω0 and Ω1 are smooth functions with support in a small neighborhood of (0, 0) and (1, 1), respectively. So, we only compute the integrals Z(5) Ω0 (s) and Z(5) Ω1 (s). We assume that Ω0 and Ω1 are smooth functions with support in a small neighborhood of (0, 0) and (1, 1), respectively. So, we only compute the integrals Z(5) Ω0 (s) and Z(5) Ω1 (s). In terms of convergence and holomorphy, around (0, 0), the factor \|1 −x2\|s42\|1 −x3\|s43 can be neglected, hence we only need an embedded resolution of x2x3(x2 −x3) = 0, which is obtained by a blow-up at the origin. It means to make the changes of variables σ0 : R2 →R2; u2 7→x2 = u2 u3 7→x3 = u2u3. 10. Meromorphic Continuation of Koba–Nielsen Amplitudes Deﬁned on Local Fields of Characteristic Zero Hence integral Z(5) Ω0 (s) becomes Hence integral Z(5) Ω0 (s) becomes Z R2 (Ω0 ◦σ0)(u2, u3)\|u2\|s12+s13+s23+1\|u3\|s13\|1 −u3\|s23g(u, s)du2du3, where g(u, s) is invertible on the support of Ω0 ◦σ0 and can thus be neglected from the point of view of convergence and holomorphy. Hence, by ([6], Lemma 3.1), we obtain the convergence conditions: Re(s12) + Re(s13) + Re(s23) + 2 > 0, Re(s23) + 1 > 0, Re(s13) + 1 > 0. (101) (101) We can take other chart of the blow-up, i.e., the change of variables We can take other chart of the blow-up, i.e., the change of variables σ′ 0 : R2 →R2; u2 7→x2 = u2u3 u3 7→x3 = u3, σ′ 0 : R2 →R2; u2 7→x2 = u2u3 u3 7→x3 = u3, with this change yields the same ﬁrst and second condition and Re(s12) + 1 > 0. (102) (102) By symmetry we can consider any of these changes of variables. Completely similarly, for the convergence of Z(5) Ω1 (s), we need also the new conditions By symmetry we can consider any of these changes of variables. Completely similarly, for the convergence of Z(5) Ω1 (s), we need also the new conditions Re(s42) + Re(s43) + Re(s23) + 2 > 0, Re(s42) + 1 > 0, Re(s43) + 1 > 0. (103) (103) onditions coming from the locally monomial integral Z(5) Ω2 (s) are already included. The conditions coming from the locally monomial integral Z(5) Ω2 (s) are already included. The integral Z(5) {3}(s) is not a multivariate local zeta function, so we take the following change of variables x2 →1 x2 , then we have dx2 → 1 \|x2\|2 dx2, and by setting eχ(x2) := 1−χ 1 x2 , we have that eχ(x2) =    1 if \|x2\| ≤ 1 2+ϵ 0 if \|x2\| ≥1 2, . Then Symmetry 2021, 13, 967 51 of 53 Z(5) {3}(s) = Z R2 eχ(x2)χ(x3)\|x2\|−s12−s42−s23−2\|x3\|s13\|1 −x2\|s42\|1 −x3\|s43 Z(5) {3}(s) = Z R2 eχ(x2)χ(x3)\|x2\|−s12−s42−s23−2\|x3\|s13\|1 −x2\|s42\|1 −x3\|s43 × \|1 x x \|s23dx dx × \|1 −x2x3\|s23dx2dx3. Since x2x3(1 −x2)(1 −x3)(1 −x2x3) is locally monomial in the support of eχ(x2)χ(x3), the only new condition is Since x2x3(1 −x2)(1 −x3)(1 −x2x3) is locally monomial in the support of eχ(x2)χ(x3), the only new condition is −Re(s12) −Re(s42) −Re(s23) −1 > 0. 10. Meromorphic Continuation of Koba–Nielsen Amplitudes Deﬁned on Local Fields of Characteristic Zero (104) (104) Completely analogously, Z(5) {2}(s) induces the extra condition Completely analogously, Z(5) {2}(s) induces the extra condition −Re(s13) −Re(s43) −Re(s23) −1 > 0. (105) (105) For the last integral, we are setting eχ(x2) := 1 −χ 1 x2 and eχ(x3) := 1 −χ 1 x3 . Thus For the last integral, we are setting eχ(x2) := 1 −χ 1 x2 and eχ(x3) := 1 −χ 1 x3 . Thus, Z(5) ∅(s) = Z R2 eχ(x2)eχ(x3)\|x2\|−s12−s42−s23−2\|x3\|−s13−s43−s23−2\|1 −x2\|s42\|1 −x3\|s43 Z(5) ∅(s) = Z R2 eχ(x2)eχ(x3)\|x2\|−s12−s42−s23−2\|x3\|−s13−s43−s23−2\|1 −x2\|s42\|1 −x3\|s43 × \|x2 −x3\|s23dx2dx3. In this case, we have the same divisor as for Z(5) {2,3}(s); the differences are the powers of \|x2\| and \|x3\| and the function eχ(x2)eχ(x3) that does not contain (1, 1) in its support. Hence, the only new condition will arise from the blow-up at the origin, namely −Re(s12) −Re(s42) −Re(s23) −Re(s13) −Re(s43) −2 > 0. (106) (106) Hence Z(5)(s) is analytic in the region deﬁned by conditions (101)–(106), i.e., Re(sij) > −1, for all ij Re(s12) + Re(s13) + Re(s23) > −2, Re(s42) + Re(s43) + Re(s23) > −2, Re(s12) + Re(s42) + Re(s23) < −1, Re(s13) + Re(s43) + Re(s23) < −1, ∑Re(sij) < −2. This region of convergence contains the open subset deﬁned by This region of convergence contains the open subset deﬁned by −2 3 < Re(sij) < −2 5 for all ij. Then, in particular, Z(5)(s) is analytic in the interval −2 3 < Re(s) < −2 5. Author Contributions: Writing—review and editing: M.B.-G., H.G.-C., E.Y.L., W.A.Z.-G. All the authors contributed to the manuscript equally. All authors have read and agreed to the published version of the manuscript. Funding: The ﬁrst author was partially supported by the University of Guadalajara. The third author was supported by a Conacyt fellowship. The fourth author was partially supported by Conacyt Grant No. 250845 (Mexico) and by the Debnath Endowed Professorship (UTRGV, USA). 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https://openalex.org/W2984262216	https://kclpure.kcl.ac.uk/portal/files/118888569/Ethnic_differences_in_the_BELLO_Accepted7November2019_GREEN_AAM.pdf	English	null	Differences in the link between insulin sensitivity and ectopic fat in men of Black African and White European ethnicity	European journal of endocrinology	2,020	cc-by	14,938	Citation for published version (APA): Bello, O., Ladwa, M., Hakim, O., Marathe, C., Shojaee-Moradie, F., Charles-Edwards, G., Peacock, J. L., Margot Umpleby, A., Amiel, S. A., & Goff, L. M. (2020). Differences in the link between insulin sensitivity and ectopic fat in men of Black African and White European ethnicity. European Journal of Endocrinology, 182(1), 91-101. Advance online publication. https://doi.org/10.1530/EJE-19-0636 Citing this paper Pl h C t g t s pape Please note that where the full-text provided on King's Research Portal is the Author Accepted Manuscript or Post-Print version this may differ from the final Published version. 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Download date: 24. Oct. 2024 Ethnic differences in the link between insulin sensitivity and 1 ectopic fat in black and white men 2 Short title: Ethnicity, insulin sensitivity and ectopic fat 3 Oluwatoyosi Bello1, Meera Ladwa1, Olah Hakim1, Chinmay Marathe1, Fariba Shojaee- 4 Moradie2, Geoff Charles-Edwards3,4, Janet L. Peacock5, A. Margot Umpleby2, Stephanie A. 5 Amiel1 and Louise M. Goff1 6 7 1. Department of Diabetes, School of Life Course Sciences, Faculty of Life Sciences & 8 Medicine, King’s College London, London, UK 9 2. Faculty of Health and Medical Sciences, University of Surrey, Guildford, UK 10 3. Medical Physics, Guy’s & St Thomas’ NHS Foundation Trust, London, UK 11 4. School of Biomedical Engineering & Imaging Sciences, King’s College London, London, 12 UK 13 5. School of Population Health and Environmental Sciences, King’s College London, London, 14 UK 15 16 Corresponding author: 17 Louise M. General rights Goff, Department of Diabetes, School of Life Course Sciences, Faculty of Life 18 Sciences & Medicine, King’s College London, Franklin-Wilkins Building, Waterloo Campus, 19 London, SE1 9NH, UK 20 Email - louise.goff@kcl.ac.uk 21 Phone - 0207 848 4273 22 Louise M. Goff ORCID ID: 0000-0001-9633-8759 23 Oluwatoyosi Bello ORCID ID: 0000-0003-3850-0425 24 25 Word count (excluding title page, abstract, references, figures and tables): 4510 26 References: 58 27 Short title: Ethnicity, insulin sensitivity and ectopic fat 3 1 ABBREVIATIONS 28 ATIS – Adipose tissue insulin sensitivity 29 BAM – Black (west) African men 30 WEM – White European men 31 Rd – Rate of disappearance 32 Ra – Rate of appearance 33 VAT – Visceral adipose tissue 34 SAT – Subcutaneous adipose tissue 35 IMCL – Intramyocellular lipids 36 IHL – Intrahepatic lipids 37 MRI – Magnetic resonance imaging 38 MRS – Magnetic resonance spectroscopy 39 HISI – Hepatic insulin sensitivity index 40 PISI – Peripheral insulin sensitivity index 41 BMI – Body mass index 42 T2D – Type 2 diabetes 43 ABBREVIATIONS 28 2 2 ABSTRACT 44 In black populations, T2D is more likely to occur within 71 the normal body mass index (BMI) range (19.5-24.9 kg/m2) (3) and at a lower waist 72 circumference (4) compared to white groups. 73 74 Insulin resistance for carbohydrate metabolism is a well-established early defect in the 75 pathogenesis of T2D (5). Resistance to the antilipolytic effect of insulin in adipose tissue has 76 also been identified as an early defect, occurring prior to the onset of hyperglycaemia (6). 77 Adipose tissue insulin resistance results in increased fatty acid release, with deposition in non- 78 adipose tissue sites as ectopic fat (7, 8). This is known to trigger and exacerbate insulin 79 resistance (9). Several theories have been proposed to explain the development of ectopic fat 80 and insulin resistance. The “spillover theory” proposes that multiple dysfunctions of 81 subcutaneous adipose tissue (SAT), including insulin resistance (10), allow fatty acids to be 82 deposited as visceral adipose tissue (VAT) (7, 11). Dysfunctional SAT combined with highly 83 lipolytic VAT leads to the release of fatty acids into the portal and peripheral circulations. The 84 “portal theory” proposes that delivery of fatty acids from VAT to the liver, via the portal 85 circulation, results in accumulation of intrahepatic lipid (IHL), which subsequently leads to the 86 development of hepatic insulin resistance (12-14). Fatty acids entering the peripheral 87 circulation are understood to lead to fat deposition within skeletal muscle cells (termed 88 intramyocellular lipids, IMCL) (13, 15). Whilst there is compelling evidence linking IMCL 89 with peripheral insulin resistance (16, 17), there is debate in this field due to observations that 90 ABSTRACT 44 Objectives: In men of black west African (BAM) and white European (WEM) ethnicity, we 45 aimed to 1) compare adipose tissue, peripheral and hepatic insulin sensitivity, and 2) investigate 46 associations between ectopic fat and insulin sensitivity by ethnicity. 47 Objectives: In men of black west African (BAM) and white European (WEM) ethnicity, we 45 aimed to 1) compare adipose tissue, peripheral and hepatic insulin sensitivity, and 2) investigate 46 associations between ectopic fat and insulin sensitivity by ethnicity. 47 Design and Methods: In overweight BAM (n=21) and WEM (n=23) with normal glucose 48 tolerance, we performed a two-step hyperinsulinaemic–euglycaemic clamp with infusion of 49 [6,6 2H2]-glucose and [2H5]-glycerol to measure whole body, peripheral, hepatic and adipose 50 tissue insulin sensitivity (lipolysis). Visceral adipose tissue (VAT), intrahepatic lipids (IHL) 51 and intramyocellular (IMCL) lipids were measured using magnetic resonance imaging and 52 spectroscopy. Associations between insulin sensitivity and ectopic fat were assessed using 53 Pearson’s correlation coefficient by ethnicity and regression analysis. 54 Results: There were no ethnic differences in whole body or tissue-specific insulin sensitivity 55 (all p>0.05). Suppression of lipolysis was inversely associated with VAT and IHL in WEM but 56 not BAM (VAT: WEM r=-0.68, p<0.01; BAM r=0.07, p=0.79. IHL: WEM r=-0.52, p=0.01; 57 BAM r=-0.12, p=0.63). IMCL was inversely associated with skeletal muscle insulin sensitivity 58 in WEM but not BAM (WEM r=-0.56,p<0.01; BAM r=-0.09, p=0.75) and IHL was inversely 59 associated with hepatic insulin sensitivity in WEM but not BAM (WEM r=-0.53, p=0.02; BAM 60 r=-0.13, p=0.62). 61 Conclusions: Ectopic fat deposition may play a lesser role in reducing insulin sensitivity in 62 men of black African ethnicity, and may not be driven by lipolysis. Resistance to storing VAT, 63 IHL and IMCL may enable men of black African ethnicity to maintain comparable insulin 64 sensitivity to white Europeans. 65 Conclusions: Ectopic fat deposition may play a lesser role in reducing insulin sensitivity in 62 men of black African ethnicity, and may not be driven by lipolysis. Resistance to storing VAT, 63 IHL and IMCL may enable men of black African ethnicity to maintain comparable insulin 64 sensitivity to white Europeans. 65 Keywords: Black African, Hepatic insulin sensitivity, Intrahepatic lipid, Intramyocellular 67 lipid, Lipolysis, Skeletal muscle insulin sensitivity 68 3 INTRODUCTION 69 The risk of developing type 2 diabetes (T2D) is disproportionately high in populations of black 70 compared to white ethnicity (1, 2). INTRODUCTION 69 105 Compared to populations of white ethnicity, black populations are reported to exhibit lower 93 ectopic fat (namely VAT and IHL) (19-23), yet large cohort studies indicate that they display 94 pronounced insulin resistance (24), creating a paradox. Studies using more sensitive measures 95 of insulin resistance at a tissue specific level, alongside measurement of ectopic fat depots 96 related to T2D are scarce in non-diabetic black populations and have been restricted to obese 97 women (25). These have reported no ethnic differences in peripheral insulin sensitivity, but 98 lower hepatic insulin sensitivity in white compared to black women. Furthermore, they report 99 that peripheral insulin sensitivity does not associate with either VAT or IMCL in black women 100 but hepatic sensitivity does associate with VAT and IHL, suggesting that VAT and IHL play a 101 key role in hepatic insulin resistance in black women (25). Similarly, in vivo studies assessing 102 adipose tissue insulin sensitivity are mainly confined to obese women. They have provided 103 inconsistent results, showing either no difference (26-28) or reduced lipolysis (29-31) in black 104 compared to white populations. 105 106 While studies in women provide persuasive evidence of ethnic distinctions in the 107 pathophysiology of T2D, gender differences in physiology (greater hyperinsulinaemia and 108 insulin resistance in women (32, 33)) and body composition (less VAT and more SAT in 109 women (34)) suggest investigations in black men are required. We aimed to assess and compare 110 whole body, skeletal muscle, hepatic and adipose tissue insulin sensitivity in normally glucose 111 tolerant black west African (BAM) and white European men (WEM), and to evaluate 112 relationships between tissue specific insulin sensitivity with VAT, IHL and IMCL, to explore 113 ethnic distinctions in the pathophysiology of type 2 diabetes. 114 While studies in women provide persuasive evidence of ethnic distinctions in the 107 pathophysiology of T2D, gender differences in physiology (greater hyperinsulinaemia and 108 insulin resistance in women (32, 33)) and body composition (less VAT and more SAT in 109 women (34)) suggest investigations in black men are required. INTRODUCTION 69 These have reported no ethnic differences in peripheral insulin sensitivity, but 98 lower hepatic insulin sensitivity in white compared to black women. Furthermore, they report 99 that peripheral insulin sensitivity does not associate with either VAT or IMCL in black women 100 but hepatic sensitivity does associate with VAT and IHL, suggesting that VAT and IHL play a 101 key role in hepatic insulin resistance in black women (25). Similarly, in vivo studies assessing 102 adipose tissue insulin sensitivity are mainly confined to obese women. They have provided 103 inconsistent results, showing either no difference (26-28) or reduced lipolysis (29-31) in black 104 compared to white populations. 105 106 While studies in women provide persuasive evidence of ethnic distinctions in the 107 pathophysiology of T2D, gender differences in physiology (greater hyperinsulinaemia and 108 insulin resistance in women (32, 33)) and body composition (less VAT and more SAT in 109 women (34)) suggest investigations in black men are required. We aimed to assess and compare 110 whole body, skeletal muscle, hepatic and adipose tissue insulin sensitivity in normally glucose 111 tolerant black west African (BAM) and white European men (WEM), and to evaluate 112 relationships between tissue specific insulin sensitivity with VAT, IHL and IMCL, to explore 113 ethnic distinctions in the pathophysiology of type 2 diabetes. 114 Compared to populations of white ethnicity, black populations are reported to exhibit lower 93 ectopic fat (namely VAT and IHL) (19-23), yet large cohort studies indicate that they display 94 pronounced insulin resistance (24), creating a paradox. Studies using more sensitive measures 95 of insulin resistance at a tissue specific level, alongside measurement of ectopic fat depots 96 related to T2D are scarce in non-diabetic black populations and have been restricted to obese 97 women (25). These have reported no ethnic differences in peripheral insulin sensitivity, but 98 lower hepatic insulin sensitivity in white compared to black women. Furthermore, they report 99 that peripheral insulin sensitivity does not associate with either VAT or IMCL in black women 100 but hepatic sensitivity does associate with VAT and IHL, suggesting that VAT and IHL play a 101 key role in hepatic insulin resistance in black women (25). Similarly, in vivo studies assessing 102 adipose tissue insulin sensitivity are mainly confined to obese women. They have provided 103 inconsistent results, showing either no difference (26-28) or reduced lipolysis (29-31) in black 104 compared to white populations. INTRODUCTION 69 The risk of developing type 2 diabetes (T2D) is disproportionately high in populations of black 70 compared to white ethnicity (1, 2). In black populations, T2D is more likely to occur within 71 the normal body mass index (BMI) range (19.5-24.9 kg/m2) (3) and at a lower waist 72 circumference (4) compared to white groups. 73 Insulin resistance for carbohydrate metabolism is a well-established early defect in the 75 pathogenesis of T2D (5). Resistance to the antilipolytic effect of insulin in adipose tissue has 76 also been identified as an early defect, occurring prior to the onset of hyperglycaemia (6). 77 Adipose tissue insulin resistance results in increased fatty acid release, with deposition in non- 78 adipose tissue sites as ectopic fat (7, 8). This is known to trigger and exacerbate insulin 79 resistance (9). Several theories have been proposed to explain the development of ectopic fat 80 and insulin resistance. The “spillover theory” proposes that multiple dysfunctions of 81 subcutaneous adipose tissue (SAT), including insulin resistance (10), allow fatty acids to be 82 deposited as visceral adipose tissue (VAT) (7, 11). Dysfunctional SAT combined with highly 83 lipolytic VAT leads to the release of fatty acids into the portal and peripheral circulations. The 84 “portal theory” proposes that delivery of fatty acids from VAT to the liver, via the portal 85 circulation, results in accumulation of intrahepatic lipid (IHL), which subsequently leads to the 86 development of hepatic insulin resistance (12-14). Fatty acids entering the peripheral 87 circulation are understood to lead to fat deposition within skeletal muscle cells (termed 88 intramyocellular lipids, IMCL) (13, 15). Whilst there is compelling evidence linking IMCL 89 with peripheral insulin resistance (16, 17), there is debate in this field due to observations that 90 athletes, who are highly insulin sensitive, present with relatively high IMCL levels (18). 91 92 4 4 Compared to populations of white ethnicity, black populations are reported to exhibit lower 93 ectopic fat (namely VAT and IHL) (19-23), yet large cohort studies indicate that they display 94 pronounced insulin resistance (24), creating a paradox. Studies using more sensitive measures 95 of insulin resistance at a tissue specific level, alongside measurement of ectopic fat depots 96 related to T2D are scarce in non-diabetic black populations and have been restricted to obese 97 women (25). INTRODUCTION 69 We aimed to assess and compare 110 whole body, skeletal muscle, hepatic and adipose tissue insulin sensitivity in normally glucose 111 tolerant black west African (BAM) and white European men (WEM), and to evaluate 112 relationships between tissue specific insulin sensitivity with VAT, IHL and IMCL, to explore 113 ethnic distinctions in the pathophysiology of type 2 diabetes. 114 5 5 MATERIALS AND METHODS 115 The participants included in this analysis were recruited as part of the South London Diabetes 117 and Ethnicity Phenotyping (Soul-Deep) study, phase II (35). The aim of the Soul-Deep II study 118 was to investigate ethnic differences in insulin sensitivity, beta-cell function and ectopic fat 119 deposition in men of Black (west) African (BAM) and white European (WEM) ethnicity. The 120 study was approved by the London Bridge National Research Ethics Committee (15/LO/1121). 121 Data collection took place between April 2016 and May 2018. Participants were recruited from 122 local GP practices, newspaper advertisements, King’s College London university staff and 123 student email, religious groups, leafleting and posters where permitted. All participants 124 provided informed consent prior to any study procedures. 125 Non-diabetic Black (west) African (BAM) and white European men (WEM) aged 18-65 years, 126 with a BMI between 20-40 kg/m2 were eligible to take part; the aim of recruitment was to 127 achieve comparable BMI and age between the ethnic groups, without targeting a specific 128 weight status. Ethnicity was defined by self-reported parental and grandparental birthplace; 129 normal glucose tolerance was confirmed by a 2-hour plasma glucose <7.8mmol/l following a 130 75g oral glucose tolerance test at screening. Participants were excluded if they were being 131 treated with any medications known to affect the study outcomes, suffering from kidney or 132 liver damage (serum creatinine >150 μmol/l or serum alanine transaminase level >2.5-fold 133 above the upper limit of the reference range), or were unwilling and/or unable to comply with 134 the study protocol. 135 Prior to each visit, participants were required not to eat after their usual carbohydrate- 136 containing evening meal (no less than 10 hours prior to study), refrain from strenuous physical 137 activity for 48 hours and alcohol for 24 hours and avoid smoking on the morning of their visit. 138 Hyperinsulinaemic–euglycaemic clamp 139 6 A two-step hyperinsulinaemic–euglycaemic clamp with a stable glucose and glycerol isotope 140 infusion was used to assess whole body and tissue specific insulin sensitivity. Upon arrival at 141 the clinical research facility in King’s College Hospital, participants were advised to empty 142 their bladder and were weighed on a body composition analyser (Tanita MC780MA) to 143 determine fat free mass and body weight for infusion calculations. MATERIALS AND METHODS 115 A cannula for blood 144 sampling was placed in the dorsum of one hand in a retrograde fashion, the hand was kept in a 145 warming unit at 55º to mimic arterialised sampling. Duplicate baseline samples were taken to 146 assess background glucose and glycerol isotopic enrichments. An infusion cannula was then 147 inserted into an antecubital fossa vein on the adjacent arm for infusions of 20% (wt/vol) 148 glucose, insulin (Actrapid, Novo Nordisk, Bagsvaerd, Denmark) bound to albumin in a 4% 149 autologous blood/saline solution, [6,6-2H2]-glucose and [2H5]-glycerol tracers (CK Gases, 150 Cambridgeshire, UK). To begin the basal phase, a primed (2.0 mg/kg), continuous infusion 151 (0.02 mg/kg−1 min−1) of [6,6-2H2]-glucose and a primed (0.12 mg/kg), continuous infusion 152 (0.0067 mg/kg−1 min−1) of [2H5]-glycerol were initiated at -120 minutes (36). Blood samples 153 were taken at -30, -20, -10 and 0 minutes for basal assessments. The clamp began at 0 minutes 154 with a primed continuous insulin infusion at a rate of 10 mU m−2 BSA min−1 (low dose insulin 155 phase) for 2 hours for assessment of adipose tissue and hepatic insulin sensitivity. For the final 156 2 hours, the [2H5]-glycerol isotope infusion was terminated, the insulin infusion rate was re- 157 primed and increased to 40 mU m−2 BSA min−1 (the high dose insulin phase) for assessment of 158 whole body and peripheral (skeletal muscle) insulin sensitivity (37, 38). Euglycaemia (5 159 mmol/l) was achieved using 20% glucose enriched with [6,6-2H2]-glucose (8 mg/g glucose 160 with low-dose insulin and 10 mg/g with high-dose insulin) to maintain a constant tracer-to- 161 tracee ratio. The glucose was given at variable rates, based on plasma glucose samples drawn 162 every 5 minutes and measured on a bedside glucose analyser (Yellow Spring Instruments, 2300 163 STAT Glucose Analyzer, Yellow Springs, OH, USA). Blood was drawn at 30, 60, 90, 100, 164 A two-step hyperinsulinaemic–euglycaemic clamp with a stable glucose and glycerol isotope 140 infusion was used to assess whole body and tissue specific insulin sensitivity. Upon arrival at 141 the clinical research facility in King’s College Hospital, participants were advised to empty 142 their bladder and were weighed on a body composition analyser (Tanita MC780MA) to 143 determine fat free mass and body weight for infusion calculations. MATERIALS AND METHODS 115 A cannula for blood 144 sampling was placed in the dorsum of one hand in a retrograde fashion, the hand was kept in a 145 warming unit at 55º to mimic arterialised sampling. Duplicate baseline samples were taken to 146 assess background glucose and glycerol isotopic enrichments. An infusion cannula was then 147 inserted into an antecubital fossa vein on the adjacent arm for infusions of 20% (wt/vol) 148 glucose, insulin (Actrapid, Novo Nordisk, Bagsvaerd, Denmark) bound to albumin in a 4% 149 autologous blood/saline solution, [6,6-2H2]-glucose and [2H5]-glycerol tracers (CK Gases, 150 Cambridgeshire, UK). To begin the basal phase, a primed (2.0 mg/kg), continuous infusion 151 (0.02 mg/kg−1 min−1) of [6,6-2H2]-glucose and a primed (0.12 mg/kg), continuous infusion 152 (0.0067 mg/kg−1 min−1) of [2H5]-glycerol were initiated at -120 minutes (36). Blood samples 153 were taken at -30, -20, -10 and 0 minutes for basal assessments. The clamp began at 0 minutes 154 with a primed continuous insulin infusion at a rate of 10 mU m−2 BSA min−1 (low dose insulin 155 phase) for 2 hours for assessment of adipose tissue and hepatic insulin sensitivity. For the final 156 2 hours, the [2H5]-glycerol isotope infusion was terminated, the insulin infusion rate was re- 157 primed and increased to 40 mU m−2 BSA min−1 (the high dose insulin phase) for assessment of 158 whole body and peripheral (skeletal muscle) insulin sensitivity (37, 38). Euglycaemia (5 159 mmol/l) was achieved using 20% glucose enriched with [6,6-2H2]-glucose (8 mg/g glucose 160 with low-dose insulin and 10 mg/g with high-dose insulin) to maintain a constant tracer-to- 161 tracee ratio. The glucose was given at variable rates, based on plasma glucose samples drawn 162 7 110, 120, 150, 180, 210, 220, 230 and 240 minutes for the assessment of plasma glucose 165 concentration and isotopic enrichment and insulin concentration (38). 166 Magnetic resonance imaging and spectroscopy for ectopic fat quantification Plasma insulin concentration was measured by 191 immunoassay using chemiluminescent technology (ADVIA Centaur System, Siemens 192 Healthcare, Camberley, UK). 193 Calculations 194 Whole body insulin sensitivity was quantified using the M value (mg/kg FFM min-1) measured 195 during the final 30 minutes of the high dose insulin phase of the clamp. It is calculated as total 196 glucose disposal corrected for deviations in plasma glucose concentration. The M value was 197 divided by mean plasma insulin concentration during the high dose insulin phase, giving M/I 198 (mg kg FFM min-1) / (pmol/l) as another assessment of whole body insulin sensitivity (37). 199 Steele’s non-steady-state equations modified for stable isotopes (43) were used to determine 200 peripheral glucose utilisation (glucose rate of disappearance, Rd (μmol /kg FFM min-1)), 201 endogenous glucose production (glucose rate of appearance, Ra (μmol /kg FFM min-1)) and 202 whole body lipolysis (glycerol Ra (μmol /kg FFM min-1) ) at basal and during the different 203 phases of the clamp. Glucose kinetic calculations were modified to incorporate the [6,6-2H2]- 204 glucose isotope enriched 20% glucose (44). Optical segment analysis was used to smooth the 205 glucose and glycerol enrichment concentrations over the clamp time course (45). 206 Peripheral insulin sensitivity was determined as the percentage increase in the rate of glucose 207 disappearance from basal to the final 30 minutes of the high dose insulin phase. The peripheral 208 i li i i i i d ( S ) l l l d h l d ( l /k i 1) / 209 Whole body insulin sensitivity was quantified using the M value (mg/kg FFM min-1) measured 195 during the final 30 minutes of the high dose insulin phase of the clamp. It is calculated as total 196 glucose disposal corrected for deviations in plasma glucose concentration. The M value was 197 divided by mean plasma insulin concentration during the high dose insulin phase, giving M/I 198 (mg kg FFM min-1) / (pmol/l) as another assessment of whole body insulin sensitivity (37). 199 Steele’s non-steady-state equations modified for stable isotopes (43) were used to determine 200 peripheral glucose utilisation (glucose rate of disappearance, Rd (μmol /kg FFM min-1)), 201 endogenous glucose production (glucose rate of appearance, Ra (μmol /kg FFM min-1)) and 202 whole body lipolysis (glycerol Ra (μmol /kg FFM min-1) ) at basal and during the different 203 phases of the clamp. Magnetic resonance imaging and spectroscopy for ectopic fat quantification Magnetic resonance imaging (MRI) was used assess visceral adipose tissue (VAT) and 168 intrahepatic lipids (IHL). Proton-magnetic resonance spectroscopy (1H-MRS) was used to 169 assess intramyocellular lipids (IMCL). Full details of this methodology can be found in (39). 170 In brief, participants arrived at the MRI unit of Guy’s Hospital, London following an overnight 171 fast. Participants were scanned in a 1.5T Siemens Aera scanner, axial 2-point Dixon MRI 172 images were acquired from the abdomen, from which fat and water images were produced. 173 Images were analysed using imaging software (HOROS V 1.1.7; www.horosproject.org; 174 accessed 21/10/2017) to quantify VAT and IHL. VAT area was assessed using a single slice at 175 the L4-5 spinal anatomical position. IHL was quantified using 2 abdominal MRI images 30 176 mm apart encompassing both the superior and inferior view of the liver. A 4-circular region of 177 interest analysis was conducted to determine the hepatic fat fraction (%) in each region. IHL 178 was calculated as the mean of all 8 regions of interest. Quantification of IMCL in the soleus 179 muscle of the right leg was derived from a 1H-MRS scan on a 1.5T Siemens system with an 180 extremity RF coil to obtain muscle images. From these images two localised proton spectra 181 were obtained, a water-suppressed lipid spectra and a lipid-suppressed water spectra. The Java- 182 Based Magnetic Resonance User Interface software was used to identify and quantify the 183 IMCL peaks expressed in arbitrary units (40). 184 Plasma glucose and glycerol isotope enrichments were measured by gas chromatography-mass 186 spectrometry on an Agilent GCMS 5975C MSD (Agilent Technologies, Wokingham, UK) 187 using selected ion monitoring to determine the tracer-to-tracee ratio. The isotopic enrichment 188 of glucose was determined as the penta-O-trimethylsilyl-D-glucose-O-methoxime derivative 189 8 8 (41). The isotopic enrichment of plasma glycerol was determined as the tert-butyl trimethylsilyl 190 (tBDMS) glycerol derivative (42). Plasma insulin concentration was measured by 191 immunoassay using chemiluminescent technology (ADVIA Centaur System, Siemens 192 Healthcare, Camberley, UK). 193 (41). The isotopic enrichment of plasma glycerol was determined as the tert-butyl trimethylsilyl 190 (tBDMS) glycerol derivative (42). Plasma insulin concentration was measured by 191 immunoassay using chemiluminescent technology (ADVIA Centaur System, Siemens 192 Healthcare, Camberley, UK). 193 (41). The isotopic enrichment of plasma glycerol was determined as the tert butyl trimethylsilyl 190 (tBDMS) glycerol derivative (42). Magnetic resonance imaging and spectroscopy for ectopic fat quantification Hepatic insulin sensitivity was also quantified during the 217 basal and low dose insulin phase using the hepatic insulin sensitivity index (HISI), which is the 218 reciprocal of the product of endogenous glucose production (glucose Ra) and mean plasma 219 insulin (46). 220 Adipose tissue insulin sensitivity was measured as the percentage suppression of whole body 221 lipolysis (glycerol Ra) from basal to the final 30 minutes of the low dose insulin phase (47). 222 Adipose tissue insulin sensitivity was also quantified during the basal and low dose insulin 223 phase using the adipose tissue insulin sensitivity index (ATIS) which is the reciprocal of the 224 product of whole body lipolysis (glycerol Ra) and basal plasma insulin (46). 225 Statistical analysis 226 The Soul-Deep II study was powered on a primary outcome of insulin secretory function (48). 227 Allowing a difference of one standard deviation to be detected with 90% power and 2-sided 228 significance, we aimed to recruit 23 per group, allowing for 20 per group to complete the 229 protocol. 230 Data were assessed for normality using the Shapiro-Wilks test and histograms. A log 10 231 transformation was performed where data were skewed. Data which were normally distributed 232 are presented as mean (SD), data which required log 10 transformation are presented as 233 geometric mean (95% CI), data which remained skewed after log transformation are presented 234 as median (lower-upper IQR). Ethnic comparisons of insulin sensitivity were assessed using 235 the independent samples t-test for normally distributed data and Mann-Whitney test for non- 236 normally distributed data. The mean difference (95% CI) and ratio of the geometric mean (95% 237 CI) are presented where appropriate. Adjustment of the insulin sensitivity measures for VAT 238 Adipose tissue insulin sensitivity was measured as the percentage suppression of whole body 221 lipolysis (glycerol Ra) from basal to the final 30 minutes of the low dose insulin phase (47). 222 Adipose tissue insulin sensitivity was also quantified during the basal and low dose insulin 223 phase using the adipose tissue insulin sensitivity index (ATIS) which is the reciprocal of the 224 product of whole body lipolysis (glycerol Ra) and basal plasma insulin (46). 225 Adipose tissue insulin sensitivity was measured as the percentage suppression of whole body 221 lipolysis (glycerol Ra) from basal to the final 30 minutes of the low dose insulin phase (47). Magnetic resonance imaging and spectroscopy for ectopic fat quantification 222 Adipose tissue insulin sensitivity was also quantified during the basal and low dose insulin 223 phase using the adipose tissue insulin sensitivity index (ATIS) which is the reciprocal of the 224 product of whole body lipolysis (glycerol Ra) and basal plasma insulin (46). 225 The Soul-Deep II study was powered on a primary outcome of insulin secretory function (48). 227 Allowing a difference of one standard deviation to be detected with 90% power and 2-sided 228 significance, we aimed to recruit 23 per group, allowing for 20 per group to complete the 229 protocol. 230 The Soul-Deep II study was powered on a primary outcome of insulin secretory function (48). 227 Allowing a difference of one standard deviation to be detected with 90% power and 2-sided 228 significance, we aimed to recruit 23 per group, allowing for 20 per group to complete the 229 protocol. 230 The Soul-Deep II study was powered on a primary outcome of insulin secretory function (48). 227 Allowing a difference of one standard deviation to be detected with 90% power and 2-sided 228 significance, we aimed to recruit 23 per group, allowing for 20 per group to complete the 229 protocol. 230 Data were assessed for normality using the Shapiro-Wilks test and histograms. A log 10 231 transformation was performed where data were skewed. Data which were normally distributed 232 are presented as mean (SD), data which required log 10 transformation are presented as 233 geometric mean (95% CI), data which remained skewed after log transformation are presented 234 as median (lower-upper IQR). Ethnic comparisons of insulin sensitivity were assessed using 235 the independent samples t-test for normally distributed data and Mann-Whitney test for non- 236 normally distributed data. The mean difference (95% CI) and ratio of the geometric mean (95% 237 CI) are presented where appropriate. Adjustment of the insulin sensitivity measures for VAT 238 Data were assessed for normality using the Shapiro-Wilks test and histograms. A log 10 231 transformation was performed where data were skewed. Data which were normally distributed 232 are presented as mean (SD), data which required log 10 transformation are presented as 233 geometric mean (95% CI), data which remained skewed after log transformation are presented 234 as median (lower-upper IQR). Ethnic comparisons of insulin sensitivity were assessed using 235 the independent samples t-test for normally distributed data and Mann-Whitney test for non- 236 normally distributed data. Magnetic resonance imaging and spectroscopy for ectopic fat quantification Glucose kinetic calculations were modified to incorporate the [6,6-2H2]- 204 glucose isotope enriched 20% glucose (44). Optical segment analysis was used to smooth the 205 glucose and glycerol enrichment concentrations over the clamp time course (45). 206 Peripheral insulin sensitivity was determined as the percentage increase in the rate of glucose 207 disappearance from basal to the final 30 minutes of the high dose insulin phase. The peripheral 208 insulin sensitivity index (PISI) was also calculated as the glucose Rd (μmol /kg FFM min-1) / 209 mean plasma insulin concentration (pmol/l) during the final 30 minutes of the high dose insulin 210 phase (46). Peripheral insulin sensitivity is predominantly driven by skeletal muscle glucose 211 uptake but also captures adipose tissue glucose uptake hence, we have used PISI as an 212 assessment of skeletal muscle insulin sensitivity. 213 9 9 Endogenous glucose production (glucose Ra) was calculated by subtracting the exogenous 214 glucose infusion rate from total glucose Ra. Hepatic insulin sensitivity was measured as the 215 percentage suppression of endogenous glucose production from basal to the final 30 minutes 216 of the low dose insulin phase (47). Hepatic insulin sensitivity was also quantified during the 217 basal and low dose insulin phase using the hepatic insulin sensitivity index (HISI), which is the 218 reciprocal of the product of endogenous glucose production (glucose Ra) and mean plasma 219 insulin (46). 220 Endogenous glucose production (glucose Ra) was calculated by subtracting the exogenous 214 glucose infusion rate from total glucose Ra. Hepatic insulin sensitivity was measured as the 215 percentage suppression of endogenous glucose production from basal to the final 30 minutes 216 of the low dose insulin phase (47). Hepatic insulin sensitivity was also quantified during the 217 basal and low dose insulin phase using the hepatic insulin sensitivity index (HISI), which is the 218 reciprocal of the product of endogenous glucose production (glucose Ra) and mean plasma 219 insulin (46). 220 Endogenous glucose production (glucose Ra) was calculated by subtracting the exogenous 214 glucose infusion rate from total glucose Ra. Hepatic insulin sensitivity was measured as the 215 percentage suppression of endogenous glucose production from basal to the final 30 minutes 216 of the low dose insulin phase (47). and IHL were made using linear multiple regression. Pearson’s correlation analyses were used 239 to assess the associations between insulin sensitivity measures and ectopic fat. Interaction by 240 ethnicity was assessed using a linear multiple regression with ethnicityectopic fat depot used 241 as the interaction term. Statistical significance was defined as p <0.05 and data analyses were 242 performed using SPSS software, version 25 (IBM Analytics, Armonk, NY, USA). 243 Magnetic resonance imaging and spectroscopy for ectopic fat quantification The mean difference (95% CI) and ratio of the geometric mean (95% 237 CI) are presented where appropriate. Adjustment of the insulin sensitivity measures for VAT 238 10 and IHL were made using linear multiple regression. Pearson’s correlation analyses were used 239 to assess the associations between insulin sensitivity measures and ectopic fat. Interaction by 240 ethnicity was assessed using a linear multiple regression with ethnicityectopic fat depot used 241 as the interaction term. Statistical significance was defined as p <0.05 and data analyses were 242 performed using SPSS software, version 25 (IBM Analytics, Armonk, NY, USA). 243 11 RESULTS 244 Participant characteristics 245 The participant characteristics of the 21 BAM and 23 WEM are displayed in Table 1; by design 246 the groups were comparable in age and BMI. There were no differences in body fat, waist 247 circumference, blood pressure, HbA1c, cholesterol, fasting and post-load glucose between 248 ethnicities; however, BAM exhibited lower fasting triglycerides compared to WEM. Data on 249 ectopic fat depots in these volunteers, as previously reported by our group (39), showed lower 250 VAT and IHL in BAM but similar IMCL (included in Table 1 for reference). 251 Whole body insulin sensitivity 252 The glucose and insulin profiles during the hyperinsulinaemic–euglycaemic clamp are shown 253 in Supplementary Figure 1. BAM exhibited a trend towards greater mean plasma insulin during 254 the high dose insulin phase (ratio of the geometric mean and 95% CI of 1.10 (1.00, 1.21), 255 p=0.05). There were no ethnic differences in whole body insulin sensitivity, measured as either 256 M value (BAM: 9.65 (2.32) vs WEM: 9.51 (3.86) mg/kg FFM min-1, p=0.89) or M/I (BAM: 257 0.0171 (0.0059) vs WEM: 0.0189 (0.0094) ((mg/kg FFM min-1) / (pmol/l)), p=0.44). 258 Associations between VAT, IHL and IMCL with whole body insulin sensitivity (measured as 259 either M value or M/I) are shown in Supplementary Table 1. When combining all participants 260 as a single cohort, there were significant inverse associations between VAT, IHL, and IMCL 261 with whole body insulin sensitivity; however, when assessing the ethnic groups separately, 262 these relationships were significant in WEM but weaker or absent in BAM. 263 Adipose tissue insulin sensitivity 264 Adipose tissue insulin sensitivity (ATIS) index did not differ by ethnicity during the basal or 265 insulin stimulated state (Table 2). Insulin mediated suppression of glycerol was used as a 266 Participant characteristics 45 The participant characteristics of the 21 BAM and 23 WEM are displayed in Table 1; by design 246 the groups were comparable in age and BMI. There were no differences in body fat, waist 247 circumference, blood pressure, HbA1c, cholesterol, fasting and post-load glucose between 248 ethnicities; however, BAM exhibited lower fasting triglycerides compared to WEM. Data on 249 ectopic fat depots in these volunteers, as previously reported by our group (39), showed lower 250 VAT and IHL in BAM but similar IMCL (included in Table 1 for reference). 251 The glucose and insulin profiles during the hyperinsulinaemic–euglycaemic clamp are shown 253 in Supplementary Figure 1. BAM exhibited a trend towards greater mean plasma insulin during 254 the high dose insulin phase (ratio of the geometric mean and 95% CI of 1.10 (1.00, 1.21), 255 p=0.05). There were no ethnic differences in whole body insulin sensitivity, measured as either 256 M value (BAM: 9.65 (2.32) vs WEM: 9.51 (3.86) mg/kg FFM min-1, p=0.89) or M/I (BAM: 257 0.0171 (0.0059) vs WEM: 0.0189 (0.0094) ((mg/kg FFM min-1) / (pmol/l)), p=0.44). 258 Associations between VAT, IHL and IMCL with whole body insulin sensitivity (measured as 259 either M value or M/I) are shown in Supplementary Table 1. When combining all participants 260 as a single cohort, there were significant inverse associations between VAT, IHL, and IMCL 261 with whole body insulin sensitivity; however, when assessing the ethnic groups separately, 262 these relationships were significant in WEM but weaker or absent in BAM. 263 Adipose tissue insulin sensitivity 264 Adipose tissue insulin sensitivity (ATIS) index did not differ by ethnicity during the basal or 265 insulin stimulated state (Table 2). Insulin mediated suppression of glycerol was used as a 266 measure of adipose tissue insulin sensitivity to lipolysis and was not significantly different by 267 ethnicity (mean difference (95% CI) -8.55 (-22.0, 4.90) %, p=0.21), Supplementary Figure 2A. 268 12 There was a trend towards lower adipose tissue insulin sensitivity to lipolysis when adjusting 269 for VAT in BAM (adjusted mean difference (95% CI) -12.4 (-26.9, 2.21) %, p=0.09). Across 270 the whole cohort, adipose tissue insulin sensitivity did not correlate with VAT, IHL or IMCL 271 (Figure 1A-C). However, when assessing WEM and BAM separately, adipose tissue insulin 272 sensitivity to lipolysis correlated with VAT and IHL in WEM but there were no significant 273 correlations in BAM (Figure 1A & B). When modelled with suppression of lipolysis, ethnicity 274 had no significant interaction with VAT (p=0.12) or IHL (p=0.58). There were no significant 275 correlations between the suppression of lipolysis and IMCL in either ethnic group (Figure 1C). 276 Peripheral insulin sensitivity 277 We found no ethnic differences in peripheral insulin sensitivity, measured as percentage 278 increase in glucose utilisation from the basal to high dose insulin phase of the clamp (BAM 279 304.82 (111.11) vs WEM 286.24 (138.44) %, p =0.63), Supplementary Figure 2B. There was 280 also no ethnic difference when accounting for the insulin concentration during the high dose 281 insulin phase, using PISI (mean difference (95% CI) -1.06 x10-2 (-3.87 x10-2 ,1.74 x10-2 ) (μmol 282 /kg FFM min-1 )/ pmol/l, p=0.43), Table 2. Adjusting PISI for VAT (which we have previously 283 reported as lower in BAM), resulted in significantly lower PISI in BAM (adjusted mean 284 difference (95% CI) -3.47 x10-2 (-5.67 x10-2 , -1.27 x10-2 ) (μmol /kg FFM min-1 )/ pmol/l, 285 p<0.01). Across the whole cohort, PISI correlated significantly with VAT and IMCL (Figure 286 1D-E). When assessing WEM and BAM separately, PISI correlated significantly with VAT 287 and IMCL in WEM; however, in BAM the association with VAT was weaker (Figure 1D) and 288 there was no association with IMCL (Figure 1E). When modelled with PISI, interactions 289 between ethnicity and VAT and between ethnicity and IMCL were not significant (p=0.11 and 290 p=0.11, respectively). Adipose tissue insulin sensitivity 264 291 13 We found no ethnic differences in the basal or insulin stimulated hepatic insulin sensitivity 293 index (HISI), Table 2. Using suppression of endogenous glucose production as a measure of 294 hepatic insulin sensitivity, we found no evidence for an ethnic difference (mean difference 295 (95% CI) -4.15 (-14.83, 6.53) %, p =0.21), Supplementary Figure 2C. Adjusting hepatic insulin 296 sensitivity for VAT, resulted in lower mean hepatic insulin sensitivity in BAM (mean 297 difference (95% CI) -10.9 (-21.2, -0.72) %, p=0.04). Adjusting hepatic insulin sensitivity for 298 IHL (which we have previously reported as lower in BAM), resulted in no ethnic difference in 299 hepatic insulin sensitivity (adjusted mean difference (95% CI) -7.33 (-17.9, 3.24) %, p=0.17). 300 Across the whole cohort, hepatic insulin sensitivity correlated with VAT and IHL (Figure 1F- 301 G). When assessing WEM and BAM separately, hepatic insulin sensitivity correlated 302 significantly with VAT in both ethnicities (Figure 1F); however, the correlation with IHL was 303 only significant in WEM (Figure 1G). When modelled with suppression of endogenous glucose 304 production, interactions between ethnicity and VAT and between ethnicity and IHL were not 305 significant (p=0.50 and p=0.66, respectively). 306 We found no ethnic differences in the basal or insulin stimulated hepatic insulin sensitivity 293 index (HISI), Table 2. Using suppression of endogenous glucose production as a measure of 294 hepatic insulin sensitivity, we found no evidence for an ethnic difference (mean difference 295 (95% CI) -4.15 (-14.83, 6.53) %, p =0.21), Supplementary Figure 2C. Adjusting hepatic insulin 296 sensitivity for VAT, resulted in lower mean hepatic insulin sensitivity in BAM (mean 297 difference (95% CI) -10.9 (-21.2, -0.72) %, p=0.04). Adjusting hepatic insulin sensitivity for 298 IHL (which we have previously reported as lower in BAM), resulted in no ethnic difference in 299 hepatic insulin sensitivity (adjusted mean difference (95% CI) -7.33 (-17.9, 3.24) %, p=0.17). 300 Across the whole cohort, hepatic insulin sensitivity correlated with VAT and IHL (Figure 1F- 301 G). When assessing WEM and BAM separately, hepatic insulin sensitivity correlated 302 significantly with VAT in both ethnicities (Figure 1F); however, the correlation with IHL was 303 only significant in WEM (Figure 1G). When modelled with suppression of endogenous glucose 304 production, interactions between ethnicity and VAT and between ethnicity and IHL were not 305 significant (p=0.50 and p=0.66, respectively). Adipose tissue insulin sensitivity 264 306 14 DISCUSSION 307 In this study we have shown that whilst BAM and WEM display comparable whole body, 308 skeletal muscle, hepatic and adipose tissue insulin sensitivity, the relationships between insulin 309 sensitivity and ectopic fat are ethnically distinct. 310 311 Resistance of adipose tissue to the antilipolytic effect of insulin is suggested to be a primary 312 abnormality in the pathophysiology of T2D that occurs before the onset of hyperglycaemia (6). 313 It has been proposed that VAT and ectopic fat accumulate as result of dysfunctional lipolysis, 314 which allows an increase in circulating fatty acids, and other adipocyte abnormalities, 315 described in the “spillover theory” (10, 49). Our study, which is the first to compare men of 316 black African and white European ethnicity, shows that there are no associations between 317 lipolysis and VAT, IHL or IMCL in BAM. Our findings agree with Albu et al. who showed a 318 relationship between suppression of lipolysis and VAT in obese white women but not in black 319 women (31). Together, these findings suggest lipolysis may not be driving the accumulation of 320 ectopic fat in black people of either gender, suggesting the “spillover theory” may not hold true 321 in this ethnic group. 322 DISCUSSION 307 This conflicting result may be due to the 335 aforementioned studies focusing on women with severe obesity whilst our participants were 336 only mildly overweight, or gender itself may explain the conflicting results, adding to the 337 evidence for gender differences in T2D pathophysiology (33). 338 339 Accumulation of IHL is proposed to be central to the development of hepatic insulin resistance. 340 The “portal theory” describes the accumulation of IHL, which develops from the flux of fatty 341 acids from VAT to the liver, via the portal vein. It is, therefore, not surprising that we found 342 lower IHL in BAM, given the lower levels of VAT that they exhibited. Whilst our data from 343 WEM corroborates the current understanding of T2D pathophysiology such that hepatic insulin 344 sensitivity was significantly associated with IHL (14), we found no evidence for this 345 relationship in BAM. This contrasts with data from studies in black women whereby IHL is 346 associated with hepatic sensitivity (25, 26). This may point to IHL being more harmful in black 347 women than men, although the obesity status of the women may also have contributed to this 348 result (33). 349 350 a so-called lower threshold. Our finding of a significant association between VAT and hepatic 332 insulin sensitivity is consistent with earlier work in obese black women (25), however, our data 333 in healthy men also show an association between VAT and skeletal muscle insulin sensitivity 334 which has not been found in women (25, 50). This conflicting result may be due to the 335 aforementioned studies focusing on women with severe obesity whilst our participants were 336 only mildly overweight, or gender itself may explain the conflicting results, adding to the 337 evidence for gender differences in T2D pathophysiology (33). 338 Accumulation of IHL is proposed to be central to the development of hepatic insulin resistance. 340 The “portal theory” describes the accumulation of IHL, which develops from the flux of fatty 341 acids from VAT to the liver, via the portal vein. It is, therefore, not surprising that we found 342 lower IHL in BAM, given the lower levels of VAT that they exhibited. Whilst our data from 343 WEM corroborates the current understanding of T2D pathophysiology such that hepatic insulin 344 sensitivity was significantly associated with IHL (14), we found no evidence for this 345 relationship in BAM. DISCUSSION 307 In this study we have shown that whilst BAM and WEM display comparable whole body, 308 skeletal muscle, hepatic and adipose tissue insulin sensitivity, the relationships between insulin 309 sensitivity and ectopic fat are ethnically distinct. 310 Resistance of adipose tissue to the antilipolytic effect of insulin is suggested to be a primary 312 abnormality in the pathophysiology of T2D that occurs before the onset of hyperglycaemia (6). 313 It has been proposed that VAT and ectopic fat accumulate as result of dysfunctional lipolysis, 314 which allows an increase in circulating fatty acids, and other adipocyte abnormalities, 315 described in the “spillover theory” (10, 49). Our study, which is the first to compare men of 316 black African and white European ethnicity, shows that there are no associations between 317 lipolysis and VAT, IHL or IMCL in BAM. Our findings agree with Albu et al. who showed a 318 relationship between suppression of lipolysis and VAT in obese white women but not in black 319 women (31). Together, these findings suggest lipolysis may not be driving the accumulation of 320 ectopic fat in black people of either gender, suggesting the “spillover theory” may not hold true 321 in this ethnic group. 322 Visceral fat, IHL and IMCL play an integral role in the development of insulin resistance and 324 T2D (12, 16, 17); however, black populations are consistently reported to exhibit lower levels 325 of VAT (19-23) despite their high T2D risk. Our finding of lower VAT in BAM is in agreement 326 with the literature. Despite this, we found comparable levels of insulin sensitivity. We 327 investigated associations between VAT and insulin sensitivity and found that both peripheral 328 and hepatic insulin sensitivity were significantly associated with VAT in both ethnicities. This 329 leads us to believe that VAT is detrimental to skeletal muscle and hepatic insulin sensitivity in 330 both ethnic groups, but that this impact occurs at lower VAT levels in BAM compared to WEM, 331 15 a so-called lower threshold. Our finding of a significant association between VAT and hepatic 332 insulin sensitivity is consistent with earlier work in obese black women (25), however, our data 333 in healthy men also show an association between VAT and skeletal muscle insulin sensitivity 334 which has not been found in women (25, 50). DISCUSSION 307 374 375 In contrast to the extensive evidence base that reports pronounced insulin resistance in 358 populations of black African ethnicity (24, 54), we showed no ethnic differences in insulin 359 sensitivity at a whole body and tissue specific level. The contrast in these findings are likely 360 due to the different methodologies used to measure insulin sensitivity. In our study we have 361 used the clamp method, which is a direct assessment of insulin sensitivity (37), while other 362 methods estimate insulin sensitivity through indirect modelling. The use of such methods in 363 black populations has been criticised because of the reduced insulin clearance and higher 364 insulin levels that they exhibit, which may lead to an underestimation of modelled insulin 365 sensitivity. Indeed, in an ethnic comparison of direct and indirect measures of insulin 366 sensitivity, Pisprasert et al. showed no difference in insulin sensitivity using the clamp, while 367 surrogate indices showed greater insulin resistance in African-Americans compared to white 368 Americans. These data suggest caution should be applied when using indirect assessments of 369 insulin resistance in black populations (55). Our findings are supported by several metabolic 370 studies using glucose clamps and isotopes, which have also found comparable insulin 371 sensitivity in healthy black and white communities (25, 26, 55-57). Our experimental design 372 also limited potential confounding factors; participants were similar in BMI, participants with 373 impaired glucose tolerance were excluded and our data collection included only men. 374 We have previously published a description of the ectopic fat status for the current set of 376 participants (39) in which we found no ethnic differences in IMCL, but significantly lower 377 VAT and IHL in BAM. In the current study we adjusted our insulin sensitivity data for VAT 378 and found lower whole body, skeletal muscle and hepatic insulin sensitivity in BAM. The 379 reduced insulin sensitivity following adjustment for VAT, and the lower VAT storage in the 380 presence of similar lipolysis, suggests that the detrimental effects of VAT occur at lower levels 381 We have previously published a description of the ectopic fat status for the current set of 376 participants (39) in which we found no ethnic differences in IMCL, but significantly lower 377 VAT and IHL in BAM. DISCUSSION 307 This contrasts with data from studies in black women whereby IHL is 346 associated with hepatic sensitivity (25, 26). This may point to IHL being more harmful in black 347 women than men, although the obesity status of the women may also have contributed to this 348 result (33). 349 Accumulation of IHL is proposed to be central to the development of hepatic insulin resistance. 340 The “portal theory” describes the accumulation of IHL, which develops from the flux of fatty 341 acids from VAT to the liver, via the portal vein. It is, therefore, not surprising that we found 342 lower IHL in BAM, given the lower levels of VAT that they exhibited. Whilst our data from 343 WEM corroborates the current understanding of T2D pathophysiology such that hepatic insulin 344 sensitivity was significantly associated with IHL (14), we found no evidence for this 345 relationship in BAM. This contrasts with data from studies in black women whereby IHL is 346 associated with hepatic sensitivity (25, 26). This may point to IHL being more harmful in black 347 women than men, although the obesity status of the women may also have contributed to this 348 result (33). 349 350 Intramuscular lipids, which accumulate as a result of skeletal muscle cells taking up fatty acids 351 from the peripheral circulation, have been shown to be correlated with skeletal muscle insulin 352 resistance (15). Whilst we saw a significant relationship between IMCL and skeletal muscle 353 insulin sensitivity in WEM, this relationship was not present in BAM. This finding agrees with 354 other studies (51-53), and suggests that skeletal muscle insulin resistance develops 355 independently of IMCL in BAM. 356 16 357 In contrast to the extensive evidence base that reports pronounced insulin resistance in 358 populations of black African ethnicity (24, 54), we showed no ethnic differences in insulin 359 sensitivity at a whole body and tissue specific level. The contrast in these findings are likely 360 due to the different methodologies used to measure insulin sensitivity. In our study we have 361 used the clamp method, which is a direct assessment of insulin sensitivity (37), while other 362 methods estimate insulin sensitivity through indirect modelling. DISCUSSION 307 The use of such methods in 363 black populations has been criticised because of the reduced insulin clearance and higher 364 insulin levels that they exhibit, which may lead to an underestimation of modelled insulin 365 sensitivity. Indeed, in an ethnic comparison of direct and indirect measures of insulin 366 sensitivity, Pisprasert et al. showed no difference in insulin sensitivity using the clamp, while 367 surrogate indices showed greater insulin resistance in African-Americans compared to white 368 Americans. These data suggest caution should be applied when using indirect assessments of 369 insulin resistance in black populations (55). Our findings are supported by several metabolic 370 studies using glucose clamps and isotopes, which have also found comparable insulin 371 sensitivity in healthy black and white communities (25, 26, 55-57). Our experimental design 372 also limited potential confounding factors; participants were similar in BMI, participants with 373 impaired glucose tolerance were excluded and our data collection included only men. 374 In contrast to the extensive evidence base that reports pronounced insulin resistance in 358 populations of black African ethnicity (24, 54), we showed no ethnic differences in insulin 359 sensitivity at a whole body and tissue specific level. The contrast in these findings are likely 360 due to the different methodologies used to measure insulin sensitivity. In our study we have 361 used the clamp method, which is a direct assessment of insulin sensitivity (37), while other 362 methods estimate insulin sensitivity through indirect modelling. The use of such methods in 363 black populations has been criticised because of the reduced insulin clearance and higher 364 insulin levels that they exhibit, which may lead to an underestimation of modelled insulin 365 sensitivity. Indeed, in an ethnic comparison of direct and indirect measures of insulin 366 sensitivity, Pisprasert et al. showed no difference in insulin sensitivity using the clamp, while 367 surrogate indices showed greater insulin resistance in African-Americans compared to white 368 Americans. These data suggest caution should be applied when using indirect assessments of 369 insulin resistance in black populations (55). Our findings are supported by several metabolic 370 studies using glucose clamps and isotopes, which have also found comparable insulin 371 sensitivity in healthy black and white communities (25, 26, 55-57). Our experimental design 372 also limited potential confounding factors; participants were similar in BMI, participants with 373 impaired glucose tolerance were excluded and our data collection included only men. DISCUSSION 307 Lower ectopic fat storage, despite similar lipolysis, may 385 point to an increased tendency towards fat oxidation over ectopic storage in BAM; further 386 studies assessing fatty acid oxidation are needed to explore this possibility. 387 studies assessing fatty acid oxidation are needed to explore this possibility. 387 388 Although one of the strengths of this study was the use of rigorous measurements of insulin 389 sensitivity and ectopic fat, we recognise that our conclusions for the associations between 390 insulin sensitivity and ectopic fat may be limited by our sample size. While our sample size is 391 comparable to other studies of this nature, it does affect the statistical adjustment for VAT/IHL 392 and interaction analysis. Our insulin sensitivity data are based on lean mass assessed by 393 bioimpedance methodology; this uses calculations which are not ethnically sensitive and could 394 potentially lead to underestimation of lean mass and thus overestimating insulin sensitivity in 395 BAM (58). Finally, although the aim of our recruitment was to achieve comparable BMIs in 396 our groups, this resulted in a tendency towards lower waist circumferences in BAM, which 397 may have also contributed to differences in the metabolic characteristics that we studied. A 398 study in which the groups are matched for waist circumference would help to elucidate these 399 effects. 400 401 In summary, our data suggest that increased lipolysis due to adipose tissue insulin resistance 402 may not be driving ectopic fat deposition in BAM. Additionally, ectopic fat accumulation in 403 the liver and skeletal muscle may play less of a role in reducing insulin sensitivity in BAM 404 compared to WEM. We provide evidence that the detrimental effects of VAT on glucose uptake 405 and the suppression of endogenous glucose production occur at a lower VAT level in BAM. 406 Although one of the strengths of this study was the use of rigorous measurements of insulin 389 sensitivity and ectopic fat, we recognise that our conclusions for the associations between 390 insulin sensitivity and ectopic fat may be limited by our sample size. While our sample size is 391 comparable to other studies of this nature, it does affect the statistical adjustment for VAT/IHL 392 and interaction analysis. DISCUSSION 307 In the current study we adjusted our insulin sensitivity data for VAT 378 and found lower whole body, skeletal muscle and hepatic insulin sensitivity in BAM. The 379 reduced insulin sensitivity following adjustment for VAT, and the lower VAT storage in the 380 presence of similar lipolysis, suggests that the detrimental effects of VAT occur at lower levels 381 17 in BAM and a resistance to storing VAT allows BAM to maintain comparable insulin 382 sensitivity to WEM. In comparison, adjusting for IHL did not explain the similar hepatic insulin 383 sensitivity and provides more evidence for an independent relationship between IHL and 384 hepatic insulin sensitivity in BAM. Lower ectopic fat storage, despite similar lipolysis, may 385 point to an increased tendency towards fat oxidation over ectopic storage in BAM; further 386 studies assessing fatty acid oxidation are needed to explore this possibility. 387 388 Although one of the strengths of this study was the use of rigorous measurements of insulin 389 sensitivity and ectopic fat, we recognise that our conclusions for the associations between 390 insulin sensitivity and ectopic fat may be limited by our sample size. While our sample size is 391 comparable to other studies of this nature, it does affect the statistical adjustment for VAT/IHL 392 and interaction analysis. Our insulin sensitivity data are based on lean mass assessed by 393 bioimpedance methodology; this uses calculations which are not ethnically sensitive and could 394 potentially lead to underestimation of lean mass and thus overestimating insulin sensitivity in 395 BAM (58). Finally, although the aim of our recruitment was to achieve comparable BMIs in 396 our groups, this resulted in a tendency towards lower waist circumferences in BAM, which 397 may have also contributed to differences in the metabolic characteristics that we studied. A 398 study in which the groups are matched for waist circumference would help to elucidate these 399 effects. 400 401 In summary, our data suggest that increased lipolysis due to adipose tissue insulin resistance 402 in BAM and a resistance to storing VAT allows BAM to maintain comparable insulin 382 sensitivity to WEM. In comparison, adjusting for IHL did not explain the similar hepatic insulin 383 sensitivity and provides more evidence for an independent relationship between IHL and 384 hepatic insulin sensitivity in BAM. DISCUSSION 307 Our insulin sensitivity data are based on lean mass assessed by 393 bioimpedance methodology; this uses calculations which are not ethnically sensitive and could 394 potentially lead to underestimation of lean mass and thus overestimating insulin sensitivity in 395 BAM (58). Finally, although the aim of our recruitment was to achieve comparable BMIs in 396 our groups, this resulted in a tendency towards lower waist circumferences in BAM, which 397 may have also contributed to differences in the metabolic characteristics that we studied. A 398 study in which the groups are matched for waist circumference would help to elucidate these 399 effects. 400 18 We conclude that current theories regarding the accumulation of ectopic fat and its impact on 407 insulin sensitivity may not apply in BAM, who display a resistance to storing visceral and 408 hepatic fat. Future work, assessing the impact of ectopic fat on insulin secretory function, is 409 vital before excluding ectopic fat as the culprit behind the increased prevalence of T2D in black 410 populations. 411 We conclude that current theories regarding the accumulation of ectopic fat and its impact on 407 insulin sensitivity may not apply in BAM, who display a resistance to storing visceral and 408 hepatic fat. Future work, assessing the impact of ectopic fat on insulin secretory function, is 409 vital before excluding ectopic fat as the culprit behind the increased prevalence of T2D in black 410 populations. 411 19 Declaration of interest: Authors declare that there is no conflict of interest that could be 412 perceived as prejudicing the impartiality of the research reported 413 Declaration of interest: Authors declare that there is no conflict of interest that could be 412 perceived as prejudicing the impartiality of the research reported 413 perceived as prejudicing the impartiality of the research reported 413 Funding: This work was supported by Diabetes UK (project grant number: 14/0004967) 414 Author Contributions: LMG, SAA, AMU, JLP designed the study; OB, OH, ML, CM 415 FSM, GCE acquired the data, OB, OH, FSM, AMU performed the data analysis, OB LMG, 416 SAA, AMU contributed to the interpretation, OG, LMG drafted the article and all authors 417 contributed to revising the intellectual content before approving the final article. JLP is 418 supported by the NIHR Biomedical Research Centre based at Guy’s and St Thomas’ NHS 419 Foundation Trust and King’s College London and is an NIHR Senior Investigator. REFERENCES 433 433 1. Health Survey for England 2004: The Health of Minority Ethnic Groups– headline tables 2004 434 [Available from: https://digital.nhs.uk/data-and-information/publications/statistical/health-survey- 435 for-england/health-survey-for-england-2004-health-of-ethnic-minorities-headline-results. 436 y g y p [Available from: https://digital.nhs.uk/data-and-information/publications/statistical/health-survey- 435 for-england/health-survey-for-england-2004-health-of-ethnic-minorities-headline-results. 436 2. Goff LM, Ladwa M, Hakim O, Bello O. Ethnic distinctions in the pathophysiology of type 2 437 diabetes: a focus on black African-Caribbean populations. Proceedings of the Nutrition Society. 438 2019:1-10. 439 3. Zhang Q, Wang Y, Huang ES. 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The Journal of clinical 464 endocrinology and metabolism. 2015;100(1):235-43. 465 13. Kabir M, Catalano KJ, Ananthnarayan S, Kim SP, Van Citters GW, Dea MK, Bergman RN. DISCUSSION 307 The views 420 expressed are those of the authors and not necessarily those of the NHS, the NIHR or the 421 Department of Health. 422 Funding: This work was supported by Diabetes UK (project grant number: 14/0004967) 414 Author Contributions: LMG, SAA, AMU, JLP designed the study; OB, OH, ML, CM 415 FSM, GCE acquired the data, OB, OH, FSM, AMU performed the data analysis, OB LMG, 416 SAA, AMU contributed to the interpretation, OG, LMG drafted the article and all authors 417 contributed to revising the intellectual content before approving the final article. JLP is 418 supported by the NIHR Biomedical Research Centre based at Guy’s and St Thomas’ NHS 419 Foundation Trust and King’s College London and is an NIHR Senior Investigator. The views 420 expressed are those of the authors and not necessarily those of the NHS, the NIHR or the 421 Department of Health. 422 Acknowledgments: The authors would like to thank: Andrew Pernet, Bula Wilson, Marcia 423 Henderson-Wilson, Marietta Stadler (Diabetes Research Group, King’s College Hospital, 424 London, UK) for their contribution to the participant metabolic assessments; Louisa Green 425 and Elka Giemza (Clinical Research Facility, Kings College Hospital, London, UK) for 426 accommodating the participants and researchers for the metabolic assessments; Tracy Dew 427 (Affinity Biomarker Labs, Imperial College, London, UK) Nicola Jackson, Lucy Coppin 428 (Diabetes and Metabolic Medicine, University of Surrey, Guildford, UK) and Anne- 429 Catherine Perz (Nutritional Science Department, King’s College London, London, UK) for 430 their assistance with the laboratory sample analysis; and the participants for their 431 commitment and patience. 432 Acknowledgments: The authors would like to thank: Andrew Pernet, Bula Wilson, Marcia 423 Henderson-Wilson, Marietta Stadler (Diabetes Research Group, King’s College Hospital, 424 London, UK) for their contribution to the participant metabolic assessments; Louisa Green 425 and Elka Giemza (Clinical Research Facility, Kings College Hospital, London, UK) for 426 accommodating the participants and researchers for the metabolic assessments; Tracy Dew 427 (Affinity Biomarker Labs, Imperial College, London, UK) Nicola Jackson, Lucy Coppin 428 (Diabetes and Metabolic Medicine, University of Surrey, Guildford, UK) and Anne- 429 Catherine Perz (Nutritional Science Department, King’s College London, London, UK) for 430 their assistance with the laboratory sample analysis; and the participants for their 431 commitment and patience. 432 20 REFERENCES 433 466 Molecular evidence supporting the portal theory: a causative link between visceral adiposity and 467 hepatic insulin resistance. 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Pisprasert V, Ingram KH, Lopez-Davila MF, Munoz AJ, Garvey WT. Limitations in the use of 594 indices using glucose and insulin levels to predict insulin sensitivity: impact of race and gender and 595 superiority of the indices derived from oral glucose tolerance test in African Americans. Diabetes 596 Care. 2013;36(4):845-53. 597 56. Raygor V, Abbasi F, Lazzeroni LC, Kim S, Ingelsson E, Reaven GM, Knowles JW. Impact of 598 race/ethnicity on insulin resistance and hypertriglyceridaemia. Diabetes & vascular disease research 599 2019;16(2):153-9. 600 57. Allister-Price C, Craig CM, Spielman D, Cushman SS, McLaughlin TL. Metabolic markers, 601 regional adiposity, and adipose cell size: relationship to insulin resistance in African-American as 602 compared with Caucasian women. International journal of obesity (2005). 2018. 603 58. Nightingale CM, Rudnicka AR, Owen CG, Donin AS, Newton SL, Furness CA, Howard EL, 604 Gillings RD, Wells JCK, Cook DG, et al. 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Adipocyte lipolysis and insulin resistance. 576 Biochimie. 2016;125:259-66. 577 50. Goedecke JH, Levitt NS, Lambert EV, Utzschneider KM, Faulenbach MV, Dave JA, West S, 578 Victor H, Evans J, Olsson T, et al. Differential effects of abdominal adipose tissue distribution on 579 insulin sensitivity in black and white South African women. Obesity (Silver Spring, Md). 580 ( ) 581 23 51. Ingram KH, Lara-Castro C, Gower BA, Makowsky R, Allison DB, Newcomer BR, Munoz AJ, 582 Beasley TM, Lawrence JC, Lopez-Ben R, et al. Intramyocellular lipid and insulin resistance: differenti 583 relationships in European and African Americans. Obesity (Silver Spring, Md). 2011;19(7):1469-75. 584 52. DeLany JP, Dubé JJ, Standley RA, Distefano G, Goodpaster BH, Stefanovic-Racic M, Coen PM 585 Toledo FGS. REFERENCES 433 Lawrence JC, Newcomer BR, Buchthal SD, Sirikul B, Oster RA, Hunter GR, Gower BA. 588 Relationship of intramyocellular lipid to insulin sensitivity may differ with ethnicity in healthy girls 589 and women. Obesity (Silver Spring, Md). 2011;19(1):43-8. 590 54. Kodama K, Tojjar D, Yamada S, Toda K, Patel CJ, Butte AJ. Ethnic differences in the 591 relationship between insulin sensitivity and insulin response: a systematic review and meta-analysis. 592 Diabetes Care. 2013;36(6):1789-96. 593 55. Pisprasert V, Ingram KH, Lopez-Davila MF, Munoz AJ, Garvey WT. Limitations in the use of 594 indices using glucose and insulin levels to predict insulin sensitivity: impact of race and gender and 595 superiority of the indices derived from oral glucose tolerance test in African Americans. Diabetes 596 Care. 2013;36(4):845-53. 597 56. Raygor V, Abbasi F, Lazzeroni LC, Kim S, Ingelsson E, Reaven GM, Knowles JW. Impact of 598 race/ethnicity on insulin resistance and hypertriglyceridaemia. Diabetes & vascular disease research. 599 2019;16(2):153-9. 600 57. Allister-Price C, Craig CM, Spielman D, Cushman SS, McLaughlin TL. Metabolic markers, 601 regional adiposity, and adipose cell size: relationship to insulin resistance in African-American as 602 compared with Caucasian women. International journal of obesity (2005). 2018. 603 58. Nightingale CM, Rudnicka AR, Owen CG, Donin AS, Newton SL, Furness CA, Howard EL, 604 Gillings RD, Wells JCK, Cook DG, et al. Are Ethnic and Gender Specific Equations Needed to Derive Fat 605 Free Mass from Bioelectrical Impedance in Children of South Asian, Black African-Caribbean and 606 White European Origin? Results of the Assessment of Body Composition in Children Study. PLOS 607 ONE. 2013;8(10):e76426. REFERENCES 433 608 609 24 Table 1:Participant characteristics 610 Characteristic BAM n=21 WEM n=23 P Age (years) a 25 (22 – 40) 29 (25 – 53) 0.18 BMI (kg/m2) 26.8 (3.6) 26.5 (4.5) 0.82 Waist Circumference (cm) b 87.5 (83.4, 91.8) 92.8 (87.1, 99.0) 0.13 Systolic BP (mm/Hg) 124.0 (11.9) 121.9 (9.1) 0.52 Diastolic BP (mm/Hg) b 70.3 (65.5, 75.5) 70.7 (67.2, 74.3) 0.91 Total Cholesterol (mmol/l) b 4.26 (3.85, 4.73) 4.65 (4.23, 5.11) 0.20 LDL (mmol/l) 2.73 (0.84) 2.99 (0.82) 0.33 HDL (mmol/l) a 1.2 (1.2 – 1.4) 1.2 (1.1 – 1.4) 0.86 Triglycerides (mmol/l) b 0.67 (0.59, 0.77) 0.99 (0.81, 1.21) <0.01 Fasting glucose (mmol/l) 5.1 (0.5) 5.2 (0.4) 0.55 2-hour post load glucose (mmol/l) 5.28 (1.13) 5.09 (1.26) 0.61 Ectopic fat depots Visceral adipose tissue (VAT), L4-5 (cm2)b 46.1 (34.4 - 61.7)c 79.0 (55.4 - 112.5) 0.02 Hepatic fat fraction (HFF) (%) 3.78 (1.13)c 6.08 (5.04) 0.04 Intramyocellular lipid (IMCL) (AU)d 0.030 (0.015) 0.030 (0.014) 0.87 Data expressed as mean (SD) for normally distributed data 611 adata expressed as median (IQR) for non-normally distributed data 612 bgeometric mean (95% CI) for log transformed data 613 csample size=20 614 dsample size; BAM=18, WEM=22 615 Table 1:Participant characteristics 610 Table 1:Participant characteristics 610 Characteristic BA 25 Table 2: Substrate kinetics before and after insulin adjustments during the basal state and the 16 hyperinsulinaemic–euglycaemic clamp 17 Table 2: Substrate kinetics before and after insulin adjustments during the basal state and the hyperinsulinaemic–euglycaemic clamp Basal state Hyperinsulinaemic–euglycaemic clamp BAM n=21 WEM n=23 P BAM n=20 WEM n=23 P Glycerol Ra μmol /kg FFM min-1 1.11 (0.71 – 2.72)a 1.55 (1.29 – 2.27)a 0.10 0.64 (0.52, 0.78)b 0.77 (0.63, 0.93)bd 0.17 Adipose tissue insulin sensitivity index; ATIS (μmol /kg FFM min-1 · pmol/l)-1 14.62 x10-3 (10.36 x10-3, 20.70 x10-3)b 12.16 x10-3 (9.06 x10-3, 16.31 x10-3)b 0.40 10.2 x10-3 (4.61 x10-3) 9.04 x10-3 (4.46 x10-3) 0.42 Glucose Ra μmol /kg FFM min-1 13.60 (1.24) 13.74 (1.33) 0.72 4.37 (3.94 – 5.64)a 3.23 (2.71 – 6.14)ac 0.38 Hepatic insulin sensitivity index; HISI (μmol /kg FFM min-1 · pmol/l)-1 1.57 x10-3 (6.61 x10-4) 1.70 x10-3 (7.18 x10-4) 0.56 1.35 x10-3 (9.23 – 16.32 x10-4) a 1.68 x10-3 (11.57 – 23.59 x10- 4)ac 0.41 Glucose Rd μmol /kg FFM min-1 - - - 51.14 (44.61 – 60.16)a 50.48 (38.43 – 67.72)a 0.87 26 Glycerol and glucose isotope kinetics derived from the basal post absorptive state and during the hyperinsulinaemic–euglycaemic clamp. REFERENCES 433 8 Glycerol Ra and Glucose Ga was derived from the low dose insulin phase (10 mU m−2 BSA min−1 ). Glucose Rd was derived during the high 9 dose insulin phase (40 mU m−2 BSA min−1 ) infusion during the hyperinsulinaemic–euglycaemic clamp. Hepatic insulin sensitivity index (HISI) 20 and peripheral insulin sensitivity index (PISI) are corrected for by insulin at the basal state and during the high dose insulin phase respectively. 21 Data expressed as mean (SD) 22 adata expressed as median (IQR) 23 bgeometric mean (95% CI) 24 Peripheral insulin sensitivity index (μmol /kg FFM min-1 )/ pmol/l - - - 9.71 x10-2 (3.43 x10-2) 10.78 x10-2 (5.32 x10-2) 0.43 Peripheral insulin sensitivity index (μmol /kg FFM min-1 )/ pmol/l 0.43 Glycerol and glucose isotope kinetics derived from the basal post absorptive state and during the hyperinsulinaemic–euglycaemic clamp. 618 Glycerol Ra and Glucose Ga was derived from the low dose insulin phase (10 mU m−2 BSA min−1 ). Glucose Rd was derived during the high 619 dose insulin phase (40 mU m−2 BSA min−1 ) infusion during the hyperinsulinaemic–euglycaemic clamp. Hepatic insulin sensitivity index (HISI) 620 and peripheral insulin sensitivity index (PISI) are corrected for by insulin at the basal state and during the high dose insulin phase respectively. 621 Data expressed as mean (SD) 622 adata expressed as median (IQR) 623 bgeometric mean (95% CI) 624 csample size; 21 625 Glycerol and glucose isotope kinetics derived from the basal post absorptive state and during the hyperinsulinaemic–euglycaemic clamp. Glycerol Ra and Glucose Ga was derived from the low dose insulin phase (10 mU m−2 BSA min−1 ). Glucose Rd was derived during the high dose insulin phase (40 mU m−2 BSA min−1 ) infusion during the hyperinsulinaemic–euglycaemic clamp. Hepatic insulin sensitivity index (HISI) and peripheral insulin sensitivity index (PISI) are corrected for by insulin at the basal state and during the high dose insulin phase respectively. Data expressed as mean (SD) adata expressed as median (IQR) 27 27 Figure 1: Associations between ectopic fat and tissue specific insulin sensitivity 630 631 632 631 632 632 28 633 634 Data presented using the Pearson correlation coefficients. Peripheral insulin sensitivity index was measured during the high dose insulin phase 635 (40 mU m-2 BSA min-1), suppression of endogenous glucose production and whole body lipolysis was measured during the low dose insulin 636 phase (10 mU m-2 BSA min-1). g g p y lack West African men; IHL, intrahepatic lipid; IMCL, intramyocellular lipid; VAT, visceral adipose tissue; WEM, intrahepatic lipid; IMCL, intramyocellular lipid; VAT, visceral adipose tissue; WEM, REFERENCES 433 637 33 633 633 634 Data presented using the Pearson correlation coefficients. Peripheral insulin sensitivity index was measured during the high dose insulin phase 635 (40 mU m-2 BSA min-1), suppression of endogenous glucose production and whole body lipolysis was measured during the low dose insulin 636 phase (10 mU m-2 BSA min-1). 637 Data presented using the Pearson correlation coefficients. Peripheral insulin sensitivity index was measured during the high dose insulin phase 635 (40 mU m-2 BSA min-1), suppression of endogenous glucose production and whole body lipolysis was measured during the low dose insulin 636 phase (10 mU m-2 BSA min-1). 637 29 Supplementary table 1: Associations between whole body insulin sensitivity and ectopic fat 638 VAT area (cm2) IHL (%) IMCL (AU) M; mg/kg FFM min-1 Whole cohort r=-0.67, p<0.01 r=-0.61, p<0.01 r=-0.36, p=0.03 BAM r=-0.46, p=0.06 r=-0.18, p=0.48 r=0.00, p=0.98 WEM r=-0.80, p<0.01 r=-0.72, p<0.01 r=-0.52, p=0.01 M/I; ((mg/kg FFM min-1) / (pmol/l)) Whole cohort r=-0.63, p<0.01 r=-0.61, p<0.01 r=-0.40, p=0.01 BAM r=-0.56, p=0.02 r=-0.34, p=0.17 r=-0.10, p=0.71 WEM r=-0.78, p<0.01 r=-0.73, p<0.01 r=-0.54, p=0.01 Correlation coefficients determined using Pearson’s correlation. VAT, IHL and IMCL were log transformed to improve normality. 639 Abbreviations: BAM, Black West African men; IHL, intrahepatic lipid; IMCL, intramyocellular lipid; VAT, visceral adipose tissue; WEM, 640 White European men 641 642 Supplementary table 1: Associations between whole body insulin sensitivity and ectopic fat 638 ciations between whole body insulin sensitivity and ectopic fat 638 Correlation coefficients determined using Pearson’s correlation. VAT, IHL and IMCL were log transformed to improve normality. 639 Abbreviations: BAM, Black West African men; IHL, intrahepatic lipid; IMCL, intramyocellular lipid; VAT, visceral adipose tissue; WEM, 640 White European men 641 642 g , g p y Abbreviations: BAM, Black West African men; IHL, intrahepatic lipid; IMCL, intramyocellular lipid; VAT, visceral adipose tissue; WEM, White European men 30 30 Supplementary figure 1: Hyperinsulinaemic–euglycaemic clamp time course 643 644 645 Data expressed as mean (SEM). Plasma glucose (A) and insulin (B) and concentrations 646 during the hyperinsulinaemic–euglycaemic clamp. Data expressed as Mean ± SD per time 647 point. 648 644 5 Data expressed as mean (SEM). Plasma glucose (A) and insulin (B) and concentrations 646 during the hyperinsulinaemic–euglycaemic clamp. Data expressed as Mean ± SD per time 647 point. REFERENCES 433 648 31 Supplementary figure 2: Percentage change in glucose and glycerol kinetics during the 649 hyperinsulinaemic–euglycaemic clamp 650 651 Supplementary figure 2: Percentage change in glucose and glycerol kinetics during the 649 hyperinsulinaemic–euglycaemic clamp 650 651 651 652 653 (A) Suppression of whole body lipolysis during low dose insulin phase, presented as mean 654 (SD) (B) Increase in peripheral glucose utilisation during high dose insulin phase, presented 655 as mean (SD) (C) Suppression of endogenous glucose production during low dose insulin 656 phase, presented as median (IQR). 657 652 652 653 (A) Suppression of whole body lipolysis during low dose insulin phase, presented as mean 654 (SD) (B) Increase in peripheral glucose utilisation during high dose insulin phase, presented 655 as mean (SD) (C) Suppression of endogenous glucose production during low dose insulin 656 phase, presented as median (IQR). 657 653 (A) Suppression of whole body lipolysis during low dose insulin phase, presented as mean 654 (SD) (B) Increase in peripheral glucose utilisation during high dose insulin phase, presented 655 as mean (SD) (C) Suppression of endogenous glucose production during low dose insulin 656 phase, presented as median (IQR). 657 32
https://openalex.org/W4285063742	https://bmcinfectdis.biomedcentral.com/track/pdf/10.1186/s12879-020-05584-5	English	null	Routine blood parameters are helpful for early identification of influenza infection in children	Research Square (Research Square)	2,020	cc-by	7,954	Zhu et al. BMC Infectious Diseases (2020) 20:864 https://doi.org/10.1186/s12879-020-05584-5 Zhu et al. BMC Infectious Diseases (2020) 20:864 https://doi.org/10.1186/s12879-020-05584-5 Open Access © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract Background: Routine blood parameters, such as the lymphocyte (LYM) count, platelet (PLT) count, lymphocyte-to- monocyte ratio (LMR), neutrophil-to-lymphocyte ratio (NLR), lymphocytes multiplied by platelets (LYMPLT) and mean platelet volume-to-platelet ratio (MPV/PLT), are widely used to predict the prognosis of infectious diseases. We aimed to explore the value of these parameters in the early identification of influenza virus infection in children. Methods: We conducted a single-center, retrospective, observational study of fever with influenza-like symptoms in pediatric outpatients from different age groups and evaluated the predictive value of various routine blood parameters measured within 48 h of the onset of fever for influenza virus infection. Results: The LYM count, PLT count, LMR and LYMPLT were lower, and the NLR and MPV/PLT were higher in children with an influenza infection (PCR-confirmed and symptomatic). The LYM count, LMR and LYMPLT in the influenza infection group were lower in the 1- to 6-year-old subgroup, and the LMR and LYMPLT in the influenza infection group were lower in the > 6-year-old subgroup. In the 1- to 6-year-old subgroup, the cutoff value of the LMR for predicting influenza A virus infection was 3.75, the sensitivity was 81.87%, the specificity was 84.31%, and the area under the curve (AUC) was 0.886; the cutoff value of the LMR for predicting influenza B virus infection was 3.71, the sensitivity was 73.58%, the specificity was 84.31%, and the AUC was 0.843. In the > 6-year-old subgroup, the cutoff value of the LMR for predicting influenza A virus infection was 3.05, the sensitivity was 89.27%, the specificity was 89.61%, and the AUC was 0.949; the cutoff value of the LMR for predicting influenza B virus infection was 2.88, the sensitivity was 83.19%, the specificity was 92.21%, and the AUC was 0.924. Conclusions: Routine blood tests are simple, inexpensive and easy to perform, and they are useful for the early identification of influenza virus infection in children. The LMR had the strongest predictive value for influenza virus infection in children older than 1 year, particularly in children older than 6 years with influenza A virus infection. Keywords: Influenza, Routine blood tests, Children, Lymphocyte-to-monocyte ratio * Correspondence: 61132318@qq.com * Correspondence: 61132318@qq.com †Ronghe Zhu and Cuie Chen are co-first authors 1Department of Pediatrics, the First Affiliated Hospital of Wenzhou Medical University, South Baixiang, Ouhai District, Wenzhou 325000, Zhejiang, China Full list of author information is available at the end of the article * Correspondence: 61132318@qq.com †Ronghe Zhu and Cuie Chen are co-first authors 1Department of Pediatrics, the First Affiliated Hospital of Wenzhou Medical University, South Baixiang, Ouhai District, Wenzhou 325000, Zhejiang, China Full list of author information is available at the end of the article Routine blood parameters are helpful for early identification of influenza infection in children Ronghe Zhu1†, Cuie Chen2†, Qiu Wang1, Xixi Zhang3, Chaosheng Lu1 and Yuanyuan Sun1* Background Background Influenza is an acute respiratory infectious disease caused by influenza viruses. One billion patients are diagnosed with seasonal influenza each year worldwide, among whom 3 to 5 million have severe cases and as many as 250,000 to 500,000 die [1]. Although most chil- dren recover spontaneously from infection, morbidity and mortality rates are higher in children with under- lying diseases who are younger than 5 years, particularly children younger than 2 years [2]. However, previously healthy children are also at risk. In the USA, the admis- sion rate of non-high-risk children for influenza was estimated to be 9 per 10,000 children younger than 5 years [3]. According to the WHO, the annual infection rate of children was as high as approximately 50% in the past 11 influenza epidemic seasons [1]. Complications such as pneumonia, myocarditis, septic shock and multiple organ dysfunction, are the main causes of death in children with influenza [4]. Early (within 48 h after infection) use of anti-influenza drugs significantly relieves symptoms, shortens the disease course, and reduces complications. Therefore, the early and rapid diagnosis of influenza and the early use of anti-influenza drugs are essential to improve the prognosis of influenza in children. © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Zhu et al. BMC Infectious Diseases (2020) 20:864 Page 2 of 10 Page 2 of 10 Zhu et al. BMC Infectious Diseases (2020) 20:864 explored the predictive value of the LMR, NLR, LYMPLT, MPV/PLT, lymphocyte (LYM) count and platelet (PLT) count for influenza infection in children. explored the predictive value of the LMR, NLR, LYMPLT, MPV/PLT, lymphocyte (LYM) count and platelet (PLT) count for influenza infection in children. Patients Children with fever and influenza-like symptoms aged 0.2 to 14 years who presented at the First Affiliated Hos- pital of Wenzhou Medical University from January 2018 to February 2020 were included in this study. All the pa- tients underwent routine blood tests and the detection of influenza virus nucleic acids using RT-PCR within 48 h of the onset of fever. Influenza-like symptoms were defined as follows: fever (temperature ≥38 °C), cough or sore throat [13]. The exclusion criteria were as follows: (1) systemic chronic diseases, such as diseases of the blood, heart, lung, liver and kidney; (2) immunodefi- ciency due to a tumor, HIV infection, or the long-term use of oral hormones or immunosuppressive agents; (3) severe or critical illness, namely, the failure of one or more organs; (4) bacterial infections, such as sepsis and suppurative tonsillitis; and (5) Epstein-Barr virus (EBV) infection. This study was approved by the Ethics Com- mittee for Clinical Research of the First Affiliated Hos- pital of Wenzhou Medical University (No. 2020–065), and all the parents signed the informed consent form. The diagnosis of influenza depends on the detection of influenza virus nucleic acids in the respiratory tract, the isolation of influenza virus or the detection of a level of serum-specific antibodies that is at least 4 times the normal level. Common detection methods include virus isolation and culture, RT-PCR and serological detection, all of which have advantages and disadvantages. Virus isolation and culture, and serological detection are time- consuming and not suitable for outpatient screening. RT-PCR takes slightly less time, but is relatively expen- sive. Viral antigen detection, such as rapid influenza detection, is rapid, simple and displays good specificity; however, the sensitivity is low and it is prone to false negative results [5]. Detection of routine blood parameters Detection of routine blood parameters Finger prick blood samples were subjected to routine blood tests. A routine analyzer (XN-350, SYSMEX, Hyogo, Kobe, Japan) was used for detection. The LYM count, monocyte (MON) count, PLT count and mean platelet volume (MPV) were recorded. Additionally, other hematological parameters were calculated: the LMR is the ratio of lymphocytes to monocytes, the NLR is the ratio of neutrophils to lymphocytes, the MPV/PLT is the MPV divided by the PLT count, and the LYMPLT is the lymphocyte count multiplied by the platelet count. Routine blood tests are the first choice in pediatric fever clinics. They are easy to perform and inexpensive. In recent years, the lymphocyte-to-monocyte ratio (LMR), neutrophil-to-lymphocyte ratio (NLR), mean platelet volume-to-platelet ratio (MPV/PLT) and lym- phocytes multiplied by platelets (LYMPLT) value have been used as new inflammatory markers to predict the prognosis of infectious diseases [6–8], tumors [9, 10] and cardiovascular diseases [11, 12]; these markers have been widely studied in clinical practice. Detection of influenza virus nucleic acids using RT-PCR A fully automatic nucleic acid extractor and the associ- ated reagents (Shanghai ZJ Bio-Tech Co., Ltd.) were used to extract all nucleic acids from pharyngeal swabs. Throat swab specimens obtained for the purpose of the influenza virus nucleic acid determination were sub- jected to RT-PCR, and influenza A and B virus nucleic acid detection kits were used (Z-RR-0097-02, Shanghai ZJ Bio-Tech Co., Ltd.). The amplification system used a final volume of 25 μl, consisting of 19 μl of a mixture of influenza A and B virus nucleic acid fluorescent probes, 1 μl of the enzyme, and 5 μl of the sample. The amplifi- cation conditions were as follows: reverse transcription at 45 °C for 10 min, predenaturation at 95 °C for 15 min, Some studies have investigated routine blood index values and influenza A infection in adults, but few studies have been conducted in children. In this study, we performed early (within 48 h of the onset of fever) measurements of routine blood parameters in children with fever and influenza-like symptoms, and further Zhu et al. BMC Infectious Diseases (2020) 20:864 Zhu et al. BMC Infectious Diseases (2020) 20:864 Page 3 of 10 (P > 0.05), only 3 patients were included in the < 1-year- old B+ group, and thus a statistical analysis was not performed (Table 1). Detection of routine blood parameters denaturation at 95 °C for 15 s, and annealing, elongation and fluorescence detection at 60 °C for 60 s for 45 cycles. All amplification reactions were performed with an ABI7500 quantitative PCR instrument (Applied Biosys- tems, Inc., USA). Patient characteristics The diagnosis of influenza infection is based on the pres- ence of influenza-like symptoms with a positive RT-PCR result for influenza A or B [14]. In the present study, 388 children with influenza A virus infection (A+ group), 169 children with influenza B virus infection (B+ group), 198 children with influenza-like symptoms who were negative for both influenza A and B viruses (A-B- group) and 259 healthy children (H group) who underwent physical exam- inations at the same time were included. All children were divided into three groups according to age: the < 1-year- old group, the 1- to 6-year-old group and the > 6-year-old group. In each age group, statistically significant differ- ences in the age and sex distributions were not observed among the four clinical groups (A+, B+, A−/B- and H) Statistical analysis Only 3 patients were included in the < 1-year-old B+ group, and thus a statistical analysis was not performed (Table 2). The red blood cell count (RBC) and hemoglobin (Hb) level in the A+ group, B+ group, A-B- group and H group were not significantly different in the subgroups of patients < 1 year old, 1–6 years old and > 6 years old (Tables 2, 3 and 4). Compared with the H group, the A+ group, B+ group, and A-B- group had lower LYM counts, PLT counts, LMRs and LYMPLT values, and higher NLRs and MPV/PLT values (Tables 2, 3 and 4). In the < 1-year-old subgroup, no significant differences in the LYM count, PLT count, LMR, NLR, LYMPLT and MPV/PLT were observed between the A+ group and A-B- group. In the 1- to 6-year-old sub- group, the LYM count, PLT count, LMR, LYMPLT and MPV/PLT were significantly different between the A+ group and the A-B- group, and the LYM count, LMR and LYMPLT were significantly different between the B+ group and the A-B- group (Fig. 1 and Table 3). In the > 6-year-old subgroup, the LYM count, LMR, NLR and LYMPLT were significantly different between the A+ group and the A-B- group; the PLT count, LMR and LYMPLT were significantly different between the B+ group and the A-B- group (Fig. 1 and Table 4). SPSS 25.0 software (SPSS Inc., Chicago, IL, USA) was used to analyze the data. Continuous variables were expressed as the mean ± standard when normally distributed or the median (interquartile range) when non-normally distrib- uted. Categorical variables were expressed as frequencies. Comparisons of continuous variables groups were per- formed with t-tests or Wilcoxon tests. The One-way ANOVA or Kruskal-Wallis test was used to analyze differ- ences in continuous variables among groups. The Pearson χ2 test or Fisher’s exact test was applied to analyze categor- ical variables. A receiver operating characteristic (ROC) curve was constructed to evaluate the diagnostic value of the LYM count, PLT count, LMR, NLR, LYMPLT and MPV/PLT for influenza A and B virus infection. Statistical significance was indicated by P < 0.05. A+ group, influenza A virus infection; B+ group, influenza B virus infection; A-B- group, influenza-like symptoms who were negative f viruses; H group, healthy children The 1- to 6-year-old a+ group The variable that best predicted positivity for influenza virus A infection based on the area under the curve (AUC) was the LMR. When the A-B- group was used as a reference, the cutoff value was 3.75, the AUC was 0.886, and the sensitivity and specificity were 81.87 and Table 1 The baseline characteristics of patients Table 1 The baseline characteristics of patients A+ group (n = 388) B+ group (n = 169) A- /B- group (n = 198) H group (n = 259) χ2/H P < 1 year old Males 11 1 12 21 0.384 0.825 Females 7 2 7 17 Mean age (y) 0.8 (0.5–0.8) 0.6 ± 0.2 0.7 (0.6–0.9) 0.6 (0.5–0.8) 2.146 0.342 1–6 years old Males 98 24 49 62 1.218 0.749 Females 95 29 53 54 Mean age (y) 3.7 (2.4–4.8) 4.0 (3.1–4.9) 3.4 (2.4–4.6) 3.4 (2.4–5.1) 3.740 0.291 > 6 years old Males 104 53 33 57 2.415 0.060 Females 73 60 44 48 Mean age (y) 8.6 (7.2–11.8) 7.9 (7.1–9.8) 8.6 (6.8–11.7) 9.4 (7.5–11.5) 6.531 0.088 A+ group, influenza A virus infection; B+ group, influenza B virus infection; A-B- group, influenza-like symptoms who were negative for both influenza A and B viruses; H group, healthy children A+ group, influenza A virus infection; B+ group, influenza B virus infection; A-B- group, influenza-like symptoms who were negative for both influenza A and B viruses; H group, healthy children Zhu et al. BMC Infectious Diseases (2020) 20:864 Zhu et al. The > 6-year-old a+ group y g p The maximum AUC was obtained for the LMR. Using the A-B- group as a reference, the cutoff value was 3.05, the AUC was 0.949, and the sensitivity and specificity were 89.27 and 89.61%, respectively. When the H group was used a reference, the cutoff value was 3.09, the AUC The 1- to 6-year-old a+ group BMC Infectious Diseases ( Page 4 of 10 Table 2 Hematological parameters of the four groups in the < 1-year-old group Parameters A+ group (1) B+ group A−/B- group (2) H group (3) F/H P WBC (109/L) 7.25 ± 2.62a 7.62 ± 2.81 6.49 ± 1.89 9.14 ± 2.07 10.939 0.000 NEU (%) 45.26 ± 13.36a 34.00 ± 16.70 37.53 ± 16.13 19.74 ± 8.22 39.214 0.000 LYM (%) 36.72 ± 12.79a 51.00 ± 19.08 45.58 ± 17.76 68.05 ± 8.34 42.247 0.000 LYM(109/L) 2.72 ± 1.49a 3.98 ± 2.63 2.88 ± 1.36 6.28 ± 1.84 41.597 0.000 MON (%) 16.44 ± 4.88a 14.00 ± 2.00 14.21 ± 3.91 7.18 ± 2.68 48.749 0.000 MON (109/L) 1.18 ± 0.53a 1.02 ± 0.26 0.89 ± 0.29 0.65 ± 0.25 21.414 0.000 NLR 1.57 ± 1.18a 0.84 ± 0.74 1.14 ± 1.03 0.31 ± 0.18 40.717 0.000 LMR 2.09 (1.50–3.02)a 3.80 ± 1.86 2.55 (1.94–3.49) 10.28 (8.15–11.62) 52.816 0.000 RBC (1012/L) 4.50 ± 0.51 4.56 ± 0.26 4.56 ± 0.32 4.44 ± 0.80 0.120 0.887 Hb (g/dl) 12.05 ± 0.10 11.93 ± 0.64 12.10 ± 0.73 12.03 ± 0.74 0.260 0.878 PLT (109/L) 284.56 ± 66.36a 287.33 ± 85.85 264.47 ± 60.26 366.39 ± 91.08 13.117 0.000 LYMPLT 781.16 ± 508.13a 1040.84 ± 461.46 741.64 ± 339.72 2379.39 ± 1134.19 45.140 0.000 MPV/PLT 0.037 ± 0.013 0.036 ± 0.014 0.038 ± 0.009 0.028 ± 0.008 9.469 0.000 WBC White blood cell count, NEU NeutrophilL, LYM Lymphocyte, MON Monocyte, NLR Neutrophil-to-lymphocyte, LMR Lymphocyte-to-monocyte, RBC Red blood cell count, Hb Hemoglobin, PLT Platelet, LYMPLT Lymphocyte platelet, MPV/PLT Mean platelet volume/platelet ratio aCompared with the H group, P < 0.05 WBC White blood cell count, NEU NeutrophilL, LYM Lymphocyte, MON Monocyte, NLR Neutrophil-to-lymphocyte, LMR Lymphocyte-to-monocyte, RBC Red blood cell count, Hb Hemoglobin, PLT Platelet, LYMPLT Lymphocyte platelet, MPV/PLT Mean platelet volume/platelet ratio aCompared with the H group, P < 0.05 were 73.58 and 84.31%, respectively. When the H group was used as the reference, the cutoff value for the LMR was 4.47, the AUC was 0.918, and the sensitivity and specificity were 86.79 and 89.66%, respectively (Fig. 3). 84.31%, respectively. The 1- to 6-year-old a+ group When the H group was used as the reference, the maximum AUC was obtained for the LYMPLT (followed by the LMR); the cutoff value of the LYMPLT was 680.48, the AUC was 0.958, and the sen- sitivity and specificity were 90.67 and 89.66%, respect- ively (Fig. 2). The 1- to 6-year-old B+ group The variable that best predicted positivity for influenza virus B based on the AUC was the LMR. When the A-B- group was used as a reference, the cutoff value was 3.71, the AUC was 0.843, and the sensitivity and specificity Table 3 Hematological parameters of the four groups in the 1- to 6-year-old group Parameters A+ group B+ group A−/B- group H group F/H P WBC (109/L) 6.55 ± 2.33ab 6.35 ± 2.50ab 7.77 ± 2.94 7.94 ± 1.77 44.353 0.000 NEU (%) 59.44 ± 15.72a 56.69 ± 14.53a 57.69 ± 14.60 38.33 ± 11.82 124.595 0.000 LYM (%) 0.048 28.43 ± 14.16a 30.94 ± 12.60a 31.13 ± 13.35 50.85 ± 12.38 74.420 0.000 LYM (109/L) 1.53 (1.05–2.11)ab 1.68 (1.28–2.37)ab 2.13 (1.44–3.00) 3.89 (3.04–4.99) 185.768 0.000 MON (%) 11.00 (8.00–14.00)a 11.00 (7.65–14.15)a 9.00 (8.00–12.00) 7.00 (6.00–8.00) 96.138 0.000 MON (109/L) 0.62 (0.47–0.89)a 0.60 (0.44–0.84)a 0.69 (0.50–0.94) 0.55 (0.44–0.65) 22.076 0.000 NLR 2.38 (1.33–3.95)a 1.86 (1.14–3.08)a 2.01 (1.1–3.71) 0.74 (0.49–1.11) 134.530 0.000 LMR 2.47 (1.58–3.38)ab 2.93 (1.95–4.07)ab 5.88 (4.01–8.71) 6.96 (5.85–9.09) 235.215 0.000 RBC (1012/L) 4.58 ± 0.34 4.59 ± 0.33 4.56 ± 0.31 4.62 ± 0.28 0.731 0.534 Hb (g/dl) 12.59 ± 0.81 12.59 ± 0.81 12.47 ± 0.73 12.63 ± 0.67 1.031 0.379 PLT (109/L) 216.99 ± 65.96)ab 225.57 ± 64.79)a 239.40 ± 65.54 322.91 ± 69.79 65.139 0.000 LYMPLT 316.14 (209.46–496.97)ab 383.61 (229.00–566.41)ab 446.81 (305.83–730.05) 1210.24 (957.75–1655.55) 217.522 0.000 MPV/PLT 0.046 (0.039–0.058)ab 0.045 (0.034–0.055)a 0.043 (0.035–0.049) 0.030 (0.025–0.035) 132.718 0.000 aCompared with the H group, P < 0.05 bCompared with the A−/B- group, P < 0.05 Table 3 Hematological parameters of the four groups in the 1- to 6-year-old group Zhu et al. The 1- to 6-year-old B+ group BMC Infectious Diseases (2020) 20:864 Page 5 of 10 Table 4 Hematological parameters of the four groups in the > 6-year-old group Parameters A+ group B+ group A−/B- group H group F/ H P WBC (109/L) 6.86 ± 2.11 6.70 ± 2.20 7.44 ± 2.68 6.90 ± 1.75 4.400 0.221 NEU (%) 69.16 ± 11.95ab 65.21 ± 12.56a 62.46 ± 15.42 48.37 ± 10.03 141.857 0.000 LYM (%) 16.20 (11.25–24.85)ab 20.20 (14.20–27.30)a 23.30 (16.00–31.55) 41.30 (35.60–47.65) 175.453 0.000 LYM(109/L) 1.22 ± 0.61ab 1.41 ± 0.67a 1.74 ± 1.01 2.74 ± 0.71 187.333 0.000 MON(%) 10.97 ± 3.80a 11.23 ± 4.23a 10.35 ± 4.26 7.09 ± 2.21 90.097 0.000 MON(109/L) 0.74 ± 0.32a 0.73 ± 0.31a 0.70 ± 0.26 0.48 ± 0.16 69.427 0.000 NLR 4.41 (2.53–6.83)ab 3.35 (2.11–5.17)a 2.63 (1.73–4.68) 1.16 (0.87–1.56) 165.854 0.000 LMR 1.59 (1.06–2.26)ab 1.94 (1.34–2.61)ab 6.15 (4.11–9.62) 5.78 (4.47–7.38) 281.453 0.000 RBC (1012/L) 4.72 ± 0.49 4.76 ± 0.55 4.69 ± 0.31 4.78 ± 0.36 1.957 0.120 Hb (g/dl) 13.29 ± 0.88 13.37 ± 0.88 13.26 ± 0.88 13.44 ± 0.95 0.819 0.484 PLT (109/L) 227.70 ± 52.55a 218.50 ± 53.60ab 235.36 ± 56.33 291.42 ± 61.12 38.839 0.000 LYMPLT 242.16 (157.50–367.07)ab 265.86 (188.80–398.38)ab 337.69 (242.28–547.83) 769.29 (576.34–998.95) 196.469 0.000 MPV/PLT 0.044 (0.037–0.055)a 0.045 (0.038–0.056)a 0.042 (0.035–0.054) 0.034 (0.031–0.040) 59.784 0.000 aCompared with the H group, P < 0.05 bCompared with the A−/B- group, P < 0.05 was used a reference, the cutoff value of the LMR was 3.48, the AUC was 0.954, and the sensitivity and specifi- city were 90.27 and 92.38%, respectively (Fig. 5). was 0.975, and the sensitivity and specificity were 90.40 and 95.24%, respectively (Fig. 4). Discussion Influenza viruses can be divided into three types accord- ing to the nucleocapsid protein and the matrix protein antigen: A, B and C. No cross-immunity exists among these types. The type with the greatest antigen variability The maximum AUC was obtained for the LMR. Using the A-B- group as a reference, the cutoff value was 2.88, the AUC was 0.924, and the sensitivity and specificity were 83.19 and 92.21%, respectively. When the H group Fig. 1 Differences in lymphocyte (LYM) count, platelet (PLT) count, lymphocyte-to-monocyte ratio (LMR), neutrophil-to-lymphocyte ratio (NLR), lymphocyteplatelet (LYMPLT) and mean platelet volume-to-platelet ratio (MPV/PLT) values in the A+, B+, A-B- and H group. * P < 0.05, ** P < 0.01 Fig. 1 Differences in lymphocyte (LYM) count, platelet (PLT) count, lymphocyte-to-monocyte ratio (LMR), neutrophil-to-lymphocyte ratio (NLR), lymphocyteplatelet (LYMPLT) and mean platelet volume-to-platelet ratio (MPV/PLT) values in the A+, B+, A-B- and H group. * P < 0.05, ** P < 0.01 Zhu et al. BMC Infectious Diseases (2020) 20:864 Zhu et al. BMC Infectious Diseases Page 6 of 10 Fig. 2 ROC curves of LYM count, PLT count, LMR, NLR, LYMPLT and MPV/PLT values in the 1- to 6 -year -old A+ group (a. the A-B- group as a reference; b. the H group as the reference) Fig. 2 ROC curves of LYM count, PLT count, LMR, NLR, LYMPLT and MPV/PLT values in the 1- to 6 -year -old A+ group (a. the A-B- group as a reference; b. the H group as the reference) children are similar to infections with other respiratory pathogens and are nonspecific, including a high fever, chills, muscle aches, sore throat, cough, and runny nose. Gastrointestinal symptoms such as vomiting, abdominal pain, and diarrhea are relatively common in children in- fected with influenza B [14]. Patients with mild symptoms typically recover within a short time, and patients with severe symptoms rapidly develop dyspnea accompanied by refractory hypoxemia and may eventually develop acute respiratory distress syndrome, septic shock, heart failure, is influenza A, which often causes regional outbreaks and epidemics and might even cause a global pandemic. Over the past 100 years, influenza A outbreaks have occurred seasonally and have caused several global pan- demics. The most serious pandemic was the Spanish H1N1 influenza pandemic in 1918, which killed 50 mil- lion people [15, 16]. Influenza B has weak antigen vari- ability, and it often causes moderate epidemics or local outbreaks. Discussion Influenza C rarely infects humans [17]. The clinical symptoms of influenza virus infections in Fig. 3 ROC curves of LYM count, PLT count, LMR, NLR, LYMPLT and MPV/PLT values in the 1- to 6 -year -old B+ group (a. the A-B- group as a reference; b. the H group as the reference) Fig. 3 ROC curves of LYM count, PLT count, LMR, NLR, LYMPLT and MPV/PLT values in the 1- to 6 -year -old B+ group (a. the A-B- group as a reference; b. the H group as the reference) Zhu et al. BMC Infectious Diseases (2020) 20:864 Page 7 of 10 Fig. 4 ROC curves of LYM count, PLT count, LMR, NLR, LYMPLT and MPV/PLT values in the > 6 -year -old A+ group (a. the A-B- group as a reference; b. the H group as the reference) Fig. 4 ROC curves of LYM count, PLT count, LMR, NLR, LYMPLT and MPV/PLT values in the > 6 -year -old A+ group (a. the A-B- group as a reference; b. the H group as the reference) 100% [19], respectively. RT-PCR has now become the “gold standard” for the diagnosis of influenza. Although RT-PCR takes less time than virus isolation and culture, it is still expensive and takes several hours, limiting its use as a routine method for screening for influenza in pediatric fever clinics. Serological testing requires two serum samples from both the acute phase and the convalescent phase. Convalescent blood samples should be collected 2–4 weeks after the onset of the disease. If the antibody level is more than 4 times higher in the convalescent phase than in the acute phase, the patient is diagnosed with influenza. Obviously, this approach is acute necrotizing encephalopathy, and multiple organ dysfunction, which are life-threatening and even fatal con- ditions [18]. Therefore, a rapid and simple index for the early diagnosis of influenza infection in children is needed. acute necrotizing encephalopathy, and multiple organ dysfunction, which are life-threatening and even fatal con- ditions [18]. Therefore, a rapid and simple index for the early diagnosis of influenza infection in children is needed. The diagnosis of influenza mainly depends on the detection of viral nucleic acids and antibodies. Virus iso- lation and culture were previously the “gold standard” for the diagnosis of influenza, but this process is time- consuming and expensive, has high technical require- ments and is difficult to perform. Discussion In the present study, a significantly lower PLT count was observed in the influenza A group than in the group of children who were negative for both influenza A and influenza B in the 1- to 6-year-old sub- group, consistent with the results reported by Fei et al. [8]. However, there was no significant difference be- tween the group infected with influenza B and the group of children who were negative for both influenza A and influenza B. Among the children older than 6 years, the PLT count was not statistically significantly different between the group infected with influenza A, the group infected with influenza B and the group of children who were negative for both influenza A and influenza B. Thus, the PLT count has predictive value only for chil- dren aged 1 to 6 years who are infected with influenza A; furthermore, its predictive value is low, even in that group, with an AUC of 0.615 and sensitivity and specifi- city of 58.03 and 63.73%, respectively. In recent years, researchers have attempted to combine the PLT count with other indicators, such as LYMPLT and MPV/PLT, for disease prediction [30]. Fei et al. [8] reported better predictive value of the LYMPLT and MPV/PLT for children aged < 6 years who were infected with influenza A, and the predictive value was greater than the LMR. However, the LMR had the highest predictive value for influenza infection, followed by the LYMPLT, PLT count, and MPV/PLT, among children over the age of 1 year. Leukocytes such as neutrophils and monocytes (MON) protect the host from influenza infection by releasing preformed cytokines, and the granule contents help hosts eliminate the threat posed by replicating viruses. Coskun O et al. [25] recommended monocytosis as a surrogate marker for infection with influenza A virus. In the present study, there was no statistically sig- nificant difference in the MON count between children older than 1 year who were infected with either influenza A or B viruses and children who were not infected with influenza viruses. The LMR is the ratio of the LYM Recently, the NLR was shown to be positively associated with systemic inflammation [6], acute pancreatitis [31], liver disease [32] and rheumatic diseases [33]. The NLR exhibits a high sensitivity for the detection of influenza virus infection [34]. Discussion RT-PCR is the most effective nucleic acid detection technology for influenza, with a sensitivity and specificity as high as 98.5 and The diagnosis of influenza mainly depends on the detection of viral nucleic acids and antibodies. Virus iso- lation and culture were previously the “gold standard” for the diagnosis of influenza, but this process is time- consuming and expensive, has high technical require- ments and is difficult to perform. RT-PCR is the most effective nucleic acid detection technology for influenza, with a sensitivity and specificity as high as 98.5 and Fig. 5 ROC curves of LYM count, PLT count, LMR, NLR, LYMPLT and MPV/PLT values in the > 6 -year -old B+ group (a. the A-B- group as a reference; b. the H group as the reference) Fig. 5 ROC curves of LYM count, PLT count, LMR, NLR, LYMPLT and MPV/PLT values in the > 6 -year -old B+ group (a. the A-B- group as a reference; b. the H group as the reference) Zhu et al. BMC Infectious Diseases (2020) 20:864 Page 8 of 10 Page 8 of 10 count to the MON count, and an LMR < 2 has been used as a surrogate marker for influenza A infection [26, 27]. Based on the findings from the present study, the LMR is the best index for the prediction of influenza virus infec- tion. At the same time, the AUCs for the prediction of influenza A and influenza B infections were higher in children aged > 6 years than in children aged 1–6 years, suggesting that the diagnostic value of the LMR for influ- enza is greater in children aged > 6 years than in children aged < 6 years. In addition, the AUC of the LMR for predicting an influenza A infection was higher than that for predicting an influenza B infection in the > 1-year-old subgroup, indicating that the predictive value of the LMR was greater for influenza A than that for influenza B. not suitable for influenza screening in outpatients. Influenza virus antigen detection, such as rapid influenza diagnostic tests, are able to be performed within 30 min, but the sensitivity is only 40–70% [5]. Routine blood tests are the most common tests performed in pediatric fever clinics and have been used as the primary method for identifying bacterial and viral infections. Discussion In recent years, researchers have performed an in-depth analysis of various routine blood parameters, and these parameters are useful for the early diagnosis and prognostic assessment of other diseases [6–12]. Influenza strains infect respiratory epithelial cells, and the attachment of the virus to cells via sialic acid recep- tors enables uptake of the virus into the cells, followed by the recognition of the virus by pattern recognition receptors (PRRs). PRRs trigger cytokine responses and protective immunity, but they might also contribute to immune pathology [20]. Lymphocytes are the main immune cells involved in the elimination of viruses. In conventional viral infections, the proportion of lympho- cytes in the circulation is usually increased. Previous studies have reported a significant decrease in LYM counts in patients infected with influenza A [8, 21, 22], but few studies have examined LYM counts in patients infected with influenza B. According to Nichols et al. [23], LYM induced self-apoptosis by regulating the ex- pression of FasL on the cell surface and released soluble FasL after influenza infection, leading to a decrease in the LYM count. In the present study, a significantly lower LYM count was observed in children with an in- fluenza A or B infection than in children aged 1–6 years with influenza-like symptoms who tested negative for influenza A and B viruses, and no significant difference in the LYM count was observed between children with influenza A and influenza B infections. In children > 6 years old infected with influenza A, the LYM count was significantly lower than in children who were not infected with influenza viruses. The LYM count was not signifi- cantly different between children infected with influenza virus B and children who were not infected with either influenza A or B viruses. Lewis et al. [24] suggested that lymphopenia is mainly due to a reduction in T cells and, to a lesser extent, B cells and has a short duration. g PLT have been reported regulate host immunity and complement responses in the initial intrinsic defense against influenza virus infection [28], and researchers have explored the different mechanisms by which virus infection can interfere with PLT production and might trigger PLT destruction [29], subsequently decreasing the PLT count. References 1. World Health Organization. Up to 650000 people died of respiratory diseases linked to seasonal FLU each year [EB/OL]. [2017-12-14]. 2. Grohskopf LA, Alyanak E, Broder KR, et al. Prevention and control of seasonal influenza with vaccines: recommendations of the advisory committee on immunization practices - United States, 2019-20 influenza season. MMWR Recomm Rep. 2019;68(3):1–21. Availability of data and materials All data generated or analysed during this study are included in this published article. Abbreviations LYM L h 7. Geun Ho O, Chung SP, Park YS, et al. Mean platelet volume to platelet count ratio as a promising predictor of early mortality in severe sepsis. Shock. 2017;47(3):323–30. LYM: Lymphocyte; PLT: Platelet; LMR: Lymphocyte-to-monocyte ratio; NLR: Neutrophil-to-lymphocyte ratio; MPV/PLT: Mean platelet volume-to- platelet ratio; AUC: Area under the curve; LYMPLT: Lymphocytes multiplied by platelets; EBV: Epstein-Barr virus; ROC: Receiver operating characteristic; A+ group: Influenza A virus infection; B+ group: Influenza B virus infection; A-B- group: Negative for both influenza A and B viruses; H group: Healthy children; RBC: Red blood cell count; Hb: Hemoglobin; PRRs: Pattern recognition receptors; MON: Monocytes 8. Fei Y, Zhang H, Zhang C. The application of lymphocyteplatelet and mean platelet volume/platelet ratio in influenza a infection in children. J Clin Lab Anal. 2019;33(9):e22995. 9. Lalosevic MS, Markovic AP, Stankovic S, et al. Combined diagnostic efficacy of neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), and mean platelet volume (MPV) as biomarkers of systemic inflammation in the diagnosis of colorectal cancer. Dis Markers. 2019;2019:6036979. 9. Lalosevic MS, Markovic AP, Stankovic S, et al. Combined diagnostic efficacy of neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), and mean platelet volume (MPV) as biomarkers of systemic inflammation in the diagnosis of colorectal cancer. Dis Markers. 2019;2019:6036979. Acknowledgements ff 10. Li K-J, Xia X-F, Meng S, et al. Predictive value of lymphocyte-to-monocyte ratio (LMR) and neutrophil-to-lymphocyte ratio (NLR) in patients with oesophageal cancer undergoing concurrent chemoradiotherapy. BMC Cancer. 2019;19(1):1004. 10. Li K-J, Xia X-F, Meng S, et al. Predictive value of lymphocyte-to-monocyte ratio (LMR) and neutrophil-to-lymphocyte ratio (NLR) in patients with oesophageal cancer undergoing concurrent chemoradiotherapy. BMC Cancer. 2019;19(1):1004. We thank the First Affiliated Hospital of Wenzhou Medical University for the help and thank all the patients included in this study. Author details 1 f 1Department of Pediatrics, the First Affiliated Hospital of Wenzhou Medical University, South Baixiang, Ouhai District, Wenzhou 325000, Zhejiang, China. 2Department of Pediatrics, Yiwu Maternity and Children Hospital, No. C100 Xinke Road, Yiwu, Jinhua 322000, Zhejiang, China. 3Department of Pediatrics, People’s Hospital of Yuhuan, No. 18 Changle Road, Yucheng Street, Yuhuan, Taizhou 317600, Zhejiang, China. Received: 6 August 2020 Accepted: 4 November 2020 Received: 6 August 2020 Accepted: 4 November 2020 Received: 6 August 2020 Accepted: 4 November 2020 Conclusions A significantly lower LMR was observed in children older than 1 year who had influenza, particularly chil- dren older than 6 years infected with influenza A, than in children without influenza. The LMR is potentially useful as an early predictor of influenza A infection in children older than 6 years, with an AUC of 0.949, a sensitivity of 89.27% and a specificity of 89.61%. 3. Jules A, Carlos G Grijalva, Yuwei Zhu, et al. Influenza-related hospitalization and ED visits in children less than 5 years: 2000-2011. Pediatrics. 2015;135(1):e66–74. 4. Demir SÖ, Atıcı S, Kadayifci EK, et al. Influenza a (H1N1)-associated severe complications; hemolytic uremic syndrome, myocarditis, acute necrotizing encephalopathy. J Infect Dev Ctries. 2019;13(1):83–6. 4. Demir SÖ, Atıcı S, Kadayifci EK, et al. Influenza a (H1N1)-associated severe complications; hemolytic uremic syndrome, myocarditis, acute necrotizing encephalopathy. J Infect Dev Ctries. 2019;13(1):83–6. 5. Simeonsson K, Moore Z. Prevention and control of influenza: no easy task. N C Med J. 2013;74(5):425 427-33. p p y 5. Simeonsson K, Moore Z. Prevent C Med J. 2013;74(5):425 427-33. 5. Simeonsson K, Moore Z. Prevention and control of influenza: no easy task. N C Med J. 2013;74(5):425 427-33. 5. Simeonsson K, Moore Z. Prevent C Med J. 2013;74(5):425 427-33. 6. Russell CD, Parajuli A, Gale HJ, et al. The utility of peripheral blood leucocyte ratios as biomarkers in infectious diseases: a systematic review and meta- analysis. J Inf Secur. 2019;78(5):339–48. Competing interests Th h h The authors have no conflicts of interest to disclose. The authors have no conflicts of interest to disclose. Ethics approval and consent to participate This study was approved by the Ethics Committee for Clinical Research of the First Affiliated Hospital of Wenzhou Medical University (No. 2020–065), and consent to participate was obtained from the parents/guardians of the minors included in this study. Funding No funds. Not applicable. Funding No funds. Not applicable. Discussion In the present study, a significant dif- ference in the NLR was not observed among the group Zhu et al. BMC Infectious Diseases (2020) 20:864 Page 9 of 10 Zhu et al. BMC Infectious Diseases (2020) 20:864 infected with influenza A, the group infected with influ- enza B and the group of children who were negative for both influenza A and influenza B in the 1–6-year-old sub- group. In the subgroup of children aged > 6 years, the NLR was significantly higher in the group infected with influenza A than in the group negative for both influenza A and influenza B. However, no significant difference was observed between the group infected with influenza B and the group of children who were negative for both influ- enza A and influenza B in the > 6-year-old subgroup. Based on our findings, the NLR has predictive value only for children aged > 6 years who are infected with influenza A, with an AUC of 0.657 and a sensitivity and specificity of 58.19 and 70.13%, respectively. In addition, no signifi- cant differences in the LYM count, PLT count, LMR, NLR, LYMPLT and MPV/PLT were observed between the group infected with influenza A and the group of chil- dren who were negative for influenza A and influenza B in the < 1-year-old subgroup. Due to the small sample size, a statistical analysis was not performed to compare the influenza B group with the other groups among children < 1 year old. Thus, the routine blood parameters displayed poor predictive value for influenza in the < 1-year-old age group. We propose that this discrepancy may be related to the immune function and development of blood cells in children, and requires further investigation. infected with influenza A, the group infected with influ- enza B and the group of children who were negative for both influenza A and influenza B in the 1–6-year-old sub- group. In the subgroup of children aged > 6 years, the NLR was significantly higher in the group infected with influenza A than in the group negative for both influenza A and influenza B. However, no significant difference was observed between the group infected with influenza B and the group of children who were negative for both influ- enza A and influenza B in the > 6-year-old subgroup. Discussion Based on our findings, the NLR has predictive value only for children aged > 6 years who are infected with influenza A, with an AUC of 0.657 and a sensitivity and specificity of 58.19 and 70.13%, respectively. In addition, no signifi- cant differences in the LYM count, PLT count, LMR, NLR, LYM*PLT and MPV/PLT were observed between the group infected with influenza A and the group of chil- dren who were negative for influenza A and influenza B in the < 1-year-old subgroup. Due to the small sample size, a statistical analysis was not performed to compare the influenza B group with the other groups among children < 1 year old. Thus, the routine blood parameters displayed poor predictive value for influenza in the < 1-year-old age group. We propose that this discrepancy may be related to the immune function and development of blood cells in children, and requires further investigation. Authors’ contributions Iida H, Kaibori M, Iida KM, et al. Ratio of mean platelet volume to platelet count is a potential surrogate marker predicting liver cirrhosis. World J Hepatol. 2018;10(1):82–7. 30. Iida H, Kaibori M, Iida KM, et al. Ratio of mean platelet volume to platelet count is a potential surrogate marker predicting liver cirrhosis. World J Hepatol. 2018;10(1):82–7. 31. Jeon TJ, Park JY. Clinical significance of the neutrophil-lymphocyte ratio as an early predictive marker for adverse outcomes in patients with acute pancreatitis. World J Gastroenterol. 2017;23(21):3883–9. 31. Jeon TJ, Park JY. Clinical significance of the neutrophil-lymphocyte ratio as an early predictive marker for adverse outcomes in patients with acute pancreatitis. World J Gastroenterol. 2017;23(21):3883–9. 32. Fan Z, EnQiang C, Yao DL, et al. Neutrophil-lymphocyte ratio predicts short term mortality in patients with hepatitis B virus-related acute-on-chronic liver failure treated with an artificial liver support system. PLoS One. 2017; 12(4):e0175332. 32. Fan Z, EnQiang C, Yao DL, et al. Neutrophil-lymphocyte ratio predicts short term mortality in patients with hepatitis B virus-related acute-on-chronic liver failure treated with an artificial liver support system. PLoS One. 2017; 12(4):e0175332. 33. Yang W, Wang X, ZhangYang W, et al. Neutrophil-lymphocyte ratio and platelet-lymphocyte ratio are 2 new inflammatory markers associated with pulmonary involvement and disease activity in patients with dermatomyositis. Clin Chim Acta. 2017;465:11–6. 34. Zhang Y, Zou P, Zhang HG, et al. Neutrophil-lymphocyte ratio as an early new marker in AIV-H7N9-infected patients: a retrospective study. Ther Clin Risk Manag. 2019;15:911–9. 34. Zhang Y, Zou P, Zhang HG, et al. Neutrophil-lymphocyte ratio as an early new marker in AIV-H7N9-infected patients: a retrospective study. Ther Clin Risk Manag. 2019;15:911–9. Authors’ contributions Pediatric influenza. Pediatr Nurs. 2009;35(6): 335–45. 18. Bramley AM, Bresee J, Finelli L. Pediatric influenza. Pediatr Nurs. 2009;35(6): 335–45. 19. Cho CH, Woo MK, Kim JY, et al. Evaluation of five rapid diagnostic kits for influenza a/B virus. J Virol Methods. 2013;187(1):51–6. 19. Cho CH, Woo MK, Kim JY, et al. Evaluation of five rapid diagnostic kits for influenza a/B virus. J Virol Methods. 2013;187(1):51–6. 20. Short KR, Kasper J, van der Stijn A, et al. Influenza virus damages the alveolar barrier by disrupting epithelial cell tight junctions. Eur Respir J. 2016;47(3):954–66. 20. Short KR, Kasper J, van der Stijn A, et al. Influenza virus damages the alveolar barrier by disrupting epithelial cell tight junctions. Eur Respir J. 2016;47(3):954–66. 21. Cunha BA, Syed U, Strollo S. Non-specific laboratory test indicators of severity in hospitalized adults with swine influenza (H1N1) pneumonia. Eur J Clin Microbiol Infect Dis. 2010;29(12):1583–8. 22. Biçer S, Sariçoban HE, Özen AO, et al. Experience of influenza a H1N1 in a paediatric emergency unit. Infez Med. 2015;23(2):125–33. 23. Nichols JE, Niles JA, Roberts NJ Jr. Human lymphocyte apoptosis after exposure to influenza a virus. J Virol. 2001;75(13):5921–9. 23. Nichols JE, Niles JA, Roberts NJ Jr. Human lymphocyte apoptosis exposure to influenza a virus. J Virol. 2001;75(13):5921–9. 24. Lewis DE, Gilbert BE, Knight V. Influenza virus infection induces functional alterations in peripheral blood lymphocytes. J Immunol. 1986;137(12):3777–81. 24. Lewis DE, Gilbert BE, Knight V. Influenza virus infection induces functional alterations in peripheral blood lymphocytes. J Immunol. 1986;137(12):3777–81. 25. Coskun O, Avci IY, Sener K, et al. Relative lymphopenia and monocytosis may be considered as a surrogate marker of pandemic influenza a (H1N1). J Clin Virol. 2010;47(4):388–9. 26. Cunha BA, Connolly JJ, Irshad N. The clinical usefulness of lymphocyte: monocyte ratios in differentiating influenza from viral non-influenza-like illnesses in hospitalized adults during the 2015 influenza a (H3N2) epidemic: the uniqueness of HPIV-3 mimicking influenza a. Eur J Clin Microbiol Infect Dis. 2016;35(1):155–8. 27. McClain MT, Park LP, Nicholson B, et al. Longitudinal analysis of leukocyte differentials in peripheral blood of patients with acute respiratory viral infections. J Clin Virol. 2013;58(4):689–95. 28. Koupenova M, Corkrey HA, Vitseva O, et al. The role of platelets in mediating a response to human influenza infection. Nat Commun. 2019; 10(1):1780. 29. Assinger A. Platelets and infection - an emerging role of platelets in viral infection. Front mmunol. 2014;5:649. 30. Authors’ contributions 11. Gong S, Gao X, Xu F, et al. Association of lymphocyte to monocyte ratio with severity of coronary artery disease. Medicine (Baltimore). 2018;97(43): e12813. 11. Gong S, Gao X, Xu F, et al. Association of lymphocyte to monocyte ratio with severity of coronary artery disease. Medicine (Baltimore). 2018;97(43): e12813. RZ: study design, managed the experiments, analyzed the results, was involved in manuscript preparation. CC: data collection, analyzed the results, and was involved in manuscript preparation. QW: data collection, analyzed the results. XZ: analyzed the results, and was involved in manuscript preparation. CL: was involved in data collection and analysis. YS: study design, data collection, analyzed the results, and manuscript writing. Statement: all authors have read and approved the manuscript, and ensure that this is the case. 12. Kose N, Akin F, Yildirim T, et al. The association between the lymphocyte-to- monocyte ratio and coronary artery disease severity in patients with stable coronary artery disease. Eur Rev Med Pharmacol Sci. 2019;23(6):2570–5. 12. Kose N, Akin F, Yildirim T, et al. The association between the lymphocyte-to- monocyte ratio and coronary artery disease severity in patients with stable coronary artery disease. Eur Rev Med Pharmacol Sci. 2019;23(6):2570–5. 13. WHO. Human infection with pandemic (H1N1) 2009 virus: updated interim WHO guidance on global surveillance. Geneva: World Health Organisation; 2009. Page 10 of 10 Zhu et al. BMC Infectious Diseases (2020) 20:864 14. Paules C, Subbarao K. Influenza. Lancet. 2017;390(10095):697–708. 14. Paules C, Subbarao K. Influenza. Lancet. 2017;390(10095):697–708 15. Van Kerkhove MD, Mumford E, Mounts AW, et al. Highly pathogenic avian influenza (H5N1): pathways of exposure at the animal-human interface, a systematic review. PLoS One. 2011;6(1):e14582. 16. Biggerstaff M, Cauchemez S, Reed C, et al. Estimates of the reproduction number for seasonal, pandemic, and zoonotic influenza: a systematic review of the literature. BMC Infect Dis. 2014;14:480. 16. Biggerstaff M, Cauchemez S, Reed C, et al. Estimates of the reproduction number for seasonal, pandemic, and zoonotic influenza: a systematic review of the literature. BMC Infect Dis. 2014;14:480. 17. Nesmith N, Williams JV, Johnson M, et al. Sensitive diagnostics confirm that influenza C is an uncommon cause of medically attended respiratory illnes in adults. Clin Infect Dis. 2017;65(6):1037–9. 17. Nesmith N, Williams JV, Johnson M, et al. Sensitive diagnostics confirm that influenza C is an uncommon cause of medically attended respiratory illness in adults. Clin Infect Dis. 2017;65(6):1037–9. 18. Bramley AM, Bresee J, Finelli L. Zhu et al. BMC Infectious Diseases (2020) 20:864 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
https://openalex.org/W1966656796	https://www.frontiersin.org/articles/10.3389/fphy.2014.00054/pdf	English	null	The effect of macromolecular crowding on mobility of biomolecules, association kinetics, and gene expression in living cells	Frontiers in physics	2,014	cc-by	16,653	The effect of macromolecular crowding on mobility of biomolecules, association kinetics, and gene expression in living cells Marcin Tabaka 1, Tomasz Kalwarczyk 1, Jedrzej Szymanski 2, Sen Hou 1,3 and Robert Holyst 1* 1 Department of Soft Condensed Matter and Fluids, Institute of Physical Chemistry of the Polish Academy of Sciences, Warsaw, Poland 2 Laboratory of Bioenergetics and Biomembranes, Nencki Institute of Experimental Biology Polish Academy of Sciences, Warsaw, Poland 3 State Key Labaratory of Medicinal Chemical Biology, Nankai University, Tianjin, China Marcin Tabaka 1, Tomasz Kalwarczyk 1, Jedrzej Szymanski 2, Sen Hou 1,3 and Robert Holyst 1* 1 Department of Soft Condensed Matter and Fluids, Institute of Physical Chemistry of the Polish Academy of Sciences, Warsaw, Poland 2 Laboratory of Bioenergetics and Biomembranes, Nencki Institute of Experimental Biology Polish Academy of Sciences, Warsaw, Poland 3 State Key Labaratory of Medicinal Chemical Biology, Nankai University, Tianjin, China We discuss a quantitative inﬂuence of macromolecular crowding on biological processes: motion, bimolecular reactions, and gene expression in prokaryotic and eukaryotic cells. We present scaling laws relating diffusion coefﬁcient of an object moving in a cytoplasm of cells to a size of this object and degree of crowding. Such description leads to the notion of the length scale dependent viscosity characteristic for all living cells. We present an application of the length-scale dependent viscosity model to the description of motion in the cytoplasm of both eukaryotic and prokaryotic living cells. We compare the model with all recent data on diffusion of nanoscopic objects in HeLa, and E. coli cells. Additionally a description of the mobility of molecules in cell nucleus is presented. Finally we discuss the inﬂuence of crowding on the bimolecular association rates and gene expression in living cells. PHYSICS PHYSICS REVIEW ARTICLE published: 18 September 2014 doi: 10.3389/fphy.2014.00054 The effect of macromolecular crowding on mobility of biomolecules, association kinetics, and gene expression in living cells Reviewed by: Reviewed by: Antonio Scialdone, Wellcome Trust Sanger Institute & EMBL-EBI, UK Andrzej Stasiak, University of Lausanne, Switzerland www.frontiersin.org Keywords: macromolecular crowding, biologistics, biomolecules diffusion, kinetics of biomolecules in vivo, gene expression *Correspondence: Marcin Tabaka, Tomasz Kalwarczyk and Robert Holyst, Department of Soft Condensed Matter and Fluids, Institute of Physical Chemistry of the Polish Academy of Sciences, Kasprzaka 44/52, Warsaw 01-224, Poland Keywords: macromolecular crowding, biologistics, biomolecules diffusion, kinetics of biomolecules in vivo, gene expression e-mail: rholyst@ichf.edu.pl; mtabaka@ichf.edu.pl; tkalwarczyk@ichf.edu.pl 1. INTRODUCTION mobility [21–24], protein folding [8, 10, 23], and protein-protein interactions [10, 11, 24]. The milieu of cytoplasm is commonly considered as a highly crowded and concentrated environment that impacts the kinet- ics and thermodynamics of biochemical reactions [1–11]. The effect of macromolecular crowding on reaction rates and equi- librium constants may arise from: (i) change of reactants’ mobil- ity [12–15], (ii) depletion interaction (excluded volume effect) that results in attraction between reactants [1, 8, 16–18], and (iii) non-speciﬁc chemical interaction with crowders [9, 10, 19, 20]. Over three decades of studies of crowded systems, both at the theoretical and experimental level, contributed signiﬁcantly to our understanding of these effects. However, the quantita- tive impact of every component seems to be still elusive in living cells. In this review, we describe recent in vivo stud- ies of biomolecules’ mobilities and their interactions. We focus mostly on differences between diffusion constants in cytoplasms of prokaryotic and eukaryotic cells and their nuclei as well as on kinetics of bimolecular protein-protein and protein-DNA interactions, with gene expression in particular. We refer to the- oretical models or in vitro systems only in the introductory parts and when necessary to make this review self-supported. Therefore, at the beginning of the review, we give a short intro- duction to our own contribution to this ﬁeld of research i.e., motion of probes in synthetic complex liquids. The material pre- sented in this paper does not overlap signiﬁcantly with many informative reviews written in the last few years that concern theoretical and in vitro experimental contributions to protein Edited by: Francesco Piazza, University of Orléans, France Reviewed by: Antonio Scialdone, Wellcome Trust Sanger Institute & EMBL-EBI, UK Andrzej Stasiak, University of Lausanne, Switzerland Francesco Piazza, University of Orléans, France 2. CURRENT STATUS OF RESEARCH 2.1. MOTION IN SYNTHETIC CROWDED ENVIRONMENT Concentration of the polymer was 20% by weight. Two characteristic length scales, ξ and Rh determined the viscosity experienced by the probe. For probes whose rp was far smaller than ξ the probe diffused like in the solvent. For probes whose rp was longer than Rh, the experienced viscosity reached the same values as the macroscopic viscosity of the solution. In the inset we showed absolute values of the diffusion coefﬁcient for the same probes in the same polymer solution. The upper dotted line corresponded to the diffusion coefﬁcient calculated according to the SSE equation (Equation 4) with η equal to the viscosity of the solvent η0. Bottom dashed line corresponded to the diffusion coefﬁcient calculated according to the SSE equation (Equation 4) with η = ηm (where ηm denoted the macroscopic viscosity). For probes whose hydrodynamic radius was few times larger than Rh, assumption that in Equation (4) η = ηm seemed reasonable. For probes whose rp ≲Rh, assumption that η = ηm failed in the same manner as the assumption that η = η0. Number averaged molecular weight of the polymer Mn = 10944 g/cm . Concentration of the polymer was 20% by weight. Two characteristic length scales, ξ and Rh determined the viscosity experienced by the probe. For probes whose rp was far smaller than ξ the probe diffused like in the solvent. For probes whose rp was longer than Rh, the experienced viscosity reached the same values as the macroscopic viscosity of the solution. In the inset we showed absolute values of the diffusion coefﬁcient for the same probes in the same polymer solution. The upper dotted line corresponded to the diffusion coefﬁcient calculated according to the SSE equation (Equation 4) with η equal to the viscosity of the solvent η0. Bottom dashed line corresponded to the diffusion coefﬁcient calculated according to the SSE equation (Equation 4) with η = ηm (where ηm denoted the macroscopic viscosity). For probes whose hydrodynamic radius was few times larger than Rh, assumption that in Equation (4) η = ηm seemed reasonable. For probes whose rp ≲Rh, assumption that η = ηm failed in the same manner as the assumption that η = η0. D = kT 6πηeffrp , (4) (4) where k denotes Boltzmann constant and T is the absolute tem- perature. 2. CURRENT STATUS OF RESEARCH 2.1. MOTION IN SYNTHETIC CROWDED ENVIRONMENT In considered polymer matrix, probes of rp < Rh expe- rience effective viscosity that is much lower than the macroscopic viscosity ηm. On the contrary, probes with rp ≫Rh experience a viscosity equal to ηm. It is noteworthy that the value of the diffusion coefﬁcient D = D0 is never reached for the physically meaningful range of probe sizes. For example, the diffusion coef- ﬁcient of the ﬂuorescent dye of rp = 0.58 nm is around 14 times smaller than that in a pure solvent (Figure 1). of living cell is sometimes described as a poroelastic material [28, 29]. Kalwarczyk et al. [14] used the concept of the length-scale dependent viscosity, represented by Equation 2, to study the vis- cosity of the cytoplasm of mammalian cells and diffusion therein. In synthetic systems, control over the diffusion rate of probes could be achieved by changes of concentration, c and of size, Rh of objects crowding the given system (see also Equations 2 and 3). In living organisms this was not the case as concentration of the crowding objects and their size were constant (at given growth conditions). In cells Equation 2 had to be rewritten as a func- tion of rp only. The Equation 2, therefore, took the following form: Diffusion coefﬁcient of the probe particles strongly depends on the structure of the ﬂuid. In Figure 2 we compare diffu- sion coefﬁcient of hypothetical probes in polymer (same as for Figure 1) and in solutions of elongated and rigid micelles of non- ionic surfactant C12E6. The ratio c/c∗for both systems is equal to 14.7 and corresponds to concentration of 14% for C12E6 and 37.5% for polymer. We calculate the diffusion coefﬁcients using Equation 2 as described in Kalwarczyk et al. [14]. Parameters a and b, differing between systems, are taken from Kalwarczyk et al. [14]. Calculated diffusion coefﬁcient of probes in surfactant systems are from 2 to 50 times higher than diffusion coefﬁcients for polymer solutions proving that the network composed of rigid micelles is more permeable for moving probes than dense polymer matrix. f rp = A exp ⎡ ⎣ ξ2 R2 h + ξ2 r2p −a/2⎤ ⎦ (5) (5) Frontiers in Physics \| Biophysics 2. CURRENT STATUS OF RESEARCH 2.1. MOTION IN SYNTHETIC CROWDED ENVIRONMENT Complex liquids, in particular polymer solutions, are often considered as systems mimicking crowded environment of the cellular interior. Until recently however there has not been a single model of diffusion in synthetic complex liquids that could be applied to living systems. We have proposed [14] the phenomenological description of the diffusion in the complex ﬂuid, either synthetic or biological, as well as the viscosity of those complex systems. The model is primarily applied to polymers [14, 25], to micellar systems [14] and recently to colloidal sys- tems [26]. In this model the viscosity is deﬁned via the ﬂuctuation dissipation theorem: D0 D = f = ηeff η0 . (1) (1) Here D0 is the diffusion coefﬁcient in a pure solvent of viscosity η0 and D is the diffusion coefﬁcient in a crowded environment. ηeff denotes the viscosity that is effectively “felt” by the particle dif- fusing in the crowded environment. Here f is the hydrodynamic drag in a crowded environment divided by the drag in water. f is a function of the length scales Reff and ξ, characterizing the whole system (the probe and the environment), given by the Equation 2: Here D0 is the diffusion coefﬁcient in a pure solvent of viscosity η0 and D is the diffusion coefﬁcient in a crowded environment. ηeff denotes the viscosity that is effectively “felt” by the particle dif- fusing in the crowded environment. Here f is the hydrodynamic drag in a crowded environment divided by the drag in water. f is a function of the length scales Reff and ξ, characterizing the whole system (the probe and the environment), given by the Equation 2: September 2014 \| Volume 2 \| Article 54 \| 1 www.frontiersin.org Tabaka et al. Macromolecular crowding FIGURE 1 \| Relative diffusion coefﬁcient (diffusion coefﬁcient divided by its value in pure water) as a function of the hydrodynamic radius rp of the hypothetical probe diffusing in the polyethylene glycol solution. Number averaged molecular weight of the polymer Mn = 10944 g/cm3. Concentration of the polymer was 20% by weight. Two characteristic length scales, ξ and Rh determined the viscosity experienced by the probe. For probes whose rp was far smaller than ξ the probe diffused like in the solvent. For probes whose rp was longer than Rh, the experienced viscosity reached the same values as the macroscopic viscosity of the solution. 2. CURRENT STATUS OF RESEARCH 2.1. MOTION IN SYNTHETIC CROWDED ENVIRONMENT In the inset we showed absolute values of the diffusion coefﬁcient for the same probes in the same polymer solution. The upper dotted line corresponded to the diffusion coefﬁcient calculated according to the SSE equation (Equation 4) with η equal to the viscosity of the solvent η0. Bottom dashed line corresponded to the diffusion coefﬁcient calculated according to the SSE equation (Equation 4) with η = ηm (where ηm denoted the macroscopic viscosity). For probes whose hydrodynamic radius was few times larger than Rh, assumption that in Equation (4) η = ηm seemed reasonable. For probes whose rp ≲Rh, assumption that η = ηm failed in the same manner as the assumption that η = η0. f (Reff, ξ) = exp b Reff ξ a , (2) FIGURE 1 \| Relative diffusion coefﬁcient (diffusion coefﬁcient divided by its value in pure water) as a function of the hydrodynamic radius rp of the hypothetical probe diffusing in the polyethylene glycol solution. Number averaged molecular weight of the polymer M = 10944 g/cm3 where Reff and ξ are deﬁned as: where Reff and ξ are deﬁned as: R−2 eff = r−2 p + R−2 h , ξ = Rg c c∗ −β . (3) (3) In Equation 3, rp and Rh are the hydrodynamic radii of the probe particle and of the polymer, respectively. ξ is the corre- lation length (a mean distance between entanglement points in polymer matrix) and Rg denotes gyration radius of the polymer coil. c and c∗are polymer concentration and the overlap con- centration, respectively. β is the exponent related to the Flory’s exponent ν: β = ν/ (3ν −1). Exponent a (cf. Equation 2) is a constant of the order of 1, and b is temperature dependent parameter [27]. FIGURE 1 \| Relative diffusion coefﬁcient (diffusion coefﬁcient divided by its value in pure water) as a function of the hydrodynamic radius rp of the hypothetical probe diffusing in the polyethylene glycol solution. 3 In Figure 1 we show the plot of the diffusion coefﬁcient of a hypothetical, globular, and inert probe in a polyethylene gly- col solution as a function of rp, calculated according to Equation 2 for a constant concentration (20%), molecular weight of the polymer (10.94 kg/mol) and temperature (25°C). The diffusion coefﬁcient takes value given by the Stokes-Sutherland-Einstein formula: Number averaged molecular weight of the polymer Mn = 10944 g/cm3. 2.2. MOTION IN THE CYTOPLASM OF EUKARYOTES Curves were generated using Equation (2) with parameters: a = 0.62, b = 2.42 for polymer solutions, and a = 0.87, b = 0.9 for surfactant solutions; for details see [14]. FIGURE 2 \| Diffusion coefﬁcient as a function of the probe’s FIGURE 3 \| Relative viscosity (η/η0) that is equivalent to the reciprocal of the diffusion coefﬁcient (D0/D) for various probes diffusing in the cytoplasm of HeLa cells. Black points correspond to the data [149–152] discussed in Kalwarczyk et al. [14] and the red solid line represent the best ﬁt (Equation 5) to those data with: ξ ≈5 nm and Rh ≈86 nm marked as red dashed and blue dotted lines, respectively, [for details please see [14]]. Reproduced with permission from reference 14. Copyright 2011 American Chemical Society. Colored points represent data published recently for carbon nanotubes (CNT) [32], green ﬂuorescent protein (GFP) [33], enhanced green ﬂuorescent protein (EGFP) [34], oligomer of ten GFP proteins (GFP10) [29], ﬂuorescent dye – 5-chloromethylﬂuorescein diacetate (CMFDA) [29], and quantum dots stabilized with bovine serum albumin protein (BSA-QD) [31]. The carbon nanotubes, having huge aspect ratio, during the motion experience the hydrodynamic friction resulting not only from translational but also from the rotational motion. Increased effective friction is responsible for the increase of the effective viscosity experienced by the nanotubes with respect to the model represented by Equation (5). We expect that the globular object of rp corresponding to rp of the nanotubes will experience the viscosity predicted by the Equation (5). The CMFDA dye (the point marked by asterisk) in the cytoplasm can react with an intracellular thiols [36] leading to underestimation of the size of the probe and apparent deviations from the predictions. FIGURE 3 \| Relative viscosity (η/η0) that is equivalent to the reciprocal of the diffusion coefﬁcient (D0/D) for various probes diffusing in the t l f H L ll Bl k i t d t th d t [149 152] to the hydrodynamic radius of the actin ﬁlaments in the cyto- plasm of HeLa cells. In Swiss 3T3 cells ξ = 7 ± 2 nm while in HeLa ξ ≈5 [14]. Although the physical meaning of ξ for living cells remains unclear its value correlates with the radius of the cytoplasmic water pores in the Swiss 3T3 cells that is equal to 7 nm Luby- Phelps et al. [30]. 2.2. MOTION IN THE CYTOPLASM OF EUKARYOTES FIGURE 2 \| Diffusion coefﬁcient as a function of the probe’s hydrodynamic radius, rp, in aqueous solution of the ﬂexible polymer polyethylene glycol, and of the micelles formed of non-ionic surfactant C12E6. The c/c∗ratio (concentration divided by the overlap concentration) for both systems was set the same and equal to 14.67, and corresponded to the concentrations of 14 and 37.5% for surfactant and for polymer, respectively. Curves were generated using Equation (2) with parameters: a = 0.62, b = 2.42 for polymer solutions, and a = 0.87, b = 0.9 for surfactant solutions; for details see [14]. FIGURE 3 \| Relative viscosity (η/η0) that is equivalent to the reciprocal of the diffusion coefﬁcient (D0/D) for various probes diffusing in the cytoplasm of HeLa cells. Black points correspond to the data [149–152] discussed in Kalwarczyk et al. [14] and the red solid line represent the best ﬁt (Equation 5) to those data with: ξ ≈5 nm and Rh ≈86 nm marked as red dashed and blue dotted lines, respectively, [for details please see [14]]. Reproduced with permission from reference 14. Copyright 2011 American Chemical Society. Colored points represent data published recently for carbon nanotubes (CNT) [32], green ﬂuorescent protein (GFP) [33], enhanced green ﬂuorescent protein (EGFP) [34], oligomer of ten GFP FIGURE 2 \| Diffusion coefﬁcient as a function of the probe’s hydrodynamic radius, rp, in aqueous solution of the ﬂexible polymer polyethylene glycol, and of the micelles formed of non-ionic surfactant C12E6. The c/c∗ratio (concentration divided by the overlap concentration) for both systems was set the same and equal to 14.67, and corresponded to the concentrations of 14 and 37.5% for surfactant and for polymer, respectively. Curves were generated using Equation (2) with parameters: a = 0.62, b = 2.42 for polymer solutions, and a = 0.87, b = 0.9 for surfactant solutions; for details see [14]. FIGURE 2 \| Diffusion coefﬁcient as a function of the probe’s hydrodynamic radius, rp, in aqueous solution of the ﬂexible polymer polyethylene glycol, and of the micelles formed of non-ionic surfactant C12E6. The c/c∗ratio (concentration divided by the overlap concentration) for both systems was set the same and equal to 14.67, and corresponded to the concentrations of 14 and 37.5% for surfactant and for polymer, respectively. 2.2. MOTION IN THE CYTOPLASM OF EUKARYOTES The same value for the radius of the water pores is reported by Moeendarbary et al. [29]. Indeed such interpreta- tion is in agreement with the interpretation of ξ for the synthetic systems (polymer solutions) as a distance between entanglement points that is proportional to the size of a water channel in the polymer network. cytoplasm of HeLa cells [32]. Equations 2 and 5 are valid only for probes of globular shape, so that the rotational diffusion of those object, occurring at much shorter time-scale, do not inﬂuence the translational motion of the probes. In D ∝F−1, where F is an effective friction given by the sum of translational, Ft, and rota- tional Fr friction. In case of globular objects Ft ≫Fr and F ≈Ft. Nanotubes are characterized by huge aspect ratio and their rota- tional diffusion in the cytoplasm is strongly hindered and affects translational motion. In general Fr is not negligible with respect to Ft and the effective friction F experienced by the nanotubes is increased by the rotational friction felt by the nanotubes. As the value of ξ in mammalian cells is around 7 nm, proteins with typical radius of 4 nm, experience the viscosity comparable to the viscosity of water. In that sense for most proteins, the cyto- plasm can not be regarded as the crowded environment. Larger probes with radius exceeding ξ, e.g., ribosomes or protein aggre- gates, experience the effective viscosity ηeff (Equations 1–5) that results from the crowding. Very recent data on diffusion in HeLa cells [29, 31–34] appear to conﬁrm, to some extent, Kalwarczyk’s predictions (cf. Figure 3). Deviations from the predictions, are observed for small ﬂuorescent dye 5-chloromethylﬂuorescein diacetate (CMFDA) [29]. This dye, inside the cell, is enzymatically cleaved and become ﬂuorescent [35]. On the other hand the chloromethyl group of the CMFDA dye can react with intracellular thiols in a reaction mediated by glutathione S-transferase [36], whose hydrodynamic radius is roughly around 2.6 nm. The CMFDA dye, therefore, can not be regarded as freely diffusive in the cellular interior and its intracellular size is probably underestimated and lead to devia- tions shown in Figure 3. Another deviation toward the higher val- ues of viscosity is observed for carbon nanotubes diffusing in the 2.2. MOTION IN THE CYTOPLASM OF EUKARYOTES with ξ and Rh as free parameters. A was a pre-exponential fac- tor of the order of one. Kalwarczyk et al. [14] used Equation 5 to describe the effective viscosity experienced by probes during their motion in the cytoplasm of Swiss 3T3 and of HeLa cells. Dependence of the effective viscosity experienced by probes on their hydrodynamic radii is depicted in Figure 3. The length scale corresponding to the biggest crowding agents, Rh was comparable Although the synthetic systems may not be good experimental models to monitor biological processes, the physics governing the motion is the same for synthetic (in vitro) as well as for biolog- ical (in vivo) systems. Therefore, polymer systems, can be used as models mimicking the viscous response of the biological sys- tem to the probe’s diffusion, despite the fact that the cytoplasm September 2014 \| Volume 2 \| Article 54 \| 2 Frontiers in Physics \| Biophysics Tabaka et al. Macromolecular crowding FIGURE 3 \| Relative viscosity (η/η0) that is equivalent to the reciprocal of the diffusion coefﬁcient (D0/D) for various probes diffusing in the cytoplasm of HeLa cells. Black points correspond to the data [149–152] discussed in Kalwarczyk et al. [14] and the red solid line represent the best ﬁt (Equation 5) to those data with: ξ ≈5 nm and Rh ≈86 nm marked as red dashed and blue dotted lines, respectively, [for details please see [14]]. Reproduced with permission from reference 14. Copyright 2011 American Chemical Society. Colored points represent data published recently for carbon nanotubes (CNT) [32], green ﬂuorescent protein (GFP) [33], enhanced green ﬂuorescent protein (EGFP) [34], oligomer of ten GFP proteins (GFP10) [29], ﬂuorescent dye – 5-chloromethylﬂuorescein diacetate (CMFDA) [29], and quantum dots stabilized with bovine serum albumin protein (BSA-QD) [31]. The carbon nanotubes, having huge aspect ratio, during the motion experience the hydrodynamic friction resulting not only from translational but also from the rotational motion. Increased effective friction is responsible for the increase of the effective viscosity experienced by the nanotubes with respect to the model represented by Equation (5). We expect that the globular object of rp corresponding to rp of the nanotubes will experience the viscosity predicted by the Equation (5). The CMFDA dye (the point marked by asterisk) in the cytoplasm can react with an intracellular thiols [36] leading to underestimation of the size of the probe and apparent deviations from the predictions. www.frontiersin.org 2.3. MOTION IN THE CELL NUCLEUS The distribution of chromatin is highly regulated with active genes located at the borders of physical domains of chromosomes [38, 39]. There are many proteins involved in the maintenance of DNA molecules and they need to reach their speciﬁc locations in the nucleus to perform their function. One of the ﬁrst measurements of diffusion in a nucleus was done by Seksek et al. [40] who determined the diffusion coefﬁcients of inert probes such as dextrans, in the cytoplasm and nucleus of MDCK epithelial and 3T3 ﬁbroblast cells. They found that the diffusion coefﬁcient decreased around 4 fold in comparison to D0 for all studied probe sizes (rp = 4–30 nm), with D/D0 = 0.25 ± 0.02 (MDCK) and 0.27 ± 0.03 (3T3). In the experiments performed with oligonucleotides [43 base pairs Oligo(dA), MW ∼13 kDa]), a slight drop from D0 = 5.7 ± 0.2 × 10−7 cm2/s (in water) to D = 5.05 ± 0.52 × 10−7 cm2/s (in the nucleus) was observed (D/D0 = 0.886), showing that for inert probes with the radius of several nm diffusion in the nucleus was unhindered. At the same time, the diffusion of the probe Oligo(dT) similar in size but capable of interaction with polyadenylated RNA, was slowed down 6 times, from 5.0 ± 0.2 × 10−7 cm2/s to 0.87 ± 0.02 × 10−7 cm2/s (D/D0 = 0.174). Even slower diffusion was observed for the range of endoge- nous polyadenylated RNA (with the sizes in the range of several thousands base pairs) where the diffusion coefﬁcient was equal to 0.06 ± 0.01 × 10−7 cm2/s [41]. The movement of polyadeny- lated RNA was diffusive and the space available for diffusing molecules was restricted to the regions free from chromatin. For the range of synthetic DNA molecules of sizes ranging from 21 to 6000 base pairs (bp) an empirical formula was found [42], describing diffusion coefﬁcients, D0, of the DNA fragments in water. D0 was related empirically to DNA size with the follow- ing formula: D0 = 4.9 × 10−6 cm2/s ×[bp size]−0.72. Thus, in this size range D0 decreased with increasing size from 53 × 10−8 to 0.81 × 10−8 cm2/s. When introduced into the nucleus, these fragments were found almost immobile, likely because of the extensive binding to almost immobile obstacles (e.g., chromatin). Inert probes such as dextrans (of Mw. 2.3. MOTION IN THE CELL NUCLEUS up to 580 kDa) with the size similar to DNA fragments diffused freely in the nucleus with D/ D0 of around 0.16 (6× slower than in water). Interestingly, dex- tran of molecular weight of 2000 kDa was found immobile to the same degree as studied DNA fragments, with the D/D0 smaller than 0.01, however no exact value of diffusion coefﬁcient was reported. Some studies interpret the results on diffusion in the nucleus with the help of anomalous diffusion, where mean square dis- placement does not change linearly with time, and is described by anomaly index, α, according to the formula r2 (t) ∼tα. There are several microscopic models which can give rise to an anoma- lous diffusion behavior but physical origins of these phenomenon are still discussed [51, 52]. In the study by Wachsmuth et al. [49] measurements per- formed with ﬂuorescent proteins revealed that the diffusion of GFP was slowed down around 5 times (in AT-1 and COS-7 cells). The results could be analyzed using two models of dif- fusion: normal diffusion with two components (fast and slow) and anomalous diffusion (one diffusion time and anomaly index) [49, 53]. The obtained translational diffusion times for the fast component were similar for both models, indicating a 5 fold decrease in diffusion coefﬁcient relative to diffusion in water. The absolute value of the diffusion coefﬁcient in cytoplasm was D = 12.5(±1.3) × 10−8 cm2/s, while the two components in the nucleus moved with diffusion coefﬁcients of D1 = 9.8(±1.6) × 10−8 cm2/s, and D2 = 1.0(±0.6) × 10−8 cm2/s. For the anoma- lous diffusion model, we have D1 = 8.7(±1.0) × 10−8 cm2/s, and α = 0.87. Here D1 is given as a time dependent diffusion coefﬁcient taken at time τ1 being the diffusion time through the focal volume of size ω; D1 = D (τ1) = ω2/4τ1, where ⟨r2 (t)⟩= 6D (t) t = 6D1 (t/τ1)α. In the same study the diffusion coefﬁcient measured for protein EGFP-b-galactosidase (MW ∼80 kDa) was lower than the one for GFP (MW 27 kDa) by a factor of 1.4. Such difference could be explained by the mere difference in size of the two proteins, conﬁrming previous observations, that the relative viscosity in the nucleus was rather size independent (at least up to Mw of around 2000 kDa). 2.3. MOTION IN THE CELL NUCLEUS Genetic material of the cell is separated from the cytoplasm in a dedicated cellular compartment called nucleus. Huge amount of genetic material (around 6 × 109 base pairs) in a form of long DNA molecules (46 in human somatic cells) is packed in the nucleus with diameter of around 10 microns. Squeezing of 46 long molecules in a small volume, requires highly efﬁcient com- paction [37]. It is achieved with the help of nucleosomes leading to higher order chromatin structure which ﬁts into the nucleus. September 2014 \| Volume 2 \| Article 54 \| 3 www.frontiersin.org Macromolecular crowding Tabaka et al. apparent that many nuclear complexes undergo constant exchange with the kinetics of the order of seconds [45–48], which has a substantial effect on their diffusional mobility. For three studied proteins, HMG-17, SF2/ASF, and ﬁbrillarin, the diffusion coefﬁcients values are 0.45 × 10−8 cm2/s, 0.24 × 10−8 cm2/s, and 0.53 × 10−8 cm2/s, respectively. Molecular weight of these proteins is only about 2 times higher than that of GFP which diffuses in the nucleus with D = 9.8(±1.6) × 10−8 cm2/s [49]. Thus, D-values for HMG-17, SF2/ASF, and ﬁbrillarin are about 20–40 times lower than values reported for free solutes in the nucleus or for GFP alone indicating that interactions of these pro- teins with nuclear components slow down the diffusion. Similar results are observed for Src peptides [50], where interactions with DNA reduced signiﬁcantly the diffusion coefﬁcients measured in vivo (from D0 = 2 ± 1 × 10−7 cm2/s to Dnucleus = 4 ± 2 × 10−9 cm2/s). Before any gene can be transcribed from the compact chromatin it needs to be untangled. The structure of the chromatin in the nucleus is highly complex, with parts of accessible and inaccessi- ble chromatin, named euchromatin (open), and heterochromatin (closed), respectively. The distribution of chromatin is highly regulated with active genes located at the borders of physical domains of chromosomes [38, 39]. There are many proteins involved in the maintenance of DNA molecules and they need to reach their speciﬁc locations in the nucleus to perform their function. Before any gene can be transcribed from the compact chromatin it needs to be untangled. The structure of the chromatin in the nucleus is highly complex, with parts of accessible and inaccessi- ble chromatin, named euchromatin (open), and heterochromatin (closed), respectively. Frontiers in Physics \| Biophysics 2.3. MOTION IN THE CELL NUCLEUS Most of errorbars does not exceed an order of magnitude. FIGURE 4 \| Effective viscosity experienced by the probes diffusing in the cell nucleus [40, 42, 44, 59, 60]. Data for DNA fragments diffusing in the nucleus of the HeLa cell [42], are represented as the dashed line and corresponds to the average value calculated for the fragments of hydrodynamic radii in the range of 5–135 nm. Data for dextran polymers as probes are represented as black squares (nucleus of the Madin-Darby Canine Kidney Epithelial Cells – MDCK) and green squares (nucleus of Swiss 3T3 cells – 3T3), respectively, [40]. Data for proteins fused with GFP and moving in the nucleus of the HeLa and of the Baby Hamster Kidney (BHK) cells are marked by yellow squares [44]. The plot shows also data for the EGFP in the nucleus of Chinese Hamster Ovary (CHO) cells [59]. The data for EGFP reveal the heterogeneous structure of the nucleus. Hinde et al. [59] measure the diffusion in high-density DNA (hdDNA) and low-density DNA (ldDNA) regions. In both cases they obtain two diffusion coefﬁcients, fast and slow (D1, D2) that differ by two orders of magnitude. On the plot we show the effective viscosity corresponding to the D1 in ldDNA, D1 in hdDNA, D2 in ldDNA, and D2 in hdDNA. The heterogeneous structure of the nucleus is also represented by the data for HP1-GFP diffusing in the nucleus of 3T3 cells [60]. Both the slow and the fast component are comparable with results of Hinde et al. [59]. The top panel shows a cartoon representation of the nucleus as a heterogeneous environment consisting of densely and loosely packed DNA regions. Errorbars not shown to keep the clarity of the ﬁgure. Most of errorbars does not exceed an order of magnitude. FIGURE 4 \| Effective viscosity experienced by the probes diffusing in the cell nucleus [40, 42, 44, 59, 60]. Data for DNA fragments diffusing in the nucleus of the HeLa cell [42] are represented as the dashed line and constitutes of the regions poor and rich in DNA. In both regions the DNA may be densely packed (heterochromatin) forming a ﬁbrinal structure, or loosely packed (eurchromatin). In the DNA poor region, the motion of small probe is not hindered and the probe diffuses in soup of proteins and other small (macro)molecules. 2.3. MOTION IN THE CELL NUCLEUS On the plot we show the effective viscosity corresponding to the D1 in ldDNA, D1 in hdDNA, D2 in ldDNA, and D2 in hdDNA. The heterogeneous structure of the nucleus is also represented by the data for HP1-GFP diffusing in the nucleus of 3T3 cells [60]. Both the slow and the fast component are comparable with results of Hinde et al. [59]. The top panel shows a cartoon representation of the nucleus as a heterogeneous environment consisting of densely and loosely packed DNA regions. Errorbars not shown to keep the clarity of the ﬁgure. Most of errorbars does not exceed an order of magnitude. nucleus to be size independent and interpret results within the model of the fractal geometry of chromatin [57], whereas other results indicate some dependence of nuclear diffusion on the size of the diffusing object [42, 56]. Interesting data were reported for the different regions of chromatin revealing differences between euchromatin and het- erochromatin. Less compact euchromatin was shown to have higher fractal dimension than heterochromatin. The diffusion in the euchromatin region was slowed down around 3 times (rel- ative to diffusion in water), whereas in heterochromatin region slowing down was almost 6 fold for the tracer molecule consist- ing of GFP multimers. The chromatin architecture was described as fractal-like on the length scales smaller than ∼100 nm, giving rise to size-independence of the relative diffusion coefﬁcient since most proteins or protein complexes were smaller than 100 nm. Another interpretation of diffusive motion of GFP in the nucleus was given by Hinde et al. [59], who used a normal diffusion model with two components in the pair correlation function anal- ysis to image diffusional barriers and passages. For the region of low DNA density (euchromatin) they obtained two different mobilities with diffusion coefﬁcient D1 = 22.8 × 10−8 cm2/s and D2 = 0.3 × 10−8 cm2/s. In the high DNA density (heterochro- matin) regions, obtained values were D1 = 23 × 10−8 cm2/s and D2 = 0.48 × 10−8 cm2/s. Thus, the diffusion characteristics of EGFP in the nucleus was rather constant and did not depend on DNA density, however relative contribution of slow com- ponent was higher in the heterochromatin region. Yet slightly different results were found in the study by Capoulade et al. [60], where a diffusional mapping in 3T3 cells was performed with the use of selective plane illumination microscope. 2.3. MOTION IN THE CELL NUCLEUS The ratio of cytoplasmic to nuclear diffusion coefﬁcients of GFP was 1.28(=12.5/9.8), similar to the value of 1.22 measured for another protein TIF-IA [54] indicating that diffusion of a given protein was often slower in the nucleus than in the cytoplasm with slightly higher degree of crowding in the nucleus, however some studies report similar degree of molecular crowding in both compartments [55]. Unhindered diffusion for inert probes in wide range of sizes rp indicated that water channel in the nucleus are larger than 30 nm. Similar behavior is observed in lamellar phases of ionic surfac- tant with water channels >150 nm [43]. Probably water channels are most ﬁlled with proteins at the volume fractions φ = 0.1– 0.2 (suggested by 2–8 fold decrease of the mobility for most proteins). Generally the diffusion of the molecules that do not bind to chromatin is slowed down 2–8 times [40, 41, 44, 49, 56–58], whereas more hindered diffusion is observed for protein that interact with chromatin. Some studies report the diffusion in the The mobility measured for several nuclear proteins tagged with GFP [44] reveal their highly dynamic nature. It became September 2014 \| Volume 2 \| Article 54 \| 4 Frontiers in Physics \| Biophysics Tabaka et al. Macromolecular crowding FIGURE 4 \| Effective viscosity experienced by the probes diffusing in the cell nucleus [40, 42, 44, 59, 60]. Data for DNA fragments diffusing in the nucleus of the HeLa cell [42], are represented as the dashed line and corresponds to the average value calculated for the fragments of hydrodynamic radii in the range of 5–135 nm. Data for dextran polymers as probes are represented as black squares (nucleus of the Madin-Darby Canine Kidney Epithelial Cells – MDCK) and green squares (nucleus of Swiss 3T3 cells – 3T3), respectively, [40]. Data for proteins fused with GFP and moving in the nucleus of the HeLa and of the Baby Hamster Kidney (BHK) cells are marked by yellow squares [44]. The plot shows also data for the EGFP in the nucleus of Chinese Hamster Ovary (CHO) cells [59]. The data for EGFP reveal the heterogeneous structure of the nucleus. Hinde et al. [59] measure the diffusion in high-density DNA (hdDNA) and low-density DNA (ldDNA) regions. In both cases they obtain two diffusion coefﬁcients, fast and slow (D1, D2) that differ by two orders of magnitude. 2.3. MOTION IN THE CELL NUCLEUS They measured the diffusion coefﬁcients for the heterochromatin pro- tein 1 (HP1α-EGFP) in the nucleus of 3T3 ﬁbroblasts. Results indicated the differences between euchromatin and heterochro- matin with respect to the fast (D1) and slow (D2) components of the diffusive pool of HP1α. Different slow diffusion coefﬁcients were obtained for euchromatin (D2 = 0.33 ± 0.16 × 10−8 cm2/ s) and for heterochromatin (D2 = 0.16 ± 0.07 × 10−8 cm2/s), whereas fast diffusion coefﬁcient D1 = 8.1 × 10−8 cm2/s was similar for both chromatin regions. Slow diffusion compo- nent (D2) was taken as a measure of interaction of HP1α- EGFP with chromatin. Weaker interaction of HP1α-EGFP with euchromatin was indicated by 2 fold higher mobility in this region. FIGURE 4 \| Effective viscosity experienced by the probes diffusing in the cell nucleus [40, 42, 44, 59, 60]. Data for DNA fragments diffusing in the nucleus of the HeLa cell [42], are represented as the dashed line and corresponds to the average value calculated for the fragments of hydrodynamic radii in the range of 5–135 nm. Data for dextran polymers as probes are represented as black squares (nucleus of the Madin-Darby Canine Kidney Epithelial Cells – MDCK) and green squares (nucleus of Swiss 3T3 cells – 3T3), respectively, [40]. Data for proteins fused with GFP and moving in the nucleus of the HeLa and of the Baby Hamster Kidney (BHK) cells are marked by yellow squares [44]. The plot shows also data for the EGFP in the nucleus of Chinese Hamster Ovary (CHO) cells [59]. The data for EGFP reveal the heterogeneous structure of the nucleus. Hinde et al. [59] measure the diffusion in high-density DNA (hdDNA) and low-density DNA (ldDNA) regions. In both cases they obtain two diffusion coefﬁcients, fast and slow (D1, D2) that differ by two orders of magnitude. On the plot we show the effective viscosity corresponding to the D1 in ldDNA, D1 in hdDNA, D2 in ldDNA, and D2 in hdDNA. The heterogeneous structure of the nucleus is also represented by the data for HP1-GFP diffusing in the nucleus of 3T3 cells [60]. Both the slow and the fast component are comparable with results of Hinde et al. [59]. The top panel shows a cartoon representation of the nucleus as a heterogeneous environment consisting of densely and loosely packed DNA regions. Errorbars not shown to keep the clarity of the ﬁgure. www.frontiersin.org 2.4. MOTION IN THE CYTOPLASM OF PROKARYOTES In Figure 5 we added the recent data [78] on diffusion in the cytoplasm of E. coli which conﬁrmed the model predictions [15]. metabolism-dependent [81, 82] because ATP synthesis inhibition causes signiﬁcant quantitative change in the mobility of these complexes. The anomaly index, α, is 0.7 ± 0.1 for RNA-protein complexes [79] and 0.39 ± 0.04 for chromosomal loci [80]. The chromosomal loci are conﬁned by virtue of their connection with chromosome and movement of the speciﬁc locus depends on motion of its neighbors. Weber et al. [80] apply the model of a polymer in viscoelastic environment and show that the conﬁnement is not solely responsible for anomality from the normal diffusion [83]. Parry et al. [82] demonstrate for protein- protein aggregates (fusion of avian reovirus protein μNS with GFP), that the value of the anomaly index, α, decreases with increasing particle size above 40 nm. Below that size the diffusion is normal. Additionally, they observe very strong probes con- ﬁnement in ATP-depleted cells (treated with 2,4-dinitrophenol, DNP) with much stronger decrease in the anomaly index and the diffusion constant of the aggregates than in untreated cells. The same conﬁnement is observed in a stationary phase of growth and under carbon starvation of cells. Parry et al. [82] interpret these results as emerging from cytoplasm properties that are characteristic of glass-forming liquids. They suggest that high macromolecular crowding in E. coli, cell conﬁnement, and macromolecule interactions are responsible for glassy dynamics. All the observed deviation of particle diffusion from normal- ity is only for macromolecules of sizes above 40 nm. On the contrary, other studies of protein aggregates [78] and large plas- mids [75] show that diffusion of these macromolecules does not deviate from normal diffusion (we include them in Figure 5). The source of these discrepancies is currently unknown and more quantitative measurements for the same conditions are needed [82]. Diffusion in the cytoplasm of E. coli is very sensitive to the size of the particle. The probes whose radii lie between ξ and Rh, experience the viscosity that increases exponentially with the particle size rp. Therefore, even small proteins (i.e., GFP whose rp = 2.8 nm) experience viscosity of around 12 times higher than the viscosity of water leading to 12 times slower diffusion. The protein with twice larger rp diffuse 35 times slower than in water. 2.4. MOTION IN THE CYTOPLASM OF PROKARYOTES Size of the water channel ξ in the cytoplasm of eukaryotes is of the order of several nanometers. Prokaryotes, however, are usually about ten times smaller than eukaryotic cells and their cytoplasm is roughly 3 times more crowded. Concentration of macro- molecules is of the order of 10% (by volume) for eukaryotes and 30% for prokaryotes. An increase in the concentration of macro- molecules is reﬂected by much lower ξ value for prokaryotes than for eukaryotes. FIGURE 5 \| The logarithm of the effective relative viscosity (η/η0 = D0/D) experienced by the probe particles in the cytoplasm o E. coli. Plot shows the literature data for the reciprocal of diffusion coefﬁcients [12, 13, 65–71, 73–77] (black pints), the ﬁt of Equation (5) to the data, and the data of Coquel et al. [78]. The yellow shaded area corresponds to the maximal error of ﬁtting. The plot is adopted from Kalwarczyk et al. [15] with a permission granted under the Creative Common license v3.0. The new data points obtained by Coquel et al. [78] are marked in green. D0 is the diffusion coefﬁcient in water of viscosity η0. FIGURE 5 \| The logarithm of the effective relative viscosity FIGURE 5 \| The logarithm of the effective relative viscosity (η/η0 = D0/D) experienced by the probe particles in the cytoplasm o E. coli. Plot shows the literature data for the reciprocal of diffusion coefﬁcients [12, 13, 65–71, 73–77] (black pints), the ﬁt of Equation (5) to the data, and the data of Coquel et al. [78]. The yellow shaded area corresponds to the maximal error of ﬁtting. The plot is adopted from Kalwarczyk et al. [15] with a permission granted under the Creative Common license v3.0. The new data points obtained by Coquel et al. [78] are marked in green. D0 is the diffusion coefﬁcient in water of viscosity η0. \| η/η0 = D0/D) experienced by the probe particles in the cyto Kalwarczyk et al. [15] observed almost 10 fold decrease of ξ in E. coli with respect to HeLa and Swiss 3T3 cells. They collected literature data [12, 13, 65–77] of diffusion coefﬁcients in the cyto- plasm of E. coli and used Equation 5 to ﬁt the data. From this ﬁt they determined ξ of around 0.5 nm and Rh ≈42 nm [15]. It was found to correspond to the radius of the DNA loops. 2.3. MOTION IN THE CELL NUCLEUS When the same probe gets into the region rich in a loosely packed DNA, (eurchromatin) its motion is drasti- cally hindered due to the hindering effect of the DNA strands. A similar but not the same situation occurs in regions rich in highly packed DNA (heterochromatin). Due to the highly packed structure of the DNA the probe may diffuse inside the ﬁbers forming the heterochromatin as is partially suggested by Hinde et al. [59]. In Figure 4 we plotted the data for the effective viscosity expe- rienced by the probes during the diffusion across the nucleus for several types of probes. The effective viscosity was calculated as the reciprocal of the relative diffusion coefﬁcient provided in Seksek et al. [40], Lukacs et al. [42], Phair and Misteli [44], and Hinde et al. [59]. Hydrodynamic radius of the DNA frag- ments was calculated from the relation taken from the work of Kalwarczyk et al. [15]: rp = 0.024M0.57 w ; where Mw corresponded to molecular weight of the DNA. Hydrodynamic radius of dex- tran polymers was calculated from the relation obtained on the basis of experiments of Ioan et al. [61]: rp = 0.072M0.45 w . Nucleus is involved in multiple functions in the living cells, ranging from most important gene expression to regulation of cell cycle and mechanotransduction. The dynamic structure of the nucleus is a subject of intense studies and signiﬁcant insight has been achieved in past decades of research [62–64]. Diffusion has been shown to be a major mechanism of protein transport Some speculations about the mechanisms leading to the dis- crepancies in the nuclear diffusion of small molecules are pre- sented below. The structure of the nucleus is heterogenous and September 2014 \| Volume 2 \| Article 54 \| 5 www.frontiersin.org www.frontiersin.org Tabaka et al. Macromolecular crowding FIGURE 5 \| The logarithm of the effective relative viscosity (η/η0 = D0/D) experienced by the probe particles in the cytoplasm o E. coli. Plot shows the literature data for the reciprocal of diffusion coefﬁcients [12, 13, 65–71, 73–77] (black pints), the ﬁt of Equation (5) to the data, and the data of Coquel et al. [78]. The yellow shaded area corresponds to the maximal error of ﬁtting. The plot is adopted from Kalwarczyk et al. [15] with a permission granted under the Creative Common license v3.0. The new data points obtained by Coquel et al. 2.3. MOTION IN THE CELL NUCLEUS [78] are marked in green. D0 is the diffusion coefﬁcient in water of viscosity η0. in the nucleus and further studies are needed to understand and quantitatively describe the dynamic structure of this organelle. We expect that the model of length-scale dependent viscosity valid in the cytoplasm of living cells can be also applied for cell nucleus. Currently available data, however do not allow for any quanti- tative conclusions. In the nucleus, ξ length scale is expected to be 30 nm and so the studies with bigger probes are required to conﬁrm this prediction. Frontiers in Physics \| Biophysics 2.5. ASSOCIATION KINETICS IN LIVING CELLS The explanation proposed by authors states that likely acceleration of association of 16-mers in vivo is an effect of presence of magnesium cations or recombination mediator proteins, but deceleration rate of 12-mers is due to buffering by ssDNA- or dsDNA-binding proteins. The viscosity reference curve determined for HeLa cells (Figure 3) point that intracel- lular macromolecular crowding does not change the mobility of the short DNA strains. The change of hybridization kinetics, as argued by Schoen et al. [84], is only due to speciﬁc interactions inside cells. It is worth noting that studies of interaction between molecules in PEG solutions should be carried with caution since this crowder is not inert and may induce unexpected sys- tem behavior. For example Hou et al. [93] performed in vitro studies of the cleavage process taking part in a bacteria’s self- defense against viruses system. Authors performed the DNA cleavage using HindIII restriction enzyme. They monitored the result of the cleavage process 1 h after the reaction started for 24 h. Next they compared results of cleavage in buffer solution with those obtained in the samples containing low molecu- lar weight polyethylene glycol (lmwPEG; Mn = 3461 g/mol) and high molecular weight polyethylene glycol (hmwPEG; Mn = 854096 g/mol). Authors observed that the cleavage process was stopped (no cleavage occurred) when the concentration of the lmwPEG exceeded 7% by weight. On the other hand in solu- tions of hmwPEG the process of cleavage proceeded without any changes. The protein-protein association kinetics was studied by Phillip et al. [85] using FRET method and microinjection. The usage of prokaryotic proteins TEM1-β-lactamase and its protein inhibitor BLIP, foreign to the HeLa cells, possibly diminished the pro- teins interactions with cytoplasmic constituents. Observed reac- tion rate constants for wild type proteins in living cells and cell extracts were only slightly decelerated when compared to in vitro values (1.9 × 105M−1s−1, 2.3 × 105M−1s−1 and 3.0 × 105M−1s−1, respectively). The association rate constants were however 4-orders of magnitude smaller than predicted for diffu- sion limited protein-protein binding. Comparison of the viscosity experienced by the HindIII enzyme suggests that the stopping of the cleavage process is not governed by the hindering effect of the crowded environment. The possible explanation of the hindering effect of the lmwPEG solutions, as suggested by Hou et al. 2.5. ASSOCIATION KINETICS IN LIVING CELLS Mobility of proteins affects kinetics of reactions. Measurements of bimolecular reaction kinetics for hybridization of short DNA strands [84] and protein-protein association [85] in living HeLa cells show that intracellular crowding changes reaction rates up to several-fold as compared with reactions in buffer. Here we discuss quantitatively these rates. q y Schoen et al. [84] studied kinetics of hybridization of short DNA chains at two different lengths 12- and 16-bp. The sequences of DNA were chosen to minimize speciﬁc interactions with other nucleic acids in cells and dsDNAs were delivered into cells by lipofection. The kinetics of opening/closing of DNA due to tem- perature oscillations driven by laser were measured by Förster res- onance energy transfer (FRET) method. Non-monotonic change of association kinetics was determined in vivo, i.e., acceleration of association of the 16-mers when compared to in vitro data (2.9 × 107M−1s−1 vs. 4.2 × 106M−1s−1) but decelerated binding kinetics for 12-mers (5.5 × 106M−1s−1 vs. 2.7 × 107M−1s−1). The same reaction was investigated in a buffer with PEG or dextran as crowders [85]. The measured reaction rate constants were also similar to diluted case. This apparent lack of change of reaction rate was attributed to the combination of two oppos- ing effects: reduction in translational mobility of the proteins and attraction between them due to depletion effect. The com- bination of both effects results in the identical association rate constants as in pure water in the absence of both effects. However, the quantitative impact of depletion interactions on protein- protein association rate in vivo is hard to calculate because the interactions strongly depend on the shape of molecules under study and also on solvent involvement in the model [17]. The matter is complicated further by presence of weak, non-speciﬁc interactions [10, 20, 92]. In vitro data point to non-diffusion limited hybridization of these DNA fragments studied by Schoen et al. [84]. Moreover, the kinetics of associations is slightly enhanced in vitro with crowding agents such as Ficoll-70 and dextrans [84] pointing that these agents cause depletion interactions and speed-up the binding. In the light of in vivo data it is clear that the reactions are modulated by speciﬁc interactions with nucleic acid bind- ing molecules and the resulting effect depends on the length of DNA probes. 2.4. MOTION IN THE CYTOPLASM OF PROKARYOTES Finally the ribosome whose rp ≈16.5 nm experience viscosity that is 450 times the viscosity of water. From the point of view of proteins the ribosome is almost immobile as its diffusion time τD (the time needed to diffuse over the distance of one diam- eter) is nearly 104 times longer than for GFP (τD = 4 ms vs. τD = 0.5 μs). Such strong dependence on the size of the particle undergoing self-diffusion can be used for determination of the unknown size of protein aggregates or the interactions of probe particle with the environment. The length-scale dependent vis- cosity can be used to determine the diffusion coefﬁcient of probes of known size in any cell. This has been done to predict in-cellular diffusion coefﬁcients of all known proteins synthesized from the genome of E. coli Kalwarczyk et al. [15]. There is not enough data for diffusion collected for the same growth conditions of cells. Therefore the data used in Kalwarczyk et al. [15] and pre- sented in Figure 5 refer to diffusion in cells treated with different stimuli (various osmotic conditions and antibiotic treatment). Mika et al. [13] shows that cell treatment and growth conditions may have a signiﬁcant impact on the diffusion processes in living cells. The results for large macromolecular complexes such as RNA- protein [79], chromosomal loci [80, 81], and protein-protein aggregates or plasmids [82], show that these objects may move subdiffusively through cytoplasm of E. coli. The movement seems September 2014 \| Volume 2 \| Article 54 \| 6 Macromolecular crowding Tabaka et al. by rotating proteins is equal to water viscosity. Theoretical and experimental studies [88–91] show also that rotations of probes of protein sizes are slightly affected in cellular-like complex liq- uids. On the contrary, probes with hydrodynamic radii surpassing the sizes of largest crowders in HeLa cytoplasm experience dur- ing rotations macroscopic viscosity [32] as described in previous sections. www.frontiersin.org 2.6. GENE EXPRESSION IN LIVING CELLS The effect of crowding was mainly caused by a shift of the equilibrium rather than reduction of protein diffusion i.e., the crowding increased probability of RNAP binding to the promoter that enhanced transcription rate. Similar behavior was observed in the synthetic systems for long dextran poly- mers [103]. The crowding effects on gene transcription was also studied by Brownian dynamics and Monte Carlo simula- tions [110]. This study revealed that for crowding at volume fractions being close to physiological values transcript abun- dances reached maximum levels, in accordance with in vitro studies for various crowders [102, 103, 111]. Ge et al. [102] used three crowding agents PEG-8000, Ficol-70, and Ficol-400 to study expression of Renilla luciferase driven by T7 promoter. They observed maximal enhancement of transcription for intermediate concentration of crowders [with maximum ∼4 fold for Ficol-70 at concentration 20% (w/v)], as compared with diluted solu- tions, but translation was inhibited by each crowder. The authors suggested that the inhibition is due to protein precipitation for PEG solutions and non-speciﬁc interaction of Ficoll molecules with translational machinery proteins. The effects of crowding by dextran polymers of 6 kDa and 2 MDa on gene expression was studied in synthetic cellular nano-systems [103]. This study revealed enhancement of the binding rate and decrease of dissoci- ation rate of T7 RNA polymerase for long dextran solutions. Also signiﬁcant increase of total gene expression rate was observed for long dextran polymers at concentration of 10% (w/v). The crowded systems exhibited higher robustness of gene expression, i.e., less susceptibility to solution composition and concentration of ions. This buffering as an effect of macromolecular crowd- ing was proposed as one of the main factors facilitating response of the cells to osmotic stress [112, 113]. Also increased gene expression efﬁciency driven by T7 RNAP at intermediate level of crowders was noticed in clay hydrogel [111]. f b d f f (l Intracellular macromolecular crowding is also expected to shrink solely the nucleoid to the observed in vivo volume due to purely entropic effects [124, 125]. Indeed, the fast real-time compaction of chromosomes to native volumes [126] is observed in systems crowded by polymers. Our predictions are obtained for exponential phase of growth of E. coli, for which nucleoid is in an expanded state. We look forward to models in which more dense chromosome structures are taken into consideration. 2.6. GENE EXPRESSION IN LIVING CELLS Gene expression is a process consisting of many reactions driven either by diffusion (regulation of initiation rate of gene expres- sion by transcription factors [99–101]) or powered by hydrolysis of ATP or GTP molecules (RNA polymerase and ribosome move- ment or histone displacement). The studies of the crowding effects on particular steps of gene expression are concentrated on in vitro systems [102, 103] with crowding agents and com- parison with dilute solutions. There is a growing number of experimental studies concerning transcription factor kinetics in bacteria [104–106] and eukaryotes [45, 46, 50, 107]. On the basis of these experiments we calculated the impact of macromolecular crowding [15] inside E. coli cell on transcrip- tion factor binding rates to speciﬁc sites [114]. We considered multilevel impact of in vivo crowding on association rates to speciﬁc sites in the facilitated diffusion model: (1) reduction of the nucleoid volume due to the presence of nucleoid-associated proteins that modify persistence length of DNA and cross-link DNA chains, (2) reduction of transcription factor three- and one- dimensional diffusion constants [15], (3) obstruction of tran- scription factor binding to non-speciﬁc DNA and its sliding due to presence of nucleoid-associated proteins [115–119]. The model predicted the similar binding rate to experimentally observed for cells in the exponential phase of growth. The lac repressor was nearly optimized for binding with respect to association and dis- sociation rates to/from non-speciﬁc DNA. We extrapolated data from lac repressor to all annotated transcription factors in E. coli assuming the same equilibrium binding constants but dif- ferent diffusivities. The assumption of equal afﬁnities in vivo resulted from the observation of similar values of binding con- stants to DNA for various transcription factors [104, 120, 121]. Similarity of in vivo equilibrium constants to these determined in dilute solutions [100, 122, 123] additionally poses a question about the effect of excluded volume interactions inside living prokaryotic cells [112]. From the model, we also obtain that the in vivo number of nucleoid-associated proteins affecting nucleoid volume is enough to shrink it to experimentally observed values. Morelli et al. [108] studied theoretically the impact of macro- molecular crowding on gene expression and its regulation. The authors parametrized the model on the basis of changes of activity coefﬁcients for hemoglobin at different concentrations [1, 109]. The crowding affects both equilibrium binding constants and diffusion of proteins. The reactions were assumed to be diffusion- limited. 2.5. ASSOCIATION KINETICS IN LIVING CELLS [93], are depletion inter- actions leading in surfactant solutions to the phase separation [94–97] or in solution of DNA leading to the condensation of DNA [93, 98]. From above we deduce that the solutions of the ﬂexible, polyethylene glycol like polymers may not be preferable as in vitro models to study the inﬂuence of cellular crowding on the in-cell processes. Recently, we have computed [86] in vivo protein-protein bind- ing rate constants determined by Phillip et al. [85] taking only into account changes in reactant mobilities. In our approach, we have combined the length scale-dependent viscosity model of HeLa cytoplasm [14] to predict translational diffusion con- stants for reacting proteins and Zhou’s model [87] for kinetics of binding of two spheres with small reactive patches. The model requires also knowledge of rotational diffusion constants for pro- teins. The best agreement with experiment is obtained under assumption that the viscosity of HeLa cytoplasm experienced Study of the folding stability of the protein chymotropsin inhibitor 2 in the lyophilized E. coli cytosol [20] showed that crowder molecules interact with the protein destabiliz- ing it. This is opposite to expected stabilizing entropic effect due to the fact that folded protein occupies less space than denaturated one. Also the cytoplasm of E. coli is highly crowded environment that should signiﬁcantly reduce reac- tion rate constants between proteins [86] as predicted by September 2014 \| Volume 2 \| Article 54 \| 7 www.frontiersin.org Macromolecular crowding Tabaka et al. the model in which only protein mobilities are taken into consideration. localization [104] revealed that single lac repressor localized its target on the nucleoid in ∼6 min, being absorbed with non- speciﬁc DNA sites 87% of the search time (diffusion constant of the protein was 0.4 µm2s−1in cells and was enhanced by the pro- tein without DNA-binding domains to 3.0 µm2s−1). Subsequent studies with double speciﬁc sites determined the scale of the cor- related motion of the repressor [105]. The results showed that the repressor when bound to DNA scanned 36 base pairs of the nucleoid DNA. In fact, crucial parameters of the facilitated dif- fusion model of the lac repressor in living prokaryotic cells were determined in these studies. Frontiers in Physics \| Biophysics 2.6. GENE EXPRESSION IN LIVING CELLS There is a lack of detailed kinetic models of transcription fac- tor facilitated diffusion in eukaryotes at present. It would be of great interest to establish the impact of the crowding and chromosome structure in nucleus on the transcription factor kinetics. Inside a cell nucleus we expect weakly crowded large water chan- nels (of sizes >30 nm), because most studies show that in a wide range of sizes (4–30 nm) the viscosity is constant in a cell nucleus, only slightly larger than the water viscosity. In lamel- lar phases of ionic surfactants similar lack of size dependence of viscosity is observed (up to sizes >150 nm) [43]. More experimental studies with larger probes are required to give clear answer about transport properties of proteins in the cell nucleus. There is a signiﬁcant difference in the diffusion of proteins between prokaryotic and eukaryotic cells. In eukaryotic cells pro- teins diffuse as in water, while in prokaryotic cells because of much higher crowding the diffusion coefﬁcient of proteins is much smaller. This property has consequences for the kinet- ics of protein-protein interactions in prokaryotic cells [86] i.e., none of such reactions would have the same reaction rate as in vitro (buffer) conditions. On the contrary in the cytoplasm of eukaryotic cells we expect that the reaction rates for protein- protein association should be similar to those measured in water. Our review also shows the importance of non-speciﬁc inter- actions with cytoplasm constituents for the interpretation of the impact of macromolecular crowding on biochemical reac- tions [20, 84]. Non-monotonic changes of reaction rates of DNA hybridization [84] and destabilization of the protein [20] can be explained only by direct interactions with cytoplasm components. Non-speciﬁc interactions of transcription factors with DNA affect the search process for operators [99]. Without those interactions LacI repressor should ﬁnd its target (one base pair, since TF shift in position by one base pair results in distinct DNA binding sequence [99, 116]) in a volume of E. coli in 7 s. In vivo and in the model including binding of LacI to non-speciﬁc sequences of DNA this time is 400 s. Thus, identiﬁcation of strong non-speciﬁc interactions is important in the study of the inﬂuence of crowd- ing environment on biological processes. 2.6. GENE EXPRESSION IN LIVING CELLS One could, for example, consider detailed models of nucleoid compaction [127, 128] and apply them in the studies of tran- scription factor kinetics. It would be interesting to know how the kinetics changes during the transition to the stationary phase, for which DNA crowding affects transcription factor distribu- tion [129]. The parametrization of the model can be carried out in vivo by the proposed method of extracting diffusion and tran- sition rates from single-molecule experiments [130]. Moreover, in-cell distributions of RNA polymerases and ribosomes are not uniform and there is a strong tendency for occupation of different regions of cell by these complexes [77]. In vivo rate of binding of transcription factor (lac repres- sor) to the speciﬁc site was measured [104, 105] in E. coli. Combination of methods such as single-particle tracking of the ﬂuorescently-labeled repressor, in vivo FCS, and detection by September 2014 \| Volume 2 \| Article 54 \| 8 Tabaka et al. Macromolecular crowding The mechanism of gene regulation in eukaryotes involves many protein factors interacting in concert with active reposi- tioning of nucleosomes [131–133]. Only recently, the kinetics of transcription factors have been studied in eukaryotes [45, 46, 50, 107] revealing basic features of the facilitated diffusion of transcription factors in nuclei. The striking feature, as com- pared with prokaryotes, is a proportion of the time spent in 3D translocation and in interaction with non-speciﬁc DNA during the search process. DNA-bound fraction of glucocorticoid recep- tor, in MCF-7 breast cancer cell line, is 12% and enhanced to 37% after induction with dexamethasone [45]. Transcription fac- tors Sox2 and Oct4, in mouse embryonic stem cells, spend 18 and 21% of the time [46] being bound with non-speciﬁc DNA, respectively. These numbers show that the target search time is dominated by 3D diffusion in mammalian cells. Moreover, the localization time of the single speciﬁc site by a single Sox2 pro- tein in the genome, as estimated by Chen et al. [46], is ∼31 days. The time is ∼7000 times slower than in case of lac repres- sor in E. coli cell [104]. Taking determined rate constants for Gal4 regulatory protein in Saccharomyces cerevisiae [107] and volume of the nucleus [134] we calculate that the value of the sin- gle target localization time is ∼14 days. Nuclear environment is markedly distinct and more complex than prokaryotic nucleoid. 2.6. GENE EXPRESSION IN LIVING CELLS Mobility data including the length-scale dependent viscosity are helpful in such identiﬁ- cation e.g., by comparison of the motion of inert probe to the motion of the probe of interest of similar size. Any differences in their motion would be a signature of interactions with cellular components. Many effects are expected to be induced due to decreasing molecule mobility by macromolecular crowding. For example, diffusion of transcription factors is predicted to enhance gene expression noise [135]. The kinetics of translation has been recently studied theoretically in E. coli [136]. The results show that slow diffusion of the tRNA in the crowded milieu of cytoplasm limits translation and consequently growth rate of cells [136]. Intriguingly, the diffusion of macromolecules is predicted to be responsible for prokaryotic and eukaryotic cell sizes [137]; as such, the sizes minimize the times of migration of macro- molecules throughout the cells. www.frontiersin.org REFERENCES 1. Minton AP. The effect of volume occupancy upon the thermodynamic activ- ity of proteins: some biochemical consequences. Mol Cell Bioch. (1983) 55:119–40. doi: 10.1007/BF00673707 1. Minton AP. The effect of volume occupancy upon the thermodynamic activ- ity of proteins: some biochemical consequences. Mol Cell Bioch. (1983) 55:119–40. doi: 10.1007/BF00673707 2. Zimmerman SB, Minton AP. Macromolecular crowding: bio- chemical, biophysical, and physiological consequences. Annu Rev Bioph Biom. (1993) 22:27–65. doi: 10.1146/annurev.bb.22.060193. 000331 2. Zimmerman SB, Minton AP. Macromolecular crowding: bio- chemical, biophysical, and physiological consequences. Annu Rev Bioph Biom. (1993) 22:27–65. doi: 10.1146/annurev.bb.22.060193. 000331 3. Minton AP. Implications of macromolecular crowding for protein assem- bly. Curr Opin Struct Biol. (2000) 10:34–9. doi: 10.1016/S0959-440X(99) 00045-7 4. Minton AP. The inﬂuence of macromolecular crowding and macromolecular conﬁnement on biochemical reactions in physiological media. J Biol Chem. (2001) 276:10577–80. doi: 10.1074/jbc.R100005200 5. Ellis RJ. Macromolecular crowding: an important but neglected aspect of the intracellular environment. Curr Opin Sruc Biol. (2001) 11:114–9. doi: 10.1016/S0959-440X(00)00172-X 6. Ellis RJ. Macromolecular crowding: obvious but underappreciated. Trends Biochem Sci. (2001) 26:597–604. doi: 10.1016/S0968-0004(01) 01938-7 7. Minton AP. Inﬂuence of macromolecular crowding upon the stability and state of association of proteins: predictions and observations. J Pharm Sci. (2005) 94:1668–75. doi: 10.1002/jps.20417 8. Zhou HX, Rivas G, Minton AP. Macromolecular crowding and conﬁne- ment: biochemical, biophysical, and potential physiological consequences. Ann Rev Biophys. (2008) 37:375–97. doi: 10.1146/annurev.biophys.37.032807. 125817 9. Minton AP. Quantitative assessment of the relative contributions of steric repulsion and chemical interactions to macromolecular crowding. Biopolymers (2013) 99:239–44. doi: 10.1002/bip.22163 10. Zhou HX. Inﬂuence of crowded cellular environments on protein fold- ing, binding, and oligomerization: biological consequences and potentials of atomistic modeling. FEBS Lett. (2013) 587:1053–61. doi: 10.1016/j.febslet. 2013.01.064 11. Phillip Y, Schreiber G. Formation of protein complexes in crowded environments–from in vitro to in vivo. FEBS Lett. (2013) 587:1046–52. doi: 10.1016/j.febslet.2013.01.007 12. Elowitz M, Surette M, Wolf P, Stock J, Leibler S. Protein mobility in the cytoplasm of Escherichia coli. J Bacteriol. (1999) 181:197–203. 13. Mika JT, van den Bogaart G, Veenhoff L, Krasnikov V, Poolman B. Molecular sieving properties of the cytoplasm of Escherichia coli and consequences of osmotic stress. Mol Microbiol. (2010) 77:200–7. doi: 10.1111/j.1365- 2958.2010.07201.x Kinetics of gene expression and its regulation in living cells has been studied only within last decade. The detailed picture of the regulation kinetics is available only for lac repressor in E. coli [104–106]. 3. CONCLUSIONS AND FUTURE PERSPECTIVES Lot of attention was devoted to depletion interactions in crowded solutions [1, 8, 16–18]. Two similar objects, immersed in solutions of solutes different in size and shape from them, attract each other. Such interaction is called the depletion interac- tion. The strength of the depletion interactions is calculated from the osmotic pressure of the solutions multiplied by the excluded volume between the object and the solute particle. For proteins in ﬂexible polymer solutions this non-speciﬁc interaction is par- ticularly strong. For 12% of PEG (Mw = 6 kg/mol) solution the osmotic pressure is of the order of 105 Pa and the excluded vol- ume in the range of tens of nm3. Thus, typical free energy change upon binding of two colloidal particles in the polymer solution is several kJ/mol. The depletion interactions strongly depend on the shape of studied molecules, change of excluded volume upon binding and incorporation of solvent to the models [17]. For pro- tein solutions the effect is probably small because all molecules have similar shape. Therefore, we do not expect in the cytoplasm The macromolecular crowding plays important role in key pro- cesses in living cells starting from life emergence [111, 138] up to regulation of cell sizes [137]. We have pointed to three different phenomena emerging as a consequence of crowding: the length-scale dependent viscosity, depletion interactions, and non-speciﬁc binding. Quantitative description of interactions inside living cells is currently based on the analysis of free diffusion inside cells. Therefore in our review we emphasize effects of in vivo macro- molecular crowding on mobility of the macromolecules and kinetics of interactions between them. We review, according to our knowledge, all studies concerning mobility of molecules in prokaryotic (E. coli) and eukaryotic (HeLa) cells. We show that the cytoplasm has transport property described by the length scale-dependent viscosity. It means that the effective viscosity experienced by moving probes depends on their size [14, 25]. September 2014 \| Volume 2 \| Article 54 \| 9 www.frontiersin.org Tabaka et al. Macromolecular crowding Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin, China within the Grant No. 20130301. Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin, China within the Grant No. 20130301. Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin, China within the Grant No. 20130301. strong depletion interactions. However denaturated proteins could in principle exert stronger depletion effect in protein solutions. AUTHOR CONTRIBUTIONS All authors contribute in manuscript writing, Marcin Tabaka and Tomasz Kalwarczyk contribute equally. 18. Marenduzzo D, Finan K, Cook PR. The depletion attraction: an underappre- ciated force driving cellular organization. J Cell Biol. (2006) 175:681–6. doi: 10.1083/jcb.200609066 REFERENCES The existing models, taking intracellular crowd- ing into account and all existing experimental data show that this transcription factor is nearly optimized for its function [105, 114, 116]. We look forward to studies of gene regulation kinetics of other transcription factors to check generalization of the predic- tions in prokaryotes and detailed models of facilitated diffusion in eukaryotes. 14. Kalwarczyk T, Ziebacz N, Bielejewska A, Zaboklicka E, Koynov K, Szymanski J, et al. Comparative analysis of viscosity of complex liquids and cytoplasm of mammalian cells at the nanoscale. Nano Lett. (2011) 11:2157–63. doi: 10.1021/nl2008218 15. Kalwarczyk T, Tabaka M, Holyst R. Biologistics-Diffusion Coefﬁcients For Complete Proteome Of Escherichia coli [Article]. Bioinformatics (2012) 28:2971–8. doi: 10.1093/bioinformatics/bts537 16. Asakura S, Oosawa F. On interaction between 2 bodies immersed in a solu- tion of macromolecules. J Chem Phys. (1954) 22:1255–6. doi: 10.1063/1. 1740347 17. Berg OG. The inﬂuence of macromolecular crowding on thermodynamic activity: solubility and dimerization constants for spherical and dumbbell- shaped molecules in a hard-sphere mixture. Biopolymers (1990) 30:1027–37. doi: 10.1002/bip.360301104 3. CONCLUSIONS AND FUTURE PERSPECTIVES Interpretation of in vivo protein-protein kinetic studies requires understanding of in-cytoplasm rotational diffusion (inversely proportional to rotational viscosity) of proteins [11, 85, 86]. Proteins upon binding have to be oriented properly with respect to each other. Typical time for proper alignment of reactive patches on proteins necessary for complex formation depends on rotations of proteins. The impact of crowding on protein rotation and rotational viscosity is expected to be signif- icant in prokaryotic cells for which ξ is an order of magnitude smaller than the typical size of proteins. The Brownian dynam- ics studies [89] of the model cytoplasm of E. coli crowded only by proteins show that the rotational viscosity does not change much with the increase of protein molecular weight. On aver- age this viscosity is 10 times larger than the viscosity of water, but smaller than the length scale dependent viscosity characteriz- ing translational motion. This difference between rotational and translational viscosity is not unphysical and arises as a conse- quence of depletion. The rotational viscosity is always smaller than the translational viscosity because in a crowded environ- ment the excluded volume effects and depletion are ubiquitous. The depletion layer i.e., a layer around a particle depleted from other particles (e.g., observed for colloids in polymer solutions) leads to non-uniform viscosity around the particle [139–146]. The viscosity changes from the solvent viscosity close to the sur- face of an object to the solution viscosity in a bulk solution over a short distance of the depletion layer (usually of the order of 0.1 to few nm). Roughly speaking the rotations of molecule experience the local solvent or almost solvent viscosity while translations the one of the solution. What remains to be inves- tigated is how hydrodynamic long-range interactions affect the rotations of macromolecules [90, 91, 147]. Certainly, the most urgent are experimental studies of the rotational diffusion con- stants as a function of protein shapes and sizes in cytoplasm of living cells or cell extracts [148]. Frontiers in Physics \| Biophysics ACKNOWLEDGMENTS A benchmark for chromatin binding measurements in live cells. Nucl Acids Res. (2012) 40:e119. doi: 10.1093/nar/gks701 26. Kalwarczyk T, Sozanski K, Jakiela S, Wisniewska A, Kalwarczyk E, Kryszczuk K, et al. Length-scale dependent transport properties of colloidal and pro- tein solutions for prediction of crystal nucleation rates. Nanoscale (2014) 6:10340–6. doi: 10.1039/C4NR00647J 49. Wachsmuth M, Waldeck W, Langowski J. Anomalous diffusion of ﬂuorescent probes inside living cell nuclei investigated by spatially-resolved ﬂuorescence correlation spectroscopy. J Mol Biol. (2000) 298:677–89. doi: 10.1006/jmbi. 2000.3692 27. Sozanski K, Wisniewska A, Kalwarczyk T, Holyst R. Activation energy for mobility of dyes and proteins in polymer solutions: from diffusion of sin- gle particles to macroscale ﬂow. Phys Rev Lett. (2013) 111:228301. doi: 10.1103/PhysRevLett.111.228301 50. Vukojevi´c V, Papadopoulos DK, Terenius L, Gehring WJ, Rigler R. Quantitative study of synthetic Hox transcription factor–DNA interactions in live cells. Proc Natl Acad Sci USA. (2010) 107:4093–8. doi: 10.1073/pnas. 0914612107 28. Zhou EH, Martinez FD, Fredberg JJ. Cell rheology: mush rather than machine. Nat Mater. (2013) 12:184–5. doi: 10.1038/nmat3574 51. Szymanski J, Weiss M. Elucidating the origin of anomalous diffusion in crowded ﬂuids. Phys Rev Lett. (2009) 103:038102. doi: 10.1103/PhysRevLett. 103.038102 29. Moeendarbary E, Valon L, Fritzsche M, Harris AR, Moulding DA, Thrasher AJ, et al. The cytoplasm of living cells behaves as a poroelastic material. Nat Mater. (2013) 12:253–61. doi: 10.1038/nmat3517 52. Ernst D, Köhler J, Weiss M. Probing the type of anomalous diffusion with single-particle tracking. Phys Chem Chem Phys. (2014) 16:7686–91. doi: 10.1039/c4cp00292j 30. Luby-Phelps K, Castle P, Taylor D, Lanni F. Hindered diffusion of inert tracer particles in the cytoplasm of mouse 3T3 cells. Proc Natl Acad Sci USA. (1987) 84:4910–3. doi: 10.1073/pnas.84.14.4910 31. Nakane Y, Sasaki A, Kinjo M, Jin T. Bovine serum albumin-coated quan- tum dots as a cytoplasmic viscosity probe in a single living cell. Anal Methods (2012) 4:1903–5. doi: 10.1039/c2ay25318f 53. 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Hui YY, Zhang B, Chang YC, Chang CC, Chang HC, Hsu JH, et al. Two-photon ﬂuorescence correlation spectroscopy of lipid-encapsulated ﬂuorescent nanodiamonds in living cells. Opt Exp. (2010) 18:5896–905. doi: 10.1364/OE.18.005896 Citation: Tabaka M, Kalwarczyk T, Szymanski J, Hou S and Holyst R (2014) The effect of macromolecular crowding on mobility of biomolecules, association kinetics, and gene expression in living cells. Front. Phys. 2:54. doi: 10.3389/fphy.2014.00054 This article was submitted to Biophysics, a section of the journal Frontiers in Physics. Copyright © 2014 Tabaka, Kalwarczyk, Szymanski, Hou and Holyst. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permit- ted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 152. Dauty E, Verkman A. Actin cytoskeleton as the principal determinant of size-dependent DNA mobility in cytoplasm. J Biol Chem. (2005) 280:7823–8. September 2014 \| Volume 2 \| Article 54 \| 14 Received: 27 June 2014; accepted: 25 August 2014; published online: 18 September 2014. Citation: Tabaka M, Kalwarczyk T, Szymanski J, Hou S and Holyst R (2014) The effect of macromolecular crowding on mobility of biomolecules, association kinetics, and gene expression in living cells. Front. Phys. 2:54. doi: 10.3389/fphy.2014.00054 This article was submitted to Biophysics, a section of the journal Frontiers in Physics. Copyright © 2014 Tabaka, Kalwarczyk, Szymanski, Hou and Holyst. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permit- ted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Received: 27 June 2014; accepted: 25 August 2014; published online: 18 September 2014. ACKNOWLEDGMENTS doi: 10.1074/jbc.M412374200 Conﬂict of Interest Statement: The authors declare that the research was con- ducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. September 2014 \| Volume 2 \| Article 54 \| 14 Frontiers in Physics \| Biophysics Frontiers in Physics \| Biophysics
https://openalex.org/W2803087809	https://europepmc.org/articles/pmc5904098?pdf=render	English	null	Author Correction: Dmrt1 is required for primary male sexual differentiation in Chinese soft-shelled turtle Pelodiscus sinensis	Scientific reports	2,018	cc-by	619	www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports should read: “Figure 6. Responses of sex-specific genes to Dmrt1 knockdown or over-expression. (a) qRT-PCR analysis showing the effects of Dmrt1 knockdown or over-expression on the mRNA expression of Amh, Sox9, Cyp19a1 and Foxl2 in embryonic gonads at stage 15, 17, 19, 21, 23, 25 and 27. Data are shown as means ± S.D., N ≥ 3. P < 0.05; P < 0.01; *P < 0.001. Data for ZZ was published before in ref.1 as Figure 3C and is reprinted here with permission. (b) Double immunofluorescence of Sox9 and γH2AX was performed in sections of control ZZ gonads, ZZ gonads with Dmrt1 knockdown, the control ZW gonads and ZW gonads with Dmrt1 over-expression at stage 27. pre-sc, precursor sertoli cell; gc, germ cells. Scale bars are 50 μm. Author Correction: Dmrt1 is required for primary male sexual differentiation in Chinese soft- shelled turtle Pelodiscus sinensis Wei Sun1, Han Cai1, Gloria Zhang2, Haiyan Zhang1, Haisheng Bao3, Li Wang3, Ji Y 4 G i Qi 1 &Ch ti G 1 xx OPEN Published: xx xx xxxx Correction to: Scientific Reports https://doi.org/10.1038/s41598-017-04938-5, published online 30 June 2017 ome of the data presented in this Article was published by the Authors previously. The Authors neglected to cite his previous paper, which is included below as ref.1 and should be cited in legend of Figure 6 as below: “Figure 6. Responses of sex-specific genes to Dmrt1 knockdown or over-expression. (a) qRT-PCR analysis showing the effects of Dmrt1 knockdown or over-expression on the mRNA expression of Amh, Sox9, Cyp19a1 and Foxl2 in embryonic gonads at stage 15, 17, 19, 21, 23, 25 and 27. Data are shown as means ± S.D., N ≥ 3. P < 0.05; P < 0.01; *P < 0.001. (b) Double immunofluorescence of Sox9 and γH2AX was performed in sections of control ZZ gonads, ZZ gonads with Dmrt1 knockdown, the control ZW gonads and ZW gonads with Dmrt1 over-expression at stage 27. pre-sc, precursor sertoli cell; gc, germ cells. Scale bars are 50 μm.” Reference 1. Wei, S. et al. Function Analysis of Dmrt1 in Male Sexual Differentiation in Pelodiscus sinensis. Scientia Sinica Vitae 45, 881–889 (2015). SCiEnTiFiC REPOrtS \| (2018) 8:6322 \| DOI:10.1038/s41598-018-23722-7 Reference l Reference 1. Wei, S. et al. Function Analysis of Dmrt1 in Male Sexual Differentiation in Pelodiscus sinensis. Scientia Sinica Vitae 45, 881–889 (2015). 1College of Biological and Environmental Sciences, Zhejiang Wanli University, Ningbo, 315100, China. 2Trinity School of Arts and Sciences, Duke University, Durham, NC, 27708, USA. 3College of Fisheries and Life Sciences, Shanghai Ocean University, Shanghai, 201306, China. 4HangZhou Aquacultural Technique Extending Centre, Hangzhou, 310001, China. Wei Sun and Han Cai contributed equally to this work. Correspondence and requests for materials should be addressed to G.Q. (email: qiangy@zwu.edu.cn) or C.G. (email: cge@zwu.edu.cn) SCiEnTiFiC REPOrtS \| (2018) 8:6322 \| DOI:10.1038/s41598-018-23722-7 SCiEnTiFiC REPOrtS \| (2018) 8:6322 \| DOI:10.1038/s41598-018-23722-7 www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per- mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2018 SCiEnTiFiC REPOrtS \| (2018) 8:6322 \| DOI:10.1038/s41598-018-23722-7 2 2
https://openalex.org/W2336101418	https://europepmc.org/articles/pmc4582289?pdf=render	English	null	Carbon loading in airway macrophages to traffic-derived particulate matter air pollution	Archives of public health	2,015	cc-by	648	Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission Authors’ details Published: 17 September 2015 doi:10.1186/2049-3258-73-S1-P19 Cite this article as: Bai et al.: Carbon loading in airway macrophages to traffic-derived particulate matter air pollution. Archives of Public Health 2015 73(Suppl 1):P19. doi:10.1186/2049-3258-73-S1-P19 Cite this article as: Bai et al.: Carbon loading in airway macrophages to traffic-derived particulate matter air pollution. Archives of Public Health 2015 73(Suppl 1):P19. 1KULeuven, Leuven, Belgium Full list of author information is available at the end of the article Methods In this follow-up study, international students undergo a first assessment shortly after their arrival in Belgium, and then 7 further assessments every 6 weeks. For compari- son, students having resided in Belgium for more than one year are examined concurrently. AM are obtained using induced sputum and the surface of carbon loading (m2) is measured in 50 AM by image analysis. Carbon loading in airway macrophages to traffic- derived particulate matter air pollution From Methods in Epidemiology Symposium Leuven, Belgium. 17 September 2015 this biomarker is suitable to estimate chronic exposure to traffic-related air pollution in individuals. This is con- firmed by the observation of a sharp decline in carbon loading in AM over a period of six weeks when moving from an area of high pollution to an area of moderate pollution. Bai et al. Archives of Public Health 2015, 73(Suppl 1):P19 http://www.archpublichealth.com/content/73/S1/P19 Bai et al. Archives of Public Health 2015, 73(Suppl 1):P19 http://www.archpublichealth.com/content/73/S1/P19 ARCHIVES OF PUBLIC HEALTH Open Access Open Access © 2015 Bai et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http:// creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/ zero/1.0/) applies to the data made available in this article, unless otherwise stated. Results Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit distributed under the terms of the Creative Commons Attribution License (http:// The preliminary data of this ongoing study indicate that the carbon loading in AM remains stable over the observation period. However, in subjects who moved from a highly polluted area (n=3, so far), a pronounced decrease in average carbon loading (2.86 to 1.33) is observed between their first second visits. Background and aims Epidemiological studies indicate that the health outcomes of exposure to urban air pollution are driven by exposure to elemental carbon (EC) emitted from traffic-related fuel combustion. One possible method for assessing chronic exposure to EC in individual subjects consists of estimat- ing the amount of carbonaceous particles present in alveolar macrophages (AM)s. However, a number of issues remain to be solved regarding this biomarker. We studied the reproducibility and the kinetics of this bio- marker by making repeated measurements over one year in healthy subjects, with some of them having moved from a highly polluted area (PM10 > 30 μg/m3) to a mod- erately polluted area (PM10 20-30 μg/m3). Authors’ details 1KULeuven, Leuven, Belgium. 2Hasselt University, Hasselt, Belgium. Authors’ details 1KULeuven, Leuven, Belgium. 2Hasselt University, Hasselt, Belgium. Conclusions The stability of the carbon loading of AM in induced sputum over a period of several months suggests that Submit your manuscript at www.biomedcentral.com/submit © 2015 Bai et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http:// creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/ zero/1.0/) applies to the data made available in this article, unless otherwise stated.
https://openalex.org/W3153587776	https://revije.ff.uni-lj.si/AndragoskaSpoznanja/article/download/9920/9397	Slovene	null	Sabina Jelenc Krašovec – lik andragoginje	AS. Andragoška spoznanja/Andragoška spoznanja	2,021	cc-by-sa	1,447	SABINA JELENC KRAŠOVEC – LIK ANDRAGOGINJE Sodeč po tem, kar so o Sabini v svojih sožalnih sporočilih napisali tisti, ki so jo poznali, med njimi še posebno kolegice in kolegi ter sodelavke in sodelavci ter drugi strokovnjaki s področja izobraževanja odraslih, tako iz Slovenije kot tudi iz tujine, je bila žal pre- zgodaj umrla Sabina Jelenc Krašovec zgled uspešne andragoginje. To lahko razberemo iz sporočil, kot so: » v mojih mislih bo vedno ostala velika andragoginja in kolegica«; »koliko znanja, energije je odšlo, kljub kratkemu življenju je med mnoge ljudi zasejala znanje, prepričanja in voljo do izobraževanja odraslih«; »bila je predana stroki, kritična, neravnodušna do tega, kar se dogaja, in zelo angažirana«; »I found her to be a serious and enthusiastic colleague, who also enjoyed life to the full; Sabina was a favourite for many of us, she will not be forgotten by those who came to know her well«. Ob teh izbra- nih strokovnih opredelitvah pa tudi ne manjka sporočil, ki govorijo o njenih osebnostnih značilnostih ter načinu življenja in delovanja: »v mislih jo imam nasmejano, energično in vedno pripravljeno na iskriv pogovor«; »kar ne morem verjeti, da ni več tako energije polne Sabine, kakor smo jo poznali«; »koliko znanja, energije, dobrote je odšlo«; »zmeraj je znala stvari narediti zemeljske, postaviti življenje v ospredje«; »vsakič me je preplavila njena silovita energija, žar, ki se je širil kot blisk«; »bila je iskrena, pozitivna, polna ener- gije in entuziazma, hkrati pa zelo pravična«; »bila je dragoceno sonce naše širše družine, akademskega in ostalega sveta, zato nikoli ne bo prenehala sijati«. Ali to pomeni, da si je Sabina izbrala pravi poklic? Je na to kazalo, ko se je po osnovni šoli vpisala na gimnazijo pedagoške smeri? Ni nujno. Tedaj so se srednje šole ravnale po zahtevah usmerjenega izobraževanja in so si kandidati morali izbrati smer, ki je vodila k določenim poklicnim področjem in usmeritvam, kot so, če naštejem nekatere, naravoslov- no in tehnično, jezikovno, pedagoško, zdravstveno ... . Poleg zgodnje usmeritve v poklic in kasneje v delo na izbranem področju je bil poglavitni namen takega usmerjanja odvra- čanje od splošnega izobraževanja, ker so odločevalci na področju vzgoje in izobraževanja ter oblikovalci družbenega razvoja in politike presodili, da se preveč učencev odloča za splošno izobraževalne smeri ter še posebej za družboslovna in humanistična področja na- mesto za tehniko in naravoslovje. Andragoška spoznanja/Studies in Adult Education and Learning, 2021, 27(1), 186-188 DOI: https://doi.org/10.4312/as/9920 Andragoška spoznanja/Studies in Adult Education and Learning, 2021, 27(1), 186-188 DOI: https://doi.org/10.4312/as/9920 Andragoška spoznanja/Studies in Adult Education and Learning, 2021, 27(1), 186-188 DOI: https://doi.org/10.4312/as/9920 In memoriam SABINA JELENC KRAŠOVEC – LIK ANDRAGOGINJE Tako naj bi odpravljali neravnovesje med področji dela in poklici v praksi, saj naj bi šole tisti čas in na splošno dajale preveč družboslovcev ter premalo naravoslovcev in tehnikov, ki jih v družbi potem primanjkuje. Sabina se je pred odločitvijo za študij želela prepričati o tem, za kakšen poklic naj bi bila najbolj primerna. Za tehnične poklice, medicino, študij s pomembnim deležem matema- tike in druga poklicna področja (izjema bi lahko bila veterina, a predvsem zato, ker je kot otrok imela rada živali) Sabina ni kazala zanimanja. Še najbližja ji je bila psihologija. 187 In memoriam Oglasila se je pri svetovalcu za visokošolske poklice, psihologu Zdenku Lapajnetu na Centru za razvoj univerze, da bi preverila ustreznost svoje poklicne namere. Svetovalec je na podlagi ugotovitev iz svetovalne obravnave njeno namero podprl in ji potrdil, da bi bila njena odločitev prava in bi bila psihologija pravi poklic zanjo. Število kandidatov, ki bi jih tisto leto sprejeli na študij psihologije, so določili tako, da so morali opraviti sprejemni preizkus, kot merilo za sprejem pa je poleg dovolj dobrega šolskega uspeha štelo število doseženih točk v vprašalniku, ki so ga morali izpolniti. V njem so bile različne zahteve, med drugim tudi uspešno rešene matematične naloge. Sabini so na koncu za sprejem na študij psihologije zmanjkale tri točke in na študij psihologije ni bila sprejeta. Sam sem bil do takega načina selekcije kandidatov zelo kritičen; menil sem, da bi psihologi morali biti sposobni presojati primernost kandidatov za delo psihologa s svojimi, psihološkimi sred- stvi (zlasti s pogovorom in opazovanjem), ne pa z rezultati nekega vprašalnika, s katerim kandidat izkazuje (šolsko) znanje ali izraža svoja stališča; če drugega ne, bi morali psiho- logi pri presoji kandidatov upoštevati mnenje svetovalca za visokošolske poklice, kakršen je bil Lapajne. Sabini torej psihologija ni bila namenjena in omogočena. Prepričan pa sem, da bi bila tudi kot psihologinja uspešna. Ker je možnost študija psihologije odpadla, se je Sabina hitro odločila za dvopredmetni študij pedagogike in sociologije. Odločila se je za andragoško usmeritev. Po končnem študiju, zlasti pri projektih, posebno raziskovalnih, ki jih je izpeljevala, se je pokazalo, da jo zlasti privlači sociološka razsežnost obravnavanih vprašanj, tako tudi izobraževanja odraslih. SABINA JELENC KRAŠOVEC – LIK ANDRAGOGINJE Prepričan sem, da je to zelo okrepilo njeno andragoško kompetentnost, ki se po mojem mnenju krepi toliko bolj, kolikor bolj se povezuje z drugimi strokami in razse- žnostmi, med njimi seveda zlasti s sociologijo, pomembno pa gotovo tudi s psihologijo, pomembnim področjem, ki jo je tudi zanimalo. Tako je andragogika postala za Sabino kar ustrezna izbira. Energija, živahnost, dinamičnost, odprtost, komunikativnost, dostopnost za srečevanje in stike so lastnosti, ki so odlikovale Sabino. Bila je živahen in zvedav otrok, ki si je s svojim vedenjem znala pridobiti naklonjenost tako staršev kot tudi drugih ljudi. Takoj se je bila pripravljena odzvati na pobude, ki so se pojavljale v njenem okolju, se kot otok vključevati v igro vrstnikov, se odzivati na družabne situacije in dejavnosti v svojem okolju, se vklju- čevati v športne, kulturne, interesne in različne druge dejavnosti, ki so obstajale v njenem okolju in so jo vabile, da se jim pridruži in se vključi vanje. Rada se je pridružila petju, ko smo v družini, zlasti med vožnjo z avtomobilom, prepevali ljudske pesmi ali znane melo- dije; z veseljem se nam je pridružila, ko smo šli na pohode v naravo, bodisi v bližnje hribe bodisi v planine bodisi na morje ali pa v neznane domače in tuje kraje. Že zelo zgodaj je postala članica plavalnega kluba in je ves čas, ko je obiskovala osnovno šolo, trenirala plavanje; pri tem je bila zelo uspešna, saj je, ko je bila stara okoli 10 let, zasedla tretje mesto v Jugoslaviji v plavanju v prsnem slogu. Ko je postal trening zelo zahteven in je bilo treba trenirati trikrat na dan (zjutraj, pred šolo, takoj po šoli in zvečer), se je pametno odločila, da bo prenehala trenirati plavanje in bo začela trenirati skoke v vodo, ki so zahte- vali manj časa. Obiskovala je likovne delavnice v Pionirskem domu in glasbeno šolo, kjer ANDRAGOŠKA SPOZNANJA/STUDIES IN ADULT EDUCATION AND LEARNING 1/2021 ANDRAGOŠKA SPOZNANJA/STUDIES IN ADULT EDUCATION AND LEARNING 1/2021 188 se je osem let učila klavir in prav toliko ali leto dlje kitaro. Zlasti kitara ji je prav prišla, da je zaigrala v družbi kot mladostnica ali kasneje ob različnih družabnih priložnostih. Občudovala je naravo in tuje kraje, kamor smo se napotili na potovanjih. Ko je postala profesorica na fakulteti, je bila med študentkami in študenti zelo priljubljena. Ves čas se je v razredu gibala med njimi in jih animirala, izogibala se je položaju predavateljice za katedrom. Študentke in študentje so jo radi prosili za mentorstvo pri diplomskih, magi- strskih in doktorskih nalogah. Direktorica ljudske univerze iz Radovljice Mateja Rozman Amon je v svojem sožalnem sporočilu o njej napisala, da jo je zelo spoštovala in da je bila Sabina »vedno srčna, bojevita in prodorna strokovnjakinja na svojem področju, brez dlake na jeziku«, direktorica Zavoda za izobraževanje in kulturo Črnomelj Nada Žagar pa: »Globoko smo jo spoštovali in cenili kot strokovnjakinjo in žensko. Vedno se bomo spominjali prisrčnih in iskrivih srečanj ob različnih priložnostih, ko ni manjkalo toplih besed, spodbud, pohval in smeha.« Še pedagoginja mag. Marija Velikonja: »Sabino še ve- dno vidim kot sproščeno, prijazno, razgledano, delavno kolegico, ki je povezovala družbo ob sebi in pogosto premalo mislila nase.« Kombinacija izbire poklicnih področij in osebnostnih značilnosti je tisto, kar je Sabini omogočilo, da je postala uspešna andragoginja. Morda bi lahko vse opisane značilnosti, ki so odlikovale Sabino, opredelili kot tisto optimalno osebnostno in vedenjsko strukturo, ki je potrebna za uspešno delovanje andragoga. Ponovimo: zanimanje za poklic s podro- čja vzgoje in izobraževanja; pri tem je andragogika, delo z odraslimi, bolj ustrezna kot pedagogika in delo z otroki, zlasti v šolah; občutljivost za vprašanja strukture, razvoja in delovanja družbe ter skupnosti, vključno s politiko in kritičnostjo do nje; poznavanje človekovega duševnega ustroja in delovanja, vključno s hierarhijo vrednot in motivov; osebnostna in vedenjska dinamičnost, živahnost, energija, odprtost, komunikativnost, do- stopnost za srečevanje in stike z ljudmi. Sabinin primer sem opisal, ker se mi zdi živo udejanjenje lika uspešne andragoginje. Zoran Jelenc
https://openalex.org/W2515998503	https://iris.uniroma1.it/bitstream/11573/893772/1/Juffe_Assessing_2016.pdf	English	null	Assessing the Cost of Global Biodiversity and Conservation Knowledge	PloS one	2,016	cc-by	13,774	Assessing the Cost of Global Biodiversity and Conservation Knowledge Diego Juffe-Bignoli1, Thomas M. Brooks2,3,4, Stuart H. M. Butchart5,6, Richard B. Jenkins7, Kaia Boe8, Michael Hoffmann1,2, Ariadne Angulo9, Steve Bachman10, Monika Böhm11, Neil Brummitt12, Kent E. Carpenter13, Pat J. Comer27, Neil Cox14, Annabelle Cuttelod7, William R. T. Darwall7, Moreno Di Marco15,16, Lincoln D. C. Fishpool5, Bárbara Goettsch7, Melanie Heath5, Craig Hilton-Taylor7, Jon Hutton1,17, Tim Johnson1, Ackbar Joolia7, David A. Keith18,29, Penny F. Langhammer19, Jennifer Luedtke9, Eimear Nic Lughadha10, Maiko Lutz10, Ian May5, Rebecca M. Miller8,20, María A. Oliveira- Miranda21, Mike Parr28, Caroline M. Pollock7, Gina Ralph13, Jon Paul Rodríguez2,21,22, Carlo Rondinini23, Jane Smart2,24, Simon Stuart1,2, Andy Symes5, Andrew W. Tordoff25, Stephen Woodley26, Bruce Young27, Naomi Kingston1 a1111 1 United Nations Environment Programme, World Conservation Monitoring Centre (UNEP-WCMC), 219 Huntingdon Road, CB3 0DL Cambridge, United Kingdom, 2 International Union for Conservation of Nature (IUCN), 28 rue Mauverney, 1196 Gland, Switzerland, 3 World Agroforestry Center (ICRAF), University of the Philippines Los Baños, Laguna 4031, Philippines, 4 Institute for Marine and Antarctic Studies, University of Tasmania, Hobart TAS 7001, Australia, 5 BirdLife International, David Attenborough Building, Pembroke Street, Cambridge CB2 3QZ, United Kingdom, 6 Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, United Kingdom, 7 IUCN Global Species Programme, The David Attenborough Building, Pembroke Street, Cambridge CB2 3QZ, United Kingdom, 8 Nature-based Solutions Group, IUCN, 28 Rue Mauverney, 1196 Gland, Switzerland, 9 IUCN Species Survival Commission, Amphibian Specialist Group, Toronto M8W 1R2, Canada, 10 Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3AB, United Kingdom, 11 Institute of Zoology, Zoological Society of London, Regent’s Park, London NW1 4RY, United Kingdom, 12 Department of Life Sciences, Natural History Museum, London SW7 5BD, United Kingdom, 13 IUCN Marine Biodiversity Unit, Global Species Programme/ Biological Sciences, Old Dominion University, Norfolk, Virginia, United States of America, 14 IUCN CI Biodiversity Assessment Unit, IUCN Global Species Programme, c/o Conservation International, 2011 Crystal Drive, Suite 500, Arlington, VA 22202, United States of America, 15 ARC Centre of Excellence for Environmental Decisions, Centre for Biodiversity and Conservation Science, The University of Queensland, 4072 Brisbane, Queensland, Australia, 16 School of Geography, Planning and Environmental Management, The University of Queensland, 4072 Brisbane, Queensland, Australia, 17 Luc Hoffmann Institute, WWF International, 1196 Gland, Switzerland, 18 Centre for Ecosystem Science, University of New South Wales, Sydney, New South Wales 2052, Australia, 19 School of Life Sciences, Arizona State University, P.O. RESEARCH ARTICLE Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by the MAVA foundation: http://en.mava-foundation.org/ (TMB), Integrated Biodiversity Assessment Tool (IBAT): https://www.ibatforbusiness.org/ (DJ), National Science Foundation’s Dimensions of Biodiversity program: http://www.nsf.gov/; award 1136586 (BY), the Rufford Foundation: http://www.rufford.org/ (MB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. diego.juffe-bignoli@unep-wcmc.org Assessing the Cost of Global Biodiversity and Conservation Knowledge Box 874501, Tempe, AZ 85287, United States of America, 20 IUCN Global Ecosystem Management Programme, The David Attenborough Building, Pembroke Street, Cambridge CB2 3QZ, United Kingdom, 21 Provita, Apdo. 47552, Caracas 1041-A, Venezuela, 22 Centro de Ecología, Instituto Venezolano de Investigaciones Científicas, Apdo. 20632, Caracas 1020-A, Venezuela, 23 Global Mammal Assessment Program, Department of Biology and Biotechnologies, Sapienza University of Rome, Viale dell'Università 32, I-00185 Rome, Italy, 24 Biodiversity Conservation Group, IUCN, 28 Rue Mauverney, 1196 Gland, Switzerland, 25 Critical Ecosystem Partnership Fund, 2011 Crystal Drive, Suite 500, Arlington, VA 22202, United States of America, 26 World Commission on Protected Areas IUCN, 64 Juniper Road, Chelsea, QC J9B1T3, Canada, 27 NatureServe, 4600 N. Fairfax Dr., Arlington, VA 22203, United States of America, 28 American Bird Conservancy, 1731 Connecticut Avenue, Washington DC 20009, United States of America, 29 New South Wales Office of Environment and Heritage, Hurstville, New South Wales 2220, Australia OPEN ACCESS Citation: Juffe-Bignoli D, Brooks TM, Butchart SHM, Jenkins RB, Boe K, Hoffmann M, et al. (2016) Assessing the Cost of Global Biodiversity and Conservation Knowledge. PLoS ONE 11(8): e0160640. doi:10.1371/journal.pone.0160640 Editor: RunGuo Zang, Chinese Academy of Forestry, CHINA Editor: RunGuo Zang, Chinese Academy of Forestry, CHINA Received: April 25, 2016 Accepted: July 24, 2016 Published: August 16, 2016 Copyright: © 2016 Juffe-Bignoli et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. The Cost of Global Biodiversity and Conservation Knowledge Considerable resources are dedicated to developing and maintaining knowledge products for biodiversity conservation, and they are widely used to inform policy and advise decision makers and practitioners. However, the financial cost of delivering this information is largely undocumented. We evaluated the costs and funding sources for developing and maintain- ing four global biodiversity and conservation knowledge products: The IUCN Red List of Threatened Species, the IUCN Red List of Ecosystems, Protected Planet, and the World Database of Key Biodiversity Areas. These are secondary data sets, built on primary data collected by extensive networks of expert contributors worldwide. We estimate that US$160 million (range: US$116–204 million), plus 293 person-years of volunteer time (range: 278– 308 person-years) valued at US$ 14 million (range US$12–16 million), were invested in these four knowledge products between 1979 and 2013. More than half of this financing was provided through philanthropy, and nearly three-quarters was spent on personnel costs. The estimated annual cost of maintaining data and platforms for three of these knowl- edge products (excluding the IUCN Red List of Ecosystems for which annual costs were not possible to estimate for 2013) is US$6.5 million in total (range: US$6.2–6.7 million). We esti- mated that an additional US$114 million will be needed to reach pre-defined baselines of data coverage for all the four knowledge products, and that once achieved, annual mainte- nance costs will be approximately US$12 million. These costs are much lower than those to maintain many other, similarly important, global knowledge products. Ensuring that biodi- versity and conservation knowledge products are sufficiently up to date, comprehensive and accurate is fundamental to inform decision-making for biodiversity conservation and sustainable development. Thus, the development and implementation of plans for sustain- able long-term financing for them is critical. Competing Interests: The authors have declared that no competing interests exist. Abstract Knowledge products comprise assessments of authoritative information supported by stan- dards, governance, quality control, data, tools, and capacity building mechanisms. 1 / 22 PLOS ONE \| DOI:10.1371/journal.pone.0160640 August 16, 2016 PLOS ONE \| DOI:10.1371/journal.pone.0160640 August 16, 2016 Development status of the four knowledge products included in this stud London; (2) Protected Planet (incorporating the World Database on Protected Areas, which documents designation of areas for conservation) developed by IUCN and UNEP-WCMC; (3) the World Database of Key Biodiversity Areas (KBAs; which documents sites contributing sig- nificantly to the global persistence of biodiversity), developed by BirdLife International, the Critical Ecosystem Partnership Fund, IUCN and several other organisations that are currently joining together to establish a KBA Partnership; and (4) the IUCN Red List of Ecosystems (which documents risk of ecosystem collapse), developed by IUCN and a team of partners, including Provita, Centre for Ecosystem Science (UNSW Australia) and NSW Office of Envi- ronment and Heritage. These four knowledge products are in different phases of development that span from established and widely used in conservation practice and policy, to initial imple- mentation (Table 1). These four knowledge products are widely applicable in conservation decision-making in at least four contexts. First, they are fundamental for tracking progress towards international bio- diversity commitments such as the Millennium Development Goals [8, 9, 10], the Aichi Biodi- versity Targets of the 2020 Strategic Plan for Biodiversity [11, 12, 13], and the emerging Sustainable Development Goals [14, 15, 7]. Notably, around a third of the indicators used to assess current status of Aichi Biodiversity Targets draw information from The IUCN Red List of Threatened Species, Protected Planet, and/or the World Database of Key Biodiversity Areas [16, 13]. Second, they are increasingly used by the private and banking sectors to assess poten- tial biodiversity risks associated with their activities [17, 18]. Third, they are extensively used to inform conservation strategies and plans, guiding, for example, investments by the Global Environment Facility and the Critical Ecosystem Partnership Fund [19, 20, 21], as well as numerous National Biodiversity Strategies and Action Plans under the Convention on Biologi- cal Diversity [22] and global and regional environmental assessments [23]. Fourth, and possi- bly most importantly, they are used to inform local land/seascape planning decisions around the world, although this is often hard to document (e.g., [24], [25], [26], [27]). While not all four knowledge products may have been used yet in all four contexts they are certainly designed to do so. However, the delivery of all four of these knowledge products faces two major challenges. Knowledge product Development of standards and processes Development of data, tools, products and capacity The IUCN Red List of Threatened Species [67, 68] Advanced, datasets comprehensive globally for many taxa and countries Adopted by IUCN Council Decision C/51/ 35 www.iucnredlist.org Protected Planet [69, 70] Advanced, datasets comprehensive globally for most countries. www.protectedplanet.nethttp://www. protectedplanet.net/ United Nations Economic and Social Council (ECOSOC) Resolution 713 (XXVII) Endorsed by IUCN Resolution WCC- 2012-Res-040 The World Database of Key Biodiversity Areas [53, 71] Datasets for some components of biodiversity (i.e. IBAs, AZE sites); other components in progress. Database, tools and capacity development for KBA identiﬁcation are being improved and expanded. Adopted by IUCN Council Decision C/88/ 25. www.birdlife.org/datazone/site www.zeroextinction.org/search.cfm IUCN Red List of Ecosystems [57, 61, 72] Some countries and regions completed. Pilot databases developed, tools and capacity building support are available; additional resources are underway. Adopted by IUCN Council Decision C/83/ 17. www.iucnredlistofecosystems.org doi:10.1371/journal.pone.0160640.t001 Table 1. Development status of the four knowledge products included in this study. A brief description of each knowledge product is available in [7]. Knowledge product Development of standards and processes Development of data, tools, products and capacity The IUCN Red List of Threatened Species [67, 68] Advanced, datasets comprehensive globally for many taxa and countries Adopted by IUCN Council Decision C/51/ 35 www.iucnredlist.org Protected Planet [69, 70] Advanced, datasets comprehensive globally for most countries. www.protectedplanet.nethttp://www. protectedplanet.net/ United Nations Economic and Social Council (ECOSOC) Resolution 713 (XXVII) Endorsed by IUCN Resolution WCC- 2012-Res-040 The World Database of Key Biodiversity Areas [53, 71] Datasets for some components of biodiversity (i.e. IBAs, AZE sites); other components in progress. Database, tools and capacity development for KBA identiﬁcation are being improved and expanded. Adopted by IUCN Council Decision C/88/ 25. www.birdlife.org/datazone/site www.zeroextinction.org/search.cfm IUCN Red List of Ecosystems [57, 61, 72] Some countries and regions completed. Pilot databases developed, tools and capacity building support are available; additional resources are underway. Adopted by IUCN Council Decision C/83/ 17. www.iucnredlistofecosystems.org doi:10.1371/journal.pone.0160640.t001 Table 1. Introduction We live in an era of unprecedented access to biodiversity and conservation data and informa- tion [1]. An enormous wealth of primary biodiversity data are collected around the world directly through field surveys or indirectly through remote sensing, and more recently through citizen science [2, 3]. To be useful, these primary data must be collated into databases, validated and processed into information that can be readily used to improve knowledge, and thus inform decision-making in policy and practice, as well as to transfer knowledge and enhance capacity building [4]. New technologies have facilitated the development of biodiversity infor- mation platforms that aim to make such information easily accessible to, and usable, by a wide range of audiences [5]. Shifting from potential usability by specialists to actual utility therefore requires the development of knowledge products. UNDP [6] describes good knowledge prod- ucts as: relevant; based on an assessment of demand, audience needs, and unbiased evaluation; timely; clearly and consistently written and presented; developed through participatory pro- cesses; and easily accessible. In the context of biodiversity and its conservation, Brooks et al. [7] identify standards, governance, quality control, data, tools, and capacity building mechanisms as common components for four knowledge products: (1) The IUCN Red List of Threatened Species (which documents species extinction risk) developed by IUCN and a consortium of Red List Partners, including BirdLife International, Botanic Gardens Conservation Interna- tional, Conservation International, Microsoft Research, NatureServe, Royal Botanic Gardens Kew, Sapienza University of Rome, Texas A&M, Wildscreen and the Zoological Society of PLOS ONE \| DOI:10.1371/journal.pone.0160640 August 16, 2016 2 / 22 The Cost of Global Biodiversity and Conservation Knowledge London; (2) Protected Planet (incorporating the World Database on Protected Areas, which documents designation of areas for conservation) developed by IUCN and UNEP-WCMC; (3) the World Database of Key Biodiversity Areas (KBAs; which documents sites contributing sig- nificantly to the global persistence of biodiversity), developed by BirdLife International, the Critical Ecosystem Partnership Fund, IUCN and several other organisations that are currently joining together to establish a KBA Partnership; and (4) the IUCN Red List of Ecosystems (which documents risk of ecosystem collapse), developed by IUCN and a team of partners, including Provita, Centre for Ecosystem Science (UNSW Australia) and NSW Office of Envi- ronment and Heritage. These four knowledge products are in different phases of development that span from established and widely used in conservation practice and policy, to initial imple- mentation (Table 1). These four knowledge products are widely applicable in conservation decision-making in at least four contexts. First, they are fundamental for tracking progress towards international bio- diversity commitments such as the Millennium Development Goals [8, 9, 10], the Aichi Biodi- versity Targets of the 2020 Strategic Plan for Biodiversity [11, 12, 13], and the emerging Sustainable Development Goals [14, 15, 7]. Notably, around a third of the indicators used to assess current status of Aichi Biodiversity Targets draw information from The IUCN Red List of Threatened Species, Protected Planet, and/or the World Database of Key Biodiversity Areas [16, 13]. Second, they are increasingly used by the private and banking sectors to assess poten- tial biodiversity risks associated with their activities [17, 18]. Third, they are extensively used to inform conservation strategies and plans, guiding, for example, investments by the Global Environment Facility and the Critical Ecosystem Partnership Fund [19, 20, 21], as well as numerous National Biodiversity Strategies and Action Plans under the Convention on Biologi- cal Diversity [22] and global and regional environmental assessments [23]. Fourth, and possi- bly most importantly, they are used to inform local land/seascape planning decisions around the world, although this is often hard to document (e.g., [24], [25], [26], [27]). While not all four knowledge products may have been used yet in all four contexts they are certainly designed to do so Table 1. Development status of the four knowledge products included in this study. A brief description of each knowledge product is available in [7]. PLOS ONE \| DOI:10.1371/journal.pone.0160640 August 16, 2016 First, none have reached their stated goals for comprehensive data coverage to build a robust PLOS ONE \| DOI:10.1371/journal.pone.0160640 August 16, 2016 3 / 22 The Cost of Global Biodiversity and Conservation Knowledge baseline. These shortfalls in coverage leave serious biases in the datasets; e.g., in geographic and taxonomic coverage for The IUCN Red List of Threatened Species and for the World Database of Key Biodiversity Areas; in taxonomic and geographic coverage for the IUCN Red List of Ecosystems; and in governance types of protected areas documented by Protected Planet. Sec- ond, their utility is dependent on their quality and currency [28]. This means they require con- tinuous maintenance and updating to reflect, for example, the impacts of changing taxonomy or protected area legislation, emerging threats or status changes resulting from conservation and development action, and to keep abreast of innovations in technology. The overall cost of such development and maintenance of these knowledge products has not been assessed comprehensively. The few studies available have focused on The IUCN Red List of Threatened Species (e.g. [29], [30], [28], [31], [32], [33]). Most of these studies typically included only the cost of conducting species extinction risk assessments, but not the costs of maintaining structures and processes associated with the delivery and publication of such assessments. There have also been a number of studies quantifying the cost of conservation actions (e.g.,[34]) and the cost of achieving conservation targets (e.g., [35], [36]), but, again, these do not include estimates of the cost of knowledge generation from primary data necessary to guide the planning and monitoring of such actions and achievement of targets. The financial cost of developing and maintaining these biodiversity and conservation knowledge products is not trivial. Quantifying this cost is fundamental for those who invest in their delivery, to ensure sustainable financing in the long term, and to allow exploration of ways to minimise these costs by improving the efficiency of data collection, validation pro- cesses, and dissemination, through, for instance, integration in unified technological platforms [28]. Given the widespread use of these knowledge products to inform policy and decision making across sectors and at global, national and regional scales, it is equally important to understand their value as a global good, and to put in perspective the relative benefit of the con- servation outcomes with respect to the investment in developing and maintaining knowledge products. There is a clear a gap in the understanding of the actual costs associated with the delivery of global biodiversity datasets, their funding sources, and the relative importance of these invest- ments. To fill this gap, we assess the costs of developing and maintaining The IUCN Red List of Threatened Species, Protected Planet, The World Database of Key Biodiversity Areas, and the IUCN Red List of Ecosystems. First, we evaluate the funding invested until 2013 and the annual costs in 2013 for each knowledge product (excluding the IUCN Red List of Ecosystems for which annual costs were not possible to estimate for 2013), along with the sources of this financing (by sector) and types of expenditure. We explain the scope of this study and we describe how the data on costs were collected and standardised. Second, we estimate the fund- ing needed to reach pre-defined baselines by 2020 for each of these knowledge products, and the estimated annual costs of maintaining such expanded data coverage. Finally, we discuss the results in the context of ensuring long-term sustainability and delivery of biodiversity and con- servation knowledge products to inform decision-making in diferent contexts, and show that the funding needed to do this is relatively low compared with other similar processes. Scope of the study Biodiversity knowledge products are compiled and maintained in diverse ways, with multiple phases of data collection and processing, each associated with a cost (Fig 1). Typically, primary biodiversity data, which underpin the knowledge products, go through several stages of data collation, compilation, validation, and quality control before knowledge products are made 4 / 22 PLOS ONE \| DOI:10.1371/journal.pone.0160640 August 16, 2016 The Cost of Global Biodiversity and Conservation Knowledge available to the wider community and general public in various forms (databases, spatial dat i di b i bli i ) Fig 1. Different stages of knowledge generation and dissemination covered by the four knowledge products for which costs are included in this review. The coloured bars show which stages are covered by each knowledge product currently which were co in the study. In 2013 only IBAs, AZE and KBAs identified through CEPF hotspot ecosystem profiling processes cover stages 4, 5 and The IUCN Red List of Ecosystems will cover stages 4 and 7 in the near future; an extensive list of technical resources is available at www.iucnredlistofecosystems.org but no spatial data or indicators are yet available. doi:10.1371/journal.pone.0160640.g001 The Cost of Global Biodiversity and Conservation Knowle Fig 1. Different stages of knowledge generation and dissemination covered by the four knowledge products for which costs are included in this review. The coloured bars show which stages are covered by each knowledge product currently which were costed in the study. In 2013 only IBAs, AZE and KBAs identified through CEPF hotspot ecosystem profiling processes cover stages 4, 5 and 7. The IUCN Red List of Ecosystems will cover stages 4 and 7 in the near future; an extensive list of technical resources is available at www.iucnredlistofecosystems.org but no spatial data or indicators are yet available. doi:10.1371/journal.pone.0160640.g001 doi:10.1371/journal.pone.0160640.g001 available to the wider community and general public in various forms (databases, spatial data, assessments, indicators, web services, publications, etc.). Here, we focus on the costs of the secondary processes that are directly and completely devoted to the delivery of the knowledge products (see Table 2 and S3 Table). We do not PLOS ONE \| DOI:10.1371/journal.pone.0160640 August 16, 2016 5 / 22 The Cost of Global Biodiversity and Conservation Knowledge Table 2. Categories, subcategories and funding sources classification used to categorise costs. Cost Category Deﬁnition Concept development Cost of the process of developing the knowledge product concept (e.g. Scope of the study development of IUCN Red List of Ecosystems Categories and Criteria) Data development and assessments Cost of compilation of primary data and assessment using the knowledge product standards (e.g. ﬁrst global bird extinction risk assessment) Maintenance of standards and systems On-going costs per year of managing and maintaining data standards and information systems such as databases and websites (e.g. maintenance of The IUCN Red List of Threatened Species database). Monitoring and indicator development On-going costs per year of repeat assessments to maintain coverage of data compiled, to derive indicators (e.g. Global Protected Area Coverage). Cost Sub-category Deﬁnition Personnel time Remunerated time of individuals Travel and workshops Travel and accommodation expenses Infrastructure Maintenance of ofﬁces, information systems information systems such as databases and websites. Publications and outreach Reports, books, leaﬂets. It does not include cost of producing scientiﬁc papers. Funding Source Examples Governments Ministry for the Environment, Germany Financial Institutions World Bank Intergovernmental organizations (IGOs) United Nations Environment Programme Multilateral donor Global Environment Facility (GEF) Non-Governmental Organizations (NGOs) Birdlife International, Conservation International, academia Philanthropy MacArthur Foundation, MAVA Foundation, Gordon and Betty Moore Foundation Private Sector Shell, Rio Tinto Voluntary time Non-remunerated time spent in reviewing assessments, providing and validating data, attending meetings and workshops, etc. doi:10.1371/journal.pone.0160640.t002 doi:10.1371/journal.pone.0160640.t002 include the costs of collection of primary data [37] which, while essential for the delivery of the knowledge product, precede work on the knowledge products themselves and are not exclusive to them. The most obvious example of primary cost is gathering data in the field. For example, we did not include the costs of establishing, mapping, digitizing, and maintaining national databases of protected areas or the cost of surveying, classifying and mapping ecosystems for the Red List of Ecosystems; rather we included the costs of aggregating these data centrally through workshops and direct contact with governmental protected area agencies, and subse- quent quality checking and revision before making them available online. Similarly, for The IUCN Red List of Threatened Species, we did not include the costs of establishing, digitizing and maintaining specimen locality databases. PLOS ONE \| DOI:10.1371/journal.pone.0160640 August 16, 2016 Scope of the study We also excluded the costs of maintaining net- works supporting data collection such as the IUCN Species Survival Commission (IUCN SSC) network of Specialist Groups and Red List Authorities which support The IUCN Red List of Threatened Species; the network of BirdLife Partners worldwide who lead identification and monitoring of Important Bird and Biodiversity Areas (the subset of KBAs identified using data for birds [26]; or the IUCN World Commission on Protected Areas (IUCN WCPA) network supporting the development of the WDPA. include the costs of collection of primary data [37] which, while essential for the delivery of the knowledge product, precede work on the knowledge products themselves and are not exclusive to them. The most obvious example of primary cost is gathering data in the field. For example, we did not include the costs of establishing, mapping, digitizing, and maintaining national databases of protected areas or the cost of surveying, classifying and mapping ecosystems for the Red List of Ecosystems; rather we included the costs of aggregating these data centrally through workshops and direct contact with governmental protected area agencies, and subse- quent quality checking and revision before making them available online. Similarly, for The IUCN Red List of Threatened Species, we did not include the costs of establishing, digitizing and maintaining specimen locality databases. We also excluded the costs of maintaining net- works supporting data collection such as the IUCN Species Survival Commission (IUCN SSC) network of Specialist Groups and Red List Authorities which support The IUCN Red List of Threatened Species; the network of BirdLife Partners worldwide who lead identification and monitoring of Important Bird and Biodiversity Areas (the subset of KBAs identified using data for birds [26]; or the IUCN World Commission on Protected Areas (IUCN WCPA) network supporting the development of the WDPA. These processes are important for numerous reasons and applications above and beyond the delivery of biodiversity and conservation knowledge products, and so including their costs PLOS ONE \| DOI:10.1371/journal.pone.0160640 August 16, 2016 6 / 22 The Cost of Global Biodiversity and Conservation Knowledge here would inflate our estimates inappropriately. However, it must be emphasized that these processes, worth many millions of dollars per year (see, e.g., [38], [39]) and are critical to proper establishment, development and ongoing maintenance of knowledge products. Compiling costs Data collection for this review was undertaken from August 2013 to November 2014. We obtained and collated costs of: 1) developing and maintaining knowledge products from the earliest year for which data on costs were available (for each product) up until December 2013; 2) annual costs of maintaining data, structures, and processes in 2013; and 3) projected future expenditure to achieve pre-defined baselines by 2020. To compile the past, present and future costs in a standardised way we classified costs by knowledge product stage, cost type, and funding source (Table 2). We contacted individuals who manage or have managed knowledge product development processes (henceforth referred to as study data providers), and asked them to provide the relevant cost data. A spreadsheet with options and specific instructions were created for collection of data in a standardised way, to facilitate subsequent analyses. The spreadsheet included drop-down lists and free-text fields which allowed study data providers to classify each cost under each of the pre-defined expendi- ture categories and subcategories, and funding sources. Study data providers were requested to provide minimum and maximum estimates where precise values were not known, currency and year of currency, and minimum and maximum volunteer time invested. All files received were then merged into a single database which included all costs collected for each of the four knowledge products. Finally, to account for inflation and standardise estimates, all costs were converted to 2014 US$ (see S1 Table). Scope of the study There- fore, the costs reported herein for knowledge product delivery represent a fraction of the total cost to populate knowledge products with data (e.g. Fig 1, stage 1). Similarly, we did not assess the costs or benefits that result from use of knowledge products which are typically incurred by end-users of the information (e.g., conservation actions such as protected area establishment and management, interventions for threatened species, changes to land use policies, etc.). Finally, we excluded the costs of national assessments which follow national standards, such as national red lists of species or designating and delineating new protected areas. Some national species assessments, for example, do contribute to the global IUCN Red Lists of Threatened Species and Ecosystems when a nationally endemic species or ecosystem type is assessed using the IUCN Categories and Criteria and the data are shared with IUCN [40, 27], but the processes and costs of generating a national red list usually occur and are borne inde- pendently of the global IUCN Red Lists [25, 41]. PLOS ONE \| DOI:10.1371/journal.pone.0160640 August 16, 2016 The Cost of Global Biodiversity and Conservation Knowledge Table 3. Summary of data collection for all four knowledge products. The table summarises which costs were collected for each of the four knowledge products and how much of the total number of assesments, available in December 2013, these represent. In cases where 100% of the costs were not col- lected, the total sum for each knowledge product was increased propotionally to reach 100%. Knowledge product Organizations that provided data for this study Units Number of units Period covered Percentage of dataset (December 2013) The IUCN Red List of Threatened Species1 IUCN, BirdLife International, University of Rome, Zoological Society of London, NatureServe, Royal Botanical Gardens Kew, and Natural History Museum London. Species assessments and associated spatial and tabular data 76,068 1985– 2013 68% Protected Planet2 UNEP-World Conservation Monitoring Centre and IUCN Protected areas spatial and tabular data 214,000 1981– 2013 100% The World Database of Key Biodiversity Areas (KBAs)3 BirdLife International, Alliance for Zero Extinction, IUCN, and CEPF. Key Biodiversity Areas spatial and tabular data 17,732 1979– 2013 88% IUCN Red List of Ecosystems4 IUCN, Provita, Instituto Venezolano de Investigaciones Cientiﬁcas (IVIC), and the Centre for Ecosystem Science, University of New South Wales, Australia Ecosystem assessments and associated spatial and tabular data 616 2004– 2013 100% 1The dataset assessed was all species published on www.iucnredlist.org by the end of 2013 including re-assessments. 2The dataset assessed was the December 2013 version of World Database on Protected Areas. 3The dataset assessed was the World Birds and Biodiversity Database. 4The dataset assessed was all ecosystem assessments completed or about to be completed by end of 2013. Table 3. Summary of data collection for all four knowledge products. The table summarises which costs were collected for each of the four knowledge products and how much of the total number of assesments, available in December 2013, these represent. In cases where 100% of the costs were not col- lected, the total sum for each knowledge product was increased propotionally to reach 100%. 1The dataset assessed was all species published on www.iucnredlist.org by the end of 2013 including re-assessments. 2The dataset assessed was the December 2013 version of World Database on Protected Areas. 3The dataset assessed was the World Birds and Biodiversity Database. 4The dataset assessed was all ecosystem assessments completed or about to be completed by end of 2013. doi:10.1371/journal.pone.0160640.t003 considerable support from the IUCN WCPA network. Addressing data gaps We anticipated that we would be unable to obtain complete data on all of the past costs for each knowledge product (particularly for processes and components dating from further in the past). While the origins of The IUCN Red List of Threatened Species date back to the early 1960s [42], very little information was available on the costs of development of The IUCN Red List of Threatened Species methodology and infrastructure prior to 1999, except for the costs of assessing all birds globally, which date from 1985. Similarly, we compiled the costs associated with the establishment of Protected Planet since 1981, when the first digital version of the World Database on Protected Areas (WDPA)–the database underpinning Protected Planet since 2008 –was created. However, the history of the WDPA goes back to 1959, when the United Nations (UN) Economic and Social Council called for a list of national parks and equiv- alent reserves in their natural state (Resolution 713 (XXVIII)), which was then developed with PLOS ONE \| DOI:10.1371/journal.pone.0160640 August 16, 2016 7 / 22 PLOS ONE \| DOI:10.1371/journal.pone.0160640 August 16, 2016 Accounting for voluntary time Voluntary time (including time donated to knowledge product delivery that is paid for by other institutions, universities, museums, governments, departments and NGOs) plays a cru- cial role in the production of biodiversity information. For example, more than ten thousand individuals through the IUCN Commissions and many more outside the Commissions, have provided voluntary input to the delivery of knowledge products [7]. These range from individ- uals compiling information to experts participating in workshops, reviewing assessments, pro- viding data, and contributing to technical committees or governing bodies. Here, we recorded volunteer time invested in each knowledge product in terms of the number of working days (one working day equalling eight hours) and considering 240 working days per year (52 weeks per year minus four weeks of holidays at five working days per week). In many instances (if not most), because it was not an official requirement in project man- agement, volunteer time invested had not even been accounted for or was not consistently recorded, making it difficult to apply a valid method. For these reasons we are likely to have underestimated substantially the total extent of volunteer time invested. We acknowledge that our estimates are uncertain, do not necessarily reflect the variation in any given process, and in some instances they may only capture a small subset of the input invested into assessments. For example, for amphibian assessments on the IUCN Red List considered in this paper, the estimate of volunteer time is solely based on workshop participation from 2001–2004 (see S2 Table and S3 Table); however, as mentioned above, this is just one form of input volunteers contribute to assessments, and it does not capture the full range of work required for these processes. Acknowledging the complexity of measuring the value of volunteer time, Salamon et al. [43] recommend the use of the replacement cost method which estimates the value of the work that the volunteer performs based on market wages. The enormous variation in expertise, experi- ence, and geographic location of individuals providing it makes assiging a monetary value to the time invested by volunteers in the development and maintanance of biodiversity knowledge products highly complex. Nevertheless, we assigned a value to volunteer time using a higher and lower estimate attempting to assign a financial value to the known volunteer contributions based on different levels of experience and expertise. Accounting for voluntary time The higher estimate was US$30.97 per hour, which is the mean hourly rate of a conservation scientist according to the U.S. Bureau of Labor Statistics [44]. The lower estimate is US$23.07 per hour, a generic volunteer time esti- mate provided by Independent Sector [45]. Two levels of uncertainty are incorporated through this approach. First, the uncertainty reflected in the maximun and minimum working days assigned in each estimate. Second, uncertainty on the monetary value assigned to those estimates. Therefore, the financial and voluntary contributions from IUCN WCPA to the development and compilation of protected areas information before the establishment of a digital database on protected areas were not accounted for. To estimate the total funds invested in knowledge products until 2013, we compared the number of units of assessment for which we obtained cost estimates (e.g., species in The IUCN Red List of Threatened Species with a known assessment cost) with the total number of assess- ment units in the database (e.g. total number of species on The IUCN Red List of Threatened Species) in December 2013 and scaled the total cost estimate proportionately (see Table 3). This assumes that the volume of available data for compilation, standards, methods and effort involved in assessments did not change through time. This is not the case especially for species assessments where the methods applied were different as the system used for assessing risk for extinction of species was updated in 1994. It is therefore likely that this have led to some under-estimation of earlier costs due to changes in criteria and increasing rigour in standards of assessment and documentation. Our data comprised 875 cost records for all four knowledge products sourced from 11 study data providers (Table 3). As anticipated, this does not represent 100% of the costs for each knowledge product (Table 3). However, we covered the costs for conducting 76,068 species assessments (relating to 39,533 species, some of them assessed multiple times) between 1985 and 2013. This represented 68% of species assessments on The IUCN Red List of Threatened Species as of December 2013, hence we proportionately rescaled the cost to match 100% of the species. Similarly, our data for KBAs from 1979–2013 accounted for 88% of existing KBAs, and we rescaled the cost to represent 100% of exisiting sites. Aside from the important exclusions outlined in these Methods–notably the exclusion of primary data generation and compilation costs by national agencies–all of the secondary known costs associated with producing Pro- tected Planet and the global IUCN Red List of Ecosystems were collected, so these were not increased other than by adjusting all the costs to account for inflation. PLOS ONE \| DOI:10.1371/journal.pone.0160640 August 16, 2016 8 / 22 The Cost of Global Biodiversity and Conservation Knowledge Reaching pre-defined baselines by 2020 higher in Europe and lower in Australia [52]. We then assessed the costs of identifying these new sites, the costs of documenting all existing sites for a wider range of taxa and under new criteria in the forth- coming KBA standard [53] drawing from the known costs of similar processes [54]. This approach is likely to result in an underestimate because IBAs are likely to contribute a smaller proportion of all KBAs, especially in freshwater [55] and marine ecosystems [56]. Moreover, the identification of KBAs for multiple taxonomic groups (see [51] and references therein), although based on the best available data at the time, has only covered a subsample of plant and invertebrate groups. • The World Database of Key Biodiversity Areas: We assessed the costs of expanding the KBA dataset, documenting existing KBAs for other taxonomic groups and criteria, and the predicted costs of a governance structure to manage the KBA program in the future. Two global networks of KBAs have been systematically identified to date: c. 12,800 Important Bird and Biodiversity Areas derived from bird data (IBAs; [49], [26]) and 587 Alliance for Zero Extinction sites derived from data for mammals, birds, a small sample of selected rep- tiles, conifers, reef-building corals, and amphibians (AZEs; [50]). In regions where KBAs have been identified for multiple taxonomic groups [51], IBAs comprised 68% of all KBAs identified [26]. We used this percentage to extrapolate from the total number of recognised IBAs to the potential total number of KBAs that may be identified globally, although we rec- ognise that IBA density varies in different regions of the world, e.g. higher in Europe and lower in Australia [52]. We then assessed the costs of identifying these new sites, the costs of documenting all existing sites for a wider range of taxa and under new criteria in the forth- coming KBA standard [53] drawing from the known costs of similar processes [54]. This approach is likely to result in an underestimate because IBAs are likely to contribute a smaller proportion of all KBAs, especially in freshwater [55] and marine ecosystems [56]. Moreover, the identification of KBAs for multiple taxonomic groups (see [51] and references therein), although based on the best available data at the time, has only covered a subsample of plant and invertebrate groups. Reaching pre-defined baselines by 2020 For each knowledge product we estimated: 1) annual maintenance costs for each year until 2020, considering inflation and annual incremental costs; 2) additional one-off costs required to reach stated target ‘baselines’ by 2020 (e.g., additional investment to improve infrastructure, new assessments to be undertaken, updating old assessments). We did this by drawing from information compiled from past and annual costs of knowledge products, plus additional future projections documented by the study data providers, specifically: • The IUCN Red List of Threatened Species: we followed Stuart et al. [30] who propose the assessment of 160,000 species based on a “subset of species broadly representative of • The IUCN Red List of Threatened Species: we followed Stuart et al. [30] who propose the assessment of 160,000 species based on a “subset of species broadly representative of PLOS ONE \| DOI:10.1371/journal.pone.0160640 August 16, 2016 9 / 22 The Cost of Global Biodiversity and Conservation Knowledge biodiversity as a whole”, including sampled assessments for selected groups [46], to enable better conservation and policy decisions at a global level. • Protected Planet: We assessed the costs of implementing the Protected Planet strategy by 2020. This includes re-structuring the Protected Planet website to improve data dissemina- tion, expanding the scope of the WDPA to record information on “other effective area-based conservation measures” [47], integrating the Global Database on Protected Area Manage- ment Effectiveness [48], and enhancing interoperability with other relevant datasets such as the other knowledge products discussed here. • The World Database of Key Biodiversity Areas: We assessed the costs of expanding the KBA dataset, documenting existing KBAs for other taxonomic groups and criteria, and the predicted costs of a governance structure to manage the KBA program in the future. Two global networks of KBAs have been systematically identified to date: c. 12,800 Important Bird and Biodiversity Areas derived from bird data (IBAs; [49], [26]) and 587 Alliance for Zero Extinction sites derived from data for mammals, birds, a small sample of selected rep- tiles, conifers, reef-building corals, and amphibians (AZEs; [50]). In regions where KBAs have been identified for multiple taxonomic groups [51], IBAs comprised 68% of all KBAs identified [26]. We used this percentage to extrapolate from the total number of recognised IBAs to the potential total number of KBAs that may be identified globally, although we rec- ognise that IBA density varies in different regions of the world, e.g. Reaching pre-defined baselines by 2020 • IUCN Red List of Ecosystems: Although there is a stated aim to complete the global IUCN Red List of Ecosystems by 2025 [57], including assessment of all terrestrial, freshwater, marine and subterranean ecosystems worldwide, for the purposes of alignment and compa- rability in our study, we regarded completion of the first global IUCN Red List of Ecosystems by 2020 as a pre-defined baseline. Indeed, if all the resources and capacity required (as esti- mated in our study) were to be made available in good time, the original 2025 target [57] could be achieved earlier. We estimated the costs for reaching this endpoint based on the costs of managing and upscaling assessment processes that are underway and the predicted cost of establishing and managing an online database for a global IUCN Red List of Ecosys- tems and putting in place governance structures and training programmes. Further informa- tion about how the projections were calculated is available in S2 Table. Personnel time is the major expense for all four knowledge products, accounting for 71% of overall costs (Fig 4). The importance of other cost sub-categories varies and depends on the processes behind each knowledge product. For example, more funds have been committed to infrastructure for knowledge products that have fully developed online platforms to access and download data, such as The IUCN Red List of Threatened Species (7% of overall costs) and Protected Planet (10%). Such systems are less well developed for The World Database of Key Biodiversity Areas (1%) and IUCN Red List of Ecosystems (2%) so far, due to their earlier stage of application. Annual costs in 2013 The delivery of knowledge products involves a series of common processes (Fig 1), but the scale of these efforts and the way the processes are managed are very different between knowl- d d t A di l l t i 2013 diff d id l f th diff t k l d Table 4. Overall funds (midpoints) and volunteer days invested in the four knowledge products between 1979 and 2013, and annual cost in 2013. The mid-point is the equidistant point between the maximum and minimum values. Full details are available in S4 Table. Knowledge product One-off costs Annual cost in 2013 Total funds Volunteer time Total funds Volunteer time The IUCN Red List of Threatened Species US$34,935,095 209 person-years US$8,788,075 US$4,785,729 2,474 days US$504,085 Protected Planet US$19,055,847 US$ 5 person-years US$242,492 US$861,000 35 days US$7,132 The World Database of Key Biodiversity Areas US$99,106,414 78 person-years US$3,269,511 US$856,414 454 days US$92,492 IUCN Red List of Ecosystems US$6,706,400 2 person-years US$80,899 Not available Not available Total US$159,803,756 293 person-years US$14,335,785 US$6,503,144 2,963 days US$603,709 doi:10.1371/journal.pone.0160640.t004 er days invested in the four knowledge products between 1979 and 2013, and annual cost in 2013. maximum and minimum values. Full details are available in S4 Table. knowledge products), followed by The IUCN Red List of Threatened Species (22%), Protected Planet (12%), and IUCN Red List of Ecosystems (4%). More than half of the funding (53%) took the form of philanthropic donations from founda- tions and individuals (Fig 2). Intergovernmental Organizations, Non-Governmental Organiza- tions and Governments have together contributed a combined total of 43% to the development and maintenance of knowledge products. The private sector, multilateral donors and financial institutions have contributed with just5% of the costs of development and maintenance of knowledge products. The relative importance of different sources of funding varies between the individual knowl- edge products (Fig 3). The IUCN Red List of Threatened Species, IUCN Red List of Ecosys- tems, and The World Database of Key Biodiversity Areas have each received more support from philanthropy than from any other single source, with governments representing an important secondary source of finance for both The IUCN Red List of Threatened Species and The World Database of Key Biodiversity Areas, while NGOs play a similar role with respect to the IUCN Red List of Ecosystems. Protected Planet has a strikingly different funding profile, being primarily funded by NGOs and Government sources, with the private sector as an important secondary funding source. This funding profile is a result of the history of Protected Planet, reflecting diverse intial number of sources supporting this knowledge product in the first decades of development, in partnership with IUCN WCPA and the subsequent contribu- tions of the Proteus Partnership (www.proteuspartners.org) in maintaining the system since 2006. Personnel time is the major expense for all four knowledge products, accounting for 71% of overall costs (Fig 4). The importance of other cost sub-categories varies and depends on the processes behind each knowledge product. For example, more funds have been committed to infrastructure for knowledge products that have fully developed online platforms to access and download data, such as The IUCN Red List of Threatened Species (7% of overall costs) and Protected Planet (10%). Such systems are less well developed for The World Database of Key Biodiversity Areas (1%) and IUCN Red List of Ecosystems (2%) so far, due to their earlier stage of application. Costs up to and including 2013 Costs up to and including 2013 In total, at least US$160 million (range: US$116—US$203 million) and at least 293 person- years of volunteer time (range: 278–308 year), valued at US$14 million (range: US$12 million– US$16 million), have been invested in the four knowledge products between 1979 and 2013 (Table 4). Total costs have been greatest in KBAs (over half of the total investment in the 10 / 22 PLOS ONE \| DOI:10.1371/journal.pone.0160640 August 16, 2016 The Cost of Global Biodiversity and Conservation Knowledge Table 4. Overall funds (midpoints) and volunteer days invested in the four knowledge products between 1979 and 2013, and annual cost in 2013. The mid-point is the equidistant point between the maximum and minimum values. Full details are available in S4 Table. Knowledge product One-off costs Annual cost in 2013 Total funds Volunteer time Total funds Volunteer time The IUCN Red List of Threatened Species US$34,935,095 209 person-years US$8,788,075 US$4,785,729 2,474 days US$504,085 Protected Planet US$19,055,847 US$ 5 person-years US$242,492 US$861,000 35 days US$7,132 The World Database of Key Biodiversity Areas US$99,106,414 78 person-years US$3,269,511 US$856,414 454 days US$92,492 IUCN Red List of Ecosystems US$6,706,400 2 person-years US$80,899 Not available Not available Total US$159,803,756 293 person-years US$14,335,785 US$6,503,144 2,963 days US$603,709 doi:10.1371/journal.pone.0160640.t004 knowledge products), followed by The IUCN Red List of Threatened Species (22%), Protected Planet (12%), and IUCN Red List of Ecosystems (4%). More than half of the funding (53%) took the form of philanthropic donations from founda- tions and individuals (Fig 2). Intergovernmental Organizations, Non-Governmental Organiza- tions and Governments have together contributed a combined total of 43% to the development and maintenance of knowledge products. The private sector, multilateral donors and financial institutions have contributed with just5% of the costs of development and maintenance of knowledge products. The relative importance of different sources of funding varies between the individual knowl- edge products (Fig 3). The IUCN Red List of Threatened Species, IUCN Red List of Ecosys- tems, and The World Database of Key Biodiversity Areas have each received more support from philanthropy than from any other single source, with governments representing an important secondary source of finance for both The IUCN Red List of Threatened Species and The World Database of Key Biodiversity Areas, while NGOs play a similar role with respect to the IUCN Red List of Ecosystems. Protected Planet has a strikingly different funding profile, being primarily funded by NGOs and Government sources, with the private sector as an important secondary funding source. This funding profile is a result of the history of Protected Planet, reflecting diverse intial number of sources supporting this knowledge product in the first decades of development, in partnership with IUCN WCPA and the subsequent contribu- tions of the Proteus Partnership (www.proteuspartners.org) in maintaining the system since 2006. Fig 2. Sources of funding (midpoints of estimates) until 2013 for all knowledge products. Others include multilateral donors and financial institutions. doi:10.1371/journal.pone.0160640.g002 for the IUCN Red List of Ecosystems was still under development, and therefore annual costs of maintaining data, structures and processes in 2013 could not be accurately estimated. for the IUCN Red List of Ecosystems was still under development, and therefore annual costs of maintaining data, structures and processes in 2013 could not be accurately estimated. PLOS ONE \| DOI:10.1371/journal.pone.0160640 August 16, 2016 Annual costs in 2013 The delivery of knowledge products involves a series of common processes (Fig 1), but the scale of these efforts and the way the processes are managed are very different between knowl- edge products. Accordingly, annual costs in 2013 differed widely for the different knowledge products: the estimated annual cost in 2013 was US$4.7 million for The IUCN Red List of Threatened Species, US$861,000 for Protected Planet, and US$856,000 for Key Biodiversity Areas (Table 4). This amounts to a total of US$6.5 million per year. In 2013, the global database PLOS ONE \| DOI:10.1371/journal.pone.0160640 August 16, 2016 11 / 22 The Cost of Global Biodiversity and Conservation Knowledge Fig 2. Sources of funding (midpoints of estimates) until 2013 for all knowledge products. Others include multilateral donors and financial institutions. d i 10 1371/j l 0160640 002 all knowledge products. Others include multilateral donors and Reaching pre-defined baslines by 2020 Overall, an additional US$103 million in one-off investments would be needed to reach pre- defined baselines for the four knowledge products by 2020 (Fig 5). However, this amount is not distributed evenly between knowledge products because they are in different stages of develop- ment. For instance, an additional US$38 million will be needed to deliver the additional c. 85,000 species assessments on The IUCN Red List of Threatened Species necessary to provide a “Barometer of Life” [30] and maintain the database structures and processes to manage these by 2020. Implementing the Protected Planet strategy will require around US$10 million, while the cost for reaching a baseline for The World Database of Key Biodiversity Areas is estimated at US$21million. Finally, US$43 million was estimated to be required to complete a global IUCN Red List of Ecosystems by 2020. Once these baselines are achieved by 2020, the annual investment required thereafter to maintain them will be US$5.4 million for The IUCN Red List of Threatened Species, US$1.9 million for Protected Planet, US$2 million for The World Data- base of Key Biodiversity Areas and US$3.7 million for the IUCN Red List of Ecosystems. 12 / 22 PLOS ONE \| DOI:10.1371/journal.pone.0160640 August 16, 2016 Fig 3. Sources of funding (midpoints of estimates) invested until 2013 for each knowledge product. doi:10.1371/journal.pone.0160640.g003 The Cost of Global Biodiversity and Conservation Knowledge The Cost of Global Biodiversity and Conservation Knowledge Fig 3. Sources of funding (midpoints of estimates) invested until 2013 for each knowledge product. doi:10.1371/journal.pone.0160640.g003 PLOS ONE \| DOI:10.1371/journal.pone.0160640 August 16, 2016 The Cost of Global Biodiversity and Conservation Knowledge Fig 4. Categories of costs (midpoints of estimates) for funding invested until 2013 for each knowledge product. doi:10 1371/journal pone 0160640 g004 Fig 4. Categories of costs (midpoints of estimates) for funding invested until 2013 for each knowledge product. Fig 4. Categories of costs (midpoints of estimates) for funding invested until 2013 for each knowledge product. Fig 4. Categories of costs (midpoints of estimates) for funding invested until 2013 for each knowledge product. 4. Categories of costs (midpoints of estimates) for funding invested until 2013 for each knowledge product. doi:10.1371/journal.pone.0160640.g004 Discussion To fill in an identified research gap, here we assess the cost of developing and maintaining four global biodiversity datasets: The IUCN Red List of Threatened Species, Protected Planet, The World Database of Key Biodiversity Areas, and the IUCN Red List of Ecosystems. We estimate that US$160 million (range: US$116–204 million), plus 293 person-years of volunteer time (range: 278–308 person-years) valued at US$ 14 million (range US$12–16 million), were invested in these four knowledge products between 1979 and 2013. In addition, US$6.5 million in total (range: US$6.2–6.7 million) were needed annually to maintain data and platforms for The IUCN Red List of Threatened Species, Protected Planet, and The World Database of Key Biodiversity Areas. Finally, we estimated that an additional US$114 million will be needed to reach pre-defined baselines of data coverage for all the four knowledge products, and that once achieved, annual maintenance costs will be approximately US$12 million. 13 / 22 PLOS ONE \| DOI:10.1371/journal.pone.0160640 August 16, 2016 PLOS ONE \| DOI:10.1371/journal.pone.0160640 August 16, 2016 Differences between biodiversity knowledge products Our results provide unique insight into the costs associated with the development and mainte- nance of some of the most extensively used biodiversity knowledge products ([58, 7] and S5 Table). The relative differences in total funds invested until 2013 and annual costs in 2013 between the four knowledge products are explained by the differences in the processes by which they are derived, as well as their differing stages of development. The IUCN Red List of Threatened Species. The costs until 2013 and annual costs in 2013 for this knowledge product include both new assessments and re-assessments (to be carried out every 10 years) (Fig 1; stage 2). Our finding that US$38 million of investment will be required to attain the 160,000 species coverage for the “Barometer of Life” is broadly consistent with Stuart et al.’s [30] estimate of US$60m, given that 20,000 previously-unassessed species have been added to The IUCN Red List of Threatened Species over the last five years. Protected Planet. The investments until 2013 and annual investment in 2013 in Protected Planet reflect global compilation, data management, and dissemination costs alone (Fig 1; PLOS ONE \| DOI:10.1371/journal.pone.0160640 August 16, 2016 14 / 22 The Cost of Global Biodiversity and Conservation Knowledge Fig 5. Estimated costs to reach pre-defined baselines by 2020 for each knowledge product. doi:10 1371/journal pone 0160640 g005 Fig 5. Estimated costs to reach pre-defined baselines by 2020 for each knowledge product. doi:10.1371/journal.pone.0160640.g005 Fig 5. Estimated costs to reach pre-defined baselines by 2020 for each knowledge product. Fig 5. Estimated costs to reach pre-defined baselines by 2020 for each knowledge product. doi:10.1371/journal.pone.0160640.g005 doi:10.1371/journal.pone.0160640.g005 stages 3–7), explaining their relatively modest costs. Now that the documentation, including boundary polygons, of protected areas managed by governments has plateaued over time [12], the bulk of the effort facing the delivery of Protected Planet will be to establish mechanisms to better document protected areas governed by non-governmental sectors, such as the various types of Indigenous and Community Conserved Areas (ICCAs) and privately protected areas, to track degazettal, downlisting and downsizing, as well as the inclusion of “other effective area-based conservation measures”, and improving user experience and access to more com- prehensive information through Protected Planet. PLOS ONE \| DOI:10.1371/journal.pone.0160640 August 16, 2016 Differences between biodiversity knowledge products The establishment, management, and asso- ciated documentation of protected areas is a national process, and, although extremely important because of their role in generating primary data for Protected Planet, these national costs have been evaluated elsewhere (e.g., [34], [59], [35]) and as explained in the Methods are beyond the scope of this study (Fig 1; stages 1 and 2). The World Database of Key Biodiversity Areas. That the KBA knowledge product had received the greatest investment until 2013 reflects the fact that this includes the costs of mainly PLOS ONE \| DOI:10.1371/journal.pone.0160640 August 16, 2016 15 / 22 The Cost of Global Biodiversity and Conservation Knowledge IBA identification within each of approximately 200 countries and territories, as well as global coordination and compilation. It is also important to note that the KBA knowledge product is, to a great extent, dependent on the other three knowledge products considered here for its application (with both Red Lists necessary to allow identification of sites holding significant levels of Threatened biodiversity, and Protected Planet supporting site delineation for pro- tected KBAs). Once KBAs have been identified, the cost of ongoing re-assessment is much lower than of initial identification and delineation (but note that this does not include the essential costs of field data collection for ongoing monitoring; [60]). The estimated US$21m cost to identify and document a comprehensive network of KBAs by 2020 is an underestimate, given the as-yet unknown extent to which limited KBAs identified so far in freshwater and marine environments are reflective of those for all biodiversity, and the fact that many sites are still to be documented for other largely terrestrial taxonomic groups. The IUCN Red List of Ecosystems. This is a new knowledge product [61, 27], so its costs until 2013 have been small and have not yet fully covered components such as database main- tenance, online dissemination, or indicator development (Fig 1; stages 4, 5, and 7), and it is not yet meaningful to derive an annual cost. This also explains the relatively large investment which will be required to deliver a baseline for achieving a global IUCN Red List of Ecosystems by 2020. PLOS ONE \| DOI:10.1371/journal.pone.0160640 August 16, 2016 Diversifying funding sources for knowledge products Perhaps our most surprising result in considering investments until 2013 is the lack of diversi- fication in funding sources. While the combined contributions of NGOs, intergovernmental organisations, and governments account for 43% of funding for knowledge products in the last four decades, more than half of the funding has come from philanthropic sources. This domi- nance reflects both a high reliance on project funding and the often catalytic role of philan- thropic funding. However, the distribution of funding sources seems heavily skewed given the extensive use and uptake of knowledge products to inform practice and policy across sectors, and users of knowledge products seem to contribute very little to their financial sustainability. In particular, contributions from the private sector could be greatly increased, given the curr- rent importance of three knowledge products (all except the Red List of Ecosystems which has not yet been incorporated in IFC’s standards) in maintaining the International Finance Corpo- ration’s Performance Standard 6, which is used in turn by all the Equator Principles Banks, and, in aggregate, guides tens of billions of dollars of annual private sector loans [17]. The establishment of the Proteus Partnership, and of the Integrated Biodiversity Assessment Tool (www.ibatforbusiness.org), which makes the knowledge products available for commercial use through data licensing, represents an initial step in this direction for support. Proportionate contributions from governments should also be increased [34], considering the importance of the knowledge products in supporting national-level planning as well as tracking progress towards countries’ international commitments and contributing to knowledge transfer [4]. By contrast, the allocation of investment until 2013 across expenditure categories is rather similar among the four knowledge products, primarily driven by personnel costs, accounting for nearly three-quarters of all expenses. This reliance on human capital is further underscored by the enormous volunteer contributions into the process, notwithstanding its underestimation here (293 person-years–US$14 million). Much of this volunteer effort comes from experts belonging to IUCN’s Species Survival Commission working on species assessments or experts from the BirdLife International partnership working on IBA identification and documentation. Diversifying funding sources for knowledge products Relatively greater investments in technology might increase efficiency of the process (e.g., increasing the role of online consultation rather than workshops for reassessing species’ 16 / 22 The Cost of Global Biodiversity and Conservation Knowledge extinction risk; [28]) but we suspect that personnel costs (and volunteer contributions) are a fundamental reflection of the human capacity needed for biodiversity and conservation assess- ments, and the data collection and management underlining them to ensure they are robust. Making knowledge products more efficient The database generated by this project incorporates valuable information to assess more in- depth aspects of the delivery of biodiversity and conservation knowledge products, such as comparing the efficiency of different processes within each knowledge product. For example, the cost of assessing species varied greatly between taxonomic groups (making it difficult to arrive at a single dollars per species assessment cost), which in part reflects different approaches applied to collate data and apply them to the IUCN Red List Categories and Crite- ria and, which have different costs [28] and the extent to which training and capacity-building actions are incorporated into the assessment process. Our data could also offer insights on cur- rent costs which could be used for the development of innovative approaches to, for example, reduce costs of data compilation and validation, allowing greater investment in interpretation, analyses and tool development. Another issue which these data would help to inform concerns exploration of the optimal allocation of resources between information and management [62, 63]. Finally, it is clear that those maintaining all four knowledge products should invest in accurate documentation of income and expenditure, as well as contributed volunteer time, including the inevitable uncertainties in estimates. Cost-effectiveness of knowledge products could probably be improved by enhancing the integration of their technological platforms and databases, as they are currently managed rela- tively independently from each other [7]. Better integrating knowledge products into a spatially explicit platform in the Integrated Biodiversity Assessment Tool would also allow users to explore more easily information from different sources available for their region of interest [57]. Likewise, the costs of managing datasets that are built on a common platform may poten- tially be lower than managing them independently. PLOS ONE \| DOI:10.1371/journal.pone.0160640 August 16, 2016 Conclusion This study provides the first comprehensive assessment on the costs of developing and main- taining global biodiversity and conservation knowledge through four widely used knowledge products. Understanding the costs of data generation is fundamentally important to ensure the long term sustainability of global biodiversity information. Without data we cannot generate information and build knowledge to make informed decisions or develop indicators to track progress towards biodiversity goals and targets. The people, structures, and processes that make this possible have a financial cost that is often taken for granted by those who benefit from such resources. Generation of authoritative biodiversity and conservation information through knowledge products is affordable if considered at a global scale and offers a good value for money compared with other similar processes. Given the relevance of these knowledge products, we argue that, as recognised by the IUCN Council, they should be regarded as global public goods, fundamental to improved development planning, and sustainable long-term funding mechanisms should be ensured for the sake of nature and humanity. Costs of knowledge products in the wider context The study did not systematically compare the costs of knowledge products with other societal investments. This would be an important consideration for future studies as it will inform fur- ther decisions on allocation of investments. Nevertheless, the costs of developing and maintain- ing these knowledge products are very low compared with similar knowledge-generation processes important for humanity. For example, the cost of completing the 2010 US Census was around US$13bn [64], while observations and infrastructure for the Global Observing Sys- tem for Climate in support of the United Nations Framework Convention on Climate Change (UNFCCC) cost between US$5-7bn in 2010 [65]. These processes, however, include the cost of collecting primary data which this study did not consider. Nevertheless, even if only 10% (e.g., 10% of 5 billion is 500 million) of these totals were invested in collating, interpreting, and pub- lishing secondary data, and 90% in collection of primary data, they dwarf the sums invested in the biodiversity and conservation knowledge products discussed here. It is clear that the devel- opment and maintenance of biodiversity knowledge products does not represent a dispropor- tionate economic cost, especially considering their global relevance to inform decisions relating international conventions and agreements, notably the CBD Aichi Biodiversity Targets and the Sustainable Development Goals. It is therefore surprising that none of these knowledge prod- ucts has long-term sustainable funding models and all are financed in an ad hoc fashion, leav- ing them critically underfunded and at risk of becoming inaccurate, outdated or unavailable. Acknowledging their value, the IUCN Council recently recognized the data underlying the PLOS ONE \| DOI:10.1371/journal.pone.0160640 August 16, 2016 17 / 22 The Cost of Global Biodiversity and Conservation Knowledge knowledge products as global public goods, and requested IUCN to engage with international finance organizations and interested governments regarding the need to invest in their devel- opment and maintenance ([66] pp. 24). This is perhaps a first step to work towards long-term sustainability. At least 4.5 million unique visitors (albeit ignoring potential overlaps between knowledge products) access these knowledge products online each year to visualise informa- tion or download tabular or spatial data (See S5 Table), further highlighting the importance of making sure these resources are updated, accurate and relevant to users. Supporting Information S1 Table. Currency conversions and accounting for inflation. Costs were provided in British Pounds (GBP), US Dollars (US$), Euros (EUR), Australian Dollars (AUD), and Swiss Francs (CHF) of the year when the investment took place. We accounted for inflation by calculating the value of the currency in 2014 and then we transformed all currencies to US Dollars (US$). (DOCX) S2 Table. Calculation of the cost of achieving pre-defined baselines by 2020. All extrapola- tions were derived through data from the sample compiled in the study and projections pro- vided by each knowledge product team. (DOCX) S2 Table. Calculation of the cost of achieving pre-defined baselines by 2020. All extrapola- tions were derived through data from the sample compiled in the study and projections pro- vided by each knowledge product team. (DOCX) S2 Table. Calculation of the cost of achieving pre-defined baselines by 2020. All extrapola- tions were derived through data from the sample compiled in the study and projections pro- vided by each knowledge product team. (DOCX) S3 Table. Summary of data collection for all four knowledge products. The table summa- rises which costs were collected for each of the four knowledge products and how much of the total number of assesments, available in December 2013, these represent. In cases where 100% of the costs were not collected, the total sum for each knowledge product was increased propo- tionally to reach 100%. (DOCX) S4 Table. Extrapolated one-off costs costs per funding source between 1979 and 2013 for all four knowledge products. Extrapolated one-off costs costs per cost categories between 1979 and 2013 for all four knowledge products. (DOCX) S5 Table. Unique visitors per year to knowledge products’ websites. (DOCX) 18 / 22 PLOS ONE \| DOI:10.1371/journal.pone.0160640 August 16, 2016 The Cost of Global Biodiversity and Conservation Knowledge Acknowledgments The authors would like to thank the funders of this study, the MAVA Foundation and the Inte- grated Biodiversity Assessment Tool (IBAT) Partnership. NatureServe acknowledges support from the National Science Foundation’s Dimensions of Biodiversity program (award 1136586). MB received continued funding from the Rufford Foundation. The Red List Unit would like to thank the MAVA Foundation, the Rufford Foundation, the Environment Agency Abu Dhabi, the European Commission and the Critical Ecosystem Partnership Fund. We are also grateful to: John Pilgrim, Trevor Sandwith, Pedro Rosabal, Kathy MacKinnon, Sarah Wyatt, Jerry Har- rison, Brian MacSharry, Yichuan Shi, Mat Jones, Soyoung Park, Sylvia Wicander, Florent Latil, Enrique Lahmann and all the many thousands of individuals and organisations who contribute to the creation and maintenance of the four knowledge products assessed. Visualization: DJB TMB SHMB RJ KB NK M. Hoffmann. Writing - original draft: DJB TMB SHMB RJ KB NK M. Hoffmann. Writing - review & editing: DJ TMB SHMB RJ KB NK M. Hoffmann AA MB NB KEC PJC NC AC WRTD MM LDCF BG M. Heath CHT JH TJ AJ DAK PFL JL ENL ML IM RMM MAOM MP CMP GR JPR CR JS SS AS AWT SW BY NK. Conceptualization: DJB TMB SHMB RJ KB NK M. Hoffmann. Formal analysis: DJB TMB SHMB RJ KB NK M. Hoffmann. Formal analysis: DJB TMB SHMB RJ KB NK M. Hoffmann. Funding acquisition: DJB TMB NK. Funding acquisition: DJB TMB NK. Funding acquisition: DJB TMB NK. Investigation: DJB TMB SHMB RJ KB NK M. Hoffmann AA MB NB KEC PJC NC AC WRTD MM LDCF BG M. Heath CHT TJ AJ DAK PFL JL ENL ML IM RMM MAOM MP CMP GR JPR CR AS AWT BY NK. Investigation: DJB TMB SHMB RJ KB NK M. Hoffmann AA MB NB KEC PJC NC AC WRTD MM LDCF BG M. Heath CHT TJ AJ DAK PFL JL ENL ML IM RMM MAOM MP CMP GR JPR CR AS AWT BY NK. Methodology: DJB TMB SHMB RJ KB NK M. Hoffmann. Methodology: DJB TMB SHMB RJ KB NK M. Hoffmann. Author Contributions Conceptualization: DJB TMB SHMB RJ KB NK M. Hoffmann. PLOS ONE \| DOI:10.1371/journal.pone.0160640 August 16, 2016 Project administration: DJB TMB NK. Resources: TMB SHMB RJ KB NK M. Hoffmann AA MB NB KEC PJC NC AC WRTD MM LDCF BG M. Heath H CHT TJ AJ DAK PFL JL ENL ML IM RMM MAOM MP CMP GR JPR CR AS AWT BY NK. Supervision: TMB SHMB RJ KB NK M. Hoffmann. Visualization: DJB TMB SHMB RJ KB NK M. Hoffmann. Visualization: DJB TMB SHMB RJ KB NK M. Hoffmann. References Juffe-Bignoli D, Burgess ND, Bingham H, Belle EMS, de Lima MG, Deguignet M, et al. Protected Planet Report 2014. 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https://openalex.org/W4386402755	https://archive.interconf.center/index.php/2709-4685/article/download/4257/4294	Ukrainian	null	Нормативна база з проектування закладів охорони здоров’я з надання психологічної і психіатричної допомоги населенню україни: сучасні санітарно-епідеміологічна та містобудівна складові, закордонний досвід	InterConf	2,023	cc-by-sa	7,580	Махнюк Валентина Михайлівна2 1 доцент, канд.мед.наук, доцент кафедри медицини катастроф та військової медицини; Вінницький національний медичний університет імені М.І.Пирогова; Україна 2 завідувач лабораторії гігієни планування та забудови населених місць, доктор медичних наук, професор, лікар вищої кваліфікаційної категорії за спеціальністю «Загальна гігієна»; Державна установа «Інститут громадського здоров’я ім. О.М. Марзєєва Національної академії медичних наук України»; Україна Національної академії медичних наук України»; Україна Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» (August 19-20, 2023). Seattle, USA No 167 Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» (August 19-20, 2023). Seattle, USA No 167 Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» (August 19-20, 2023). Seattle, USA Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» (August 19-20, 2023). Seattle, USA Анотація. В статті проведено аналіз нормативних документів санітарного та містобудівного законодавств з проєктування, розміщення, та експлуатації закладів охорони здоров’я психіатричного профілю в Україні. Вивчена та проаналізована нормативно-правова база європейського санітарного та містобудівного законодавств щодо гігієнічних вимог до розміщення ЗОЗ. Через відсутність будь-якого сучасного нормативного документу з проєктування та будівництва ЗОЗ в Україні, який враховує європейські стандарти, міжнародний досвід, розроблений проєкт «Концепції («Дорожня карта») формування нормативної бази проектування та будівництва споруд і комплексів закладів охорони здоров’я» та прийнятий новостворений ДБН В.2.2-10:2022 «Заклади охорони здоров’я. Основні положення», якій необхідний як в мирний час, так і для відновлення руйнацій від бойових дій закладів охорони здоров’я при проєктуванні/реконструкції/ ЗОЗ. DOI 10.51582/interconf.19-20.08.2023.024 DOI 10.51582/interconf.19-20.08.2023.024 Нормативна база з проектування закладів охорони здоров’я з надання психологічної і психіатричної допомоги населенню україни: сучасні санітарно- епідеміологічна та містобудівна складові, закордонний досвід Ключові слова: санітарно-містобудівні нормативні документи заклади охорони здоров’я психіатричного профілю базові міжнародні правила Дорожня карта ДБН В.2.2-10:2022 This work is distributed under the terms of the Creative Commons Attribution-ShareAlike 4.0 International License (https://creativecommons.org/licenses/by-sa/4.0/). This work is distributed under the terms of the Creative Commons Attribution-ShareAlike 4.0 International License (https://creativecommons.org/licenses/by-sa/4.0/). 293 MEDICINE AND PHARMACY Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» (August 19-20, 2023). Seattle, USA No 167 Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» (August 19-20, 2023). Seattle, USA Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» (August 19-20, 2023). Seattle, USA (August 19-20, 2023). Seattle, USA MEDICINE AND PHARMACY Аналізуючи нормативні документи санітарного та містобудівного законодавств з проєктування, розміщення, та експлуатації ЗОЗ ПП встановлено, що профільні (спеціалізовані) нормативні документи в Україні відсутні [1,2]. Аналізуючи нормативні документи санітарного та містобудівного законодавств з проєктування, розміщення, та експлуатації ЗОЗ ПП встановлено, що профільні (спеціалізовані) нормативні документи в Україні відсутні [1,2]. Нормативні вимоги до будівництва та функціонування ЗОЗ загального профілю в Україні, здійснюється відповідно до наступних нормативних документах санітарного та містобудівного законодавств (таблиця 1 ). Нормативні вимоги до будівництва та функціонування ЗОЗ загального профілю в Україні, здійснюється відповідно до наступних нормативних документах санітарного та містобудівного законодавств (таблиця 1 ). Таблиця 1 Перелік нормативних документів санітарного та містобудівного законодавств України щодо розміщення та експлуатації ЗОЗ загального профілю № п/п Назва нормативного документа Призначення нормативного документа QR-код 1 Державні санітарні норми і правила «Санітарно- протиепідемічні вимоги до закладів охорони здоров‘я, що надають первинну медичну (медико- санітарну) допомогу. ДСанПіН 2.3-183-2013» Санітарно-гігієнічні вимоги до закладів первинної ланки 2 Державні санітарні норми та правила "Гігієнічні вимоги до розміщення, облаштування, обладнання та експлуатації перинатальних центрів. ДСанПіН № 55-12" Встановлює санітарно- гігієнічні вимоги до перинатальних центрів 3 «Державні санітарні правила планування та забудови населених пунктів. ДСП № 173- 96» Встановлює санітарно- гігієнічні вимоги до планування території громадських закладів ц Перелік нормативних документів санітарного та містобудівного законодавств України щодо розміщення та експлуатації ЗОЗ загального профілю 294 MEDICINE AND PHARMACY Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» (August 19-20, 2023). Seattle, USA No 167 Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» (August 19-20, 2023). Seattle, USA Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» (August 19-20, 2023). Seattle, USA MEDICINE AND PHARMACY Продовження табл. 1 Продовження табл. 1 4 Державні санітарні правила і норми «Гігієнічні вимоги до влаштування та експлуатації рентгенівських кабінетів і проведення рентгенологічних процедур. ДСанПіН 6.6.3-150-2007» Встановлює санітарно- гігієнічні вимоги до рентген.кабінетів 5 «Державні санітарні норми і правила при роботі з джерелами електромагнітних полів. ДСанПіН 3.3.6-096-2002» Встановлює санітарно- гігієнічні вимоги до медичного обладнання ЗОЗ 6 Державні санітарні правила «Основні санітарні правила забезпечення радіаційної безпеки України. ОСП № 54- 2005» Встановлює норми радіаційної безпеки 7 Державні гігієнічні нормативи ДГН 6.6.1. - 6.5.061-2000 "Норми радіаційної безпеки України (НРБУ-97)" Встановлює норми радіаційної безпеки 8 «Державні санітарні норми допустимих рівнів шуму в приміщеннях громадських будинків. ДСН № 463- 19» Встановлює допустимі рівні шуму 9 «Санітарні норми мікроклімату виробничих приміщень ДСН 3.3.6.042-99» Встановлює нормативні гігієнічні показники мікроклімату на робочих місцях This work is distributed under the terms of the Creative Commons Attribution-ShareAlike 4.0 International License (https://creativecommons.org/licenses/by-sa/4.0/). 295 Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» (August 19-20, 2023). Seattle, USA Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» (August 19-20, 2023). Seattle, USA MEDICINE AND PHARMACY Продовження табл. 1 Продовження табл. 1 10 Державні санітарні норми та правила «Полімерні та полімервмісні матеріали, вироби і конструкції, що застосовуються у будівництві та виробництві меблів. Гігієнічні вимоги. ДСанПіН 8.2.1-181- 2012» Встановлює вимоги до якості оздоблювальних матеріалів, поверхонь приміщень 11 «Державні санітарно- протиепідемічні правила і норми щодо поводження з медичними відходами. ДСанПіН № 325-2015» Встановлює санітарно- протиепідемічні правила і норми поводження з медичними відходами 12 «Державні санітарні норми та правила утримання території населених місць. ДСанПіН № 145-2011» Встановлює санітарні норми і правила санітарного очищення територій населених місць 13 Гігієнічні регламенти «Гранично допустимі концентрації хімічних і біологічних речовин в атмосферному повітрі населених місць», затверджені наказом МОЗ від 14.01.2020 р. № 52 Встановлює гігієнічні регламенти якості атмосферного повітря 14 Гігієнічні регламенти «Орієнтовно безпечних рівнів впливу хімічних і біологічних речовин в атмосферному повітрі населених місць» затверджені наказом МОЗ від 14.01.2020 р. № 52 Встановлює гігієнічні регламенти якості атмосферного повітря Встановлює вимоги до якості оздоблювальних матеріалів, поверхонь приміщень 296 MEDICINE AND PHARMACY Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» (August 19-20, 2023). Seattle, USA No 167 Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» (August 19-20, 2023). Seattle, USA Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» (August 19-20, 2023). Seattle, USA 297 This work is distributed under the terms of the Creative Commons Attribution-ShareAlike 4.0 International License (https://creativecommons.org/licenses/by-sa/4.0/). This work is distributed under the terms of the Creative Commons Attribution-ShareAlike 4.0 International License (htt :// r ti r /li /b /4 0/) MEDICINE AND PHARMACY Продовження табл. 1 Продовження табл. 1 15 Державні санітарні норми та правила "Гігієнічні вимоги до води питної, призначеної для споживання людиною. ДСанПіН 2.2.4-171- 10" Встановлює гігієнічні вимоги до води питної 16 Державні будівельні норми «Водопостачання. Зовнішні мережі та споруди В.2.5- 74:2013» Встановлює гігієнічні вимоги до води питної 17 Державні будівельні норми «Захист територій, будинків і споруд від шуму ДБН В.1.1-31:2013» Встановлює вимоги щодо захисту від шуму 18 Державні будівельні норми «Інклюзивність будівель і споруд. Основні положення. ДБН В.2.2-40:2018» Встановлює вимоги до будівель для забезпечення умов доступу маломобільних груп населення 19 Державні будівельні норми "Опалення, вентиляція та кондиціонування. ДБН В.2.5-67:2013" Встановлює вимоги до опалення, вентиляції, кондиціонування приміщень ЗОЗ 20 Державні будівельні норми «Природне і штучне освітлення. ДБН В.2.5-28:2018» Встановлює санітарно- гігієнічні вимоги до природного та штучного освітлення на робочих місцях, в т.ч. у ЗОЗ В таблиці 1 наведено 20 нормативних документів санітарного законодавства України, з них 7 ДСанПіНів вузькоспеціалізованого напрямку та 5 нормативних документів містобудівного законодавства [3]. 297 Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» (August 19-20, 2023). Seattle, USA Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» (August 19-20, 2023). Seattle, USA MEDICINE AND PHARMACY This work is distributed under the terms of the Creative Commons Attribution-ShareAlike 4.0 International License (https://creativecommons.org/licenses/by-sa/4.0/). MEDICINE AND PHARMACY ДБН В.2.2-10-2001 «Будинки і споруди. Заклади охорони здоров’я» був створений у 2001 р. і ґрунтувався на нормативний базі санітарного та містобудівного законодавства до 2000 р. Зокрема, зазначений ДБН В.2.2-10-2001 «Будинки і споруди. Заклади охорони здоров’я» був взаємопов’язаний з нормативним документом - «Санитарными правилами устройства, оборудования и эксплуатации больниц, родильных домов и других лечебных стационаров СанПиН 5179-90», затверженими Головним державним санітарним лікарем СРСР 29.06.1990 р.[4-6]. Радянський нормативний документ «Санитарные правила устройства, оборудования и эксплуатации больниц, родильных домов и других лечебных стационаров. СанПиН 5179-90» на сьогодні скасований розпорядженням Кабінету Міністрів України від 20.01.2016 р. № 94-р. Окрім цього документу, скасовані всі нормативно-правові акти (понад 300 документів), які були видані до прийняття Акту проголошення незалежності України, тобто нормативно-правові акти Союзу РСР та Української РСР. Будівництво та експлуатація стаціонарних лікарень в Україні здійснювалась на основі попереднього вітчизняного напрацьованого досвіду та використанні окремих елементів планувальних рішень та організаційних санітарно- протиепідемічних заходів, запозичених із практики інших країн ближнього та дальнього зарубіжжя. Була гостра потреба у розробці сучасного нормативного документу санітарного законодавства України для сучасних стаціонарних лікарень, які є базовими закладами вторинної ланки за реформою галузі охорони здоров’я. До того ж вузькоспеціалізовані лікарні психоневрологічного профілю за медичною реформою відносяться до третинного рівня за профілем надання медичних послуг – з одного боку, за наявністю стаціонарів відносяться до вторинного рівня – з іншого, і тому основні санітарно- протиепідемічні та гігієнічні вимоги є спільними як для вузькоспеціалізованих лікарень, так і для лікарень загального типу з відділеннями психіатричного профілю (ПП). На сьогодні для забезпечення санітарно-гігієнічних та протиепідемічних умов праці медпрацівників та умов соціалізації для осіб, які мають психічні розлади, необхідно створювати новий тип (дизайн) лікувально-профілактичного закладу з внутрішнім арт-еколого-терапевтичним простором та розробляти новітні нормативні документи з проектування цих 298 Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» (August 19-20, 2023). Seattle, USA No 167 Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» (August 19-20, 2023). Seattle, USA Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» (August 19-20, 2023). Seattle, USA MEDICINE AND PHARMACY 299 This work is distributed under the terms of the Creative Commons Attribution-ShareAlike 4.0 International License (https://creativecommons.org/licenses/by-sa/4.0/). MEDICINE AND PHARMACY закладів. В Україні на сьогодні реконструкцій або будівництво нових закладів охорони психічного здоров’я не здійснюються, а тільки проводяться поточні ремонти діючих ЗОЗ ПП. закладів. В Україні на сьогодні реконструкцій або будівництво нових закладів охорони психічного здоров’я не здійснюються, а тільки проводяться поточні ремонти діючих ЗОЗ ПП. В ході дослідження була вивчена та проаналізована нормативно-правова база європейського санітарного та містобудівного законодавств щодо гігієнічних вимог до розміщення ЗОЗ, яка охоплює наступні країни: Республіка Польща, Республіка Чехія, Румунія, Республіка Молдова, Франція та Україна. Досліджувані нормативні документи були відібрані на офіційних інтернет-ресурсах профільних міністерств та відомств європейських країн, де зазначається дата прийняття документу, статус його чинності, а також наведено повний текст документу. Нами здійснено переклад оригінального варіанту документів з іноземних мов на українську мову для можливості його читання та аналізу. За результатами аналізу нормативних документів санітарного та містобудівного законодавства країн ЄС та України в частині вимог до проєктування, розміщення та експлуатації ЗОЗ встановлено наступне. Для порівняльної оцінки європейських нормативів з вітчизняними нормативами в частині проектування, розміщення та експлуатації ЗОЗ відібрані нормативні документи санітарного та містобудівного законодавств країн ЄС, які містять основні положення з цих питань, що представлені в таблиці 2 Таблиця 2 б Нормативні документи санітарного та містобудівного законодавств країн ЄС щодо будівництва ЗОЗ № Нормативний документ (назва мовою оригіналу) Нормативний документ (назва перекладена на українську мову) дата прийняття документу QR-код Республіка Польща 1 «Krajowe ramy strategiczne Policy paper dla ochrony zdrowia na lata 2014– 2020» Національний стратегічний документ МОЗ Республіки Польща про «Захист здоров‘я на 2014- 2020 рр.» 07.2014 р. Нормативні документи санітарного та містобудівного законодавств країн ЄС щодо будівництва ЗОЗ This work is distributed under the terms of the Creative Commons Attribution-ShareAlike 4.0 International License (https://creativecommons.org/licenses/by-sa/4.0/). 299 MEDICINE AND PHARMACY Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» (August 19-20, 2023). Seattle, USA No 167 Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» (August 19-20, 2023). Seattle, USA (August 19-20, 2023). Seattle, USA MEDICINE AND PHARMACY Продовження табл. 2 2 ROZPORZĄDZENIE MINISTRA ZDROWIA w sprawie szczegółowych wymagań, jakim powinny odpowiadać pomieszczenia i urządzenia podmiotu wykonującego działalność leczniczą Постанова МОЗ Республіки Польща про «Детальні вимоги, яким повинні відповідати приміщення та обладнання суб'єкта медичної діяльність» 26.03.2019 р. 3 ROZPORZĄDZENIE MINISTRA ZDROWIA zmieniające rozporządzenie w sprawie świadczeń gwarantowanych z zakresu podstawowej opieki zdrowotnej Постанова МОЗ Республіки Польща про «Внесення змін до положення про гарантовані пільги у сфері первинної медичної допомоги» 05.01.2023 р. 4 «W sprawie warunków technicznych, jakim powinny odpowiadać budynki i ich usytuowanie» Постанова Міністерства інфраструктури Республіки Польща про «Технічні умови, яким повинні відповідати будівлі та їх розташування» 12.04.2002 р. Республіка Чехія 5 Vyhláška č. 92/2012 Sb.Vyhláška o požadavcích na Постанова МОЗ Республіки Чехія «Про вимоги до мінімального технічного та матеріального 15.03.2012 р. 300 MEDICINE AND PHARMACY Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» (August 19-20, 2023). Seattle, USA No 167 Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» (August 19-20, 2023). Seattle, USA Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» 301 This work is distributed under the terms of the Creative Commons Attribution-ShareAlike 4.0 International License (https://creativecommons.org/licenses/by-sa/4.0/). MEDICINE AND PHARMACY MEDICINE AND PHARMACY Продовження табл. 2 minimální technické a věcné vybavení zdravotnických zařízení a kontaktních pracovišť domácí péče оснащення закладів охорони здоров’я та контактних центрів домашньої допомоги» Румунія 6 Asistența medicală primară Lege 95/2006 Закон, який регулює медичну практику - з питань реформи охорони здоров’я 14.04.2006 р. № 95 7 Ordinul nr. 1338/2007 pentru aprobarea Normelor privind structura funcțională a cabinetelor medicale și de medicină dentară Наказ Міністерства громадського здоров‘я Румунії «Про затвердження норм щодо функціональної структури медичного та стоматологічно го кабінетів» 22.08.2007 р. № 1338/2007 8 GUVERNUL HOTĂRÎRE Nr. HG 696/2018 din 2018-07-11 pentru aprobarea Regulamentului sanitar privind gestionarea deșeurilor rezultate din activitatea medical Рішення уряду Румунії «Про затвердження санітарного регламенту поводження з відходами результати від медичної діяльності» 07.11.2018 р. № HG 696 Продовження табл. 2 This work is distributed under the terms of the Creative Commons Attribution-ShareAlike 4.0 International License (https://creativecommons.org/licenses/by-sa/4.0/). 301 MEDICINE AND PHARMACY Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» (August 19-20, 2023). Seattle, USA No 167 Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» (August 19-20, 2023). Seattle, USA (August 19-20, 2023). Seattle, USA MEDICINE AND PHARMACY 303 This work is distributed under the terms of the Creative Commons Attribution-ShareAlike 4.0 International License (https://creativecommons.org/licenses/by-sa/4.0/). MEDICINE AND PHARMACY Продовження табл. 2 Республіка Молдова 9 Hotărîrea Guvernului nr. 663 din 23.07.2010 pentru aprobarea Regulamentului sanitar privind condiţiile de igienă pentru instituţiile medico- sanitare. Рішення Уряду від про затвердження Санітарного регламенту щодо гігієнічних умов медико-санітарних закладів 23.07.2010 р. № 663 Франція 10 Code de la santé publique Ministère des Solidarités et de la Santé Кодекс громадської охорони здоров‘я 02.07.2020 р. 11 Règlement de s écurité contre l'incendie re latif aux étab lissements rec evant du publi c Livre II : Dispositions applicables aux établissements des quatre premières categories Titre deux : Dispositions particulières Arrêté du 10 décembre 2004 Правила пожежної безпеки для громадських закладів (Установи типу U – Установи по догляду) 10.09.2004 р. Продовження табл. 2 This work is distributed under the terms of the Creative Commons Attribution-ShareAlike 4.0 International License (https://creativecommons.org/licenses/by-sa/4.0/). 302 MEDICINE AND PHARMACY Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» (August 19-20, 2023). Seattle, USA No 167 Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» (August 19-20, 2023). Seattle, USA Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» This work is distributed under the terms of the Creative Commons Attribution-ShareAlike 4.0 International License (https://creativecommons.org/licenses/by-sa/4.0/). MEDICINE AND PHARMACY MEDICINE AND PHARMACY MEDICINE AND PHARMACY Продовження табл. 2 Chapitre IX : Établissements du type U - Établissements de soins Ministère des Solidarités et de la Santé 12 Arrêté du 25 avril 2003 relatif à la limitation du bruit dans les établissements de santé Ministère des Solidarités et de la Santé Наказ про обмеження шуму в медичних установах 25.04.2003 р. 13 Loi n° 2005-102 du 11 février 2005 pour l'égalité des droits et des chances, la participation et la citoyenneté des personnes handicapées. Ministère des Solidarités et de la Santé Закон «Про рівні права та можливості, участь та громадянство для людей з обмеженими можливостями» 11.02.2005 р. № 2005-102 14 Les locaux des professionnels de santé: réussir l’accessibilité Être prêt pour le 1er janvier 2015 Ministère des Solidarités et de la Santé Керівництво для забезпечення доступності приміщень закладів охорони здоров‘я для людей з особливими потребами 01.01.2015 р. Продовження табл. 2 Поряд з національними документами досліджуваних країн, які подані в таблиці Х, при проектуванні і будівництві ЗОЗ ПП 303 This work is distributed under the terms of the Creative Commons Attribution-ShareAlike 4.0 International License (https://creativecommons.org/licenses/by-sa/4.0/). 303 Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» (August 19-20, 2023). Seattle, USA Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» (August 19-20, 2023). Seattle, USA MEDICINE AND PHARMACY використовуються базові міжнародні правила - International Health Facility Guidelines (Міжнародні Директиви для медичних закладів), Design Guidance for Psychiatric Intensive Care Units (2017), Low Secure Units (NAPICU) Design in Mental Health Network (Керівництво з проектування відділень інтенсивної психіатричної терапії), IBC R A Mental of the International Code Family (Міжнародний будівельний кодекс) і посилання та посібники Architecture for psychiatric environments and therapeutic spaces (Архітектура для психіатричних середовищ і терапевтичних просторів), Psychiatric Facility (Психіатричний заклад) NIKA. For VA Office of Construction & Facilities Management (CFM) (Управління будівництва та управління об’єктами) [7-12]. використовуються базові міжнародні правила - International Health Facility Guidelines (Міжнародні Директиви для медичних закладів), Design Guidance for Psychiatric Intensive Care Units (2017), Low Secure Units (NAPICU) Design in Mental Health Network (Керівництво з проектування відділень інтенсивної психіатричної терапії), IBC R A Mental of the International Code Family (Міжнародний будівельний кодекс) і посилання та посібники Architecture for psychiatric environments and therapeutic spaces (Архітектура для психіатричних середовищ і терапевтичних просторів), Psychiatric Facility (Психіатричний заклад) NIKA. MEDICINE AND PHARMACY For VA Office of Construction & Facilities Management (CFM) (Управління будівництва та управління об’єктами) [7-12]. ( р уд ц у р ) [ ] Враховуючи актуальність розробки новітньої нормативної бази з проектування ЗОЗ ПП нами у рамках угоди про наукове співробітництво між Національним авіаційним університетом та Державною установою «Інститут громадського здоров’я ім. О.М. Марзєєва Національної академії медичних наук України» від 27.10.2021 р. № 802/16 опрацьовано проєкт «Концепції («Дорожня карта») формування нормативної бази проектування та будівництва споруд і комплексів закладів охорони здоров’я», з метою розробки еколого-гігієнічної та санітарно-епідеміологічної складової для його вдосконалення та ефективної реалізації на рівні територіальних громад [13-15]. У зв’язку з відсутністю будь-якого сучасного нормативного документу з проєктування та будівництва закладів охорони здоров’я, який враховує європейські стандарти, міжнародний досвід, розроблений проєкт «Концепції («Дорожня карта») формування нормативної бази проектування та будівництва споруд і комплексів закладів охорони здоров’я» (далі - Концепція) є першим концептуальним документом на рівні держави, який координує створення нової нормативної бази проектування та будівництва споруд і комплексів закладів охорони здоров’я. Саме розробка еколого-гігієнічної, санітарно- епідеміологічної, економічної та правової складової до проєтування та будівництва закладів охорони здоров’я та включення їх до зазначеної Концепції сприятиме покращенню умов доступності, перебування пацієнтів в запроєктованих 304 MEDICINE AND PHARMACY Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» (August 19-20, 2023). Seattle, USA No 167 Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» (August 19-20, 2023). Seattle, USA Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» (August 19-20, 2023). Seattle, USA MEDICINE AND PHARMACY 305 This work is distributed under the terms of the Creative Commons Attribution-ShareAlike 4.0 International License (https://creativecommons.org/licenses/by-sa/4.0/). This work is distributed under the terms of the Creative Commons Attribution-ShareAlike 4.0 International License (https://creativecommons.org/licenses/by-sa/4.0/). Commons Attribution-ShareAlike 4.0 International License MEDICINE AND PHARMACY Зазначена Концепція передбачає розвиток нормативного забезпечення щодо проектування та будівництва об’єктів ЗОЗ ПП з урахуванням чинних міжнародних вимог та досвіду технічно розвинутих країн, що дозволить підвищити професійний рівень працівників, зайнятих у цій сфері, та якість проектної документації об’єктів, що будуються. Зазначена Концепція передбачає розвиток нормативного забезпечення щодо проектування та будівництва об’єктів ЗОЗ ПП з урахуванням чинних міжнародних вимог та досвіду технічно розвинутих країн, що дозволить підвищити професійний рівень працівників, зайнятих у цій сфері, та якість проектної документації об’єктів, що будуються. У новітньому Законі України від 06 вересня 2022 року № 4142 «Про систему громадського здоров’я» новелою є положення статті 27 «Медико-санітарні вимоги безпеки для здоров'я і життя людини у містобудівній діяльності», якою передбачено у режимах забудови територій, визначених для містобудівних потреб, встановлених у генеральних планах населених пунктів, планах зонування та детальних планах територій, в тому числі при розміщенні закладів охорони здоров’я, створення безпечних умов для життя і здоров'я шляхом реалізації планувальних заходів щодо попередження та усунення шкідливого впливу факторів середовища життєдіяльності. Другою частиною цієї статті передбачено в обов’язковому порядку врахування при нормативному регулюванні планування та забудови територій вимог державних медико-санітарних нормативів та правил [16]. У новітньому Законі України від 06 вересня 2022 року № 4142 «Про систему громадського здоров’я» новелою є положення статті 27 «Медико-санітарні вимоги безпеки для здоров'я і життя людини у містобудівній діяльності», якою передбачено у режимах забудови територій, визначених для містобудівних потреб, встановлених у генеральних планах населених пунктів, планах зонування та детальних планах територій, в тому числі при розміщенні закладів охорони здоров’я, створення безпечних умов для життя і здоров'я шляхом реалізації планувальних заходів щодо попередження та усунення шкідливого впливу факторів середовища життєдіяльності. Другою частиною цієї статті передбачено в обов’язковому порядку врахування при нормативному регулюванні планування та забудови територій вимог державних медико-санітарних нормативів та правил [16]. Саме на виконання ст.27 Закону України «Про систему громадського здоров’я» та положень прийнятого Закону України «Про внесення змін до Закону України «Про будівельні норми» щодо удосконалення нормування у будівництві» від 03.10.2019 р. № 156-IX, яким запроваджено параметричний підхід у проектуванні закладів охорони здоров’я, пропонується включити до першого розділу «Нормативні посилання» наступний перелік державних санітарних норм і правил, які містять нормативні параметри для внутрішньолікарняного середовища та для прилікарняної території: ДСП 173-96 Державні санітарні правила планування та забудови населених пунктів; ДСП 6.177- This work is distributed under the terms of the Creative Commons Attribution-ShareAlike 4.0 International License (https://creativecommons.org/licenses/by-sa/4.0/). This work is distributed under the terms of the Creative Commons Attribution-ShareAlike 4.0 International License (https://creativecommons.org/licenses/by-sa/4.0/). MEDICINE AND PHARMACY закладах охорони здоров’я та ефективної роботи медичного персоналу, обумовлює актуальність зазначеного дослідження на рівні держави та територіальних громад. При прийнятті зазначеної Концепції, запропоновано приділити увагу реформуванню містобудівної галузі та охороні здоров’я, посилаючись на новітнє законодавство, нову вітчизняну нормативну базу містобудівного та санітарного законодавства, міжнародні правила International Health Facility Guidelines (Міжнародні Директиви для медичних закладів), Design Guidance for Psychiatric Intensive Care Units (2017), Low Secure Units (NAPICU) Design in Mental Health Network (Керівництво з проектування відділень інтенсивної психіатричної терапії), IBC R A Mental of the International Code Family (Міжнародний будівельний кодекс) щодо санітарних правил і норм стосовно проектування, організації та експлуатації, що є корисними для проектувальників. проектувальників. В Україні продовжується здійснення реформування системи охорони здоров’я, основна мета якого – забезпечення громадян України рівним доступом до якісних медичних послуг, в результаті змін орієнтування системи так, щоб у центрі її був пацієнт. Реформування системи охорони здоров’я здійснюється за такими пріоритетними напрямами: медична реформа; громадське здоров’я; спеціалізована медицина; доступні та якісні лікарські засоби. За кожним із напрямів реалізовуються завдання, спрямовані на забезпечення фінансування системи охорони здоров’я, підвищення заробітної плати медичних працівників, створення ефективної системи контролю якості надання медичної допомоги, розробка та оновлення національних галузевих стандартів та протоколів лікування, впровадження державної оцінки медичних технологій, створення конкурентного середовища для медичних закладів, розвиток добровільного медичного страхування, розвиток державно-приватного партнерства у сфері охорони здоров’я, залучення приватного капіталу, пільгове оподаткування за рахунок податку на прибуток та ін. Для реалізації завдань реформування системи охорони здоров’я зазначеною Концепцією запропоновано створення сучасної повноцінної мережі закладів охорони здоров’я. Проектування та будівництво закладів охорони здоров’я в 305 MEDICINE AND PHARMACY Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» (August 19-20, 2023). Seattle, USA No 167 Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» (August 19-20, 2023). Seattle, USA Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» (August 19-20, 2023). Seattle, USA MEDICINE AND PHARMACY тому чіслі ЗОЗ ПП повинно здійснюватися на підставі аналізу попиту на медичні послуги для населення відповідної територіальної громади, на території якої заплановане будівництво/реконструкція ЗОЗ ПП. Нами запропоновано для включення в Концепцію участь громадськості у вирішенні питань проектування та будівництва ЗОЗ ПП, що відповідає вимогам Європейської Хартії, міжнародній програмі ВООЗ “Здорові Міста” та новітньому законодавству України . MEDICINE AND PHARMACY 306 MEDICINE AND PHARMACY Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» (August 19-20, 2023). Seattle, USA No 167 Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» (August 19-20, 2023). Seattle, USA Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» (August 19-20, 2023). Seattle, USA MEDICINE AND PHARMACY MEDICINE AND PHARMACY 2005-09-02 Основні санітарні правила забезпечення радіаційної безпеки України (ОСПУ-2005); ДСП 9.9.5-080-02 «Правила влаштування і безпеки роботи в лабораторіях (відділах, відділеннях) мікробіологічного профілю; ДСН 3.3.6.037-99 Державні санітарні норми виробничого шуму, ультразвуку та інфразвуку; ДСН 239-96 Державні санітарні норми і правила захисту населення від впливу електромагнітних випромінювань; ДСН 463-2019 Державні санітарні норми допустимих рівнів шуму в приміщеннях житлових та громадських будинків і на території житлової забудови; ДСанПіН 9.9.5-153-2008 Організація роботи лабораторій при дослідженні матеріалу, що містить біологічні патогенні агенти І-ІV груп патогенності молекулярно- генетичними методами; ДСанПіН 145-2011 Державні санітарні норми та правила утримання територій населених місць; ДСанПіН 6.6.3-150-2007 Гігієнічні вимоги до влаштування та експлуатації рентгенівських кабінетів і проведення рентгенологічних процедур; ДСанПіН 259-2013 Санітарно- протиепідемічні вимоги до закладів охорони здоров'я, що надають первинну медичну (медико-санітарну) допомогу; ДСанПіН 325-2015 Державні санітарно-протиепідемічні правила і норми щодо поводження з медичними відходами; ДСанПіН 784-2011 Гігієнічні вимоги до розміщення, облаштування, обладнання та експлуатації перинатальних центрів; ДСанПіН 552-2014 Дезінфекція, передстерилізаційне очищення та стерилізація медичних виробів в закладах охорони здоров'я; ДСанПіН 2.2.4- 171-10 Гігієнічні вимоги до води питної, призначеної для споживання людиною; ДСанПіН 8.2.1-181-2012 Полімерні та полімервмісні матеріали, вироби і конструкції, що застосовуються у будівництві та виробництві меблів. Гігієнічні вимоги; ДСанПіН 3.3.6.096-2002 «Державні санітарні норми та правила при роботі з джерелами електромагнітних полів» та ін. 2005-09-02 Основні санітарні правила забезпечення радіаційної безпеки України (ОСПУ-2005); ДСП 9.9.5-080-02 «Правила влаштування і безпеки роботи в лабораторіях (відділах, відділеннях) мікробіологічного профілю; ДСН 3.3.6.037-99 Державні санітарні норми виробничого шуму, ультразвуку та інфразвуку; ДСН 239-96 Державні санітарні норми і правила захисту населення від впливу електромагнітних випромінювань; ДСН 463-2019 Державні санітарні норми допустимих рівнів шуму в приміщеннях житлових та громадських будинків і на території житлової забудови; ДСанПіН 9.9.5-153-2008 Організація роботи лабораторій при дослідженні матеріалу, що містить біологічні патогенні агенти І-ІV груп патогенності молекулярно- генетичними методами; ДСанПіН 145-2011 Державні санітарні норми та правила утримання територій населених місць; ДСанПіН 6.6.3-150-2007 Гігієнічні вимоги до влаштування та експлуатації рентгенівських кабінетів і проведення рентгенологічних процедур; ДСанПіН 259-2013 Санітарно- протиепідемічні вимоги до закладів охорони здоров'я, що надають первинну медичну (медико-санітарну) допомогу; ДСанПіН 325-2015 Державні санітарно-протиепідемічні правила і норми щодо поводження з медичними відходами; ДСанПіН 784-2011 Гігієнічні вимоги до розміщення, облаштування, обладнання та експлуатації перинатальних центрів; ДСанПіН 552-2014 Дезінфекція, передстерилізаційне очищення та стерилізація медичних виробів в закладах охорони здоров'я; ДСанПіН 2.2.4- 171-10 Гігієнічні вимоги до води питної, призначеної для споживання людиною; ДСанПіН 8.2.1-181-2012 Полімерні та полімервмісні матеріали, вироби і конструкції, що застосовуються у будівництві та виробництві меблів. 307 This work is distributed under the terms of the Creative Commons Attribution-ShareAlike 4.0 International License (https://creativecommons.org/licenses/by-sa/4.0/). This work is distributed under the terms of the Creative Commons Attribution-ShareAlike 4.0 International License (https://creativecommons.org/licenses/by-sa/4.0/). MEDICINE AND PHARMACY захворювань, викликаних дією асбесту . захворювань, викликаних дією асбесту . З метою підвищення рівня безпеки пацієнтів, медперсоналу та відвідувачів громадян від воєнних злочинів, які вчиняються збройними силами російських терористичних військ, Законом України «Про внесення змін до деяких законодавчих актів України щодо забезпечення вимог цивільного захисту під час планування та забудови територій» від 29.07.2022 р. № 2486-IX обґрунтовано необхідність влаштування у цокольних і підвальних поверхах ЗОЗ ПП захисних споруд цивільного захисту (сховище, приторадіаційне укриття, споруди подвійного призначення) з урахуванням вимог інженерно-технічних заходів. При розробці нових державних будівельних норм потребує врахування з проєктування та будівництва будинків і споруд закладів охорони здоров’я [17]. захворювань, викликаних дією асбесту . З метою підвищення рівня безпеки пацієнтів, медперсоналу та відвідувачів громадян від воєнних злочинів, які вчиняються збройними силами російських терористичних військ, Законом України «Про внесення змін до деяких законодавчих актів України щодо забезпечення вимог цивільного захисту під час планування та забудови територій» від 29.07.2022 р. № 2486-IX обґрунтовано необхідність влаштування у цокольних і підвальних поверхах ЗОЗ ПП захисних споруд цивільного захисту (сховище, приторадіаційне укриття, споруди подвійного призначення) з урахуванням вимог інженерно-технічних заходів. При розробці нових державних будівельних норм потребує врахування з проєктування та будівництва будинків і споруд закладів охорони здоров’я [17]. Відповідно до наказу Міністерства внутрішніх справ України «Про затвердження вимог з питань використання та обліку фонду захисних споруд цивільного захисту» від 09.07.2018 року № 579, зареєстрованого в Міністерстві юстиції України 30.07.2018 р. за № 879/32331, зазначені захисні споруди цивільного захисту входитимуть до фонду захисних споруд цивільного захисту матимуть статус будівель подвійного призначення та можуть використовуватись не тільки для пацієнтів, медичних працівників, а й для населення прилеглої житлової забудови територіальної громади з метою збереження їх здоров’я та захисту життя [18]. У зазначеній Концепції нами запропоновано підсилити доступність, ефективність медичних послуг, їх економічної, епідеміологічної складової шляхом запровадження у медичному процесі новітніх європейських медичних технологій: обладнування ЗОЗ системами пневматичної пошти для переміщення по медичному закладу рецептів, результатів аналізів, інструментів, витратних матеріалів, лікувальних засобів, препаратів крові тощо; встановлення великогабаритного високотехнологічного медичного обладнання (МРТ, КТ), що потребує врахування нових планувальних та інженерно-технічних рішень при проектуванні закладів охорони здоров’я. MEDICINE AND PHARMACY Гігієнічні вимоги; ДСанПіН 3.3.6.096-2002 «Державні санітарні норми та правила при роботі з джерелами електромагнітних полів» та ін. У статті 27 Закону України «Про систему громадського здоров’я» на виконання вимог міжнародного та європейського законодавства запроваджена пряма заборона виробництва і використання азбесту незалежно від виду, а також азбестовмісних виробів і матеріалів у технологічних процесах під час здійснення будівельно-монтажних робіт на будь-яких об'єктах, що потребує обов’язкового виконання при будівництві закладів охорони здоров’я з метою попередження онкологічних 307 Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» (August 19-20, 2023). Seattle, USA Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» (August 19-20, 2023). Seattle, USA (August 19-20, 2023). Seattle, USA MEDICINE AND PHARMACY This work is distributed under the terms of the Creative Commons Attribution-ShareAlike 4.0 International License (https://creativecommons.org/licenses/by-sa/4.0/). MEDICINE AND PHARMACY Зазначене впровадження забезпечуватиме право пацієнтів на доступність медичної допомоги, включаючи діагностичні і лікувальні процедури та лікарські засоби, що відповідають міжнародним стандартам, що відповідає Європейській Хартії Прав Пацієнтів У зазначеній Концепції нами запропоновано підсилити доступність, ефективність медичних послуг, їх економічної, епідеміологічної складової шляхом запровадження у медичному процесі новітніх європейських медичних технологій: обладнування ЗОЗ системами пневматичної пошти для переміщення по медичному закладу рецептів, результатів аналізів, інструментів, витратних матеріалів, лікувальних засобів, препаратів крові тощо; встановлення великогабаритного високотехнологічного медичного обладнання (МРТ, КТ), що потребує врахування нових планувальних та інженерно-технічних рішень при проектуванні закладів охорони здоров’я. Зазначене впровадження забезпечуватиме право пацієнтів на доступність медичної допомоги, включаючи діагностичні і лікувальні процедури та лікарські засоби, що відповідають міжнародним стандартам, що відповідає Європейській Хартії Прав Пацієнтів This work is distributed under the terms of the Creative Commons Attribution-ShareAlike 4.0 International License (https://creativecommons.org/licenses/by-sa/4.0/). 308 MEDICINE AND PHARMACY Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» (August 19-20, 2023). Seattle, USA No 167 Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» (August 19-20, 2023). Seattle, USA Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» (August 19-20, 2023). Seattle, USA MEDICINE AND PHARMACY This work is distributed under the terms of the Creative Commons Attribution-ShareAlike 4.0 International License (https://creativecommons.org/licenses/by-sa/4.0/). 309 This work is distributed under the terms of the Creative Commons Attribution-ShareAlike 4.0 International License (https://creativecommons.org/licenses/by-sa/4.0/). This work is distributed under the terms of the Creative Commons Attribution-ShareAlike 4.0 International License (https://creativecommons.org/licenses/by-sa/4.0/). MEDICINE AND PHARMACY в Україні (п. 10.) [19,20]. Для розробки нових ДБН В.2.2-10:2022 були використано наступні законодавчі документи: Кодекс цивільного захисту України (зі змінами від 2022), Закон України «Про внесення змін до деяких законодавчих актів України щодо забезпечення вимог цивільного захисту під час планування та забудови територій» (2022), Закон України «Про систему громадського здоров’я» (2022), Закон України «Про внесення змін до Закону України “Про будівельні норми” щодо удосконалення нормування у будівництві» (2019), оновлені міжнародні правила International Health Facility Guidelines (Міжнародні Директиви для медичних закладів), Design Guidance for Psychiatric Intensive Care Units (2017). Low Secure Units (NAPICU) Design in Mental Health Network (Керівництво з проектування відділень інтенсивної психіатричної терапії), IBC R A Mental of the International Code Family (Міжнародний будівельний кодекс), щодо санітарних правил і норм стосовно проєктування, організації та експлуатації ЗОЗ та ЗОЗ ПП, національні стандарти України, гармонізовані з міжнародними та європейськими нормативними документами – всього понад 50 документів ДБН В.2.2-10:2022 встановлюють основні положення до проєктування ЗОЗ та ЗОЗ ПП (будівель і споруд усіх типів закладів охорони здоров’я у цілому та приміщень, що використовуються для надання медичної та реабілітаційної допомоги у сфері охорони здоров’я) при новому будівництві, реконструкції і капітальному ремонті та розроблені нові гігієнічні критерії до проєктування та облаштування ЗОЗ та ЗОЗ ПП, а також вимоги до новітніх медичних технологій (телемедицина), інклюзивності зовнішнього та внутрішнього обємно-планувального простору, забезпечення вимог безпеки для здоров’я як пацієнтів з психічними розладами, відвідувачів (родичів), так і медпрацівників, які враховували нормативну базу ЄС та досвід європейських країн, включені в зазначені ДБН В.2.2-10:2022. Враховуючи наслідки повномасштабної війни, щодо психічного здоров’я населення України, попередження професійних захворювань серед медичних працівників ЗОЗ ПП (предиктори розвитку емоційного вигорання), що обґрунтовано результатами наших досліджень, розширено перелік медичних 309 This work is distributed under the terms of the Creative Commons Attribution-ShareAlike 4.0 International License (https://creativecommons.org/licenses/by-sa/4.0/). 309 MEDICINE AND PHARMACY Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» (August 19-20, 2023). Seattle, USA No 167 Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» (August 19-20, 2023). Seattle, USA Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» (August 19-20, 2023). Seattle, USA MEDICINE AND PHARMACY MEDICINE AND PHARMACY Seattle, USA Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» (August 19-20, 2023). Seattle, USA (August 19-20, 2023). Seattle, USA MEDICINE AND PHARMACY MEDICINE AND PHARMACY 1. Санітарно-епідеміологічна складова, яка базується на закордонному досвіді європейських країн, реформуванні медичної галузі, вимогах цивільного захисту, включена до «Концепціі («Дорожньої карти») формування нормативної бази проектування та будівництва споруд і комплексів закладів охорони здоров’я» і є надзвичайно актуальною для відбудови медичної інфраструктури в умовах воєнного та післявоєнного стану в Україні. Створена «Дорожня карта» з урахуванням розробленої нами санітарно-гігієнічної складової до основного за ієрархічною структурою нормативного документу ДБН «Будинки і споруди. Заклади охорони здоров’я» та низки інших будівельних норм, передбачає оновлення нормативної бази з проєктування та будівництва закладів охорони здоров’я, в т. ч. ЗОЗ ПП, на рівні територіальних громад з урахуванням чинних міжнародних вимог та найкращого світового досвіду. 2. В новоствореному ДБН врахована спеціалізація ЗОЗ ПП направлена на забезпечення оптимальних умов перебування пацієнтів з психічними розладами в умовах стаціонару, умов праці медичних працівників, умов відвідувачів. 2. В новоствореному ДБН врахована спеціалізація ЗОЗ ПП направлена на забезпечення оптимальних умов перебування пацієнтів з психічними розладами в умовах стаціонару, умов праці медичних працівників, умов відвідувачів. 2. В новоствореному ДБН врахована спеціалізація ЗОЗ ПП направлена на забезпечення оптимальних умов перебування пацієнтів з психічними розладами в умовах стаціонару, умов праці медичних працівників, умов відвідувачів. This work is distributed under the terms of the Creative Commons Attribution-ShareAlike 4.0 International License (https://creativecommons.org/licenses/by-sa/4.0/). MEDICINE AND PHARMACY приміщень в ЗОЗ та ЗОЗ ПП за рахунок приміщень для психологічного розвантажування медперсоналу. приміщень для В ДБН В.2.2-10:2022 нами впроваджено санітарно-гігієнічні вимоги до проєктування захисних споруд цивільного захисту ЗОЗ та ЗОЗ ПП щодо безпеки пацієнтів, медперсоналу та відвідувачів ЗОЗ в умовах воєнних бойових дій. В цих захисних спорудах цивільного захисту ЗОЗ та ЗОЗ ПП підлягають укриттю хворі, медичний та обслуговуючий персонал закладів охорони здоров’я, які не підлягають евакуації в безпечне місце і перебувають у зонах можливих значних руйнувань населених пунктів, що відповідає новелам Кодексу цивільного захисту України та Закону України «Про внесення змін до деяких законодавчих актів України щодо забезпечення вимог цивільного захисту під час планування та забудови територій» (2022). Новостворений ДБН В.2.2-10:2022 слугує становленню нової нормативної бази містобудівного законодавства та законодавства в галузі охорони здоров’я при проєктуванні/реконструкції/ або відновленні внаслідок руйнації від бойових дій закладів охорони здоров’я у воєнний та післявоєнний періоди України та сприятиме підвищенню якості менеджменту в сучасній охороні здоров’я територіальних громад країни. Новостворений ДБН В.2.2-10:2022 слугує становленню нової нормативної бази містобудівного законодавства та законодавства в галузі охорони здоров’я при проєктуванні/реконструкції/ або відновленні внаслідок руйнації від бойових дій закладів охорони здоров’я у воєнний та післявоєнний періоди України та сприятиме підвищенню якості менеджменту в сучасній охороні здоров’я територіальних громад країни. Метою створення медичної складової новітніх державних будівельних норм України ДБН В.2.2-10:2022 «Заклади охорони здоров’я. Основні положення» є забезпечення ефективного менеджменту охорони здоров’я територіальних громад України при проєктуванні/реконструкції/ або відновленні внаслідок руйнації від бойових дій закладів охорони здоров’я у воєнний та післявоєнний періоди України [21]. Реалії сьогодення свідчать про нагальну потребу у відбудові пошкоджених 1218 об’єктів ЗОЗ ( 540 лікарень, з них 173 – повністю знищено) внаслідок повномасштабного вторгнення рф в Україну. Планом відновлення України упродовж 2023- 2025 р.р., затвердженим Урядом країни, передбачено ремонт та повна відбудова ЗОЗ, які зазнали пошкоджень та тих, які були повністю знищені []. Саме санітарно-епідеміологічна складова до нормативної містобудівної бази України з проектування ЗОЗ та ЗОЗ ПП з урахуванням закордонного досвіду слугує її вдосконаленню та є детермінантою громадського здоров’я [22,23]. Висновки. 310 MEDICINE AND PHARMACY Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» (August 19-20, 2023). Seattle, USA No 167 Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» (August 19-20, 2023). 311 This work is distributed under the terms of the Creative Commons Attribution-ShareAlike 4.0 International License (https://creativecommons.org/licenses/by-sa/4.0/). This work is distributed under the terms of the Creative Commons Attribution-ShareAlike 4.0 International License (https://creativecommons.org/licenses/by-sa/4.0/). MEDICINE AND PHARMACY Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» (August 19-20, 2023). Seattle, USA No 167 Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» (August 19-20, 2023). Seattle, USA Proceedings of the 5th International Scientific and Practical Conference «Scientific Goals and Purposes in XXI Century» (August 19-20, 2023). Seattle, USA MEDICINE AND PHARMACY References: [1] Machniuk V.M., Hopperia V.G., Polka O.O., Pavlenko N.P., Ocheretyana G.V. Hygiene and ecology in state regulation of urban planning // Edited by the corresponding member of NAMS of Ukraine N.S.Polky. Видавництво МВЦ «Медінформ». К.: 2020. 131 р. 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https://openalex.org/W4313909908	https://www.frontiersin.org/articles/10.3389/fimmu.2022.1075335/pdf	English	null	Editorial: Irradiation technologies for vaccine development	Frontiers in immunology	2,023	cc-by	2,724	Editorial on the Research Topic Irradiation technologies for vaccine development Vaccine development is of high priority in the control of infectious diseases. The impact of vaccination on health is immense; except for the improvement of drinking water quality, no other approach has had such a major effect on mortality reduction and population growth (Rodrigues and Plotkin). However, despite an increase in our knowledge on host-pathogen interactions and the advancement in various cutting-edge technologies in vaccine design, there is still a dearth of effective vaccines against many human and animal diseases. The need to design and generate vaccines in a shorter period against emerging and re-emerging pathogens that are difﬁcult to control by other means is critical for human and animal welfare. The efforts to control current SARS-CoV-2 pandemic is a prime example. Inactivated whole virus vaccines were the ﬁrst vaccines developed and applied against SARS-CoV-2 and they are still widely used (around 50% of total vaccines delivered), indicating the value of this traditional method of vaccine development (1). At present, chemical inactivation is the most common method to kill pathogens for vaccine preparation. However, in the last decade the use of irradiation (gamma-, X-ray-, electron beam-irradiation) has been considered as a potential, valid alternative for vaccine development. Inactivation by irradiation has some potentially important advantages over chemical inactivation. The compilation of this Research Topic will attract attention to the state-of-the art of irradiation technology in vaccine development. © 2023 Wijewardana, Ulbert and Cattoli. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. The two mini reviews that appear in this collection give a comprehensive overview of the technology including historical developments. Despite the fact that irradiation technology is still primarily in the research and development phase, there is an increasing interest in this area as illustrated in the review paper by Bhatia and Pillai that provides a representative list of 24 patents that have been ﬁled for the creation of irradiated vaccines for human and animal bacterial, viral, and protozoan diseases. In the second review paper, Unger et al. TYPE Editorial PUBLISHED 09 January 2023 DOI 10.3389/fimmu.2022.1075335 OPEN ACCESS EDITED AND REVIEWED BY Anke Huckriede, University Medical Center Groningen, Netherlands Viskam Wijewardana 1, Sebastian Ulbert 2 and Giovanni Cattoli 1* 1Animal Production and Health Laboratory, Joint Food and Agriculture Organization (FAO)/International Atomic Energy Agency (IAEA) Centre of Nuclear Techniques in Food and Agriculture, International Atomic Energy Agency, Vienna, Austria, 2Department of Vaccines and Infection Models, Fraunhofer Institute for Cell Therapy and Immunology, Leipzig, Germany KEYWORDS gamma irradiation, e-beam irradiation, livestock, vaccine, zoonotic diseases, immune responses Editorial on the Research Topic Editorial on the Research Topic Irradiation technologies for vaccine development Editorial on the Research Topic Irradiation technologies for vaccine development Editorial on the Research Topic Irradiation technologies for vaccine development discuss the development of irradiated vaccines against livestock diseases with special reference to initiatives from the Animal Production and Health section (APH) of the Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture at the International Atomic Energy Agency. In this paper, information on the dose of irradiation used in various vaccine preparations is also provided. Both these articles show the science behind the ionizing Frontiers in Immunology frontiersin.org 01 10.3389/ﬁmmu.2022.1075335 Wijewardana et al. antigens to CD4 T helper (Th) lymphocytes while inefﬁcient in presenting antigens through MHC class I which is needed to activate CD8 (T cytotoxic) lymphocytes (5). Therefore, traditional inactivated vaccines most often do not yield a pronounced cell- mediated immunity. However, irradiation-inactivated Salmonella gallinarum provided an immune response skewed towards Th1 type (higher IgG2b and IgG3 levels) compared to a formalin inactivated vaccine which led to a protection level similar to a live attenuated vaccine when the challenge was done in chicken (Ji et al.). In another irradiated bacterial vaccine experiment in chicken, Dessalegn et al. showed intranasal or intraocular delivery of gamma irradiated Pasteurella multocida provided 100% protection reinforced with higher levels of secretory IgA. Inactivated pathogens are easily cleared by cilia and mucus in the intranasal mucosa unlike live attenuated organisms which can replicate. However, if the structure is maintained along with membrane integrity, this will aid adherence and allow the antigen presenting cells to take up the vaccine antigens. Several groups here investigated the structural integrity following sterilizing irradiation of pathogens when used as vaccine candidates. Electron micrography data showed a high degree of structural integrity when optimum irradiation conditions were used to inactivate Inﬂuenza virus (Bortolami et al.) African Swine Fever virus (ASFV) (Pikalo et al.) or Salmonella gallinarum (Ji et al.). However, in the case of ASFV, although the irradiated vaccine elicited antibodies when delivered intra-muscularly, there was no protection induced. Since it was shown above that several irradiated vaccines do provide better protection when administered through mucosal rather than parenteral routes, the authors could investigate alternative delivery of the vaccine in future experiments. radiation and its effect on microorganisms. Depending on the dose applied, ionizing radiation can irreparably damage the microbes. Interestingly, the radiation dose can be calibrated in a way that exposed microorganisms are unable to replicate (i.e. to cause infection) but still retain their metabolic activity, resulting in so- called “metabolically active, non-replicating” microorganisms (Hieke and Pillai). Editorial on the Research Topic Irradiation technologies for vaccine development The residual functional proteins that yield metabolic activity in these microorganisms induce a broader immune response in the host when vaccinated. This approach was adopted to develop a metabolically active, non-replicating sporozoite vaccine (PfSPZ vaccine) to prevent Plasmodium falciparum malaria which is now being tested at phase 2 clinical trials globally (2). While traditionally gamma irradiation was used for the inactivation of pathogens when developing irradiated vaccines, recent technological advances have paved the way for the use of electron (e)-beam or other irradiation techniques in an effort to move away from the use of radioactive material. Moreover, by employing new radio-protectant compounds such as manganese ions (Mn2+) and trehalose, immunogenic epitopes have been better preserved during irradiation inactivation (3). The antigenic variation of the HA1 domain and its resulting antigenic drift has led to reformulating seasonal inﬂuenza vaccines with new strains every year (4). This urge for the development of a universal inﬂuenza vaccine that must provide long-lasting cross- protective immunity that can induce both B and T cell responses. In veterinary medicine, the availability of safe and effective avian inﬂuenza vaccines suitable for mass applications (e.g. aerosol, drinking water) would facilitate the prevention and control of this disease in poultry. Within this collection there are three articles which aim to investigate if an irradiated inﬂuenza vaccine could perform better than chemically inactivated vaccines and/or induce a broader protection. Bortolami et al. investigated the protective efﬁcacy of a H9N2 avian inﬂuenza vaccine prototype. In this experiment, birds were vaccinated with an irradiated or a chemical inactivated formulation. The irradiated vaccine group performed as well as the chemical inactivated vaccine group upon challenge when the vaccines were given parenterally. Interestingly, when the vaccine was applied by the mucosal route, the irradiated preparation provided 100% protection at a challenge dose of 3 logs while the chemically inactivated one did not. This indicates the possibility of developing mucosal immunity through irradiated inactivated vaccines which is very difﬁcult to achieve with traditional chemical inactivation. Motamedi Sedeh et al. give further support to this aspect. In their H9N2 avian inﬂuenza experiments, both methods of inactivation and routes of administration provided similar levels of protection, but cell-mediated immune responses were more pronounce for the irradiated vaccine formulated with trehalose and given through the mucosal route. In a third article by Singleton et al., the conditions for irradiation were investigated in an H1N1 inﬂuenza vaccine experiment. 4. Petrova VN, Russell CA. The evolution of seasonal inﬂuenza viruses. Nat Rev Microbiol (2018) 16:47–60. doi: 10.1038/nrmicro.2017.118 5. BlumJS, Wearsch PA, Cresswell P. Pathways of antigen processing. Annu Rev Q15 Immunol (2013) 31:443–73. doi: 10.1146/annurev-immunol-032712-095910 6. Fertey J, Bayer L, Grunwald T, Pohl A, Beckmann J, Gotzmann G, et al. Pathogens inactivated by low-energy-electron irradiation maintain antigenic properties and induce protective immune responses. Viruses (2016) 8(11). doi: 10.3390/v8110319 7. di Pasquale A, Preiss S, da Silva FT, Garç on N. Vaccine adjuvants: From 1920 to 2015 and beyond. Vaccines (2015) 3:320–43. doi: 10.3390/vaccines3020320 Acknowledgments We thank the authors, reviewers and external editors that contributed to this Research Topic. We also thank Dr William Dundon of the International Atomic Energy Agency for editing language of this editorial. Publisher’s note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their afﬁliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Author contributions All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication. Editorial on the Research Topic Irradiation technologies for vaccine development There is increasing understanding of the role of vaccine adjuvants and how the formulation of modern vaccines can be better tailored towards the desired clinical beneﬁts. Thus discovery of novel adjuvants that could activate speciﬁc immune pathways will aid in the quest for developing vaccines against challenging pathogens (7). Conﬂict of interest The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. In summary, this Research Topic highlights some of the latest developments, innovations and understanding of the use of irradiation technologies for vaccine development. It also raised scientiﬁc and technical questions that need to be answered in future research including the underlying mechanisms involved in the remaining metabolic activity in lethally irradiated microbial cells, generation of better cell mediated immunity compared to chemical inactivation, use of LEEI in bulk vaccine preparations, the best route to deliver irradiated vaccines, discovery of novel radio-protectant compounds to preserve vaccine antigenicity and stabilization of Editorial on the Research Topic Irradiation technologies for vaccine development They show not only protection from the vaccine strain but also cross-protection through a gamma irradiated vaccine. Interestingly the induction of higher neutralizing antibodies and more effective cytotoxic T cell responses were correlated with higher temperatures during the irradiation process. y p One of the bottlenecks which holds back the scaling up and commercial production of irradiated vaccines is the safety and containment requirements for gamma irradiators sourced by radio isotopes. More and more groups are now investigating the use of e- beam or x-ray technologies to produce irradiated vaccines (6). Low- energy electron irradiation (LEEI) which consists of electrons accelerated with up to 500 kilo electron volts (keV) very rapidly delivers high doses necessary for pathogen inactivation, but only requires minimal shielding. Finkensieper et al. showed vaccination with three doses of LEEI inactivated tick-borne encephalitis virus provided complete protection from infection and induced higher antibody titers and avidities as compared to the formalin inactivated virus. E-beam technology can also help in producing biological therapeutics. E-beam irradiated inactivated human rotavirus (HRV) was used as antigen in chickens to produce antibodies against HRV. These egg yolk antibodies and serum derived IgY were effective at neutralizing HRV in-vitro (Skrobarczyk et al.). In LEEI processes, determination of the absorbed electron dose is challenging due to the limited, material-dependent penetration depth of the accelerated electrons into the matter. As a solution for this, Schopf et al. proposed the use of bacterial suspensions as biological indicators for electron beam doses. The use of gamma irradiators has been seen as a hindrance in scaling up of irradiated vaccines against viruses and bacteria, which require relatively high doses. In contrast, eukaryotic cells are several orders of magnitudes more susceptible to ionizing radiation, and consequently a biopharmaceutical production process for attenuation by gamma irradiation has been developed (James et al.) to produce In classical antigen presentation, the exogenous antigen is degraded via the endosome pathway and is loaded onto major histocompatibility complex (MHC) class II molecules that presents Frontiers in Immunology frontiersin.org 02 Wijewardana et al. 10.3389/ﬁmmu.2022.1075335 irradiated vaccine formulations. Research and technical innovation are also needed to transfer irradiation technologies for vaccine development from an applied research sector to production and marketing. PfSPZ vaccine against malaria. This process was used for several hundred irradiation events to produce the PfSPZ vaccine candidate in the last 13 years which generated multiple lots released for pre-clinical studies and clinical trials. 3. Gayen M, Gupta P, Morazzani EM, Gaidamakova EK, Knollmann-Ritschel B, Daly MJ, et al. Deinococcus Mn2+-peptide complex: A novel approach to alphavirus vaccine development. Vaccine (2017) 35(29):3672–81. doi: 10.1016/j.vaccine.2017.05.016 2. Oneko M, Steinhardt LC, Yego R, Wiegand RE, Swanson PA, Kc N, et al. Safety, immunogenicity and efﬁcacy Q13 of PfSPZ vaccine against malaria in infants in western Kenya: a double-blind, randomized, placebo-controlled phase 2 trial. Nat Med (2021) 27:1636–45. doi: 10.1038/s41591-021-01470-y Editorial on the Research Topic Irradiation technologies for vaccine development By studying another unicellular parasite, Kangethe et al. performed experiments to identify genes that are involved in disease establishment by gamma irradiation of Trypanosoma evansi. By subjecting parasites to a lower-dose of irradiation than that needed to stop replication, the genes responsible for the repair of the radiation-induced damage and thus potential virulent factors were identiﬁed. The authors propose a strategy for a candidate vaccine by deleting some of these virulent genes in the parasite. Radiation-induced mutations were also applied to attenuate the virulence of Salmonella spp. to develop live vaccine strains (Ji et al.). The selected mutant strain (ATOMSal-L6) was almost 10,000 times less virulent than its parent strain. Moreover, attenuation was maintained for over 10 passages. ATOMSal-L6 induced protective immunity upon intramuscular vaccination of mice. Finally, Porﬁri et al. explored the immunomodulatory landscape of replication deﬁcient metabolically active Lactobacilli produced through gamma irradiation to be used as novel vaccine adjuvants. There is increasing understanding of the role of vaccine adjuvants and how the formulation of modern vaccines can be better tailored towards the desired clinical beneﬁts. Thus discovery of novel adjuvants that could activate speciﬁc immune pathways will aid in the quest for developing vaccines against challenging pathogens (7). PfSPZ vaccine against malaria. This process was used for several hundred irradiation events to produce the PfSPZ vaccine candidate in the last 13 years which generated multiple lots released for pre-clinical studies and clinical trials. By studying another unicellular parasite, Kangethe et al. performed experiments to identify genes that are involved in disease establishment by gamma irradiation of Trypanosoma evansi. By subjecting parasites to a lower-dose of irradiation than that needed to stop replication, the genes responsible for the repair of the radiation-induced damage and thus potential virulent factors were identiﬁed. The authors propose a strategy for a candidate vaccine by deleting some of these virulent genes in the parasite. Radiation-induced mutations were also applied to attenuate the virulence of Salmonella spp. to develop live vaccine strains (Ji et al.). The selected mutant strain (ATOMSal-L6) was almost 10,000 times less virulent than its parent strain. Moreover, attenuation was maintained for over 10 passages. ATOMSal-L6 induced protective immunity upon intramuscular vaccination of mice. Finally, Porﬁri et al. explored the immunomodulatory landscape of replication deﬁcient metabolically active Lactobacilli produced through gamma irradiation to be used as novel vaccine adjuvants. 1. Dolgin E. Omicron thwarts some of the world’s most-used COVID vaccines. Nature (2022) 601:311. doi: 10.1038/d41586-022-00079 1. Dolgin E. Omicron thwarts some of the world’s most-used COVID vaccines. Nature (2022) 601:311. doi: 10.1038/d41586-022-00079 2. Oneko M, Steinhardt LC, Yego R, Wiegand RE, Swanson PA, Kc N, et al. Safety, immunogenicity and efﬁcacy Q13 of PfSPZ vaccine against malaria in infants in western Kenya: a double-blind, randomized, placebo-controlled phase 2 trial. Nat Med (2021) 27:1636–45. doi: 10.1038/s41591-021-01470-y 3. Gayen M, Gupta P, Morazzani EM, Gaidamakova EK, Knollmann-Ritschel B, Daly MJ, et al. Deinococcus Mn2+-peptide complex: A novel approach to alphavirus vaccine development. Vaccine (2017) 35(29):3672–81. doi: 10.1016/j.vaccine.2017.05.016 References 1. Dolgin E. Omicron thwarts some of the world’s most-used COVID vaccines. Nature (2022) 601:311. doi: 10.1038/d41586-022-00079 03 Frontiers in Immunology frontiersin.org
https://openalex.org/W3209043395	https://zenodo.org/record/3533948/files/Agim%20Ibraimi.pdf	English	null	THE ROLE OF L1 IN CLASSROOM (LEXICAL APPROACH IN ELT)	DOAJ (DOAJ: Directory of Open Access Journals)	2,019	cc-by	1,876	October 2019 e-ISSN: 1857-8187  p-ISSN: 1857-8179 October 2019 e-ISSN: 1857-8187  p-ISSN: 1857-8179 Research Article Research Article Research Article Agim Ibraimi Southeast European University – Tetovo. North Macedonia. ELT Classes have tried and continue to try new methods and techniques so as to achieve the best possible results in ELT classrooms. Various language teaching methods have treated translation (L1) in ELT classrooms differently. With the introduction of Communicative Approach, translation and interference fell out of favour. But according to Lexical Approach, an approach which is being widely used in ELT classrooms in the world, the importance of translation or L1 in ELT classes is far from being trivial. Seen from a lexical view of language, translation and interference became fruitful again. In the past, language was considered as composed of words, but Lexical Approach has argued that, besides words, language consists of chunks (lexis). With language chunking in mind, we will consider the idea of L1 (translation) role in ELT. In this research paper will elaborate translation inevitability in classroom, L1-L2 comparison, the role the lexis play in acquiring the language, interference and L1 awareness. THE ROLE OF L1 IN CLASSROOM (LEXICAL APPROACH IN ELT) Language Teaching Keywords: translation, interference, approach, lexical, chunks, acquiring, lexis. Abstract THE ROLE OF L1 IN CLASSROOM (LEXICAL APPROACH IN ELT) Keywords: translation, interference, approach, lexical, chunks, acquiring, lexis. Agim Ibraimi Abstract ELT Classes have tried and continue to try new methods and techniques so as to achieve the best possible results in ELT classrooms. Various language teaching methods have treated translation (L1) in ELT classrooms differently. With the introduction of Communicative Approach, translation and interference fell out of favour. But according to Lexical Approach, an approach which is being widely used in ELT classrooms in the world, the importance of translation or L1 in ELT classes is far from being trivial. Seen from a lexical view of language, translation and interference became fruitful again. In the past, language was considered as composed of words, but Lexical Approach has argued that, besides words, language consists of chunks (lexis). With language chunking in mind, we will consider the idea of L1 (translation) role in ELT. In this research paper will elaborate translation inevitability in classroom, L1-L2 comparison, the role the lexis play in acquiring the language, interference and L1 awareness. Anglisticum Journal (IJLLIS),Volume: 8 \| Issue: 10 \| II. L2 and L1 are not Identical In Lexical Approach there is an implicit assumption that the relationship between L1 and L2 is more similar than different, that the human mind handles languages in certain non-language –specific ways. However there are some differences between acquiring L1 and L2. Two differences cannot be ignored during language learning process. By leaning a second language you are able to mediate yourself and your world through language in general. L2 allows you to mediate self and worlds in wider contexts, in other words L2 is a second way of doing something you can already do. The second difference is that acquiring L2 mirrors acquiring L1 in that you relate L2 words to L2 words, or L2 words to external world (what is this called in English?), but you also or inevitably relate L2 words and expressions to L1 (How do you say..... in English?); in short, you translate. October 2019 e-ISSN: 1857-8187  p-ISSN: 1857-8179 communities do not invent or coin new words all the time; on the contrary, there are Expressions which the non-natives need to acquire as wholes. They have to be remembered as such wholes so they are available for subsequent use in exactly the same form. Learners have an instinctive understanding that languages have what we call equivalents, correct translations, which are totally different form word-for-word translations. III. Translation and Lexis By lexis, as mentioned above, we mean words, collocations fixed and semi-fixed expressions that inevitably are encountered during learning language process. To prevent word-for word translation during learning, teachers should pay more attention to developing the skills to chunk language. In my opinion here is where teachers‘ help is need to guide students and help understand and become aware that the language to be learnt should be viewed as composed of phrases, collocations and idioms, which, in most cases, cannot be understood if translated word- for-word. I. Translation is Inevitable Until recently language learning has been considered as a process where the learner has to learn the language (words) and its grammar. But Lexical Approach challenges this view alluding that language composes of chunks which, when combined, produce a continuous coherent text. These chunks are of different kinds but only four of them are considered as fundamental. The first kind of these kinds consists of single words while all others are multi-part items. Since we saw above that language is no more composed only of words (vocabulary) and grammar, then teachers should pay attention to chunks during the learning process in ELT. When we say chunks, by this we mean, they have to pay attention to lexis: words, collocations (co- occurrence of words: community service...) and fixed expressions (social greetings, politeness phrases, idioms) Lexical approach encourages identification of chunks and recognition that word-for-word equivalence is often impossible. Having pedagogical chunking in mind, we need to consider the role learners‘ L1 would play in classroom. According to this approach, translation became once again a very fruitful tool when seen in the context of lexical view. Through translation has fallen out of use, teachers still encounter questions like How do you say...... in English? or Can you say.....? These learners‘ question reveals two points; when students can not express their selves in English, you naturally fall back into L1, and search for a translation in L1. This is way translation is considered as an instinctive part the brain approaches language learning. The second point that we may infer from the above questions is that learners when posing these questions let us know that they have a feeling for the lexis- they understand that Page \| 74 Anglisticum Journal (IJLLIS),Volume: 8 \| Issue: 10 \| 3.1. The value of translation Since language is and should be considered as composed of lexis (chunks) not only words alone, there are several reasons why translation should occupy the right place in ELT: 1. It is inevitable that learners use L1 as a source, and that they make both helpful and unhelpful assumptions based on their experience of L1. 1. It is inevitable that learners use L1 as a source, and that they make both helpful and unhelpful assumptions based on their experience of L1. 2. Comparing L2 and L1 is not always helpful for learners seeking to master the contrasts internal to English. But if this equation becomes L2 lexical item=L1 lexical item then it is helpful as correctly identified chunks do have equivalents in other languages. 2. Comparing L2 and L1 is not always helpful for learners seeking to master the contrasts internal to English. But if this equation becomes L2 lexical item=L1 lexical item then it is helpful as correctly identified chunks do have equivalents in other languages. Page \| 75 Page \| 75 Anglisticum Journal (IJLLIS),Volume: 8 \| Issue: 10 \| 3.3. L1 awareness as a source According to Lexical Approach, it is unhelpful to teach structure by comparing L1 and L2. A few grammar mistakes, whether the result of interference or not, impede communication and thus merit no more than occasional correction. In contrast, lexical mistakes do matter and activities which aim at raising awareness of untrue parallels is valuable. Increased learners‘ awareness of certain lexical parallels can be helpful. Most learners have not thought closely about features of their mother-tongue. Therefore it‘s helpful to show learners ways their own language and the language they are learning are lexically similar:  collocation is a feature of all languages  different genres contain different kinds of collocation  Fixed expressions exist in their language as well as in English and have immediately recognisable pragmatic meaning.  Expressions suitable for use to one person may not be suitable if used to a different person: all languages have ways of making formality. October 2019 e-ISSN: 1857-8187  p-ISSN: 1857-8179 3. Searching, discussing and recording appropriate L1 lexical item=L2 lexical item is useful and efficient procedure, but using this technique properly requires a kind of language awareness which a few learners will employ without help form a teacher. 3. Searching, discussing and recording appropriate L1 lexical item=L2 lexical item is useful and efficient procedure, but using this technique properly requires a kind of language awareness which a few learners will employ without help form a teacher. 4. The argument behind using translation in ELT is that the techniques and processes made in search for equivalents exactly mirror the processes every student uses when trying to express something for which the language does not come to mind automatically. 3.2. Interference can be helpful Learners do posses a mental picture and a perception of the structure or L2. This knowledge has been built from different sources such as exposure appropriate reading and listening in L2 and by analogy with L1. Most, if not all, these processes by which this method develops are subconsciously that is why in this context we want to use this natural tendency of learners to assume that features of L1 apply to L2. Though up to now the language used to describe the ―interference of L1‖ carries negative connotations, many analogies do work and are positive aids to L2 acquisition. The teachers‘ task is to raise learners‘ awareness of the effects, so helping them both to avoid the unhelpful and to benefit from helpful, parallels. October 2019 e-ISSN: 1857-8187  p-ISSN: 1857-8179 unit=L2 lexical unit should be encouraged as this helps student remember and memorise more easily the language they are learning, especially when dealing with adult learners who rarely learn concepts, they learn language, opposite to young learners. Lexical approach focuses on lexis and chunks, and very often these needs to be paralleled with L1 so as to inculcate them into learners‘ brain. By focusing on similarities and differences between L1 and L2 we make them aware of features that both languages have or don‘t have in common. 1. Lewis, M. (1998). Implementing the lexical approach, Putting theory into practice, 11a Church Road, Hove, BN3 2EB, England. 2. https://www.oebv.at/system/files/celum/371891_developing_sense.pdf (withdrawn on 06.08.2019) 3. Willis, D. (2003). Rules, Patterns and Words, Grammar and Lexis in English Language Teaching published by Press Syndicate of the University of Cambridge, United Kingdom. 4. Lipka, L. (2002). English Lexicology; lexical structure, word semantics and word formation. IV. Conclusion As can be seen from the above said, we can find a lot of positive features and contributions that L1 or translation may bring to our work in ELT classroom. This inevitability of L1 interference that takes place in our mind should not and cannot be ignored during ELT classroom processes. Word-for-word translation should be discourages, but on the other hand, L1-lexial Page \| 76 Anglisticum Journal (IJLLIS),Volume: 8 \| Issue: 10 \| Anglisticum Journal (IJLLIS),Volume: 8 \| Issue: 10 \| References 4. Lipka, L. (2002). English Lexicology; lexical structure, word semantics and word formation. Page \| 77 Page \| 77 Page \| 77 Anglisticum Journal (IJLLIS),Volume: 8 \| Issue: 10 \|
https://openalex.org/W2092142218	https://journals.plos.org/plosmedicine/article/file?id=10.1371/journal.pmed.1000362&type=printable	English	null	Efficacy of Oseltamivir-Zanamivir Combination Compared to Each Monotherapy for Seasonal Influenza: A Randomized Placebo-Controlled Trial	PLoS medicine	2,010	cc-by	10,797	Efficacy of Oseltamivir-Zanamivir Combination Compared to Each Monotherapy for Seasonal Influenza: A Randomized Placebo-Controlled Trial Xavier Duval1,2,3, Sylvie van der Werf4,5,6, Thierry Blanchon7,8, Anne Mosnier9, Maude Bouscambert- Duchamp10,11, Annick Tibi12,13, Vincent Enouf4, Ce´cile Charlois-Ou14, Corine Vincent2,3,15, Laurent Andreoletti16,17, Florence Tubach2,3,18, Bruno Lina10,11, France Mentre´ 2,3,15, Catherine Leport14,19, and the Bivir Study Group" 1 Inserm CIC 007, APHP, Hoˆpital Bichat, Paris, France, 2 Inserm U738, Paris, France, 3 Universite´ Paris Diderot, Paris 7, UFR de Me´decine, site Bichat, Paris, France, 4 Institut Pasteur, Centre National de Re´fe´rence des virus influenzae (Re´gion-Nord), Unite´ de Ge´ne´tique Mole´culaire des Virus a` ARN, Paris, France, 5 CNRS URA3015, Paris, France, 6 Universite´ Paris Diderot, Paris 7, UFR Sciences du Vivant, Paris, France, 7 Inserm UPMC UMR-S 707, Faculte´ de me´decine Pierre et Marie Curie, Paris, France, 8 Universite´ Pierre et Marie Curie, Paris 6, UFR de Me´decine, U707, Paris, France, 9 Re´seau des Groupes Re´gionaux d’Observation de la Grippe (GROG), Coordination nationale, Paris, France, 10 Hospices Civils de Lyon, Centre National de Re´fe´rence des virus influenzae (Re´gion-Sud), GHE, Bron, France, 11 Universite´ Lyon 1, VirPatH, CNRS FRE 3011, Lyon, France, 12 APHP- Agence Ge´ne´rale des Equipements et Produits de Sante´, Unite´ Essais Cliniques, Paris, France, 13 Universite´ Paris Descartes, Paris 5, Faculte´ de Pharmacie, Paris, France, 14 Universite´ Paris Diderot, Paris 7, UFR de Me´decine, site Bichat, Laboratoire de Recherche en Pathologie Infectieuse, Paris, France, 15 APHP, Hoˆpital Bichat, Unite´ de Biostatistiques, Paris, France, 16 Hoˆpital Robert Debre´, Unite´ de Virologie me´dicale, Reims, France, 17 Unite´ de Virologie Me´dicale et Mole´culaire Faculte´ de Me´decine Universite´ Champagne-Ardenne IFR53/EA-4303, Reims, France, 18 APHP Hoˆpital Bichat, De´partement d’Epide´miologie, Biostatistiques et Recherche Clinique, Paris, France, 19 APHP, Unite´ de Coordination des Risques Epide´miques et Biologiques, Paris, France Abstract Background: Neuraminidase inhibitors are thought to be efficacious in reducing the time to alleviation of symptoms in outpatients with seasonal influenza. The objective of this study was to compare the short-term virological efficacy of oseltamivir-zanamivir combination versus each monotherapy plus placebo. Methods and Findings: We conducted a randomized placebo-controlled trial with 145 general practitioners throughout France during the 2008–2009 seasonal influenza epidemic. Patients, general practitioners, and outcome assessors were all blinded to treatment assignment. Adult outpatients presenting influenza-like illness for less than 36 hours and a positive influenza A rapid test diagnosis were randomized to oseltamivir 75 mg orally twice daily plus zanamivir 10 mg by inhalation twice daily (OZ), oseltamivir plus inhaled placebo (O), or zanamivir plus oral placebo (Z). Treatment efficacy was assessed virologically according to the proportion of patients with nasal influenza reverse transcription (RT)-PCR below 200 copies genome equivalent (cgeq)/ml at day 2 (primary outcome), and clinically to the time to alleviation of symptoms until day 14. Overall 541 patients (of the 900 planned) were included (OZ, n = 192; O, n = 176; Z, n = 173), 49% male, mean age 39 years. In the intention-to-treat analysis conducted in the 447 patients with RT-PCR-confirmed influenza A, 46%, 59%, and 34% in OZ (n = 157), O (n = 141), and Z (n = 149) arms had RT-PCR,200 cgeq/ml (213.0%, 95% confidence interval [CI] 223.1 to 22.9, p = 0.025; +12.3%, 95% CI 2.39–22.2, p = 0.028 for OZ/O and OZ/Z comparisons). Mean day 0 to day 2 viral load decrease was 2.14, 2.49, and 1.68 log10 cgeq/ml (p = 0.060, p = 0.016 for OZ/O and OZ/Z). Median time to alleviation of symptoms was 4.0, 3.0, and 4.0 days (+1.0, 95% CI 0.0–4.0, p = 0.018; +0.0, 95% CI 23.0 to 3.0, p = 0.960 for OZ/O and OZ/Z). Four severe adverse events were observed. Nausea and/or vomiting tended to be more frequent in the combination arm (OZ, n = 13; O, n = 4; and Z, n = 5 patients, respectively). Conclusions: In adults with seasonal influenza A mainly H3N2 virus infection, the oseltamivir-zanamivir combination appeared less effective than oseltamivir monotherapy, and not significantly more effective than zanamivir monotherapy. Despite the theoretical potential for the reduction of the emergence of antiviral resistance, the lower effectiveness of this combination calls for caution in its use in clinical practice. Combined Oseltamivir-Zanamivir in Influenza Citation: Duval X, van der Werf S, Blanchon T, Mosnier A, Bouscambert-Duchamp M, et al. (2010) Efficacy of Oseltamivir-Zanamivir Combination Co Each Monotherapy for Seasonal Influenza: A Randomized Placebo-Controlled Trial. PLoS Med 7(11): e1000362. doi:10.1371/journal.pmed.1000362 Academic Editor: Benjamin J. Cowling, The University of Hong Kong, Hong Kong Received April 29, 2010; Accepted September 22, 2010; Published November 2, 2010 Copyright: 2010 Duval et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, wh unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by a research grant from the French Ministry of Health. The sponsor was: De´partement a` la Recherche Clin Developpement, Assistance Publique a` Hopitaux de Paris (Programme hospitalier de recherche clinique, AOM 06060 and AOM 08209). The funder ha study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: XD has had a conference invitation from GSK and lecture fees from Roche and Gilead. AM has membership in the ministr advisory board on influenza; involvement in some epidemiological studies partially or fully granted by Roche and GSK, and travel grants from participation in scientific meetings. SVDW has had a conference invitation from GSK; research grant from GSK on unrelated subject; joined patent from with GSK on unrelated subject; travel grants for meetings from GSK; contribution to clinical trial financed by Roche; member of the advisory com influenza of the French ministry of health; is a member of ESWI; is a member of the scientific committee of the GEIG; and is vice-president of the GRO FM received fees from Roche, preclinical pharmacokinetic department, for a course on MONOLIX in December 2008. BL has had paid consultancy membership (Roche, GSK, Novartis, BioCryst, MedImmune), has had research grants from Roche and Sanofi-Pasteur, and had received travel grants an for speaking or participation at meetings (Roche, Sanofi-Pasteur). Abbreviations: cgeq, copies genome equivalent; CI, confidence interval; IQR, interquartile range; ITT, intention to treat; NIC, National Influenza oseltamivir; OZ, oseltamivir-zanamivir combination; RT, reverse transcription; SD, standard deviation; Z, zanamivir. E-mail: catherine.leport@univ-paris-diderot.fr " Membership of the BIVIR Study Group is provided in the Acknowledgments. Citation: Duval X, van der Werf S, Blanchon T, Mosnier A, Bouscambert-Duchamp M, et al. (2010) Efficacy of Oseltamivir-Zanamivir Combination Compared to Each Monotherapy for Seasonal Influenza: A Randomized Placebo-Controlled Trial. PLoS Med 7(11): e1000362. Combined Oseltamivir-Zanamivir in Influenza doi:10.1371/journal.pmed.1000362 Citation: Duval X, van der Werf S, Blanchon T, Mosnier A, Bouscambert-Duchamp M, et al. (2010) Efficacy of Oseltamivir-Zanamivir Combination Compared to Each Monotherapy for Seasonal Influenza: A Randomized Placebo-Controlled Trial. PLoS Med 7(11): e1000362. doi:10.1371/journal.pmed.1000362 Academic Editor: Benjamin J. Cowling, The University of Hong Kong, Hong Kong Academic Editor: Benjamin J. Cowling, The University of Hong Kong, Hong Kong Received April 29, 2010; Accepted September 22, 2010; Published November 2, 2010 Received April 29, 2010; Accepted September 22, 2010; Published November 2, 2010 Copyright: 2010 Duval et al. This is an open-access article distributed under the terms of the Creative Commons unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: 2010 Duval et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by a research grant from the French Ministry of Health. The sponsor was: De´partement a` la Recherche Clinique et au Developpement, Assistance Publique a` Hopitaux de Paris (Programme hospitalier de recherche clinique, AOM 06060 and AOM 08209). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: XD has had a conference invitation from GSK and lecture fees from Roche and Gilead. AM has membership in the ministry of health advisory board on influenza; involvement in some epidemiological studies partially or fully granted by Roche and GSK, and travel grants from Roche for participation in scientific meetings. SVDW has had a conference invitation from GSK; research grant from GSK on unrelated subject; joined patent from institution with GSK on unrelated subject; travel grants for meetings from GSK; contribution to clinical trial financed by Roche; member of the advisory committee on influenza of the French ministry of health; is a member of ESWI; is a member of the scientific committee of the GEIG; and is vice-president of the GROG network. FM received fees from Roche, preclinical pharmacokinetic department, for a course on MONOLIX in December 2008. BL has had paid consultancy and board membership (Roche, GSK, Novartis, BioCryst, MedImmune), has had research grants from Roche and Sanofi-Pasteur, and had received travel grants and honoraria for speaking or participation at meetings (Roche, Sanofi-Pasteur). Abstract Trial registration: http://www.ClinicalTrials.gov NCT00799760 Trial registration: http://www.ClinicalTrials.gov NCT00799760 Please see later in the article for the Editors’ Summary. Please see later in the article for the Editors’ Summary. PLoS Medicine \| www.plosmedicine.org November 2010 \| Volume 7 \| Issue 11 \| e1000362 1 PLoS Medicine \| www.plosmedicine.org Introduction test for influenza A (Clearview Exact Influenza A & B) performed by the practitioner. Enrolment of women required a negative urine pregnancy test. Exclusion criteria were vaccination against influenza during the 2008–2009 season, recent exacerbations of chronic obstructive pulmonary disease (COPD), asthma or severe chronic disease, previous history of depression, and prior inclusion in this trial. Prior to inclusion, patients gave informed written consent. The protocol was approved by the Ethics Committee of Ile de France 1 (Texts S1 and S2). Neuraminidase inhibitors (oseltamivir [O], zanamivir [Z]) are thought to be efficacious as compared to placebo in outpatients with uncomplicated seasonal influenza [1–6], both clinically in terms of reduction in duration of symptoms, as well as in terms of a reduction in viral shedding. In 2008, they were considered an important strategy to limit the impact of an influenza pandemic both individually, by reducing morbidity and mortality, and collectively, by slowing spread of the virus to allow time for vaccine production, the cornerstone of influenza control [2–4,7]. It was hypothesized that the widespread use of a single antiviral might result in the emergence of resistant strains whose subsequent spread could dramatically reduce the effectiveness of antiviral therapy. The combination of two antiviral agents, if well tolerated, and if producing at least additive antiviral activity, theoretically offers several advantages: reducing disease severity, viral shedding, and viral excretion period, thereby also lowering the attack rate and risk of selection of resistant viruses, specifically in individuals with prolonged viral shedding, such as immunocompromised patients [8,9]. Indeed, mathematical modelling showed a reduc- tion in risk of emergence of resistant strains during early phases of a pandemic, associated with use of two antivirals as compared to single antiviral therapy [9]. Finally, another theoretical advantage of combining two drugs would be to ensure optimal treatment of all circulating influenza virus types, subtypes, or variants, as susceptibility of influenza viruses has been shown to vary, and seasonal H1N1 viruses naturally resistant to oseltamivir, which remain susceptible to zanamivir, emerged in 2008 [10]. Among antivirals active against influenza virus, the combination of neuraminidase inhibitors is attractive, because both compounds are licensed for seasonal influenza, they are delivered to the respiratory tract by distinct means (directly through a diskhaler for zanamivir, after gastrointestinal absorption and hepatic metabo- lism for oseltamivir), and key mutations associated with resistance are different for each drug. Combined Oseltamivir-Zanamivir in Influenza Abbreviations: cgeq, copies genome equivalent; CI, confidence interval; IQR, interquartile range; ITT, intention to treat; NIC, National Influenza Centre; O, oseltamivir; OZ, oseltamivir-zanamivir combination; RT, reverse transcription; SD, standard deviation; Z, zanamivir. " Membership of the BIVIR Study Group is provided in the Acknowledgments. PLoS Medicine \| www.plosmedicine.org November 2010 \| Volume 7 \| Issue 11 \| e1000362 PLoS Medicine \| www.plosmedicine.org 2 Combined Oseltamivir-Zanamivir in Influenza Virological Analysis In 2006, in the context of pandemic planning, we designed a double-placebo randomized controlled trial in patients presenting with seasonal influenza-like illness to compare the oseltamivir- zanamivir combination to each of the monotherapies plus placebo. The trial was conducted in France, during the winter of 2008– 2009. Because of the emergence of the pandemic 2009 (H1N1) virus in humans, in April 2009 in North America, and its subsequent worldwide spread, the independent data-monitoring committee requested that we terminate the trial early and analyze the results earlier than planned, given the possible impact of the results on antiviral treatment management during the pandemic [11]. g y Nasal swabs placed into a transport medium (Virocult, Elitech) were transported at 4uC by special courier to the nearest National Influenza Centre (NIC) (Hospices Civils de Lyon, Lyon, or Pasteur Institute, Paris, France). Upon arrival, the swab samples were eluted into 2 ml of transport medium, processed for real-time reverse transcription (RT)-PCR analyses and inoculated onto MDCK cells for virus isolation and subsequent subtyping using a standard hemagglutination inhibition assay. For RT-PCR analy- ses, RNA extraction from 200 ml of specimen was performed using the QIAmp virus RNA mini kit (Qiagen) with RNA elution into a final volume of 60 ml. All real-time RT-PCR assays were performed in a final volume of 15 mL with 5 mL RNA, 0. mM of each primer, 0.2 mM probe, and 0.8 ml enzyme mix (Super- ScriptIII platinum one-step quantitative RT-PCR system, Invitro- gen). Type A influenza virus RNA was detected by a real-time RT-PCR targeting the conserved matrix gene using GRAM/7Fw (59-CTTCTAACCGAGGTCGAAACGTA-39) and GRAM/ 161Rv (59-GGTGACAGGATTG GTCTTGTCTTTA-39) prim- ers and GRAM probe/52/+ (59[Fam]-TCAGGCC CCTCAA- AGCCGAG-[BHQ-1]39) probe. The quality of the specimens was assessed by real-time RT-PCR targeting the GAPDH cellular gene [12]. Amplification was performed on a LightCycler 480 (Roche Diagnostics) (NIC, Pasteur Institute, Paris) or an ABI 7500 (Applied Biosystems) (NIC, Lyon). Cycling conditions are available Introduction However, negative interactions cannot not be ruled out owing to the possible competition between these two drugs, which target the same binding pocket in the neuraminidase. Study Procedures At enrolment (day 0), a nasal swab for virological analysis was performed using a standard operating procedure (sample kit plus instructional video). Patients were allocated to treatment by a randomization list, with an arm ratio of 1:1:1, balanced by practitioner. A computer random number generator was used to select random permuted blocks of size 3. This randomisation code was given to the central hospital pharmacy that prepared blinded treatment units in conformity with good manufacturing practices (GMP). Each general practitioner received six treatment units and was told to distribute them by order of inclusion of his patients in the trial. Allocation was concealed through the similarity of all the containers and the impossibility for the GP to identify the treatment arm when opening the container. The three treatments were (1) oseltamivir capsule for oral use plus inhaled zanamivir, (2) oseltamivir plus inhaled placebo, (3) zanamivir plus oral placebo. Oseltamivir dosage was 75 mg orally twice daily; zanamivir dosage was 10 mg by oral inhalation using the commercialized GlaxoSmithKline Diskhaler, twice daily. Active drugs and placebo were kindly provided by Roche and Glaxo-SmithKline laborato- ries. A visiting nurse performed a nasal swab for virological analysis on day 2. Patients returned to their general practitioner at day 7 for a follow-up examination, and were contacted by phone on day 14. Patients, general practitioners assigning the patients, and outcome assessors (practitioners, virologists, patients), were blinded to treatment assignment throughout the study and statisticians until the end of the analysis. PLoS Medicine \| www.plosmedicine.org Combined Oseltamivir-Zanamivir in Influenza Combined Oseltamivir-Zanamivir in Influenza compared to 55% in each of monotherapy arms on the basis of the extrapolation of the results of previous trials [2,4]. Samples of 300 subjects per arm had 90% power to detect this difference, with a two-sided test and a type I error of 0.025 because of the two tests (factoring 20% lack of follow-up). Proportions of response at day 2 were compared between the combination therapy arm (OZ) and each monotherapy arm (O or Z) separately using two tests with a type I error of 0.025 because of the two tests. Patients without a day 2 sample were considered treatment failures. Mean decreases of log10 viral load were compared using a t-test in patients who had both day 0 and day 2 samples assuming a value of 0.5 cgeq/ml when RT-PCR was negative. For clinical endpoints nonparametric tests were used. Times to resolution of illness and symptoms score at the end of treatment were compared using Wilcoxon tests. If time to symptom alleviation was missing it was imputed to be 14 d, i.e., the end of the trial; 95% confidence intervals (95% CIs) of median differences were estimated by bootstrap. Probability of symptoms alleviation versus day of treatment was estimated using the Kaplan Meier method and was compared between groups with the log-rank test. Proportions of clinical events and of patients with alleviation of symptoms were compared using Fisher’s exact tests. As an exploratory analysis, 95% CIs for differences of response between the two monotherapies were also estimated. All analyses were performed using SAS software, version 9.1 (SAS Institute). upon request. Quantified synthetic RNA transcripts corresponding to the M and GAPDH genes were used as controls in parallel [13]. g p To take into account the variability in the quantity of cells collected by nasal swab, we calculated a normalized influenza viral load for each specimen; this normalized viral load was defined as the ratio of the M RT-PCR and GAPDH RT-PCR multiplied by the average GAPDH RT-PCR at day 0 to express results in copies of genome equivalent/ml (cgeq/ml). The virological response was defined as a normalized viral load below 200 cgeq/ml at day 2. Virological Samples Out of the 541 enrolled patients, 447 (83%) had a RT-PCR laboratory confirmation of influenza A virus infection on the day 0 specimen, with a mean viral load of 4.38 log10 cgeq/ml (interquartile range [IQR] 3.75–5.30). All the day 0 specimens were GAPDH RT-PCR positive with a mean value of 3.88 log10 copies/ml. Patients Out of the 900 patients initially planned, a total of 541 patients were enrolled by 145 general practitioners. They were randomly assigned to oseltamivir plus zanamivir (OZ, n =192), oseltamivir plus inhaled placebo (O, n =176), zanamivir plus oral placebo (Z, n= 173) (Figure 1). Mean age was 39 y (standard deviation [SD] =13), 49% were male, 14% had preexisting chronic diseases, mean fever was 38.2uC (SD =0.8). The mean duration of illness before enrolment was 25 h (SD = 10). Other characteristics of patients appeared well balanced in the three arms (Table 1). The rate of fully compliant patients was not significantly different among the three arms (84% for the OZ arm, 88% for the O arm, and 85% for the Z arm). According to the protocol, the intention-to-treat (ITT) analysis was performed on two populations: (1) all enrolled patients (primary objective), and (2) enrolled patients with an influenza A virus infection confirmed by RT-PCR on day 0 (influenza A- infected population). Combined Oseltamivir-Zanamivir in Influenza This threshold was determined according to results on specimens from patients with a positive influenza A rapid test from winter 2008– 2009 included in the French influenza surveillance network (GROG), and analysed by the 2NICs, and because it resulted in 5% false positive and 5% false negative results with respect to virus isolation (Table S1). It was validated by the independent data- monitoring committee prior to any analysis. Sensitivity of the qRT- PCR was assessed using serial dilutions of quantified synthetic transcripts corresponding to the target genes (Text S3). To assess comparability of the data between the two NICs, specimens were exchanged showing excellent concordance. The threshold of the qRT-PCR used was set well above the limit of detection. PLoS Medicine \| www.plosmedicine.org Endpoints, Analyzed Populations The primary efficacy endpoint was the proportion of patients with RT-PCR,200 cgeq/ml on day 2 of treatment. Given the viral shedding kinetics in patients with seasonal influenza receiving neuraminidase inhibitors, the day 2 virological endpoint was considered to be best suited to measurement of virological effects [2,4]. Other endpoints were (1) the decrease of log10 viral load between days 0 and 2 in the patients with confirmed influenza A on day 0 and available samples both at days 0 and 2; (2) the time to resolution of illness; (3) the number of patients with alleviation of symptoms at the end of treatment (day 5); (4) the symptoms score at the end of treatment; (5) the incidence of secondary complications of influenza such as otitis, bronchitis, sinusitis, pneumonia, and the use of antibiotics; (6) the occurrence of adverse events in all participants having received at least one dose. Clinical Response Oral temperature was recorded and severity of seven symptoms (nasal stuffiness, sore throat, cough, muscle aches, tiredness or fatigue, headache, and feverishness) was rated by the patient twice daily (morning and evening) up to day 5 and then once daily on a four-point scale (0, none; 1, mild; 2, moderate; 3, severe) [2–4,14]. The time to resolution of illness was defined as time from study drug initiation to time of symptom alleviation. Symptom alleviation was defined as the first 24-h period during which the above seven symptoms were absent or only mild as previously described [3,4]. Influenza-related clinical events were defined as incidence of a secondary complication (such as pneumonitis or otitis) independently of any antibiotic initiation, and/or occurrence of exacerbation of a preexisting chronic disease. Patients reported treatment compliance using a self-administered questionnaire; full compliance during the day 0 to day 2 period was considered when 100% of planned drug intakes had been completed. Early Study Termination y y Recruitment was interrupted on March 15th 2009 at the end of the epidemic period in France after the enrolment of the first 541 patients and prior to the emergence of the 2009 H1N1 pandemic later in 2009. A second recruitment period was planned for the next winter (i.e., 2009–2010) to reach the number of 900 patients required. However, due to the emergence of the 2009 H1N1 pandemic virus, the independent data-monitoring committee, without any prior knowledge of the results, recommended terminating the trial and conducting an early analysis on May 6th 2009. This decision was based firstly on the need to rapidly provide results on efficacy and tolerance of combined oseltamivir- zanamivir therapy, and secondly, on the inadvisability of pooling the results of the two winters, one with a seasonal virus (winter 2008–2009, mainly H3N2) and the other one with a novel pandemic virus of a different subtype (winter 2009–2010). Patients From January 7th to March 15th 2009 (period of the winter 2008–2009 influenza epidemic in France), we enrolled throughout France adults 18 y old and older who consulted their general practitioner within 36 h of influenza symptoms onset (following the first influenza symptoms reported by the patient), with a temperature greater than or equal to 38uC (reported or observed by the practitioner), one or more respiratory symptoms (cough, sore throat), one or more general symptoms (headache, dizziness, myalgia, sweats and or chills, fatigue), and a positive nasal rapid November 2010 \| Volume 7 \| Issue 11 \| e1000362 3 Sample Size and Statistical Analysis Trial flow chart. doi:10.1371/journal.pmed.1000362.g001 301 patients did not undergo randomization N 173 assigned oseltamivir−zanamivir and included in intention to treat analysis N=447 149 patients with day 0 positive influenza A detection by RT−PCR included in influenza A infected intention to treat analysis 157 patients with day 0 positive influenza A detection by RT−PCR included in influenza A infected intention to treat analysis Figure 1. Trial flow chart. doi:10.1371/journal.pmed.1000362.g001 Figure 1. Trial flow chart. doi:10.1371/journal.pmed.1000362.g001 Figure 1. Trial flow chart. doi:10.1371/journal.pmed.1000362.g001 g doi:10.1371/journal.pmed.1000362.g001 O comparison, treatment effect comparison: 29.9%, [95% CI 219.9 to 0.2]), and 40.5% in the zanamivir monotherapy arm (p = 0.020, for the OZ versus Z comparison; treatment effect comparison: +12.1%, [95% CI 2.02–22.3]) (Table 2). Sample Size and Statistical Analysis Sample size evaluation assumed that virological response was obtained in 70% of patients in the oseltamivir-zanamivir arm, November 2010 \| Volume 7 \| Issue 11 \| e1000362 4 Combined Oseltamivir-Zanamivir in Influenza N=414 141 patients with both day 0 and day 2 nasal swabs 134 patients with both day 0 and day 2 nasal swabs 139 patients with both day 0 and day 2 nasal swabs 15 day 2 nasal swabs missing 6 day 2 nasal swabs missing 9 day 2 nasal swabs missing 1 withdrawal before day 2 1 withdrawal before day 2 1 withdrawal before day 2 N=447 157 patients with day 0 positive influenza A detection by RT−PCR included in influenza A infected intention to treat analysis 141 patients with day 0 positive influenza A detection by RT−PCR included in influenza A infected intention to treat analysis 149 patients with day 0 positive influenza A detection by RT−PCR included in influenza A infected intention to treat analysis 32 with negative day 0 influenza RT−PCR 31 with negative day 0 influenza RT−PCR 21 with negative day 0 influenza RT−PCR 3 day 0 nasal swabs missing 4 day 0 nasal swabs missing 3 day 0 nasal swabs missing N=541 192 assigned oseltamivir−zanamivir and included in intention to treat analysis 176 assigned oseltamivir−zanamivir and included in intention to treat analysis 173 assigned oseltamivir−zanamivir and included in intention to treat analysis 541 patients enrolled 301 patients did not undergo randomization 842 patients screened N=414 141 patients with both day 0 and day 2 nasal swabs 134 patients with both day 0 and day 2 nasal swabs 139 patients with both day 0 and day 2 nasal swabs 15 day 2 nasal swabs missing 6 day 2 nasal swabs missing 9 day 2 nasal swabs missing 1 withdrawal before day 2 1 withdrawal before day 2 1 withdrawal before day 2 N=447 157 patients with day 0 positive influenza A detection by RT−PCR included in influenza A infected intention to treat analysis 141 patients with day 0 positive influenza A detection by RT−PCR included in influenza A infected intention to treat analysis 149 patients with day 0 positive influenza A detection by RT−PCR included in influenza A infected intention to treat analysis 32 with negative day 0 influenza RT−PCR 31 with negative day 0 influenza RT−PCR 21 with negative day 0 influenza RT−PCR 3 day 0 nasal swabs missing 4 day 0 nasal swabs missing 3 day 0 nasal swabs missing N=541 192 assigned oseltamivir−zanamivir and included in intention to treat analysis 176 assigned oseltamivir−zanamivir and included in intention to treat analysis 173 assigned oseltamivir−zanamivir and included in intention to treat analysis 541 patients enrolled 301 patients did not undergo randomization 842 patients screened Figure 1. PLoS Medicine \| www.plosmedicine.org Virological Endpoints Primary endpoint. In the ITT analysis, considering the 541 enrolled patients with positive influenza A rapid test, the proportion of patients with a RT-PCR,200 cgeq/ml on day 2 of treatment was 52.6% in the oseltamivir-zanamivir arm, 62.5% in the oseltamivir monotherapy arm (p = 0.055, for the OZ versus In the ITT analysis, considering the 447 influenza RT-PCR- confirmed patients, the proportions were 45.9% in the oseltamivir- PLoS Medicine \| www.plosmedicine.org November 2010 \| Volume 7 \| Issue 11 \| e1000362 PLoS Medicine \| www.plosmedicine.org 5 Combined Oseltamivir-Zanamivir in Influenza Table 1. Characteristics of the 541 patients enrolled in the study and of the 447 influenza A-infected patients according to treatment arms. Virological Endpoints Patients Characteristics Combined Oseltamivir and Zanamivir Oseltamivir Plus Placebo Zanamivir Plus Placebo All patients included in the study n = 541 (%) n = 192 (35.5%) n = 176 (32.5%) n = 173 (32.0%) Age (y): mean (SD) 38.7 (13.2) 39.5 (13.1) 39.9 (13.8) [Age range] [18.3–73.2] [18.1–76.3] [18.0–84.2] n male (%) 91 (47.6%) 92 (52.3%) 86 (49.7%) n smoker (%) 34 (17.8%) 25 (14.2%) 26 (15.0%) n comorbidities (%) 27 (14.1%) 27 (15.3%) 23 (13.3%) n fever at enrolment$38uC (%) 123 (69.9%) 118 (73.3%) 117 (75.5%) n initiation of treatment#24 h after onset of symptoms (%) 92 (47.9%) 85 (48.3%) 101 (58.4%) Symptoms score per patienta Mean (SD) 15.2 (2.8) 14.9 (3.2) 15.1 (3.2) % of maximal score: mean (SD)b 72.4% (13.4) 71.0% (15.2) 72.1% (15.4) Influenza A–infected patients n = 447 (%) n = 157 (35.1%) n = 141 (31.6%) n = 149 (33.3%) Age (y): mean (SD) 38.7 (13.2) 39.5 (13.0) 40.1 (14.1) [Age range] [18.3–73.2] [18.1–76.3] [18.0–84.2] n male (%) 76 (48.7%) 73 (51.8%) 77 (51.7%) n smoker (%) 22 (14.1%) 15 (10.7%) 20 (13.4%) n comorbidities (%) 21 (13.4%) 20 (14.2%) 20 (13.4%) n fever$38uC at enrolment (%) 101 (67.8%) 95 (70.9%) 104 (75.9%) n initiation of treatment#24 h after onset of symptoms (%) 72 (45.9%) 68 (48.2%) 86 (57.7%) Symptoms score per patienta Mean (SD) 15.6 (2.7) 15.3 (3.2) 15.5 (3.1) % of maximal score: mean (SD)b 74.2% (12.8) 72.7% (15.2) 73.8% (15.0) Influenza virus subtype H1N1 9 (5.7%) 5 (3.5%) 7 (4.7%) H3N2 136 (86.6%) 130 (92.2%) 129 (86.6%) Not determined 12 (7.6%) 6 (4.3%) 13 (8.7%) aSum of the severity of the seven day 0 influenza symptoms (feverishness, nasal stuffiness, sore throat, cough, muscle aches, tiredness-fatigue, and headache) using a four-point scale [2,14]. bThe score is expressed as a percentage of the maximal score of 21. doi:10.1371/journal.pmed.1000362.t001 Table 1. Characteristics of the 541 patients enrolled in the study and of the 447 influenza A-infected patients according to treatment arms. stics of the 541 patients enrolled in the study and of the 447 influenza A-infected patients according to Clinical Endpoints zanamivir arm, 58.9% in the oseltamivir monotherapy arm (p = 0.025 for the OZ versus O comparison; treatment effect comparison: 213.0%, [95% CI 223.1 to 22.9]), and 33.6% in the zanamivir monotherapy arm (p = 0.028 for the OZ versus Z comparison; treatment effect comparison: +12.3%, [95% CI 2.39– 22.2]) (Table 2). The same trends were observed in the 382 fully compliant influenza A-infected patients (Table S3), and in the 395 patients with H3N2 infection with proportions of 42.4% in the oseltamivir-zanamivir arm, 58.6% in the oseltamivir monotherapy arm, and 30.3% in the zanamivir monotherapy arm. zanamivir arm, 58.9% in the oseltamivir monotherapy arm (p = 0.025 for the OZ versus O comparison; treatment effect comparison: 213.0%, [95% CI 223.1 to 22.9]), and 33.6% in the zanamivir monotherapy arm (p = 0.028 for the OZ versus Z comparison; treatment effect comparison: +12.3%, [95% CI 2.39– 22.2]) (Table 2). The same trends were observed in the 382 fully compliant influenza A-infected patients (Table S3), and in the 395 patients with H3N2 infection with proportions of 42.4% in the oseltamivir-zanamivir arm, 58.6% in the oseltamivir monotherapy arm, and 30.3% in the zanamivir monotherapy arm. p The median time to resolution of illness in the 541 enrolled patients was 3.5 d in the oseltamivir-zanamivir arm, 3.0 d in the oseltamivir monotherapy arm (p = 0.015 for the OZ versus O comparison; treatment effect comparison: +0.5%, [95% CI 0.0– 1.5]), and 4.0 d in the zanamivir monotherapy arm (p = 0.78 for the OZ versus Z comparison; treatment effect comparison: 20.5, [95% CI 21.0 to 0.5]) (Table 3). In the 447 influenza A-infected patients, this figure was 4.0 d in the oseltamivir-zanamivir arm, 3.0 d in the oseltamivir monotherapy arm (p = 0.018 for the OZ versus O comparison; treatment effect comparison: +1.0, [95% CI 0.0–4.0]), and 4.0 d in the zanamivir monotherapy arm (p = 0.96 for the OZ versus Z comparison; treatment effect comparison: +0.0, [95% CI 23.0 to 3.0]). Figure 2 presents the time to resolution of illness in the 447 patients, also showing by the log- rank test a significantly shorter time in the oseltamivir monother- apy arm. The time to resolution of illness was significantly shorter in patients with day 2 viral load below 200 cgeq/l (3.5 d) than in patients with viral load above 200 cgeq/l (7 d; p = 0.0014). The Other virological endpoints. Clinical Endpoints In the 414 influenza RT-PCR confirmed patients with both day 0 and day 2 available specimens, the day 2 to day 0 decrease was 2.14 log10 cgeq/ml in the oseltamivir-zanamivir arm, 2.49 log10 cgeq/ml in the oseltamivir monotherapy arm, (p = 0.060 for the OZ versus O comparison; treatment effect comparison 20.35, [95% CI 20.8 to 0.07]), and 1.68 log10 cgeq/ml in the zanamivir monotherapy arm (p = 0.016 for the OZ versus Z comparison; treatment effect comparison: +0.46, [CI 95% 0.03–0.9]) (Table 2). Other virological endpoints. In the 414 influenza RT-PCR confirmed patients with both day 0 and day 2 available specimens, the day 2 to day 0 decrease was 2.14 log10 cgeq/ml in the oseltamivir-zanamivir arm, 2.49 log10 cgeq/ml in the oseltamivir monotherapy arm, (p = 0.060 for the OZ versus O comparison; treatment effect comparison 20.35, [95% CI 20.8 to 0.07]), and 1.68 log10 cgeq/ml in the zanamivir monotherapy arm (p = 0.016 for the OZ versus Z comparison; treatment effect comparison: +0.46, [CI 95% 0.03–0.9]) (Table 2). PLoS Medicine \| www.plosmedicine.org November 2010 \| Volume 7 \| Issue 11 \| e1000362 6 Combined Oseltamivir-Zanamivir in Influenza Table 2. Virological and clinical response according to treatment arms in the 541 enrolled patients, between day 0 and day 2 (ITT analysis). Clinical Endpoints Other clinical outcomes showed similar trends (Tables 2, 3, and S3). Analysis of the different antiviral regimens’ efficacy was based on a primary virological endpoint, which we hypothesized could be a sensitive and a more specific indicator than a primary clinical endpoint. Clinical endpoints, used as primary endpoints in previous studies, were used as secondary endpoints in the present study [1,5]. Clinical endpoints, which are based on a global assessment of both general (mainly immunologically linked) and respiratory (mainly virologically linked) symptoms, are probably not the best way to monitor the virological effect of treatment, because clinical symptoms are not exclusive to influenza. We thus considered that a difference in viral shedding rate would be the best indicator of the virological effects of combined therapy, and consequently a valuable surrogate. Our initial hypothesis was that the combination of two antivirals may reduce the rate of resistant virus emergence (for a naturally susceptible pandemic virus and a nonimmune population). In addition, we hypothesized that for cases of infection with susceptible seasonal influenza viruses, this could not be easily shown, owing first to the rarity of this phenomenon in adults, and second, to the necessity of monitoring virus excretion for several days, whereas for cases of influenza due to H1N1 viruses, which are naturally resistant to oseltamivir, the question was not relevant. Given the viral shedding kinetics in patients with seasonal influenza receiving neuraminidase inhibitors, the day 2 virological endpoint was considered to be best suited to quantify virological effects. The 200 cgeq/l threshold was chosen, as it was the best compromise in terms of specificity and sensitivity as compared to standard culture. Of note, the same trends were observed when a 100 cgeq/l or a 1,000 cgeq/ml cut-off was used to define virological success (Table S2). Furthermore, the study was designed to be statistically two-sided to take into account the possibility that the combination would perform worse than either Tolerance Four serious adverse events occurred during the study, one of which was considered unrelated to study drugs (acute bacterial pneumonia at day 3 in a patient receiving oseltamivir-zanamivir combination). Two adverse events also occurred in patients receiving the oseltamivir-zanamivir combination: severe head- aches leading to interruption of therapy and facial oedema following the first administration, disappearing within 24 h postdrug interruption. The remaining patient experienced repeat- ed vomiting after oseltamivir monotherapy drug administration. All four patients completely recovered. Other nonserious adverse events reported in more than 1% of the total population were in the OZ, O, and Z arms, respectively, nausea and/or vomiting (in 13, 4, and 5 patients), diarrhoea (in 2, 1, and 5 patients), and rash (in 1, 2, and 2 patients). PLoS Medicine \| www.plosmedicine.org Clinical Endpoints Type of Response Virological and Clinical Response Variables Combined Oseltamivir and Zanamivir Oseltamivir Plus Placebo O+Z versus O Zanamivir Plus Placebo O+Z versus Z O versus Z p- Value Difference [95% CI] p- Value Difference [95% CI] Difference [95% CI]a n patients 192 176 173 Virological Primary virological endpoint Day 2 influenza RT-PCR,200 cgeq/ml (% patients) 52.6% 62.5% 0.055 29.9% [219.9 to 0.2] 40.5% 0.020 +12.1% [2.02–22.3] +22.0% [12.1–32.0] Clinical Time to resolution of illness in days (median, IQR) 3.5 [2.5–14] 3.0 [2–7] 0.015 +0.5 [0.0–1.5] 4.0 [2.5–14] 0.78 20.5 [21.0 to 0.5] 21.0 [21.5 to 20.5] n (%) of patients with alleviation of symptoms at end of treatment 111 (57.8%) 122 (69.3%) 0.023 211.5% [221.3 to 21.7] 100 (57.8%) 1.00 +0.0% [210.1 to 10.1] +11.5% [1.7–21.3] Symptoms score at end of treatment (median, IQR) 3 [2–5] 2 [1–4] 0.0006 +1.0 [0.0–1.0] 3 [1–6] 0.79 +0.0 [21.0 to 0.0] 21.0 [22.0 to 21.0] n (%) of patients with clinical event during treatment 26 (13.5%) 15 (8.5%) 0.14 +5.0% [21.3 to 11.4] 23 (13.3%) 1.00 +0.3% [26.7 to 7.2] 24.8% [211.2 to 1.6] Initiation of antibiotics 17 (8.9%) 10 (5.7%) — 13 (7.5%) — — Pneumonia 2 (1.0%) 1 (0.6%) — 0 (0.0%) — — Other 21 (10.9%) 14 (8.0%) — 22 (12.7%) — — aExploratory analysis. doi:10.1371/journal.pmed.1000362.t002 Table 2. Virological and clinical response according to treatment arms in the 541 enrolled patients, between day 0 and day 2 (ITT analysis). than zanamivir monotherapy in adults with seasonal influenza A virus infection. median symptoms score at day 5 (end of treatment) was 3 in the oseltamivir-zanamivir arm, 2 in the oseltamivir monotherapy arm (p = 0.013 for the OZ versus O comparison; treatment effect comparison: +1, [95% CI 0.0–1.0]), and 3 in the zanamivir monotherapy arm (p = 0.93 for the OZ versus Z comparison; treatment effect comparison: +0.0, [95% CI 21.0 to 0.0]) (Tables 2 and 3). Other clinical outcomes showed similar trends (Tables 2, 3, and S3). median symptoms score at day 5 (end of treatment) was 3 in the oseltamivir-zanamivir arm, 2 in the oseltamivir monotherapy arm (p = 0.013 for the OZ versus O comparison; treatment effect comparison: +1, [95% CI 0.0–1.0]), and 3 in the zanamivir monotherapy arm (p = 0.93 for the OZ versus Z comparison; treatment effect comparison: +0.0, [95% CI 21.0 to 0.0]) (Tables 2 and 3). Discussion This large publicly funded clinical trial examined the effect of combination neuraminidase inhibitor antiviral therapy in influ- enza, as compared to each monotherapy plus placebo. It showed that, during the prepandemic winter of 2009 with a predomi- nance of H3N2 viruses (more than 85%) in France, the oseltamivir-zanamivir combination seemed less effective than oseltamivir monotherapy, and not significantly more effective November 2010 \| Volume 7 \| Issue 11 \| e1000362 7 Combined Oseltamivir-Zanamivir in Influenza Table 3. Virological and clinical response according to treatment arms in the 447 influenza A-infected patients between day 0 and day 2 (ITT analysis). Discussion Type of Response Virological and Clinical Response Variables Combined Oseltamivir and Zanamivir Oseltamivir Plus Placebo O+Z versus O Zanamivir Plus Placebo O+Z versus Z O versus Z p- Value Difference [95% CI] p- Value Difference [95% CI] Difference [95% CI]a n patients 157 141 149 Virological Primary virological endpoint Day 2 influenza RT-PCR,200 cgeq/ml (%) 45.9% 58.9% 0.025 213.0% [223.1 to 22.9] 33.6% 0.028 +12.3% [2.39–22.2] +25.3% [15.5–35.2] n patients 141 134 139 Other virological endpoints in patients with available day 0 and day 2 nasal swabs (n = 414) Mean (SD) viral load at day 0 (log 10 cgeq/ml) 4.36 (1.36) 4.57 (1.32) 4.34 (1.37) Mean (SD) viral load at day 2 (log 10 cgeq/ml) 2.22 (1.12) 2.08 (1.17) 2.66 (1.35) Mean (SD) viral load decrease between day 0 and 2 (log 10 cgeq/ml) 2.14 (1.54) 2.49 (1.52) 0.060 20.35 [20.8 to 0.07] 1.68 (1.68) 0.016 +0.46 [0.03–0.9] +0.81 [0.4–1.3] n patients 157 141 149 Clinical Time to resolution of illness in days (median, IQR) 4.0 [2.5–14] 3.0 [2–7] 0.018 +1.0 [0.0–4.0] 4.0 [2.5–14] 0.96 +0.0 [23.0 to 3.0] 21.0 [24.0 to 0.0] n (%) of patients with alleviation of symptoms at end of treatment 87 (55.4%) 95 (67.4%) 0.043 212.0% [221.8 to 22.1] 84 (56.4%) 0.91 21.0% [211.1 to 9.2] +11.0% [1.1 to 20.9] Symptoms score at end of treatment (median, IQR) 3 [2–5] 2 [1–4] 0.013 +1.0 [0.0–1.0] 3 [1–6] 0.93 +0.0 [21.0 to 0.0] 21.0 [22.0 to 20.5] n (%) of patients with clinical event during treatment 19 (12.1%) 10 (7.1%) 0.17 +5.0% [21.0 to 11.0] 18 (12.1%) 1.00 +0.02% [26.6 to 6.7] 25.0% [211.0 to 1.0] Initiation of antibiotics 14 (8.9%) 7 (5.0%) — — 10 (6.7%) — — — Pneumonia 2 (1.3%) 1 (0.7%) — — 0 (0.0%) — — — Other 15 (9.6%) 9 (6.4%) — — 17 (11.4%) — — — aExploratory analysis Table 3. Virological and clinical response according to treatment arms in the 447 influenza A-infected patients between day 0 and day 2 (ITT analysis). drug alone because of the theoretical concern of antagonism at the receptor level. be metabolized, thus delaying arrival of the active drug at the infection site (tmax = 4 h), inhaled zanamivir is delivered directly to the primary site of influenza virus replication. The hypothesis that zanamivir is more likely to occupy the catalytic pocket first, thus preventing the action of oseltamivir, must be tested. PLoS Medicine \| www.plosmedicine.org November 2010 \| Volume 7 \| Issue 11 \| e1000362 PLoS Medicine \| www.plosmedicine.org Discussion According to this hypothesis, the combination would be largely reduced to a zanamivir monotherapy. The oseltamivir-zanamivir combination seemed, both virolog- ically and clinically, significantly less effective than the oseltamivir monotherapy. This result seems robust because (1) it was found using a double-blind placebo methodology, (2) there was overall concordance both among virological endpoints, and between virological and clinical endpoints, (3) it was confirmed over the three different subgroups of subjects included in the global population (541 enrolled patients, 447 influenza A-infected patients, 382 influenza A-infected and fully compliant patients). This lower clinical and virological response to the combination may suggest a negative effect of zanamivir on oseltamivir, as in the absence of interactions the effect of the combination should at least be additive [15]. A negative interaction at the level of binding at the catalytic pocket of the neuraminidase is an explanation that should be further investigated in vitro for both seasonal H3N2 and H1N1 viruses. Recent in vitro data showing the lack of synergy between oseltamivir and zanamivir, and some antagonism at higher concentrations of zanamivir on pandemic H1N1 2009 virus, are in agreement with this hypothesis [16]. Furthermore, contrary to oseltamivir, which upon digestive absorption needs to Whereas the results of the primary virological endpoints indicated a superiority of the oseltamivir-zanamivir combination to zanamivir monotherapy, clinical results were not significantly different, suggesting that oseltamivir adds essentially nothing to zanamivir monotherapy. This view is concordant with the above hypothesis of the predominant catalytic site occupation by zanamivir when the combination is administered. As an exploratory analysis, oseltamivir showed a significantly higher clinical and virological efficacy as compared to zanamivir. This finding could be the consequence of a suboptimal treatment regimen in the zanamivir arm, since the IC50 values for the A(H3N2) viruses of the 2008–2009 season were 2- to 3-fold higher for zanamivir as compared to oseltamivir, but remained within the range for susceptible strains (GROG surveillance; NICs, unpub- lished data). The virological result is confirmed by the longer time November 2010 \| Volume 7 \| Issue 11 \| e1000362 November 2010 \| Volume 7 \| Issue 11 \| e1000362 8 Combined Oseltamivir-Zanamivir in Influenza 0 1 2 3 4 5 6 7 8 9 10 0.00 0.25 0.50 0.75 1.00 End of symptom (days) Proportion of population O + Z O + P Z + P Figure 2. Discussion Proportion of the 447 influenza A-infected patients with alleviation of symptoms when treated with combined oseltamivir-zanamivir (plain line), oseltamivir plus placebo (dotted line), or zanamivir plus placebo (dashed line). Log-rank test for oseltamivir-zanamivir versus oseltamivir-placebo: p = 0.025 and for oseltamivir-zanamivir versus zanamivir-placebo: p = 0.036). Alleviation of symptoms defined by the presence of no symptoms of nasal stuffiness, sore throat, cough, muscle aches, tiredness-fatigue, feverishness, and headache or only mild ones, for at least 24 h. doi:10.1371/journal.pmed.1000362.g002 0 1 2 3 4 5 6 7 8 9 10 0.00 0.25 0.50 0.75 1.00 Proportion of population O + Z O + P Z + P End of symptom (days) Figure 2. Proportion of the 447 influenza A-infected patients with alleviation of symptoms when treated with combined oseltamivir-zanamivir (plain line), oseltamivir plus placebo (dotted line), or zanamivir plus placebo (dashed line). Log-rank test for oseltamivir-zanamivir versus oseltamivir-placebo: p = 0.025 and for oseltamivir-zanamivir versus zanamivir-placebo: p = 0.036). Alleviation of symptoms defined by the presence of no symptoms of nasal stuffiness, sore throat, cough, muscle aches, tiredness-fatigue, feverishness, and headache or only mild ones, for at least 24 h. doi:10.1371/journal.pmed.1000362.g002 to alleviation of the influenza symptoms in patients receiving zanamivir. mentioned above, day 2 sampling was chosen to show the virological effect. However, this is probably not the best moment to look for resistance emergence induced by drug selective pressure, as it has been shown to occur later in the course of treatment [18–20]. Nevertheless, we looked for neuraminidase inhibitor resistance using a standard fluorimetric test in the 65 patients with day 2 positive viral culture; none of them carried a resistant virus, except for one patient infected with an H1N1 virus resistant to oseltamivir but susceptible to zanamivir, as were all H1N1 viruses circulating during the study period. However, the absence of resistance at day 2 does not rule out any further (post– day 2) resistance selection. Finally, this trial was conducted in adult outpatients, which prevents any extrapolation of the results to adults with severe presentation necessitating hospitalisation, and to children, who usually have more prolonged viral shedding. We chose the outpatient adult population because it seemed to be the most homogenous and the easiest in which to test our hypothesis. Discussion As the present study was conducted before the 2009 H1N1 pandemic during an influenza season where A(H3N2) viruses predominated, the impact of prepandemic seasonal A(H1N1) oseltamivir-resistant viruses on the results is expected to be negligible. We observed the same trends after excluding patients infected with seasonal H1N1 from our population. Of note, A(H3N2) viruses are to date sensitive to both drugs. It remains to be determined to what extent the present results can be extrapolated to susceptible viruses of other subtypes, e.g., H1N1, and in particular to the pandemic H1N1 2009 virus, which displays significant differences in the catalytic pocket of the neuraminidase [17], or to a mixed viral season with H3N2 and H1N1 cocirculating viruses. We must acknowledge several limitations to our study. First, this preliminary analysis was conducted on a partial set of data after enrolment of 541 patients instead of the 900 initially planned. However, it is highly unlikely that the lower response of the combination as compared to oseltamivir would have been reversed if all originally planned 900 patients had been enrolled. Second, as previous randomized clinical trials had shown the superiority of each monotherapy as compared to placebo in terms of time to symptom alleviation and viral shedding, it was decided, on the basis of ethical reasons, that the study would not comprise a double placebo arm. Third, the proportion of patients with unavailable viral swab on day 2 was higher in the combination arm. As the missing value equals failure, this may have biased the results in the combination arm towards reduced performance. Indeed, in the analysis of the 414 patients with available day 0 and day 2 nasal swabs, the same trends were observed. Fourth, the virological response was assessed only in one site (nose) and at one time (day 2), which prevents extrapolation of the results to the entire virological response over time and throughout the respiratory tract. However, clinical endpoints completed the picture, giving information on the overall response. Fifth, as Despite the theoretical potential for the reduction of the emergence of antiviral resistance, the lower efficiency of the oseltamivir-zanamivir combination found in this study calls for caution in its use in clinical practice. Thus, also considering the superiority of oseltamivir monotherapy over zanamivir monother- apy observed in this trial, oseltamivir should be the recommended primary anti-influenza treatment during influenza seasons with predominant H3N2 viruses naturally susceptible to oseltamivir. Discussion These results would need to be confirmed for the 2009 H1N1 pandemic virus and in the coming years, for future circulating influenza viruses. PLoS Medicine \| www.plosmedicine.org Combined Oseltamivir-Zanamivir in Influenza Table S2 Virological response in the 447 influenza A infected patients for thresholds of 100 and 1000 cgeq/mL. Found at: doi:10.1371/journal.pmed.1000362.s002 (0.04 MB DOC) Independent data-monitoring committee (advice on trial conduct and interruption): Cheˆne G, Hannoun C, Vittecoq D. Monitoring and statistical analysis: Boucherit S, Dornic Q, Quintin C, Vincent C, and Atlanstat, Studypharm clinical research organisations. Table S3 Virological and clinical response according to treatment arms in the 382 influenza A infected patients and fully compliant between day 0 and day 2 Intention to treat analysis. Found at: doi:10.1371/journal.pmed.1000362.s003 (0.06 MB DOC) Table S3 Virological and clinical response according to treatment arms in the 382 influenza A infected patients and fully compliant between day 0 and day 2 Intention to treat analysis. Combined Oseltamivir-Zanamivir in Influenza Clinical investigators (responsible for patient enrolment and follow-up): Alea JR, Aleonard JL, Arditti L, Baranes C, Beaujard J, Beaurain C, Behar M, Beignot-Devalmont P, Biquet D, Blanchard M, Blot E, Bodin X, Bouaniche H, Boulet L, Bourgeois O, Bretillon F, Breton N, Broyer F, Buffler P, Camper E, Carissimo P, Carrera J, Causse P, Cayet JP, Cayron P, Cazard C, Chaix C, Chazerans D, Cheftel JA, Codron G, Cooren G, Coutrey L, Crappier JJ, Daugenet C, Dauzat C, Defreyn F, Delamare G, Delsart D, Demure P, Desmarchelier P, Domenech A, Dubois D Laroy G, Dubrana E, Dumond P, Dumont A, Durel G, Elle´ P, Evellin F, Eyraud P, Fhal G, Fournillou JC, Galesne Herceg G, Gastan G, Geoffray B, Giagnorio P, Goguel J, Granger JF, Guenee P, Haushalter B, Huber C, Hureau JP, Jacob L, Jami A, Jordan E, Jourde P, Journet L, Julien D, Jusserand JT, Korsec P, Laforest G, Lalanne G, Le Duff N, Le Guen-Naas A, Le Hir A, Lebois S, Leclerc S, Leclere V, Lejay D, Lejoly JM, Lemoine C, Lepine C, Lepoutre B, Leprince P, Lhoumeau P, Lognos B, Lustig G, Mannessier B, Marlier M, Marmor P, Martocq G, Massot J, Meme B, Mercier P, Mercier G, Mesnier PL, Meyrand G, Mongin G, Montavont J, Morlon P, Pantea D, Parisot J, Partouche H, Pertusa MC, Petot A, Peyrol Y, Piketty B, Poignant G, Pradere H, Rabaud D, Rachine L, Ragon B, Rambaud J, Richard P, Rigaill P, Robinson D, Rosenberg S, Ruetsch M, Sacareau D, Saint Lannes M, Saugues M, Sauvage P, Schaupp T, Schmitt C, Sellam A, Severin JF, Simian B, Specht L, Szmuckler I, Tetaud D, Trehou P, Triantaphylides JC, Triot P, Uge P, Urbain F, Urbina JC, Vailler P, Vallez V, Varnier H, Venot N, Verhun R, Vogel JY, Zanuttini-Vogt C Zeline V Found at: doi:10.1371/journal.pmed.1000362.s003 (0.06 MB DOC) Text S1 Methods. Choice of threshold for virological endpoint and sensitivity analysis. Found at: doi:10.1371/journal.pmed.1000362.s004 (0.03 MB DOC) Text S2 Trial protocol. Found at: doi:10.1371/journal.pmed.1000362.s005 (0.59 MB PDF) Text S3 CONSORT checklist. Found at: doi:10.1371/journal.pmed.1000362.s006 (0.22 MB DOC) Acknowledgments We are grateful to Marc Guerrier for his fruitful support in the ethical aspects of the research, and to Philippe Ravaud for stimulating discussion. We are indebted to Mathilde Benassaya, David Briand, Fre´de´rique Cuvelier, Se´bastien Le Gal, Vanessa Roca (NIC Northern France), and to Re´da Ihsani and Audrey Bonnetin (NIC Southern France) for excellent technical assistance. We thank all patients for their active participation in the study. We are grateful to Marc Guerrier for his fruitful support in the ethical aspects of the research, and to Philippe Ravaud for stimulating discussion. We are indebted to Mathilde Benassaya, David Briand, Fre´de´rique Cuvelier, Se´bastien Le Gal, Vanessa Roca (NIC Northern France), and to Re´da Ihsani and Audrey Bonnetin (NIC Southern France) for excellent technical assistance. We thank all patients for their active participation in the study. J Zanuttini-Vogt C, Zeline V. References 9. Wu JT, Leung GM, Lipsitch M, Cooper BS, Riley S (2009) Hedging against antiviral resistance during the next influenza pandemic using small stockpiles of an alternative chemotherapy. PLoS Med 6: e1000085. doi:10.1371/journal. pmed.1000085. 1. Burch J, Corbett M, Stock C, Nicholson K, Elliot AJ, et al. (2009) Prescription of anti-influenza drugs for healthy adults: a systematic review and meta-analysis. Lancet Infect Dis 9: 537–545. 2. Hayden FG, Osterhaus AD, Treanor JJ, Fleming DM, Aoki FY, et al. (1997) Efficacy and safety of the neuraminidase inhibitor zanamivir in the treatment of influenzavirus infections. GG167 Influenza Study Group. N Engl J Med 337: 874–880. 10. Sheu TG, Deyde VM, Okomo-Adhiambo M, Garten RJ, Xu X, et al. (2008) Surveillance for neuraminidase inhibitor resistance among human influenza A and B viruses circulating worldwide from 2004 to 2008. Antimicrob Agents Chemother 52: 3284–3292. 3. Nicholson KG, Aoki FY, Osterhaus AD, Trottier S, Carewicz O, et al. (2000) Efficacy and safety of oseltamivir in treatment of acute influenza: a randomised controlled trial. Neuraminidase Inhibitor Flu Treatment Investigator Group. Lancet 355: 1845–1850. 11. Dawood FS, Jain S, Finelli L, Shaw MW, Lindstrom S, et al. (2009) Emergence of a novel swine-origin influenza A (H1N1) virus in humans. N Engl J Med 360: 2605–2615. 12. Wong SC, Chan JK, Lee KC, Lo ES, Tsang DN (2005) Development of a quantitative assay for SARS coronavirus and correlation of GAPDH mRNA with SARS coronavirus in clinical specimens. J Clin Pathol 58: 276–280. 4. Treanor JJ, Hayden FG, Vrooman PS, Barbarash R, Bettis R, et al. (2000) Efficacy and safety of the oral neuraminidase inhibitor oseltamivir in treating acute influenza: a randomized controlled trial. US Oral Neuraminidase Study Group. JAMA 283: 1016–1024. 13. Duchamp MB, Casalegno JS, Gillet Y, Frobert E, Bernard E, et al. (2010) Pandemic A(H1N1)2009 influenza virus detection by real time RT-PCR: is viral quantification useful? Clin Microbiol Infect 16: 317–321. p J 5. Jefferson T, Jones M, Doshi P, Del Mar C (2009) Neuraminidase inhibitors for preventing and treating influenza in healthy adults: systematic review and meta- analysis. BMJ 339: b5106. 14. Hayden FG, Treanor JJ, Fritz RS, Lobo M, Betts RF, et al. (1999) Use of the oral neuraminidase inhibitor oseltamivir in experimental human influenza: randomized controlled trials for prevention and treatment. JAMA 282: 1240–1246. y 6. Puhakka T, Lehti H, Vainionpaa R, Jormanainen V, Pulkkinen M, et al. Scientific committee (trial design, trial conduct, and data interpretation): Scientific committee (trial design, trial conduct, and data interpretation): Steering committee: Leport C (principal investigator), Andreoletti L, Blanchon T, Carrat F, Duval X, Guimfack A, Lina B, Loubie`re S, Mentre´ F, Mosnier A, Tibi A, Tubach F, van der Werf S. , , , , , , F, Mosnier A, Tibi A, Tubach F, van der Werf S. Clinical study manager: Charlois-Ou C. Other members: Bouscambert-Duchamp M, Bricaire F, Cohen JM, Enouf V, Flahault A, Moatti JP, Vincent C, Vogel JY. Other members: Bouscambert-Duchamp M, Bricaire F, Cohen JM, Enouf V, Flahault A, Moatti JP, Vincent C, Vogel JY. Invited members (informed of study design but did not participate in any decision-making study discussions; supplied drugs and placebos): Eid Z (GSK), Peurichard C (GSK), Pecking M Invited members (informed of study design but did not participate in any decision-making study discussions; supplied drugs and placebos): Eid Z (GSK), Peurichard C (GSK), Pecking M (Roche), Dantin S (Roche), Gysembergh-Houal A (AP-HP). drugs and placebos): Eid Z (GSK), Peurichard C (GSK), Pecking M (Roche), Dantin S (Roche), Gysembergh-Houal A (AP-HP). Author Contributions ICMJE criteria for authorship read and met: SvdW TB AM AT VE CCO CV LA FT BL FM CL. Agree with the manuscript’s results and conclusions: XD SvdW TB AM MBD AT VE CCO CV LA FT BL FM CL. Designed the experiments/the study: XD SvdW TB AM VE CCO LA BL FM CL. Analyzed the data: XD SvdW TB AM CV LA FT BL FM CL. Collected data/did experiments for the study: XD SvdW TB AM MBD VE CCO FT BL FM CL. Enrolled patients: CCO. Wrote the first draft of the paper: XD SvdW TB BL FM CL. Contributed to the writing of the paper: XD SvdW TB AM MBD AT VE CCO LA FT BL FM CL. Enrolled investigators and took part in the epidemiological surveillance which gave the go-ahead to the study: AM. Responsible for data monitoring: FT. Obtained funding: FM CL. Supporting Information Table S1 Proportion of false positive and false negative results for various viral load thresholds compared to viral isolation in the sample of GROG patients. Found at: doi:10.1371/journal.pmed.1000362.s001 (0.04 MB DOC) Table S1 Proportion of false positive and false negative results for various viral load thresholds compared to viral isolation in the sample of GROG patients. Found at: doi:10.1371/journal.pmed.1000362.s001 (0.04 MB DOC) PLoS Medicine \| www.plosmedicine.org November 2010 \| Volume 7 \| Issue 11 \| e1000362 9 Combined Oseltamivir-Zanamivir in Influenza Bivir Study Group membership: Scientific committee (trial design, trial conduct, and data interpretation): 20. Stephenson I, Democratis J, Lackenby A, McNally T, Smith J, et al. (2009) Neuraminidase inhibitor resistance after oseltamivir treatment of acute influenza A and B in children. Clin Infect Dis 48: 389–396. 17. Russell RJ, Haire LF, Stevens DJ, Collins PJ, Lin YP, et al. (2006) The structure of H5N1 avian influenza neuraminidase suggests new opportunities for drug design. Nature 443: 45–49. 19. Gubareva LV, Kaiser L, Matrosovich MN, Soo-Hoo Y, Hayden FG (2001) Selection of influenza virus mutants in experimentally infected volunteers treated with oseltamivir. J Infect Dis 183: 523–531. Combined Oseltamivir-Zanamivir in Influenza 18. Aoki FY, Boivin G, Roberts N (2007) Influenza virus susceptibility and resistance to oseltamivir. Antivir Ther 12: 603–616. 19. Gubareva LV, Kaiser L, Matrosovich MN, Soo-Hoo Y, Hayden FG (2001) Selection of influenza virus mutants in experimentally infected volunteers treated with oseltamivir. J Infect Dis 183: 523–531. 20. Stephenson I, Democratis J, Lackenby A, McNally T, Smith J, et al. (2009) Neuraminidase inhibitor resistance after oseltamivir treatment of acute influenza A and B in children. Clin Infect Dis 48: 389–396. 17. Russell RJ, Haire LF, Stevens DJ, Collins PJ, Lin YP, et al. (2006) The structure of H5N1 avian influenza neuraminidase suggests new opportunities for drug design. Nature 443: 45–49. 18. Aoki FY, Boivin G, Roberts N (2007) Influenza virus susceptibility and resistance to oseltamivir. Antivir Ther 12: 603–616. References The combination of two neuraminidase inhibitors is feasible as both oseltamivir and zanamivir are licensed for seasonal influenza and have different key mutations associated with resistance to each drug. otitis, bronchitis, sinusitis, and pneumonia. In the study, patients, general practitioners, and outcome assessors were all blinded to treatment assignments. Due to the emergence of the H1N1 pandemic in 2009, the study’s independent data-monitoring committee requested that the researchers terminate the trial early and analyze the results earlier than planned. otitis, bronchitis, sinusitis, and pneumonia. In the study, patients, general practitioners, and outcome assessors were all blinded to treatment assignments. Due to the emergence of the H1N1 pandemic in 2009, the study’s independent data-monitoring committee requested that the researchers terminate the trial early and analyze the results earlier than planned. 541 patients (of the 900 planned) were enrolled in the study (192 in group 1; 176 in group 2; and 173 in group 3) of whom 447 were infected with influenza A. Overall the oseltamivir- zanamivir combination was both virologically and clinically significantly less effective than the oseltamivir monotherapy. In addition, the clinical effects of the oseltamivir-zanamivir combination on time to resolution of symptoms were not significantly different from that of zanamivir monotherapy, suggesting that oseltamivir does not add clinical benefit to zanamivir monotherapy. What Do These Findings Mean? The results of this study essentially show that in France during the Winter of 2009 prepandemic (of which 85% was due to of H3N2 virus), in adults with seasonal influenza A virus infection, the combination of oseltamivir and zanamivir was less effective than oseltamivir monotherapy and not significantly more effective than zanamivir monotherapy. These results call for caution in the use of the oseltamivir-zanamivir combination in treatment of adult outpatients. In addition, as the clinical and virological effects of oseltamivir monotherapy over zanamivir monotherapy were superior in this trial, oseltamivir should be the recommended treatment during influenza seasons with predominant H3N2 viruses. However, the results of this study should be confirmed in the coming years on future circulating influenza viruses. Why Was This Study Done? As yet, there have been no robust randomized controlled trials that compare the effectiveness of monotherapy with either oseltamivir or zanamivir with the effectiveness of a oseltamivir-zanamivir combination. Such a study would be important for influenza pandemic planning. What Did the Researchers Do and Find? References (2003) Zanamivir: a significant reduction in viral load during treatment in military conscripts with influenza. Scand J Infect Dis 35: 52–58. 15. Gubareva LV, Kaiser L, Hayden FG (2000) Influenza virus neuraminidase inhibitors. Lancet 355: 827–835. 7. The MIST (Management of Influenza in the Southern Hemisphere Trialists) Study Group (1998) Randomised trial of efficacy and safety of inhaled zanamivir in treatment of influenza A and B virus infections. Lancet 352: 1877–1881. 7. The MIST (Management of Influenza in the Southern Hemisphere Trialists) Study Group (1998) Randomised trial of efficacy and safety of inhaled zanamivir in treatment of influenza A and B virus infections. Lancet 352: 1877–1881. 16. Nguyen JT, Hoopes JD, Le MH, Smee DF, Patick AK, et al. (2010) Triple combination of amantadine, ribavirin, and oseltamivir is highly active and synergistic against drug resistant influenza virus strains in vitro. PLoS One 5: e9332. doi:10.1371/journal.pone.0009332. 8. Poland GA, Jacobson RM, Ovsyannikova IG (2009) Influenza virus resistance to antiviral agents: a plea for rational use. Clin Infect Dis 48: 1254–1256. 8. Poland GA, Jacobson RM, Ovsyannikova IG (2009) Influenza virus resistance to antiviral agents: a plea for rational use. Clin Infect Dis 48: 1254–1256. PLoS Medicine \| www.plosmedicine.org November 2010 \| Volume 7 \| Issue 11 \| e1000362 November 2010 \| Volume 7 \| Issue 11 \| e1000362 10 PLoS Medicine \| www.plosmedicine.org Combined Oseltamivir-Zanamivir in Influenza PLoS Medicine \| www.plosmedicine.org November 2010 \| Volume 7 \| Issue 11 \| e1000362 PLoS Medicine \| www.plosmedicine.org 11 Combined Oseltamivir-Zanamivir in Influenza Editors’ Summary Background. In the last few years, use of the neuraminidase inhibitors, oseltamivir and zanamivir, has been considered a key strategy for limiting the impact of influenza both in individuals (by reducing morbidity and mortality) and collectively (by slowing the virus’ spread to buy time for vaccine production, the cornerstone of influenza control). However, there are concerns that widespread use of a single antiviral drug may lead to resistant strains, which could dramatically reduce its effectiveness in future. Theoretically, if well tolerated, and if producing at least additive antiviral activity, the combination of two antiviral agents could offer several advantages such as reducing disease severity and reducing the viral shedding period, which in turn could lead to lower infection rates and reduced resistance especially in immunocompromised patients. Importantly, combining two drugs could ensure optimal treatment of all types of circulating influenza virus and subtypes or variants. References The researchers conducted a randomized, placebo-controlled trial within 145 general practitioners throughout France during the seasonal influenza epidemic in 2008–2009. Adults who visited their general practitioner with symptoms of an influenza-like illness for less than 36 hours and who had a positive influenza A rapid test were randomized to one of three arms: (1) oral oseltamivir 75 mg twice daily plus zanamivir 10 mg by inhalation twice daily, (2) oral oseltamivir 75 mg twice daily plus inhaled placebo, or (3) zanamivir 10 mg by inhalation twice daily plus oral placebo. The effects of the drugs or combination of drugs was assessed virologically, by looking at the proportion of patients with nasal influenza reverse transcription (RT)-PCR below a particular level on day 2 of treatment. Clinical measures of effectiveness included the time to resolution of illness, the number of patients with alleviation of symptoms at the end of treatment, and the incidence of secondary complications of influenza such as Additional Information. Please access these Web sites via the online version of this summary at http://dx.doi.org/10. 1371/journal.pmed.1000362. N Wikipedia has information on H3N3 influenza A virus (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages) N The World Health Organization has a global alert and response site on seasonal influenza N Patient UK provides information about antivirals for influenza N Answers.com has information about oseltamivir and about zanamivir PLoS Medicine \| www.plosmedicine.org November 2010 \| Volume 7 \| Issue 11 \| e1000362 PLoS Medicine \| www.plosmedicine.org 12
W3117794832.txt	https://www.biorxiv.org/content/biorxiv/early/2020/03/06/2020.01.13.905323.full.pdf	en		Hypercluster: a python package and SnakeMake pipeline for flexible, parallelized unsupervised clustering optimization	bioRxiv (Cold Spring Harbor Laboratory)	2,020	cc-by	6,575	bioRxiv preprint doi: https://doi.org/10.1101/2020.01.13.905323; this version posted March 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 2 3 4 5 Hypercluster: a python package and SnakeMake pipeline for flexible, parallelized 6 unsupervised clustering optimization 7 8 Lili Blumenberg1,2, Kelly V. Ruggles1,2* 9 10 1 11 10016, USA 2Department of Medicine, New York University School of Medicine, New 12 York, NY 10016, USA Institute of Systems Genetics, New York University School of Medicine, New York, NY 13 14 *Corresponding author 15 Email: Kelly.Ruggles@nyulangone.org 16 17 18 19 20 21 22 23 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.13.905323; this version posted March 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 24 Full title: Hypercluster: a python package and SnakeMake pipeline for flexible, 25 parallelized unsupervised clustering optimization 26 Short title: Hypercluster: a tool for unsupervised clustering optimization 27 28 29 Abstract Unsupervised clustering is a common and exceptionally useful tool for large 30 biological datasets. However, clustering requires upfront algorithm and hyperparameter 31 selection, which can introduce bias into the final clustering labels. It is therefore 32 advisable to obtain a range of clustering results from multiple models and 33 hyperparameters, which can be cumbersome and slow. To streamline this process, we 34 present hypercluster, a python package and SnakeMake pipeline for flexible and 35 parallelized clustering evaluation and selection. Hypercluster is available on bioconda; 36 installation, documentation and example workflows can be found at: 37 https://github.com/ruggleslab/hypercluster. 38 39 40 Author summary Unsupervised clustering is a technique for grouping similar samples within a 41 dataset. It is extremely common when analyzing big data from patient samples, or high 42 throughput techniques like single cell RNA-seq. When researchers use unsupervised 43 clustering, they have to select parameters that affect the final result—for instance, how 44 many groups they expect to find or what the smallest group is allowed to be. Some 45 methods require setting even less intuitive parameters. For most applications, it is 46 extremely challenging to guess what the values of these parameters should be; 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.13.905323; this version posted March 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 47 therefore to prevent introducing bias into the final results, researchers should test many 48 different parameters and methods to find the best groups. This process is cumbersome, 49 slow and challenging to perform in a reproducible way. We developed hypercluster, a 50 tool that automates this process, make it much faster, and presenting the results in a 51 reproducible and helpful manner. 52 53 54 Introduction Unsupervised clustering is commonly used for the interpretation of ‘omics 55 datasets. It provides an objective and intuitive measure of similarity and difference 56 between samples. Clustering can be used to determine biologically relevant subgroups 57 of samples, find co-regulated molecular features, or provide objective support for the 58 phenotypic similarity of biological perturbations. Moreover, clustering is a key step in the 59 analysis of many emerging sequencing-based technologies. For example, a 60 fundamental challenge in the analysis of single-cell measurement data, in particular 61 single cell RNA-seq (scRNA-seq), is determining robust clusters of phenotypically 62 similar cells (1–3). Clustering is also increasingly being used alongside traditional 63 diagnostic techniques to establish new classifications of patient samples into disease- 64 relevant subgroups (4–7) and for patient subgroup classification and risk stratification 65 (6,8–12). The near-future of personalized medicine relies on researchers identifying 66 robust unsupervised clustering-based disease subtypes. Therefore, it is essential that 67 high-quality clustering results are easily and robustly obtainable, without user-selected 68 hyperparameters introducing bias and impeding rapid analysis. 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.13.905323; this version posted March 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 69 Currently, researchers robustly employing unsupervised clustering must choose 70 specific algorithms and hyperparameters that are appropriate to their experiment type 71 and data. Although some efforts have been made to advise researchers on optimal 72 selection of both (13), biological datasets vary between batches, days, labs and 73 researchers, underlining the importance of context- and experiment-dependent analysis 74 tuning. Software packages for automatic hyperparameter tuning and model selection for 75 regression and classification machine learning techniques exist, notably auto-sklearn 76 from AutoML (14), but there are not yet packages for automated unsupervised 77 clustering optimization. 78 Typically, the effect of hyperparameter choice on the quality of clustering results 79 cannot be described with a convex function, meaning that when searching the 80 landscape of hyperparameter choices there are often local maxima that may appear to 81 be the optimal results if broad choices of hyperparameters are not considered. 82 Therefore it is unlikely that a sequential approach using for instance, gradient descent 83 from a single initialized set of hyperparameters, would be able to select the optimal 84 parameters for the majority of clustering challenges (15). Exhaustive (i.e. grid) search is 85 the most likely to obtain optimal results from unsupervised clustering. However, grid 86 search can be slow and cumbersome to perform for the multiple hyperparameters and 87 clustering algorithms that are available from most clustering packages. 88 Here we present hypercluster, a python package and SnakeMake pipeline for 89 parallelized clustering calculations and comparison. The hypercluster package allows 90 users to calculate results from multiple hyperparameters using one or many algorithms, 91 then easily calculate and visualize evaluation metrics for each result (16). The 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.13.905323; this version posted March 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 92 accompanying SnakeMake pipeline allows parallelization on a single computer, across 93 a high performance computing cluster, or on cloud based services (17,18), speeding up 94 optimization, especially for large datasets. In addition, our pipeline has all the 95 advantages of the SnakeMake framework, e.g. easily adding new datasets to analyze, 96 keeping track of progress and simplified bug tracking. Currently, hypercluster can 97 compare all clustering algorithms and evaluation metrics from scikit-learn (19), as well 98 as non-negative matrix factorization (NMF) (20), Louvain and Leiden clustering (21,22). 99 In addition, hypercluster can be extended to employ user-supplied clustering algorithm 100 or evaluation metrics. Given a metric to maximize, hypercluster identifies “best” labels 101 and optionally provides comparisons of labeling results. Even if no single metric can be 102 used to select the best hyperparameters, hypercluster provides several visualizations 103 that help users pick labels by balancing many metrics or picking the most reproducible 104 clusters. Hypercluster provides researchers with a python package and pipeline for 105 flexible, parallelized, distributed and user-friendly algorithm selection and hyper- 106 parameter tuning for unsupervised clustering. 107 108 Design and Implementation 109 Requirements and structure 110 The hypercluster package uses scikit-learn (19), python-igraph (23), leidenalg 111 (24) and louvain-igraph (25) to assign cluster labels and uses scikit-learn and custom 112 metrics to compare clustering algorithms and hyperparameters to find optimal clusters 113 for any given input data (Fig. 1). Hypercluster requires python3, pandas (26), numpy 114 (27), scipy (28), matplotlib (29), seaborn (30), scikit-learn (19), python-igraph (23), 5 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.13.905323; this version posted March 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 115 leidenalg (24), louvain-igraph (25) and SnakeMake (17). Hypercluster can be run 116 independently of SnakeMake, as a standalone python package. Inputs, outputs and an 117 example workflow are described below, but additional example workflows are provided 118 at https://github.com/ruggleslab/hypercluster/tree/master/examples. 6 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.13.905323; this version posted March 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 119 120 Fig. 1 Hypercluster workflow schematic 7 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.13.905323; this version posted March 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 121 a) Clustering algorithms and their respective hyperparameters are user-specified. 122 Hypercluster then uses those combinations to create exhaustive configurations, and if 123 selected a random subset is chosen. 124 b) Snakemake is then used to distribute each clustering calculation into different jobs. 125 c) Each set of clustering labels is then evaluated in a separate job by a user-specified 126 list of metrics. 127 d) All clustering results and evaluation results are aggregated into tables. Best labels 128 can also be chosen by a user-specified metric. 129 130 131 Modes Hypercluster takes pandas DataFrames as input. For local running, 132 AutoClusterer and MultiAutoClusterer objects can be instantiated with default or user- 133 defined values. To run through hyperparameters for a dataset, users simply provide a 134 pandas DataFrame to the “fit'' method on either object. Users evaluate the labeling 135 results by running the “evaluate” method. 136 config.yml 137 SnakeMake allows users to parallelize clustering calculations. To configure the 138 SnakeMake pipeline, users edit a config.yml file (Table 1). In that file, users can specify 139 input and output directories and files (Table 1, lines 1-3, 5-7) and the hyperparameter 140 search space (Fig 1A, Table 1, line 18). Users can specify whether to use exhaustive 141 grid search or random search; if random search is selection, they can specify probability 142 weights for each hyperparameter (Table 1, line 9). Snakemake then schedules 143 performing each clustering algorithm and evaluating the results as a separate job (Fig. 8 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.13.905323; this version posted March 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 144 1B). Users can specify which evaluation metrics to apply (Fig. 1C, Table 1, line 10) and 145 add keyword arguments to tune several steps in the process (Table 1, lines 4, 8-9, 11- 146 16). Clustering and evaluation results are then aggregated into final tables (Fig. 1D). 147 Other than the location and names of the input files, everything has a predefined default 148 that allows the pipeline to be used “out of the box.” Users can reference the 149 documentation and examples for more information. Table 1 Parameters in SnakeMake configuration file config.yml parameter Explanation Path to folder in which 1 input_data_folder input data can be found. List of prefixes of data 2 input_data_files files. File name of gold_standard_file, must 3 gold_standard_file be in input_data_folder pandas.read_csv keyword 4 read_csv_kwargs arguments for input data. Path to folder into which 5 output_folder results should be written. Name of subfolder to put 6 intermediates_folder intermediate results. Name of subfolder to put 7 clustering_results aggregated results. Additional arguments to 8 clusterer_kwargs pass to clusterers. Additonal keyword arguments for the hypercluster.AutoClusterer 9 generate_parameters_addtl_kwargs class. Names of evaluation 10 evaluations metrics to use. Additional kwargs per 11 eval_kwargs evaluation metric function. Which metric to maximize 12 metric_to_choose_best to choose the labels. 13 metric_to_compare_labels Which metric to use to Example /input_data ['input_data1', 'input_data2'] {'input_data': 'gold_standard_file.txt'} {'test_input': {'index_col':[0]}} /results clustering_intermediates clustering KMeans: {'random_state':8}} {‘KMeans’: {'random_search': true) ['silhouette_score', 'number_clustered'] {'silhouette_score': {'random_state': 8}} silhouette_score adjusted_rand_score 9 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.13.905323; this version posted March 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 14 compare_samples 15 output_kwargs 16 heatmap_kwargs 17 optimization_parameters 150 151 compare label results to each other. Whether to made a table and figure with counts of how often each two samples are in the same cluster. pandas.to_csv and pandas.read_csv keyword arguments for output tables. Arguments for seaborn.heatmap for pairwise visualizations. Which algorithms and corresponding hyperparameters to try. "true" {'evaluations': {'index_col':[0]}, 'labels': {'index_col':[0]}} {'vmin':-2, 'vmax':2} {'KMeans': {'n_clusters': [5, 6, 7]}} Table 1 Line-by-line explanation of the config.yml for SnakeMake 152 153 154 Input data and execution After specifying the config.yml file, users provide a data table with samples to be 155 clustered as the rows and features as the columns, with the location specified in the 156 config.yml file (Table 1, line2). Users can then simply run “snakemake -s 157 hypercluster.smk --configfile config.yml” in the command line, with any additional 158 SnakeMake flags appropriate for their system. Applying the same configuration to new 159 files or adjusting algorithms and hyperparameter options simply requires editing the 160 config.yml file and rerunning SnakeMake. 161 Extending hypercluster 162 163 Currently, hypercluster can optimize any clustering algorithm and calculate any evaluation available in scikit-learn (19,31), as well as NMF, Louvain and Leiden 10 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.13.905323; this version posted March 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 164 clustering. Additional clustering classes and evaluation metric functions can be added 165 by users in the additional_clusterer.py and additional_metrics.py files, respectively, if 166 written to accommodate the same input, outputs and methods (see 167 additional_clusterers.py and additional_metrics.py for examples). 168 Outputs 169 By default, hypercluster outputs a yaml file containing all configurations of the 170 clustering algorithms and hyperparameters that are being searched. For each set of 171 labels, it generates a file containing labels and a file containing evaluations. It also 172 outputs aggregated tables of all labels and evaluations. Finally, given a metric to 173 maximize, hypercluster writes files containing the optimal labels. Optionally, 174 hypercluster will also output a table and heatmap of pairwise comparisons of labeling 175 similarities with a user-specified metric (Figure S1). This figure is particularly useful for 176 finding labels that are robust to differences in hyperparameters. It can also optionally 177 output a table and heatmap showing how often each pair of samples were assigned the 178 same cluster (Figure S2). 11 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.13.905323; this version posted March 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 179 180 Figure S1 Pairwise label comparisons 181 Automatically generated heatmap showing pairwise comparison of labeling 182 automatically generated using hypercluster of breast cancer samples. Colors represent 183 adjusted rand index between labels. 12 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.13.905323; this version posted March 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 184 185 Figure S2 Pairwise sample comparisons 186 Automatically generated pairwise comparison of breast cancer samples. Color indicates 187 the number of times two samples were assigned the same cluster. 188 13 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.13.905323; this version posted March 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 189 Results 190 Unsupervised clustering of RNA-seq on breast cancer patient samples 191 To illustrate the utility of hypercluster in a disease-relevant context, we applied 192 our method to RNA-seq data from 526 breast cancer patient samples from the Cancer 193 Genome Atlas (TCGA) (32), a dataset that has been previously used for benchmarking 194 clustering algorithms (33). As demonstrated, RNA-seq can be used to classify breast 195 cancer patients into four major PAM50 subtypes (Basal-like, LuminalA, LuminalB, and 196 Her2-enriched), which are based on the expression of 50 specific genes (7,34,35). We 197 removed genes with any missing values and subset to the 500 most variable genes as 198 input for all available algorithms with ranges of hyperparameter conditions. We then 199 compared the sample clustering results from our 500 gene clustering compared with 200 subtypes defined by the PAM50 classifier. This workflow is available on the github 201 examples folder (https://github.com/liliblu/hypercluster/tree/dev/examples). 202 Hypercluster automatically outputs a visualization of evaluation metrics for all 203 hyperparameter combinations (Fig. 2A), which allows users to quickly see how 204 changing hyperparameters affects clustering result quality. These results highlight how 205 evaluation metrics are not generally convex over ranges of hyperparameters (e.g. 206 silhouette score as n_clusters changes with the KMeans algorithm (Fig. 2A) 207 demonstrating the utility of the exhaustive grid search approach. In addition, our pipeline 208 optionally creates a pairwise comparison of labeling, with a specified user metric (Figure 209 S1) to make it easier to understand how robust and consistent labeling is across 210 algorithms and parameters. 211 14 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.13.905323; this version posted March 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 212 213 Fig. 2 Visualizations of clustering metrics from breast cancer RNA-seq 214 a) Example automatic output from hypercluster, showing z-scored evaluation values of 215 evaluation metrics for each clustering algorithm and hyperparameter set, including 216 those in 2E. Evaluations applied to clustering from TCGA breast cancer samples. 217 b) Effect of varying resolution (left) and k for shared nearest neighbor matrix (right) on 218 silhouette score, an inherent metric measuring clustering quality, for Louvain clustering. 219 c) Effect of resolution and choice of k on various evaluation metrics for both Louvain 220 (top) and Leiden (bottom) clustering. 221 222 Labels and evaluation results are easily accessible for further custom analyses. 223 To demonstrate a possible downstream workflow that hypercluster facilitates, we 224 investigated results from Louvain and Leiden clustering, which are commonly used in 225 scRNA-seq analysis, on the same breast cancer RNA-Seq dataset (36)). Louvain and 226 Leiden clustering are community detection algorithms for networks, usually generated 227 from shared K-nearest-neighbor adjacency matrices. We varied resolution, which 228 affects the number of members in final communities, and the k defining how many 15 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.13.905323; this version posted March 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 229 nearest neighbors are measured for constructing the adjacency graph (Fig. 2B, C). 230 Resolution and k have significant effects on labeling results and their corresponding 231 evaluations. Interestingly, increasing resolution appears to have opposite effects on 232 clustering quality (e.g. as measured by silhouette score) depending on k, with a large 233 spread of silhouette scores dependent on k at low resolution, converging to similar 234 silhouette scores at higher resolution (Fig. 2B, C). These results highlight the 235 importance of simultaneous tuning of multiple hyperparameters. Plots like those in Fig. 236 2B, showing the effect of varying each parameter individually on evaluation metrics, can 237 be automatically generated by the visualize_for_picking_best_labels function or listing 238 evaluations in the “screeplot_evals” section of a config.yml file. 239 To observe if clustering on 500 variable genes can recapitulate PAM50 240 classification, we identified results that best match PAM50 subtypes according to the 241 adjusted rand score while labeling all samples (Fig. 3). By this metric, the best labels 242 were generated by NMF clustering (37) with n_clusters=4 (Fig. 3A-C). These labels that 243 do diverge from the PAM50 classification correspond to a subset of Luminal A samples 244 that cluster with Luminal B samples (Fig. 3D). Hypercluster allows researchers to 245 compare different algorithms and hyperparameter combinations in a reproducible and 246 convenient way. 16 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.13.905323; this version posted March 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 247 17 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.13.905323; this version posted March 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 248 Fig. 3 Exploration of NMF clustering results on breast cancer RNA-seq 249 a) Automatically generated heatmap of evaluation metrics for NMF clustering results. 250 b) PCA projection of 200 random samples colored by labels assigned by NMF 251 clustering. Background color indicates similarity to PAM50 labeling calculated by 252 adjusted rand index. 253 c) PCA projection of samples colored by PAM50 subtypes and most similar NMF 254 clustering labels. 255 d) Heatmap of 125 most variable genes with PAM50 and NMF;n_cluster=4 labels 256 indicated on the top. 257 258 259 Exploration of bone marrow microenvironment scRNA-seq To demonstrate hypercluster’s utility for analysis of single cell data sets, we 260 analyzed scRNA-seq from a study investigating the hematopoietic stem cell 261 microenvironment (38) and performed comparative analysis of several clustering 262 algorithms in parallel on a high performance computing cluster utilizing a Slurm 263 scheduler (39). We used normalized expression data from untreated cells sorted for 264 mesenchymal stromal and vascular endothelial, and osteoblast markers, subset to the 265 2000 most variable genes from the seurat object containing the data (36,38). We then 266 used hypercluster to explore the labeling results from all available clustering algorithms 267 and ranges of relevant hyperparameters. Hypercluster was then used to evaluate labels 268 with every available metric, including metrics that measure inherent labeling quality, as 269 well as comparing new labels to cell types identified in the original study (Fig. 4A). The 270 approach that best recapitulated the published labels was clustering with 18 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.13.905323; this version posted March 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 271 MiniBatchKMeans with 12 clusters (Fig. 4B-D). These labels differed from published 272 labels largely from swapping cells in the P1 and P2 groups (Fig. 4B), which are both 273 LEPR+ subgroups, that were shown to be very similar in the original paper (38). While 274 the original labels were generated using community detection methods like Louvain and 275 Leiden clustering, those methods performed poorly compared to others (Figure S3), 276 likely due to differences in data pre-processing. Varying the number of clusters has 277 variable effects on evaluation results (Fig. 4A, 4C, Figure S3), again highlighting the 278 importance of an exhaustive approach. 19 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.13.905323; this version posted March 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 279 20 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.13.905323; this version posted March 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 280 Fig. 4 Clustering and evaluation of scRNA-seq data 281 a) Evaluation metrics for clustering conditions, automatically generated by hypercluster 282 for single cell RNA-seq data. 283 b) Comparison of published labels with best matching calculated labels, 284 MiniBatchKMeans;n_clusters=12. Legend shows mismatched clusters for the best 285 labels on top and clusters with high correspondence to published clusters in the bottom 286 section. 287 c) PCA projection of 700 random cells labeled by MiniBatchKMeans across 288 hyperparameters. 289 d) PCA projection of cells colored by published labels. 21 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.13.905323; this version posted March 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 290 291 Figure S3 Full evaluations of scRNA-seq clustering 292 Automatically generated full evaluation metric table from clustering of scRNA-seq stem 293 cell niche cells. 22 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.13.905323; this version posted March 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 294 295 296 Discussion Defining groups of molecularly similar patient samples is key to personalizing 297 medical prognosis, diagnosis and treatment strategies, making unsupervised clustering 298 a workhorse for researchers advancing personalized medicine. It is therefore essential 299 that unsupervised clustering is rigorous and not biased by arbitrary hyperparameter 300 selection. While extremely high quality open-source tools such as scikit-learn make 301 unsupervised clustering accessible to many, exhaustively and reproducibly comparing 302 hyperparameters is still challenging; hypercluster solves these issues. 303 Nearly every step in data analysis pipelines require hyperparameter selection, 304 during which biased or arbitrary parameter selection can greatly impact results. Further, 305 data preprocessing, involving the filtering of datasets to remove low quality or low 306 coverage samples or features (e.g. removing genes with very few reads in RNA-seq), 307 also greatly impacts downstream clustering results. Hypercluster provides a workflow to 308 address the former issue, allowing for comprehensive evaluation of multiple 309 hyperparameters and clustering algorithms simultaneously. The package auto-sklearn 310 (14) provides functionality for automating pre-processing of data tables, which could 311 easily be incorporated upstream of hypercluster to automate the latter. In addition to the 312 simple command line functions, we have also employed SnakeMake for parallelization, 313 a workflow management system already widely used for pipeline optimization (40–46). 314 If unsupervised clustering is a downstream analytic method of interest, 315 determining which parameters to select can be cumbersome, and possibly inaccurate, 316 without a clustering optimization tool like hypercluster. While it is not always clear how 23 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.13.905323; this version posted March 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 317 to choose hyperparameters or algorithms in a consistent way (e.g. when two different 318 conditions optimize for different metrics), it is essential to at least understand if the 319 labels one obtains are robust to small changes in algorithm or hyperparameter choice 320 (e.g. as shown in Figure S1). Our package greatly improves the ability of researchers to 321 gain this understanding. In addition to assisting researchers in choosing 322 hyperparameters, hypercluster aids computational biologists who are benchmarking 323 new clustering algorithms, evaluation metrics and pre- or post-processing steps (3). In 324 conclusion, hypercluster streamlines the use of unsupervised clustering to derive 325 biologically relevant structure within data. Most importantly, it eases the prioritization of 326 rigor and reproducibility for researchers using these techniques. 327 328 Acknowledgements 329 We thank the members of Ruggles and Fenyö labs for their helpful discussions and 330 input. We would like to thank MacIntosh Cornwell for his advice with the SnakeMake 331 pipeline. We would also like to thank Joseph Copper Devlin for his help and advice with 332 implementing Louvain and Leiden clustering. 333 Availability of data and materials 334 Hypercluster is released on pip (pip install hypercluster) and conda (conda install -c 335 bioconda hypercluster). Development versions and installation instructions can be found 336 at our github (https://github.com/liliblu/hypercluster/), tutorials and examples, including 337 all of the code used to create the figures in this paper, can be found here: 338 https://github.com/ruggleslab/hypercluster/tree/master/examples, and documentation 339 can be found here: https://hypercluster.readthedocs.io/en/latest/. Hypercluster is written 24 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.13.905323; this version posted March 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 340 in python and was developed and tested on MacOS and Linux. Requirements are listed 341 on the github and in the documentation. Hypercluster is available with the MIT licence. 342 343 Financial Disclosure 344 This work has been supported by the National Cancer Institute (NCI) through CPTAC 345 award U24 CA210972. The funders had no role in study design, data collection and 346 analysis, decision to publish, or preparation of the manuscript. 347 348 Authors' contributions 349 Conceptualization, Project Administration, Writing: LB and KVR. Data Curation, Formal 350 analysis, Investigation, Methodology, Software, Validation, Visualization: LB. Funding 351 acquisition, Resources, Supervision: KVR. 352 353 Competing interests 354 The authors declare no competing interests. 355 356 Related manuscripts 357 The authors do not have other related or duplicate manuscripts. 358 359 References 360 1. Kiselev VY, Andrews TS, Hemberg M. Publisher Correction: Challenges in 361 unsupervised clustering of single-cell RNA-seq data. 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https://openalex.org/W3011295534	https://biblio.ugent.be/publication/8662128/file/8662129	English	null	Striatal Dopamine D2-Muscarinic Acetylcholine M1 Receptor–Receptor Interaction in a Model of Movement Disorders	Frontiers in pharmacology	2,020	cc-by	12,404	ORIGINAL RESEARCH published: 13 March 2020 doi: 10.3389/fphar.2020.00194 Striatal Dopamine D2-Muscarinic Acetylcholine M1 Receptor–Receptor Interaction in a Model of Movement Disorders René A. J. Crans1,2, Elise Wouters1, Marta Valle-León2,3, Jaume Taura2,3, Caio M. Massari2,4, Víctor Fernández-Dueñas2,3, Christophe P. Stove1† and Francisco Ciruela2,3† René A. J. Crans1,2, Elise Wouters1, Marta Valle-León2,3, Jaume Taura2,3, Caio M. Massari2,4, Víctor Fernández-Dueñas2,3, Christophe P. Stove1† and Francisco Ciruela2,3† 1 Laboratory of Toxicology, Department of Bioanalysis, Ghent University, Ghent, Belgium, 2 Unitat de Farmacologia, Departament Patologia i Terapèutica Experimental, Facultat de Medicina, IDIBELL-Universitat de Barcelona, L’Hospitalet de Llobregat, Barcelona, Spain, 3 Institut de Neurociències, Universitat de Barcelona, Barcelona, Spain, 4 Programa de Poìs-graduação em Bioquiìmica, Centro de Ciencias Bioloìgicas, Universidade Federal de Santa Catarina, Florianoìpolis, Brazil Keywords: D2R, M1R, sumanirole, VU0255035, striatum, Parkinson’s disease Edited by: Luis F. Callado, University of the Basque Country, Spain Parkinson’s disease (PD) is a neurodegenerative disorder characterized by motor control deﬁcits, which is associated with the loss of striatal dopaminergic neurons from the substantia nigra. In parallel to dopaminergic denervation, there is an increase of acetylcholine within the striatum, resulting in a striatal dopaminergic– cholinergic neurotransmission imbalance. Currently, available PD pharmacotherapy (e.g., prodopaminergic drugs) does not reinstate the altered dopaminergic–cholinergic balance. In addition, it can eventually elicit cholinergic-related adverse effects. Here, we investigated the interplay between dopaminergic and cholinergic systems by assessing the physical and functional interaction of dopamine D2 and muscarinic acetylcholine M1 receptors (D2R and M1R, respectively), both expressed at striatopallidal medium spiny neurons. First, we provided evidence for the existence of D2R–M1R complexes via biochemical (i.e., co-immunoprecipitation) and biophysical (i.e., BRET1 and NanoBiT R⃝) assays, performed in transiently transfected HEK293T cells. Subsequently, a D2R–M1R co-distribution in the mouse striatum was observed through double- immunoﬂuorescence staining and AlphaLISA R⃝immunoassay. Finally, we evaluated the functional interplay between both receptors via behavioral studies, by implementing the classical acute reserpine pharmacological animal model of experimental parkinsonism. Reserpinized mice were administered with a D2R-selective agonist (sumanirole) and/or an M1R-selective antagonist (VU0255035), and alterations in PD-related behavioral tasks (i.e., locomotor activity) were evaluated. Importantly, VU0255035 (10 mg/kg) potentiated the antiparkinsonian-like effects (i.e., increased locomotor activity and decreased catalepsy) of an ineffective sumanirole dose (3 mg/kg). Altogether, our data suggest the existence of putative striatal D2R/M1R heteromers, which might be a relevant target to manage PD motor impairments with fewer adverse effects. Reviewed by: Luca Ferraro, University of Ferrara, Italy Melissa L. Perreault, University of Guelph, Canada Correspondence: Christophe P. Stove christophe.stove@ugent.be Francisco Ciruela fciruela@ub.edu †These authors have contributed equally to this work Reviewed by: Luca Ferraro, University of Ferrara, Italy Melissa L. Perreault, University of Guelph, Canada Correspondence: Christophe P. Stove christophe.stove@ugent.be Francisco Ciruela fciruela@ub.edu †These authors have contributed equally to this work Specialty section: This article was submitted to Neuropharmacology, a section of the journal Frontiers in Pharmacology Received: 13 December 2019 Accepted: 11 February 2020 Published: 13 March 2020 Specialty section: This article was submitted to Neuropharmacology, a section of the journal Frontiers in Pharmacology Received: 13 December 2019 Accepted: 11 February 2020 Published: 13 March 2020 INTRODUCTION M4Rs, whereas M1Rs are expressed by both D1R-MSNs and D2R- MSNs. Thereby, within the striatum, tonically active cholinergic interneurons (ChIs), which constitute 1% to 2% of the total striatal neuronal population (Bolam et al., 1984; Pisani et al., 2007), release acetylcholine (ACh) through widely arborizing axons with large terminal ﬁelds that modulate the MSNs via M1Rs and M4Rs (Graybiel, 1990; Mesulam et al., 1992; Contant et al., 1996). Interestingly, the modulation of MSNs with a selective M1R antagonist resulted in antiparkinsonian-like eﬀects in a number of rat models of movement disorders (Xiang et al., 2012). In addition, the blockade of M1R, M4R, or ChI signaling improved the motor functions in 6-hydroxydopamine–lesioned mice (Ztaou et al., 2016). Furthermore, systemic administration of scopolamine (a non-selective mAChR antagonist) modulated the DA turnover and reduced D2R aﬃnity of raclopride in monkey brains (Tsukada et al., 2000). These studies suggest an intense neuronal interaction between dopaminergic and cholinergic systems, where normal motor functions may require a ﬁne-tuned and coordinated control (Di Chiara et al., 1994; Calabresi et al., 2000; Zhang et al., 2002). The extent to which both neurotransmission systems speciﬁcally integrate at a molecular and/or functional level is of high interest for the development of novel multimodal pharmacological therapies to manage PD (Fuxe et al., 2012). Parkinson’s disease (PD) is a common movement disorder that is clinically characterized by motor control deﬁcits, such as bradykinesia, muscular rigidity, resting tremors, and postural instability (Mhyre et al., 2012). Approximately, 1% of the population older than 60 years is aﬀected by PD. The major pathophysiological PD hallmark is the loss of dopaminergic neurons projecting from the substantia nigra pars compacta (Hisahara and Shimohama, 2011; Dexter and Jenner, 2013), which leads to dopamine (DA) depletion within the striatum. L-3,4-dihydroxyphenylalanine (L-DOPA) is an eﬀective DA replacement strategy, which eﬃciently reverses motor control deﬁcits at the early stages of the disorder. However, long- term L-DOPA therapy (>5–10 years) is commonly associated with adverse motor complications, such as dyskinesia and eﬃcacy ﬂuctuations, thus reducing the patient’s quality of life (Jenner, 2003; Kalia and Lang, 2015). Currently, DA receptor agonists (i.e., pramipexole and ropinirole) are considered the ﬁrst choice in PD therapy, as monotherapy or adjuvants to L-DOPA (Fox et al., 2011; Fox et al., 2018). Again, these agonists are eﬀective at the early stages, but they eventually fail reducing motor complications (Jenner, 2003; Hisahara and Shimohama, 2011). INTRODUCTION Interestingly, before L-DOPA was extensively prescribed, anticholinergics were the ﬁrst-line therapeutics in PD (Carlsson et al., 1957; Katzenschlager et al., 2003). The cholinergic system plays a pivotal role in regulating striatal functions by modulating the excitability of GABAergic medium spiny neurons (MSNs), which constitute nearly 95% of the striatal neuronal population (Lv et al., 2017). Nowadays, anticholinergics (i.e., biperiden) are eventually used as adjuvant drugs in PD management, besides their adverse eﬀects (i.e., nausea, cognitive impairments, dry mouth, urinary retention, and blurred vision). Importantly, some of these adverse eﬀects are likely due to a lack of muscarinic acetylcholine receptor (mAChR) subtype selectivity, because both M2R and M3R are blocked (Chen and Swope, 2007; Pedrosa and Timmermann, 2013). Recently, cholinergic modulation of striatal functions has gained renewed interest because of the development of compounds targeting speciﬁc mAChR subtypes (Xiang et al., 2012; Shen et al., 2015; Ztaou et al., 2016; Lv et al., 2017; Chambers et al., 2019). Here, we describe a novel interaction between the D2R and M1R in the striatum, which may eventually harmonize with those previously described for D2R (Cabello et al., 2009; Lukasiewicz et al., 2010; Borroto-Escuela et al., 2013; Bonaventura et al., 2014; Fernandez-Duenas et al., 2015; Rico et al., 2017; Vasudevan et al., 2019). In addition, we evaluated the antiparkinsonian eﬃcacy of a combined D2R agonist (i.e., sumanirole) and M1R antagonist (i.e., VU0255035) treatment using the reserpine animal model of experimental parkinsonism. To our knowledge, this study is the ﬁrst to demonstrate a molecular interaction and a functional interplay between D2R and M1R. Citation: Crans RAJ, Wouters E, Valle-León M, Taura J, Massari CM, Fernández-Dueñas V, Stove CP and Ciruela F (2020) Striatal Dopamine D2-Muscarinic Acetylcholine M1 Receptor–Receptor Interaction in a Model of Movement Disorders. Front. Pharmacol. 11:194. doi: 10.3389/fphar.2020.00194 March 2020 \| Volume 11 \| Article 194 1 Frontiers in Pharmacology \| www.frontiersin.org Striatal D2R/M1R in Parkinson’s Disease Crans et al. M4Rs, whereas M1Rs are expressed by both D1R-MSNs and D2R- MSNs. Thereby, within the striatum, tonically active cholinergic interneurons (ChIs), which constitute 1% to 2% of the total striatal neuronal population (Bolam et al., 1984; Pisani et al., 2007), release acetylcholine (ACh) through widely arborizing axons with large terminal ﬁelds that modulate the MSNs via M1Rs and M4Rs (Graybiel, 1990; Mesulam et al., 1992; Contant et al., 1996). Interestingly, the modulation of MSNs with a selective M1R antagonist resulted in antiparkinsonian-like eﬀects in a number of rat models of movement disorders (Xiang et al., 2012). In addition, the blockade of M1R, M4R, or ChI signaling improved the motor functions in 6-hydroxydopamine–lesioned mice (Ztaou et al., 2016). Furthermore, systemic administration of scopolamine (a non-selective mAChR antagonist) modulated the DA turnover and reduced D2R aﬃnity of raclopride in monkey brains (Tsukada et al., 2000). These studies suggest an intense neuronal interaction between dopaminergic and cholinergic systems, where normal motor functions may require a ﬁne-tuned and coordinated control (Di Chiara et al., 1994; Calabresi et al., 2000; Zhang et al., 2002). The extent to which both neurotransmission systems speciﬁcally integrate at a molecular and/or functional level is of high interest for the development of novel multimodal pharmacological therapies to manage PD (Fuxe et al., 2012). Plasmid Construction Plasmid Construction The plasmids pFLAG-D2R, pHA-M1R, pD2R-Rluc, pM1R-YFP, and pEYFP were a kind gift of Dr. Kjell Fuxe (Karolinska Institutet, Stockholm, Sweden). The sequence encoding the human M1R (NM_000738.3) was polymerase chain reaction– ampliﬁed using primers containing speciﬁc restriction sites (HindIII and EcoRI, 5′-GCTTAAGCTTATGAACACTTCAG-3′ and 5′- TCGAGAATTCGCGCATTGGC-3′) and cloned into the HindIII/EcoRI sites of the NanoBiT R⃝vector NB MCS1 (Promega, Madison, WI, United States). The construct was veriﬁed by restriction digest and Sanger sequencing (Euroﬁns Genomics, Ebersberg, Germany). This resulted in the fusion of the split NanoLuciferase (NL) fragment LargeBiT (LgBiT; 18 kDa) to the C-terminus of M1R. The constructs of cannabinoid types 1 and 2 receptors (CB1R and CB2R, respectively) and D2R fused with LgBiT or Small BiT (SmBiT; 1 kDa) were previously developed and described by our research group (Cannaert et al., 2016; Wouters et al., 2019a). ) Five distinct mAChR subtypes (M1R–M5R) have been identiﬁed, which are classiﬁed into two groups, based on pharmacological and molecular characteristics. The excitatory M1-like receptors (M1R, M3R, and M5R) transduce their signals via Gq/11 proteins, whereas the inhibitory M2-like receptors (M2R and M4R) are coupled to Gi/o proteins (Zhang et al., 2002; Bordia and Perez, 2019). All subtypes are present in the striatum, with M1R and M4R being highly expressed and modulating the excitability of GABAergic MSNs (Hersch et al., 1994; Yan et al., 2001). In general, two types of MSNs have been distinguished: (i) dopamine D2 receptors (D2Rs) expressing MSNs (i.e., D2R-MSNs), which belong to the striatal indirect pathway (Gagnon et al., 2017); and (ii) dopamine D1 receptors (D1Rs) containing MSNs (i.e., D1R-MSNs) constituting the striatal direct pathway. The D1R-MSNs express postsynaptic March 2020 \| Volume 11 \| Article 194 Frontiers in Pharmacology \| www.frontiersin.org 2 Striatal D2R/M1R in Parkinson’s Disease Crans et al. antibody (clone M2; Sigma-Aldrich) or mouse anti-HA antibody (clone 16B12; Abcam, Cambridge, United Kingdom). After 1.5 h of rotation, 20 µL of washed immobilized Protein-A UltraLink R⃝ Resin (#53139; Thermo Fisher Scientiﬁc) was added to the IPs, and the rotation continued for 1.5 h at 4◦C. Then, the beads were washed three times with RIPA buﬀer supplemented with the freshly added protease inhibitors. The proteins were eluted and denatured from the beads by heating the samples for 10 min at 37◦C in RIPA buﬀer and 4 × Laemmli supplemented with freshly added 10% β-mercaptoethanol. Co-immunoprecipitation HEK293T cells were transfected with 5 µg of the constructs containing pFLAG-D2R and/or pHA-M1R. When necessary, 5 µg of the empty vector pcDNA3.1 was co-transfected to maintain a total amount of 10 µg DNA per 10-cm dish. After 48 h, cells were washed three times with ice-cold phosphate- buﬀered saline (PBS; 1.47 mM KH2PO4, 8.07 mM Na2HPO4, 137 mM NaCl, 0.27 mM KCl with pH 7.2), harvested, and centrifuged, after which the pellet was stored at −80◦C until further use. The cells were homogenized in ice-cold 50 mM Tris–HCl (pH 7.4) with the Polytron at setting six for two periods of 10 s. Subsequently, the homogenates were transferred to 1.5-mL Eppendorf and centrifuged at 12,000 × g for 30 min at 4◦C. Then, all supernatant was removed, and the pellets were lysed in radioimmunoprecipitation assay (RIPA) buﬀer [150 mM NaCl, 25 mM Tris–HCl (pH 7.5), 1% sodium deoxycholate, 1% NP-40, and 0.1% sodium dodecyl sulfate (SDS)], supplemented with freshly added protease inhibitors (2.5 g/mL aprotinin, 1 mM PEFA-block, 10 g/mL leupeptin), for 1 h while rotating at 4◦C. The samples were centrifuged at 12,000 × g for 20 min at 4◦C. Next, the supernatant of each sample was transferred to a new Eppendorf, and the protein concentrations were determined using the bicinchoninic acid (BCA) assay (Pierce Biotechnology, Rockford, IL, United States). Thereafter, all samples were diluted with RIPA buﬀer to obtain equal protein concentrations with a ﬁnal volume of 500 µL. An amount of 10% for each sample (i.e., lysate) was denatured at 37◦C for 10 min in 4 × Laemmli [5% SDS, 50% glycerol, 65 mM Tris–HCl (pH 6.8) and 0.2% bromophenol blue], supplemented with freshly added 10% β-mercaptoethanol. The lysates were loaded onto a 10% polyacrylamide 10-well gel and resolved via SDS–polyacrylamide gel electrophoresis (SDS-PAGE). Subsequently, the proteins were blotted onto a nitrocellulose membrane (Amersham Protran 0.45 NC; GE Healthcare Life Sciences, Freiburg, Germany) and subjected to immunoblot analysis, as described below. The other 90% of each sample [i.e., immunoprecipitates (IPs)] was used for immunoprecipitation through adding 2 µg mouse anti-FLAG Plasmid Construction All IP eluates were subjected to SDS-PAGE electrophoresis and immunoblotting, as described above. Cell Culture and Transient Transfection Human embryonic kidney 293T (HEK293T; American Type Culture Collection, Manassas, VA, United States) cells were maintained in Dulbecco modiﬁed Eagle medium (DMEM; Thermo Fisher Scientiﬁc, Pittsburg, PA, United States) supplemented with GlutaMAX, 10% fetal bovine serum (FBS; Merck KgaA, Darmstadt, Germany), streptomycin (100 µg/mL), and penicillin (100 µ/mL) in a controlled environment (37◦C, 98% humidity, and 5% CO2). Prior to transfection, cells were cultured in 10-cm dishes (co-immunoprecipitation) or six- well plates [Bioluminescence Resonance Energy Transfer1 (BRET1) and NanoLuciferase Binary Technology (NanoBiT R⃝) assays] in 10 or 2 mL DMEM supplemented with 10% FBS, respectively. The HEK293T cells were transiently transfected using the polyethylenimine (Sigma-Aldrich, St. Louis, MO, United States) method. In all assays, medium was refreshed with DMEM + 10% FBS after 5 h. Immunoblots containing lysates or IPs were blocked in PBS with Licor blocking buﬀer (1:1; LI-COR Biosciences, Lincoln, NE, United States) at room temperature (RT) for 1 h. Then, the immunoblots were incubated with rabbit anti-HA (1:2,000, #GTX29110; Genetex, Irvine, CA, United States) or rabbit anti- FLAG (1:1000, #PA1-984B; Thermo Fisher Scientiﬁc) antibodies in 1:1 Licor blocking buﬀer-PBST (PBS with 0.05% Tween 20) overnight at 4◦C. The blots were washed three times with PBST for 10 min at RT. Next, the blots were incubated with donkey anti–rabbit secondary antibodies (1:15,000), conjugated to IRDye680RD or IRDye800CW (LI-COR Biosciences), for 1 h at RT. After incubation, the blots were washed three times with PBST and two times with PBS, each for 10 min at RT, protected from the light. Protein bands were visualized by the Odyssey imaging system (LI-COR Biosciences). Frontiers in Pharmacology \| www.frontiersin.org Bioluminescence Resonance Energy Transfer1 Assay y HEK293T cells were transfected with a constant amount of pD2R-Rluc (200 ng) and increasing amounts of pM1R-YFP or pEYFP (0–1,000 ng). Equal DNA ratios were maintained with co-transfection of the empty vector pcDNA3.1, which equilibrated the total amount of transfected DNA. Forty- eight hours posttransfection, the cells were washed three times with PBS, detached, and resuspended in Hanks balanced salt solution (HBSS; Thermo Fisher Scientiﬁc). An aliquot was used to determine the protein concentrations via the BCA assay, to control the number of cells. All cell suspensions were diluted to a density corresponding to a ﬁnal protein concentration of 600 ng/µL. Cell suspensions (corresponding to 20 µg protein) were distributed in duplicates into white and black 96-well microplates (#3600 and #3650; Corning, Stockholm, Sweden) for BRET1 and ﬂuorescence measurements, respectively. The substrate, h-coelenterazine (Molecular Probes, Eugene, OR, United States), was added at a 5 µM ﬁnal concentration. After 1 min (BRET1) and 10 min (Rluc total), the signals were measured using the ClarioSTAR microplate reader (BMG Labtech, Ortenberg, Germany) through the sequential integration of signal detection at 475 nm (445–505 nm) and 530 nm (500–560 nm). The net BRET1 ratio was expressed as a ratio of the light intensity at 530 nm over 475 nm by subtracting the background signal, which was detected when D2R-Rluc was only expressed with pcDNA3.1. The BRET1 curve was obtained by ﬁtting the data points to a non-linear regression equation March 2020 \| Volume 11 \| Article 194 Frontiers in Pharmacology \| www.frontiersin.org 3 Striatal D2R/M1R in Parkinson’s Disease Crans et al. assuming a single binding site using GraphPad Prism version 6.00 (San Diego, CA, United States). assuming a single binding site using GraphPad Prism version 6.00 (San Diego, CA, United States). of Health (NIH), Bethesda, MD, United States]. These mice were anesthetized and perfused intracardially with 50 to 200 mL of ice-cold 4% formaldehyde solution (Sigma-Aldrich) in PBS. Subsequently, the brains were postﬁxed in 4% formaldehyde solution overnight at 4◦C. D2R KO and WT littermate ﬁxed mouse brains were a kind gift from Dr. Jean-Martin Beaulieu (Centre de recherche en Santé Mentale de Québec, Québec, QC, Canada). Coronal brain sections (50 µm) were made with the Vibratome 1200S (Leica Lasertechnik GmbH, Heidelberg, Germany). Finally, the slices were collected and stored in antifreeze solution (30% glycerol, 30% ethylene glycol in PBS with pH 7.2) at -20◦C until further processing. NanoLuciferase Binary Technology R⃝ Assay y HEK293T cells were transfected with constructs encoding for pM1R-LgBiT (200 ng) and pD2R-SmBiT (200 ng). As negative controls, the cells were transfected with a combination of pM1R- LgBiT and pCB1R-SmBiT or pD2R-SmBiT and pCB2R-LgBiT, each with DNA concentrations of 200 ng. The functionality of the CB1R-SmBiT and CB2R-LgBiT constructs was demonstrated before (Cannaert et al., 2016). In all conditions, the construct encoding for the ﬂuorescent protein Venus was co-transfected (5% of the total DNA transfected). The NanoBiT R⃝assay was performed as described previously (Wouters et al., 2019a). Brieﬂy, 48 h posttransfection, the cells were washed two times with PBS, detached, and centrifuged for 5 min at 1,000g at RT. Protein concentrations were determined on an aliquot via the BCA assay, and cell suspensions, normalized for cell number (via a corresponding protein concentration of 600 ng/µL), were diluted in HBSS. Following a 20-fold dilution of the Nano-Glo R⃝ Live Cell reagent (#N2011; Promega) containing the luminescent substrate furimazine in aqueous Nano-Glo LCS dilution buﬀer, 25 µL of the diluted substrate was added to the wells of a 96-well plate containing 100 µL cell suspension. Fluorescence (508–548 nm) or luminescence (440–480 nm) emission was measured with the ClarioSTAR microplate reader in black or white 96-well plates (#3650 and #3600; Corning), respectively. The luminescence data were normalized for the measured ﬂuorescence signals to avoid signal ﬂuctuations due to variations in transfection eﬃciencies. Animals Caesarean derived 1 (CD-1) mice (Janvier Labs, Le Genest- Saint-Isle, France), D2R knockout (D2R KO) CD-1, and M1R knockout (M1R KO) C57BL/6J mice were generated as described previously (Fisahn et al., 2002; Taura et al., 2017). Animals were housed and tested in compliance with the guidelines described in the Guide for the Care and Use of Laboratory Animals (Clark et al., 1997) and following the European Communities Council Directive (2010/63/EU), FELASA, and ARRIVE guidelines. The animals were conventionally housed in groups of four or ﬁve in a temperature-controlled (22◦C) and humidity-controlled (66%) environment under a 12-/12-h light–dark cycle, where food and water intake was ad libitum. The study protocol was approved by the Ethical Committee on Animal Use and Care of the University of Barcelona (CEEA/UB). All eﬀorts were made to minimize animal suﬀering and the number of animals used in this study. Behavioral tests were performed with wild-type (WT) mice aged 5 months, weighing 40 to 55 g, between 12:00 and 18:00. AlphaLISA R⃝Immunoassay The AlphaLISA R⃝immunoassay was performed as previously described (Fernandez-Duenas et al., 2019). Brieﬂy, WT and D2R KO animals were euthanized by cervical dislocation, followed by dissection of striata on an ice-cold plate. Then, striatum was rapidly homogenized in ice-cold 50 mM Tris–HCl (pH 7.4) with a Polytron at setting six for three periods of 10 s. The homogenate was centrifuged at 1,000 × g for 10 min, and the supernatant was transferred to a new Eppendorf. The protein concentrations were determined with the BCA assay, and the membrane fractions were centrifuged at 12,000 × g for 30 min. The pellets were resuspended in assay buﬀer [20 mM MgCl2, 130 mM NaCl, 0.2 mM EDTA, 0.1 mg/mL saponin, and 0.5% immunoglobulin G (IgG)–free bovine serum albumin] to a ﬁnal protein concentration of 1.5 µg/µL. Donkey anti–guinea pig IgGs (#706-005-148; Jackson ImmunoResearch Bioluminescence Resonance Energy Transfer1 Assay The coronal brain slices of WT, D2R KO, and M1R KO mice were washed three times with PBS and permeabilized with 0.3% Triton X-100 in PBS for 2 h at RT. Then, blocking was performed by incubating the slices with washing solution (PBS with 0.05% Triton X-100) containing 5% normal donkey serum (NDS; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, United States) for 2 h at RT. Subsequently, the slices were incubated overnight at 4◦C with rabbit anti-M1R polyclonal (1:300, #mAChR-M1-Rb-Af340; Frontier Institute Co., Ltd., Shinko-nishi, Ishikari, Hokkaido, Japan) and guinea pig anti-D2R polyclonal (1:300, #D2R- GP-Af500; Frontier Institute Co., Ltd.) antibodies in washing solution with 1% NDS. In parallel, overnight incubations of WT brain slices only in washing solution served as additional negative controls. After overnight incubation, the slices were washed three times with washing solution containing 1% NDS for 10 min at RT. Next, slices were incubated with Alexa Fluor R⃝488–conjugated donkey anti–guinea pig (1:400, #706- 545-148; Jackson ImmunoResearch Laboratories) and Cy3- conjugated donkey anti–rabbit (1:400, #711-166-152, Jackson ImmunoResearch Laboratories) antibodies in washing solution with 1% NDS for 2 h at RT. Then, the slices were washed three times with washing solution for 10 min at RT and stained with 4,6-diamidino-2-phenylindole (DAPI; 1 µg/mL, #D9542; Sigma- Aldrich) for 15 min at RT. Finally, slices were washed twice with washing solution, twice with PBS for 10 min at RT, and preserved in Vectashield (#H-1000; Vector Laboratories, Burlingame, CA, United States). Images were captured with a Zeiss laser scanning microscope 880 (Carl Zeiss AG, Jena, Germany). RESULTS D2R–M1R Interaction in HEK293T Cells The ability of D2R and M1R to physically interact in living cells was assessed by biochemical and biophysical assays. First, co-immunoprecipitation experiments were performed in transiently transfected HEK293T cells. Interestingly, when HA- M1R was immunoprecipitated from FLAG-D2R and HA-M1R co-transfected HEK293T cells, a speciﬁc immunoreactive band of 90 to 100 kDa corresponding to FLAG-D2R was detected (Figure 1A, IPs). It is important to note that this band was not observed when the cells were transfected with a single receptor plus an empty plasmid or from an extract mix of separate transfected cells. Moreover, the D2R and M1R constructs were properly expressed in the whole setup (Figure 1A, lysates). These results indicate that D2R and M1R are expressed within the same membrane context and are prone to interact. Statistical Analysis The number of biological replicates (n) in each experimental condition is indicated in the ﬁgure legends. Data of behavioral studies are expressed as the mean ± SEM; all the other data are presented as the mean ± SD. Numerical data were imported to GraphPad Prism version 6.00 for Windows (GraphPad Software, La Jolla, CA, United States). Statistical analysis of cellular or tissue data was performed using the Mann–Whitney U test or the non- parametric analysis of variance (ANOVA) by ranks of Kruskal– Wallis test followed by the Dunn multiple-comparisons post hoc test. Normal distributions of the behavioral data were inferred through the D’Agostino–Pearson normality test. Subsequently, behavioral data were analyzed with the one-way ANOVA or the two-way repeated-measures ANOVA followed by the Tukey or Dunnett multiple-comparisons post hoc test. p ≤0.05 was considered as statistically signiﬁcant. Locomotor Activity Tests Mice were administered subcutaneously (s.c.) with reserpine (3 mg/kg; Sigma-Aldrich) or vehicle (saline with 5% Tween 20, s.c.) 20.5 ± 2 h before the test. Then, mice were administered with vehicle [saline with 5% dimethyl sulfoxide (DMSO) and 5% Tween 20, i.p.], sumanirole (1, 3, or 10 mg/kg, i.p.; Sigma- Aldrich) and/or VU0255035 (10 mg/kg, i.p.; Tocris Biosciences, Bristol, United Kingdom) 10 min before each locomotor activity test. The mice were evaluated for drug-induced locomotor activity as described previously (Taura et al., 2017). Brieﬂy, non-habituated mice were placed in the center of an activity ﬁeld apparatus (30 × 30 cm, surrounded by four 50-cm-high black walls) equipped with a camera above to record activity. Exploratory behavior of the animals was recorded for 85 min. The distance traveled was analyzed using the Spot tracker function from ImageJ (NIH). All locomotor activity tests were performed in a sound attenuated room, illuminated by light of 15 lux. After each trail, the apparatus was cleaned with 70% alcohol and rinsed with water. Double Immunoﬂuorescence Staining Double Immunoﬂuorescence Staining M1R KO mice were kindly provided by Dr. Adrian James Mogg (Eli Lilly and Company Ltd., Windlesham, United Kingdom) with permission of Dr. Jurgen Wess {National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes March 2020 \| Volume 11 \| Article 194 Frontiers in Pharmacology \| www.frontiersin.org 4 Striatal D2R/M1R in Parkinson’s Disease Crans et al. Laboratories) were conjugated to the acceptor beads (#6762001; Perkin Elmer, Waltham, MA, United States), according to the manufacturer’s instructions. Subsequently, 10 µL of each striatal membrane in assay buﬀer was distributed in triplicate into a white 384-well plate (384 Well Small Volume HiBase Microplates; Greiner Bio-one, Kremsmünster, Austria) and stored for 1 h at 4◦C. Subsequently, the membranes were incubated with rabbit anti-M1R polyclonal (10 nM, #mAChR-M1-Rb-Af340; Frontier Institute Co., Ltd.) and guinea pig anti-D2R polyclonal (10 nM, #D2R-GP-Af500; Frontier Institute Co., Ltd.) antibodies in assay buﬀer overnight at 4◦C. In the WT-negative controls, only the anti-M1R antibody was added, whereas the D2R KO–negative controls were incubated with assay buﬀer overnight at 4◦C. Next, acceptor beads (40 µg/mL) were added to each well for 1 h. Then, the anti–rabbit IgG alpha donor beads (40 µg/mL, #AS105D; Perkin Elmer) were added and mixed with the acceptor beads by pipetting up and down. Any prolonged light exposure was avoided. Finally, after 1-h incubation, the donor beads were excited (640–720 nm), and acceptor beads emission (597– 633 nm) was measured with the ClarioSTAR microplate reader. before the test and 22 ± 2 h after reserpine treatment. The tremulous jaw movements (TJMs) were measured with hand- operated counters, as described previously (Massari et al., 2017). Brieﬂy, the mice were placed individually in a glass cylinder (13-cm diameter) and allowed to habituate for 10 min. Mirrors were placed under and behind the cylinder to allow observation when the animal faced away from the observer. Tremulous jaw movements were deﬁned as rapid vertical deﬂections of the lower jaw that resembled chewing, but were not directed to any particular stimulus (Salamone et al., 1998). The incidence of these oral movements was measured continuously for 10 min, but were discounted during grooming. Horizontal Bar Test Catalepsy was induced in mice by the administration of reserpine (3 mg/kg, s.c.) overnight (20.5 ± 2 h). Vehicle, sumanirole (1, 3, or 10 mg/kg, i.p.) and/or VU0255035 (10 mg/kg, i.p.) was administered, and 1.5 h later, catalepsy was measured as described previously (Massari et al., 2017; Taura et al., 2017). Brieﬂy, using a stopwatch with a cutoﬀtime of 120 s, the duration of an abnormal upright posture was measured, in which the forepaws of the mouse were placed on a horizontal wooden bar (0.6-cm diameter) that was located 4.5 cm above the ﬂoor. Subsequently, the existence of D2R–M1R complexes was veriﬁed by means of BRET1 saturation assays. Accordingly, HEK293T cells were co-transfected with a constant amount of the D2R-Rluc construct and increasing concentrations of M1R- YFP or YFP plasmids (Figure 1B). A positive BRET signal was observed when D2R-Rluc and M1R-YFP were co-expressed, due to the energy transfer between Rluc and YFP. Conversely, in cells co-expressing D2R-Rluc and YFP, no BRET1 signal was observed. Overall, the BRET1 data demonstrated that D2R and Tremulous Jaw Movements The SmBiT and LgBiT parts of the NanoLuciferase fragments were fused to the C-terminus of the indicated receptor. The constructs were overexpressed via transient transfection in HEK293T cells. Results are presented as mean ± SD (n = 3). Statistical signiﬁcance was tested using the non-parametric ANOVA by ranks of Kruskal–Wallis followed by the Dunn multiple-comparisons post hoc test, p ≤0.05. M1R are in close proximity (<10 nm), thus supporting the existence of D2R–M1R complexes in living cells (Cottet et al., 2012; Dacres et al., 2012). cells expressing either M1R (19 ± 3.5) or D2R (9 ± 1.7), along with HaloTag-SmBiT or HaloTag-LgBiT, respectively. Altogether, our results are compatible with the formation of D2R/M1R heteromer formation by ectopically expressed M1R and D2R in HEK293T cells. Finally, we implemented the complementation-based NanoBiT R⃝assay to further validate the D2R–M1R interaction in HEK293T cells (Figure 1C). This assay utilizes two inactive fragments of a split NL, which, when fused to two interacting proteins, come into close proximity and reassemble into a functional protein (Wouters et al., 2019b). As shown in Figure 1C, co-expression of M1R and D2R fused to the large and small subunits of a split NL (M1R-LgBiT and D2R-SmBiT, respectively) yielded a high luminescent signal (Figure 1C) when compared to HEK293T cells expressing either constructs for M1R and CB1R (M1R-LgBiT + CB1R-SmBiT) or D2R and CB2R (D2R-SmBiT + CB2R-LgBiT), as previously reported (Wouters et al., 2019a). In addition, very low signals were observed in Tremulous Jaw Movements Mice were administered reserpine (3 mg/kg, s.c.) or vehicle (saline with 5% Tween 20, s.c.). Subsequently, vehicle (saline with 5% DMSO and 5% Tween 20, i.p.), sumanirole (1-10 mg/kg, i.p.), and/or VU0255035 (10 mg/kg, i.p.) was administered 1.5 h March 2020 \| Volume 11 \| Article 194 Frontiers in Pharmacology \| www.frontiersin.org 5 Striatal D2R/M1R in Parkinson’s Disease Crans et al. FIGURE 1 \| D2R–M1R interaction in transiently transfected HEK293T cells. (A) Co-immunoprecipitation. HEK293T cells were harvested and lysed 48 h after transfection. The lysates were used for immunoblotting (IB) with anti-FLAG and anti-HA antibodies to demonstrate D2R and M1R expression, respectively (left panels). The rest of the samples (IPs) was subjected to immunoprecipitation with a mouse anti-HA antibody. The co-immunoprecipitate was conﬁrmed via the detection of FLAG-D2R upon IB with rabbit anti-FLAG and rabbit anti-HA antibodies (right panel; boxed lane). Data shown are representative of three independent experiments. (B) BRET1 saturation curve. The BRET1 signal in HEK293T cells co-expressing a constant amount of D2R-Rluc and increasing amounts of M1R-YFP (n = 5) or YFP (n = 3) constructs was measured 48 h posttransfection. The BRET1 saturation curve is derived from all independent experiments. (C) NanoBiT R⃝ complementation assay. The SmBiT and LgBiT parts of the NanoLuciferase fragments were fused to the C-terminus of the indicated receptor. The constructs were overexpressed via transient transfection in HEK293T cells. Results are presented as mean ± SD (n = 3). Statistical signiﬁcance was tested using the non-parametric ANOVA by ranks of Kruskal–Wallis followed by the Dunn multiple-comparisons post hoc test, p ≤0.05. FIGURE 1 \| D2R–M1R interaction in transiently transfected HEK293T cells. (A) Co-immunoprecipitation. HEK293T cells were harvested and lysed 48 h after transfection. The lysates were used for immunoblotting (IB) with anti-FLAG and anti-HA antibodies to demonstrate D2R and M1R expression, respectively (left panels). The rest of the samples (IPs) was subjected to immunoprecipitation with a mouse anti-HA antibody. The co-immunoprecipitate was conﬁrmed via the detection of FLAG-D2R upon IB with rabbit anti-FLAG and rabbit anti-HA antibodies (right panel; boxed lane). Data shown are representative of three independent experiments. (B) BRET1 saturation curve. The BRET1 signal in HEK293T cells co-expressing a constant amount of D2R-Rluc and increasing amounts of M1R-YFP (n = 5) or YFP (n = 3) constructs was measured 48 h posttransfection. The BRET1 saturation curve is derived from all independent experiments. (C) NanoBiT R⃝ complementation assay. Co-distribution of D2R and M1R in the Mouse Striatum Once the existence of D2R–M1R complexes in a heterologous expressing system was demonstrated, we aimed to verify whether this interaction might also occur in native tissue. To this end, we ﬁrst analyzed D2R and M1R expression in mouse striatum by double-immunoﬂuorescence staining. The speciﬁcity of the anti-D2R and anti-M1R antibodies was veriﬁed by using striatal slices from D2R- and M1R-deﬁcient mice (D2R KO and March 2020 \| Volume 11 \| Article 194 Frontiers in Pharmacology \| www.frontiersin.org 6 Striatal D2R/M1R in Parkinson’s Disease Crans et al. M1R KO, respectively) (Supplementary Figures S1 and S2). High-magniﬁcation images of the dorsal striatum from WT mice showed a high degree of D2R and M1R co-distribution (Figure 2A, arrows). Subsequently, to further demonstrate a close proximity (<200 nm) between both receptor types, we applied an AlphaLISA R⃝immunoassay, as described previously (Fernandez- Duenas et al., 2019). Brieﬂy, striatal membrane extracts were ﬁrst incubated with speciﬁc primary antibodies against the receptor, which can be recognized by secondary antibodies tagged with beads able to engage in an energy transfer after the production of a singlet oxygen (Fernandez-Duenas et al., 2019). A signiﬁcant higher energy transfer was observed in the WT compared to its corresponding negative control [WT vs. WT (one Prim Ab); p ≤0.05, Figure 2B]. In addition, analysis of striatal D2R KO tissue did not result in a signiﬁcant diﬀerence in signal with or without adding primary antibodies (Figure 2B). These results support the existence of the interaction (or at least the very close proximity) between D2R and M1R in native tissue, namely, the mouse striatum. activity. Accordingly, we tested the eﬀects of a combined drug treatment regimen (D2R agonist + M1R antagonist) in a well- known model of movement disorder, i.e., the reserpinized mouse (Leao et al., 2015; Leal et al., 2016). The drugs used were the D2R- selective agonist sumanirole and the M1R-selective antagonist VU0255035. Sumanirole was chosen as it shows 200-fold more selectivity for D2R than for other DA receptors subtypes and as it has been used both in human patients and animal models of PD (McCall et al., 2005; Stephenson et al., 2005; Barone et al., 2007). Similarly, the competitive orthosteric antagonist VU0255035 has a 75-fold higher selectivity for M1R over other mAChR subtypes (Sheﬄer et al., 2009). In addition, both compounds have already been tested individually in reserpine-treated animals (McCall et al., 2005; Xiang et al., 2012). Co-distribution of D2R and M1R in the Mouse Striatum First, we evaluated the eﬀects of the D2R agonist sumanirole. Mice were treated with reserpine (3 mg/kg, s.c., overnight) and, thereafter, with the selective D2R agonist. Interestingly, sumanirole only promoted an increase in locomotion at the highest dose (10 mg/kg) (Supplementary Figure S3). Similarly, only at 10 mg/kg, sumanirole blocked the cataleptic eﬀects induced by reserpine, while a slight but non-signiﬁcant reduction of TJMs was observed (Supplementary Figure 3). Thus, based on these data, we selected 3 mg/kg of sumanirole (i.e., subthreshold dose) for further multimodal experiments in combination with the M1R antagonist VU0255035. A dosage of 10 mg/kg of VU0255035 was selected based both on a pilot study and its pharmacokinetic proﬁle. According to Sheﬄer et al. (2009), 10 mg/kg VU0255035 (i.p.) was suﬃcient to cross the blood- brain barrier, with maximal M1R inhibition after 30 min, with an elimination half-life of ∼2.5 h in the brain. In addition, this concentration was also reported to not impair contextual fear conditioning, a model for hippocampus-dependent learning (Sheﬄer et al., 2009). Multimodal D2R Agonist and M1R Antagonist Treatment of Reserpinized Mice The data obtained in HEK293T cells and striatal slices support the notion that D2R and M1R might physically interact in the striatum. Therefore, we hypothesized that this receptor–receptor interaction might constitute a molecular target for multimodal pharmacological interventions ﬁnely controlling striatal motor FIGURE 2 \| Co-distribution of D2R and M1R in the mouse striatum. (A) Double-immunoﬂuorescence staining. Representative images of speciﬁc immunoreactivities with anti-D2R and anti-M1R antibodies in the dorsal striatum of wild-type (WT) CD-1 mice. Overlapping immunoﬂuorescence signals are indicated with arrows. Images shown are representative of two independent experiments. Scale bar = 50 µm. (B) AlphaLISA R⃝immunoassay. Speciﬁc signal obtained from striatal WT and D2R KO mice, with or without adding one or two primary antibodies. Results are presented as mean ± SD (n = 4). Statistical signiﬁcance was tested using the Mann–Whitney U test, p ≤0.05. In animals that received the combined treatment with VU0255035 and sumanirole (VU + SUM; 10 and 3 mg/kg, i.p.) we observed a signiﬁcant (p ≤0.05) reversal of the reserpine- induced akinesia (Figure 3). In contrast, in none of the animals treated with VU or SUM alone the akinetic status was reversed (Figure 3A). Our ﬁndings suggest a ﬁne balance in locomotor activity between reserpine-induced akinesia and VU + SUM treatment. It is interesting to note that the VU + SUM– administered animals showed an increase in locomotor activity after ∼25 min, which is in accordance with the pharmacokinetic proﬁle of VU (Figure 3B). Thus, signiﬁcant diﬀerences in locomotion were observed between the VU + SUM–treated group compared to the groups receiving a single treatment. In line with the results obtained while evaluating locomotion, a signiﬁcant reduction in reserpine-induced catalepsy was observed in VU + SUM–treated mice compared to those that were administered a single agent (Figure 3C). However, while the simultaneous VU0255035 and sumanirole administration reduced TJMs as compared to vehicle, when compared to single administered animals, no diﬀerences were found with VU0255035-treated reserpinized mice (Figure 3D). Therefore, a low dose of sumanirole was unable to potentiate the VU0255035- mediated TJMs reduction. This lack of sumanirole-mediated potentiation of VU0255035 eﬀect might be due to the fact FIGURE 2 \| Co-distribution of D2R and M1R in the mouse striatum. (A) FIGURE 2 \| Co-distribution of D2R and M1R in the mouse striatum. (A) Double-immunoﬂuorescence staining. Multimodal D2R Agonist and M1R Antagonist Treatment of Reserpinized Mice Representative images of speciﬁc immunoreactivities with anti-D2R and anti-M1R antibodies in the dorsal striatum of wild-type (WT) CD-1 mice. Overlapping immunoﬂuorescence signals are indicated with arrows. Images shown are representative of two independent experiments. Scale bar = 50 µm. (B) AlphaLISA R⃝immunoassay. Speciﬁc signal obtained from striatal WT and D2R KO mice, with or without adding one or two primary antibodies. Results are presented as mean ± SD (n = 4). Statistical signiﬁcance was tested using the Mann–Whitney U test, p ≤0.05. March 2020 \| Volume 11 \| Article 194 Frontiers in Pharmacology \| www.frontiersin.org 7 Striatal D2R/M1R in Parkinson’s Disease Crans et al. FIGURE 3 \| Effect of the combinatorial treatment of suboptimal dosages of sumanirole and VU0255035 on reserpine-induced motor disturbances in mice. Mice treated with saline (control mice = Ctrl), VEH (saline with 5% Tween, i.p.), VU (VU0255035, 10 mg/kg, i.p.), SUM (sumanirole, 3 mg/kg. i.p.), or VU + SUM (VU0255035, 10 mg/kg and sumanirole, 3 mg/kg, i.p.) after reserpine administration (3 mg/kg, s.c., 20.5 ± 2 h) were evaluated via the (A,B) locomotor activity test, (C) horizontal bar test, and (D) for tremulous jaw movements (TJMs). (A) The total distance traveled (cm) was measured for 85 min. Results are presented as mean ± SEM (n = 8–9 animals). Statistical signiﬁcance was tested using one-way ANOVA followed by the Dunnett post hoc test with VEH, VU, and SUM compared to VU + SUM animals, p ≤0.05. (B) The distance traveled (cm) was measured every 5 min for 85 min. Results are presented as mean ± SEM (n = 8–9 animals). Statistical signiﬁcance was tested using two-way repeated-measures ANOVA followed by the Tukey post hoc test with VEH, VU and SUM compared to VU + SUM animals, p ≤0.05, p ≤0.01, and p ≤0.001. (C) Reserpine-induced catalepsy in mice evaluated via the horizontal bar test with a cutoff value of 120 s. Results are presented as mean ± SEM (n = 8–13 animals). Statistical signiﬁcance was tested using one-way ANOVA followed by the Tukey post hoc test with VEH, VU, and SUM compared to VU + SUM animals, p ≤0.05, *p ≤0.001, and p ≤0.0001. (D) Reserpine-induced orofacial dyskinesia evaluated by TJM frequency for 10 min. Results are presented as mean ± SEM (n = 9–13 animals). Multimodal D2R Agonist and M1R Antagonist Treatment of Reserpinized Mice Statistical signiﬁcance was tested using one-way ANOVA followed by the Tukey post hoc test with VEH, VU, and SUM compared to VU + SUM animals, p ≤0.01. FIGURE 3 \| Effect of the combinatorial treatment of suboptimal dosages of sumanirole and VU0255035 on reserpine-induced motor disturbances in mice. Mice treated with saline (control mice = Ctrl), VEH (saline with 5% Tween, i.p.), VU (VU0255035, 10 mg/kg, i.p.), SUM (sumanirole, 3 mg/kg. i.p.), or VU + SUM (VU0255035, 10 mg/kg and sumanirole, 3 mg/kg, i.p.) after reserpine administration (3 mg/kg, s.c., 20.5 ± 2 h) were evaluated via the (A,B) locomotor activity test, (C) horizontal bar test, and (D) for tremulous jaw movements (TJMs). (A) The total distance traveled (cm) was measured for 85 min. Results are presented as mean ± SEM (n = 8–9 animals). Statistical signiﬁcance was tested using one-way ANOVA followed by the Dunnett post hoc test with VEH, VU, and SUM compared to VU + SUM animals, p ≤0.05. (B) The distance traveled (cm) was measured every 5 min for 85 min. Results are presented as mean ± SEM (n = 8–9 animals). Statistical signiﬁcance was tested using two-way repeated-measures ANOVA followed by the Tukey post hoc test with VEH, VU and SUM compared to VU + SUM animals, p ≤0.05, p ≤0.01, and p ≤0.001. (C) Reserpine-induced catalepsy in mice evaluated via the horizontal bar test with a cutoff value of 120 s. Results are presented as mean ± SEM (n = 8–13 animals). Statistical signiﬁcance was tested using one-way ANOVA followed by the Tukey post hoc test with VEH, VU, and SUM compared to VU + SUM animals, p ≤0.05, *p ≤0.001, and p ≤0.0001. (D) Reserpine-induced orofacial dyskinesia evaluated by TJM frequency for 10 min. Results are presented as mean ± SEM (n = 9–13 animals). Statistical signiﬁcance was tested using one-way ANOVA followed by the Tukey post hoc test with VEH, VU, and SUM compared to VU + SUM animals, p ≤0.01. this functional interplay grounded the utility of A2AR blockade in PD treatment, which recently ended with the approval of a selective A2AR antagonist, istradefylline (Nourianz), as an adjuvant drug in PD treatment. Multimodal D2R Agonist and M1R Antagonist Treatment of Reserpinized Mice Interestingly, while a variety of D2R oligomer complexes has been described (Marcellino et al., 2008; Triﬁlieﬀet al., 2011; Bonaventura et al., 2014; Borroto- Escuela et al., 2014; Hasbi et al., 2017), few studies exist for M1R (Goin and Nathanson, 2006; Hern et al., 2010). In the present study, we have observed, for the ﬁrst time, the existence of striatal D2R and M1R complexes. In addition, we provide data supporting a novel multimodal antiparkinsonian treatment, consisting of the use of low D2R agonist doses in combination with M1R antagonists. Thus, our results may prompt further investigating these receptor complexes as interesting targets to modulate dopaminergic neurotransmission in dopamine-related diseases (i.e., PD) (Hersch et al., 1994; Surmeier et al., 2007; Fuxe et al., 2012). that 10 mg/kg VU0255035 already induced a slight, but not signiﬁcant (p = 0.1712), reduction in TJMs. Of course, it would be reasonable to speculate that M1Rs located within neuronal circuits controlling distinct behavioral responses might have diﬀerent eﬃcacies. Overall, our data support the use of low D2R agonist doses in combination with an M1R antagonist as a novel multimodal antiparkinsonian pharmacotherapy. Frontiers in Pharmacology \| www.frontiersin.org DISCUSSION In the last years, G protein-couped receptor (GPCR) oligomers have gained interest as novel putative targets for several diseases. One of the most well-characterized D2R-containing oligomers is the D2R/A2AR heteromer in the striatum, where reciprocal antagonistic interactions both at the binding and eﬀector levels occur between these receptors (Ferré et al., 2018). Importantly, In the last years, G protein-couped receptor (GPCR) oligomers have gained interest as novel putative targets for several diseases. One of the most well-characterized D2R-containing oligomers is the D2R/A2AR heteromer in the striatum, where reciprocal antagonistic interactions both at the binding and eﬀector levels occur between these receptors (Ferré et al., 2018). Importantly, March 2020 \| Volume 11 \| Article 194 Frontiers in Pharmacology \| www.frontiersin.org 8 Striatal D2R/M1R in Parkinson’s Disease Crans et al. As described for the D2R/A2AR heteromer, both M1R and D2R are expressed at postsynaptic membranes of striatopallidal MSNs. Thus, the avidity of D2R to heteromerize with a named GPCR (i.e., A2AR, mGlu5R or M1R) within this speciﬁc subcellular domain may depend on the absolute expression of speciﬁc protomers and the relative aﬃnities shown for each receptor–receptor interaction. Importantly, the density of each individual D2R containing oligomer may be altered in disease conditions, which may constitute a putative pathological ﬁngerprint. Precisely, we recently reported that D2R/A2AR heteromers would be increased in the caudate from human postmortem PD patients (Fernandez-Duenas et al., 2019). This fact would negatively aﬀect dopaminergic neurotransmission, thus providing the rationale for using A2AR antagonists in PD (see above). Of note, whether the decrease in heteromer formation is a cause or a consequence of PD pathology, or even treatment, needs to be further elucidated. Here, we demonstrated the existence of D2R–M1R complexes in the striatum and its potential pharmacotherapeutic usefulness using an animal model of PD. However, further studies should be conducted to determine: (i) D2R/M1R heteromer status in human PD striatum (i.e., increase or decrease in the proportion of D2R and M1R protomers forming homomers or heteromers) and (ii) the molecular and functional interplay with other striatal D2R-containing oligomers (i.e., D2R/A2AR heteromers). Certainly, establishing the D2R-containing heteromer status in PD could determine the design of selective combined pharmacotherapeutic strategies restoring the unbalanced dopaminergic neurotransmission associated with PD. our results, using a suboptimal concentration of sumanirole, are not in line with the ﬁndings of another study, which also used a reserpine animal model (McCall et al., 2005). DISCUSSION The discrepancy could be owing to diﬀerences in species (mice vs. rats), reserpine inductions (3 mg/kg vs. 5 mg/kg + AMPT), administration routes of sumanirole (i.p. vs. s.c.), and/or time of reserpine pretreatments (20.5 vs. 18 h). Nevertheless, a long-term eﬀect in locomotion at high sumanirole doses was demonstrated in both studies, which has been suggested to be the result of postsynaptic D2R activation (McCall et al., 2005). On the other hand, the administered dose of the competitive orthosteric M1R antagonist VU0255035 results in maximal receptor inhibition, with a high brain penetration after 30 min, without impairment in hippocampus-dependent learning tasks (Sheﬄer et al., 2009). The combined treatment increased locomotor activity and decreased the time of catalepsy and the amount of TJMs in our animal model, whereas the reduction in TJMs was mostly due to the M1R antagonist (Lees, 2005; Pedrosa and Timmermann, 2013). ) The dysregulation of dopaminergic or cholinergic systems has been linked to movement disorders, such as dystonia, Huntington disease, or PD (Pisani et al., 2007). Nowadays, at the early stages, PD therapy is commonly initiated with D2R agonists, which do not require carrier-mediated transport or produce potentially toxic metabolites and free radicals (Hagan et al., 1997; Jenner, 2003). However, D2R agonists may elicit severe adverse eﬀects such as valvular heart disease or psychiatric disturbances (Lees, 2005; Hisahara and Shimohama, 2011; Pedrosa and Timmermann, 2013), which are probably induced by activating D3Rs and D4Rs (Rich et al., 1995; McCall et al., 2005). Despite its high D2R selectivity, sumanirole has not demonstrated a clinical improvement over ropinirole (Barone et al., 2007; Singer et al., 2007). However, as suggested by the present study, sumanirole remains a valuable tool in lead optimization, drug discovery, and animal models, where the novel D2R–M1R interaction may provide a rationale to target speciﬁc receptor subtypes in the treatment of PD. In addition, reducing the amount of D2R agonist by supplementing an M1R selective antagonist (i.e., VU0255035) in a multimodal pharmacological approach may allow achieving an eﬀective treatment and induce less adverse eﬀects. In our study, the functional interplay between D2R and M1R was demonstrated by the co-administration of a D2R agonist and an M1R antagonist to reserpinized mice, which is an animal model mimicking parkinsonian motor and non-motor impairments (Leao et al., 2015; Leal et al., 2016). The major disadvantage of this model is the lack of dopaminergic neurons degeneration and protein aggregation. Frontiers in Pharmacology \| www.frontiersin.org SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphar.2020. 00194/full#supplementary-material The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphar.2020. 00194/full#supplementary-material FIGURE S1 \| Validation of the anti-D2R and anti-M1R antibodies via double immunoﬂuorescence staining in mice brains. Images of coronal slices from mice brains representing the dorsal striatum, corpus callosum and cortex with staining of D2R-positive cells (green), M1R-positive cells (red) and DAPI-positive nuclei (blue). Minimal signal intensities were observed with the anti-D2R and anti-M1R antibodies in the D2R and M1R KO mice, respectively. Data shown are representative of two independent experiments. Scale bar = 100 µm. FIGURE S1 \| Validation of the anti-D2R and anti-M1R antibodies via double immunoﬂuorescence staining in mice brains. Images of coronal slices from mice brains representing the dorsal striatum, corpus callosum and cortex with staining of D2R-positive cells (green), M1R-positive cells (red) and DAPI-positive nuclei (blue). Minimal signal intensities were observed with the anti-D2R and anti-M1R antibodies in the D2R and M1R KO mice, respectively. Data shown are representative of two independent experiments. Scale bar = 100 µm. ACKNOWLEDGMENTS The authors thank Dr. Benjamín Torrejón-Escribano, from the CCiT-UB from Bellvitge Campus of the University of Barcelona, for the technical support during the confocal microscopy imaging. In conclusion, here we demonstrated, for the ﬁrst time, an interaction between D2R and M1R. Interestingly, our results suggest an extensive integration of dopaminergic and cholinergic neurotransmission systems in the striatum, where inhibition by DA is predicted to facilitate locomotor activity, and activation by ACh inhibits locomotion via striatopallidal MSNs (Di Chiara et al., 1994). Using reserpinized mice as a model, we demonstrated the eﬀectiveness of a multimodal treatment, combining a suboptimal dosage of the selective D2R agonist sumanirole and the M1R-speciﬁc antagonist VU0255035. Overall, further functional exploitation of this novel D2R–M1R interaction (i.e., identifying the functional ﬁngerprint of this putative new heterodimer in native tissue) may provide beneﬁcial opportunities in PD treatment. DATA AVAILABILITY STATEMENT All datasets generated for this study are included in the article/Supplementary Material. FIGURE S2 \| Validation of the anti-D2R and anti-M1R antibodies via Western Blotting. (A) The anti-D2R antibody used in our study demonstrates speciﬁcity for D2R in striatal tissue. Extracts of the striatum from D2R KO, D2R heterozygous (HET), and wild-type (WT) CD-1 littermates were loaded on 10% SDS-PAGE. The anti-α-Tubulin antibody was used to control for equal loading of the samples. (B) The anti-M1R antibody used in the study demonstrates speciﬁcity for M1R in striatal tissue. Striatal extracts from M1R KO and wild-type (WT) with C57BL/6J background were loaded on 10% SDS-PAGE. The anti-α-Tubulin antibody was used to control for equal loading of the samples. kDa = kilodalton. DISCUSSION We thank Centres de Recerca de Catalunya (CERCA) Programme/Generalitat de Catalunya for IDIBELL institutional support. RC was also supported by an EMBO Short-Term Fellowship (Grant number 6735) and an FWO Travel Grant for a Long Stay Abroad (Grant number V420718N). antagonizing M1R mainly has an excitatory eﬀect on GABAergic MSNs, but no or only a partial eﬀect at the subthalamic nucleus and substantia nigra pars reticulata (Xiang et al., 2012; Lv et al., 2017). Interestingly, mice lacking M1R have increased locomotor activity (Gerber et al., 2001; Miyakawa et al., 2001). These M1R KO mice also have increased extracellular dopamine levels in the striatum, which suggests that inhibiting M1R positively aﬀects PD treatment (Gerber et al., 2001). Moreover, M1R KO mice were shown to maintain contextual fear recognition, which indicates that M1R might not be involved in the initial stability of memory or in its formation in the hippocampus (Miyakawa et al., 2001; Anagnostaras et al., 2003). Accordingly, the main beneﬁt to target M1R over other mAChRs is due to its selective role in controlling locomotor activity, whereas its input is less critical for cognitive processes (Miyakawa et al., 2001). la Marató de TV3 (Grant number 20152031), Ministerio de Ciencia, Innovación y Universidades–Agencia Estatal de Investigación/FEDER (SAF2017-87349-R) and Generalitat de Catalunya (2017 SGR 1604 and 2017 SGR 595). We thank Centres de Recerca de Catalunya (CERCA) Programme/Generalitat de Catalunya for IDIBELL institutional support. RC was also supported by an EMBO Short-Term Fellowship (Grant number 6735) and an FWO Travel Grant for a Long Stay Abroad (Grant number V420718N). ETHICS STATEMENT The animal study was reviewed and approved by the Ethical Committee on Animal Use and Care of the University of Barcelona (CEEA/UB). FIGURE S3 \| Sumanirole dosage-response of reserpine-induced motor disturbances in mice. The mice were treated with VEH (saline and 5% Tween, i.p.), or 1, 3, or 10 mg/kg SUM (sumanirole, 1, 3, 10 mg/kg, respectively, i.p.) after reserpine administration (3 mg/kg, s.c., 20.5 ± 2 h), and evaluated via the (A,B) locomotor activity test, (C) horizontal bar test and (D) for tremulous jaw movements (TJMs). (A) The total distance traveled (cm) was measured for 85 min. Results are presented as mean ± SEM (n = 7–8 animals). Statistical signiﬁcance was tested using one-way ANOVA, followed by the Dunnett post hoc test, with VEH, 1 SUM, and 3 SUM compared to 10 SUM animals, ∗∗p ≤0.01. (B) The distance traveled (cm) was measured every 5 min for 85 min. Results are presented as mean ± SEM (n = 7–8 animals). Statistical signiﬁcance was tested using two-way repeated-measures ANOVA followed by the Tukey post hoc test, with VEH, 1 SUM, and 3 SUM compared to 10 SUM animals, ∗p ≤0.05, ∗∗p ≤0.01, ∗∗∗p ≤0.001 and ∗∗∗∗p ≤0.0001. (C) Reserpine-induced catalepsy in mice evaluated via the horizontal bar test, with cut-off value of 120 s. Results are presented as mean ± SEM (n = 7–8 animals). Statistical signiﬁcance was tested using one-way ANOVA followed by the Tukey post hoc test, ∗∗p ≤0.01. (D) Reserpine-induced orofacial dyskinesia evaluated by TJMs for 10 min. Results are presented as mean ± SEM (n = 7–8 animals). Statistical signiﬁcance was tested using one-way ANOVA followed by the Tukey post hoc test, ∗∗p ≤0.01. AUTHOR CONTRIBUTIONS RC performed and designed the experiments, analyzed the data and wrote the manuscript. EW performed the NanoBiT R⃝ assay, MV-L performed the AlphaLISA R⃝assay. JT and CM performed in vivo experiments. VF-D designed the experiments and wrote the manuscript. CS supervised the project and wrote the manuscript. FC supervised the project, designed experiments and wrote the manuscript. DISCUSSION Nevertheless, reserpine- treated rodents have been successfully applied to predict the eﬃcacy of many dopaminergic and non-dopaminergic drugs (e.g., benzotropine), which are clinically in use for PD management. The high predictive validity of this model results in the maintenance of its position as a valid choice to discover novel therapeutics in an early preclinical stage (Duty and Jenner, 2011). Other advantages are its low toxicity, low cost, and its reproducibility among laboratories (Leao et al., 2015). Furthermore, the reserpine animal model was one of the ﬁrst models used to demonstrate the therapeutic eﬃcacy of L-DOPA, which still remains the criterion standard in PD therapy (Carlsson et al., 1957). Now, D2R selective agonists are also included in the pharmacotherapeutic munition in PD management. Of note, although the full D2R agonist sumanirole has a high aﬃnity for D2R, it also has a moderate aﬃnity for the serotonin 5-HT1A receptor (Ki = 95 nM) (Heier et al., 1997; Wuts, 1999; McCall et al., 2005). However, according to Weber et al. (2010), the suboptimal sumanirole concentration applied in our study should not result in 5-HT1A receptor oﬀ- target eﬀects (Weber et al., 2010). It is worth mentioning that Muscarinic acetylcholine receptors play important roles in cognitive, motor, behavioral, sensory, and autonomic processes. Thus, non-selective blockade of mAChRs is associated with important side eﬀects, including cognitive deﬁcits. While scopolamine, a non-selective mAChR antagonist, robustly increased locomotor activity in reserpinized akinetic rats, it induced learning and memory impairments (Sheﬄer et al., 2009; Xiang et al., 2012). Importantly, most cognitive adverse eﬀects observed with anticholinergic therapies are likely due to the result of M2R and M3R blockade (Fornari et al., 2000; Wess et al., 2007). Conversely, the selective M1R blockade has been shown to exhibit some antiparkinsonian activity, although without the full eﬃcacy as observed with non-selective anticholinergics (Xiang et al., 2012; Lv et al., 2017; Chambers et al., 2019). This is probably due to activation of other mAChRs, which also have important roles in the motor circuits of the basal ganglia (e.g., M4R). Indeed, March 2020 \| Volume 11 \| Article 194 Frontiers in Pharmacology \| www.frontiersin.org 9 Striatal D2R/M1R in Parkinson’s Disease Crans et al. la Marató de TV3 (Grant number 20152031), Ministerio de Ciencia, Innovación y Universidades–Agencia Estatal de Investigación/FEDER (SAF2017-87349-R) and Generalitat de Catalunya (2017 SGR 1604 and 2017 SGR 595). REFERENCES Dexter, D. T., and Jenner, P. (2013). 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Involvement of striatal cholinergic Interneurons and M1 and M4 muscarinic receptors in motor symptoms of Parkinson’s disease. J. Neurosci. 36, 9161–9172. doi: 10.1523/JNEUROSCI.0873-16.2016 Frontiers in Pharmacology \| www.frontiersin.org REFERENCES J., Saini, D. K., and Stove, C. P. (2019b). Luminescence- and ﬂuorescence-based complementation assays to screen for GPCR oligomerization: current state of the art. Int. J. Mol. Sci. 20:2958. doi: 10.3390/ijms20122958 Pedrosa, D. J., and Timmermann, L. (2013). Review: management of Parkinson’s disease. Neuropsychiatr. Dis. Treat. 9, 321–340. doi: 10.2147/NDT.S32302 Wuts, P. G. (1999). Synthesis of PNU-95666E. Curr. Opin. Drug Discov. Devel. 2, 557–564. Pisani, A., Bernardi, G., Ding, J., and Surmeier, D. J. (2007). Re-emergence of striatal cholinergic interneurons in movement disorders. Trends Neurosci. 30, 545–553. doi: 10.1016/j.tins.2007.07.008 Xiang, Z., Thompson, A. D., Jones, C. K., Lindsley, C. W., and Conn, P. J. (2012). Roles of the M1 muscarinic acetylcholine receptor subtype in the regulation of basal ganglia function and implications for the treatment of Parkinson’s disease. J Pharmacol Exp Ther 340 595 603 doi: 10 1124/jpet 111 187856 Rich, S. S., Friedman, J. H., and Ott, B. R. (1995). Risperidone versus clozapine in the treatment of psychosis in six patients with Parkinson’s disease and other akinetic-rigid syndromes. J. Clin. Psychiatry 56, 556–559. J. Pharmacol. Exp. Ther. 340, 595–603. doi: 10.1124/jpet.111.187856 March 2020 \| Volume 11 \| Article 194 Frontiers in Pharmacology \| www.frontiersin.org 12 Striatal D2R/M1R in Parkinson’s Disease Crans et al. Conﬂict of Interest: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Copyright © 2020 Crans, Wouters, Valle-León, Taura, Massari, Fernández-Dueñas, Stove and Ciruela. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. March 2020 \| Volume 11 \| Article 194 Frontiers in Pharmacology \| www.frontiersin.org 13
https://openalex.org/W4301457302	https://hal.archives-ouvertes.fr/hal-01865363/file/Liot_20759.pdf	English	null	Pore cross-talk in colloidal filtration	HAL (Le Centre pour la Communication Scientifique Directe)	2,017	cc-by	6,136	To cite this version: Olivier Liot, Akash Singh, Patrice Bacchin, Paul Duru, Jeffrey Morris, et al.. Pore cross-talk in colloidal filtration. Scientific Reports, 2018, 8 (12460), pp.1-7. 10.1038/s41598-018-30389-7. hal- 01865363 HAL Id: hal-01865363 https://hal.science/hal-01865363v1 Submitted on 31 Aug 2018 Submitted on 31 Aug 2018 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Open Archive Toulouse Archive Ouverte Open Archive Toulouse Archive Ouverte Open Archive Toulouse Archive Ouverte Open Archive Toulouse Archive Ouverte OATAO is an open access repository that collects the work of Toulouse researchers and makes it freely available over the web where possible This is an author’s version published in: http://oatao.univ-toulouse.fr/20759 ǦƤ Olivier Liot ͷǡ͸ǡͼǡͷǡBa͹ǡͺǡ ơ ǤMorrisͻ & i ͷ ǡƤǤ Ƥ Ǧǡƪ ƤǤ ȋȌǡ ǲǦǳǤ ǡơ ǡǤǦǦ Ǥ Received: 23 April 2018 Accepted: 27 July 2018 Published: xx xx xxxx A colloidal suspension fowing through a pore network ofen results in fouling or clogging. In industrial (oil recovery1, inkjet printing2, fltration), biological (artery diseases3, detection of cells4) and natural (water infltra- tion in soils5, precipitation inside rocks6) processes, the phenomenon of particle accumulation is involved. Recent improvements in visualization of suspended particles in model pores have led to new insight into the physical parameters at play in particle capture and clogging in pores7. Tere are several diferent clogging mechanisms. Size exclusion or sieving occurs when particles block a pore smaller than their diameter8. If the pore size is larger than the particle, clogging can occur by two routes, either through particles forming an arch at the entrance of the pore9 or progressively adhering to walls and previously deposited particles, leading to blockage of the pore10,11. During the last decade, following an early study10 which described clogging of pores by smaller particles, a num- ber of studies have focused on determining the pore-scale mechanisms involved in this form of pore blockage (e.g.12–17). Other studies have proposed explanations of clogging using transition-state theory18 or by relating it to jamming phenomena19. j g p Prior to the advent of pore-scale investigations, which have been greatly facilitated by microfuidic technology, numerous studies were made at a more macroscopic membrane scale, where the usual focus was on the “fltration cake”20–24. Since a typical fltration membrane consists of a large number of closely-spaced pores, clog formation at one pore could afect its neighbours, and hence the macroscopic behavior of the membrane. Considered in this way, there is a notable lack of information related to clog formation at the pore scale, with connection to the membrane scale by consideration of interactions between pores. In this work, we address this gap of knowledge at an intermediate scale, by considering in detail the time evolution of the clog formation process at pore scale, in a short one-dimensional (1-D) array of pores. We describe the interaction between pores as “cross-talk”. ǦƤ While one recent paper18 shows that a fltration cake can overhang neighbouring pores and infuence the clog formation, there is, to our knowledge, no direct analysis of the pore cross-talk phenomenon. Yet it could have a dramatic impact on the understanding of fltration process of suspensions at macroscale, such as possible preferential locations of cake formation. In this work, we present observations of cross-talk when a Brownian suspension fows through a 1-D microfl- tration device. Te fow is driven by a fxed pressure diference, not a fxed fow rate, and this is a key point of our study. We measure a clogging growth rate as a function of the number of already clogged pores and we propose a model based on a local increase of colloid concentration close to clogged pores to explain the observations. To cite this version: Any correspondence concerning this service should be sent to the repository administrator: tech-oatao@listes-diff.inp-toulouse.fr ǤǤȀ Ten nanoslits of width w = 5 µm, length L = 50 µm, depth h = 830 nm, and center-to-center spacing δ = 20 µm are etched in silicon. Tese nanoslits connect much larger inlet and outlet rectangular microchannels acting as res- ervoirs (depth 23 µm, width 100 µm and millimetric in the third direction). Te device is covered with a 170 µm -thick borosilicate glass plate. Te design is presented in Fig. 1 (lef) which shows that the nanoslits connect ͷLAAS-CNRS, Université de Toulouse, CNRS, Toulouse, France. ͸Fédération FERMaT, INP, Toulouse, France. ͹Laboratoire de Génie Chimique, Université de Toulouse, CNRS, Toulouse, France. ͺInstitut de Mécanique des Fluides de Toulouse, Université de Toulouse, CNRS, Toulouse, France. ͻLevich Institute and Chemical Engineering, CUNY City College of New York, New York, USA. ͼPresent address: Institut Lumière Matière, CNRS, Villeurbanne, France. Correspondence and requests for materials should be addressed to O.L. (email: olivier.liot@ens-lyon.fr) SCIENTIFIC REPORTS \| (2018) 8:12460 ȁǣͷͶǤͷͶ͹;ȀͺͷͻͿ;ǦͶͷ;Ǧ͹Ͷ͹;ͿǦͽ 1 www.nature.com/scientificreports/ Figure 1. Sketch of the model pores and micrograph of some clogs. Lef: top view of the chip design with zoom on nanoslits. Microchannels are represented in blue, nanoslits in red. Inset: side view of the nanoslits (not to scale). Right: image of the development of three adjacent clogs at the entrance of pores. Red lines delimit the nanoslits. For (a) to (f) the corresponding times are t = 1333, 2000, 2666, 3000, 3333, 4000 s. Figure 1. Sketch of the model pores and micrograph of some clogs. Lef: top view of the chip design with zoom on nanoslits. Microchannels are represented in blue, nanoslits in red. Inset: side view of the nanoslits (not to scale). Right: image of the development of three adjacent clogs at the entrance of pores. Red lines delimit the nanoslits. For (a) to (f) the corresponding times are t = 1333, 2000, 2666, 3000, 3333, 4000 s. Figure 2. Time evolution of clogs area during a single acquisition. Drawn lines highlight the zone where clog growth rate is estimated. Figure 2. Time evolution of clogs area during a single acquisition. Drawn lines highlight the zone where clog growth rate is estimated. corners of the cross-section of the microchannels. Te channels are flled with a suspension of dp = 250 nm diam- eter polystyrene particles (density 1.05 g.cm−3). Te particles are carboxylate-modifed and dispersed in a solu- tion of monovalent phosphate bufered saline (PBS) diluted to an ionic strength I = 3 mM. Te zeta potential ζp is measured by laser Doppler electrophoresis, ζp = −69 mV (pH = 7.5). Te volume fraction of the suspension is φ0 = 3.8 × 10−5. A pressure diference of ∆P = 20 ± 0.02 mbar is applied across the length of the nanoslits using a controller device. Experiments are made in dead-end and slow crossfow fltration (with velocity 0 to 9 µm/s at 2 µm from the entrance of the pores). p Te clogging dynamics are observed using wide feld fuorescence microscopy with a 40× magnifcation and 1.4 numerical aperture objective. Since the characteristic time for clogging is found to be about one hour we acquire images of the clog growth process at a frequency of 90 frames per minute. Figure 1 (right) shows an example of the development of three adjacent clogs. Te contour of the aggregated particle mass is detected using custom Python scripts. From this contour analysis, we are able to determine the projected area of each clog in the feld of view. An experimental difculty is caused by the very low fow rate involved: the total fow rate through the ten pores before clogging is about 5 nL.min−1, well below the sensitivity limit of commercial fow rate sensors. Tracking of particles inside nanoslits while acquiring clog development is also technically difcult. Timescales are indeed very diferent (20 ms is the typical residence time of particles in the nanoslits versus a typical clog growth of one hour). Moreover the brightness of one single particle compared to a clog of hundreds of colloids makes hard to capture simultaneously individual colloids and clogs. Perfect watertight ftting of the chip to the pressure controller must be ensured. Also, the chips can ofen not be retrieved afer an experiment: a clogged chip is ofen discarded. We note that this balance between transport mechanisms is the one classically put forward to explain the exist- ence of a stationary concentration polarization layer in fltration of colloidal suspensions25,26. An analogous equi- librium (fuid fow-induced drag forces vs difusiophoretic fow-induced ones) has been observed recently27. Note that in the case of an experiment performed with a fxed fow rate (in contrast with the present fxed pressure drop confguration), no saturation of the clog size would be observed: particles would continue to accumulate indefnitely on the clog18. y g In the present paper, neither a precise description of the clogging dynamics at the pore scale nor a quantitative description of the saturation mechanisms are the objectives of the study (note studies of clogging at pore scale in conditions similar to that of the experiments have already been performed11,12,14). We rather focus on the follow- ing observation: when a clog begins its rapid growth afer other pores have reached saturation, the growth rate is larger: e.g., compare the green and black curves in Fig. 2. Te goal of the paper is to describe quantitatively and to model this observation. We quantitatively defne clog growth rate (with dimensions of area/time) as the average of the derivative of the measured area with time in the zone starting from the beginning of the fast growth part where its evolution is linear, see Fig. 2 (more details on the data processing are given as Supplementary materials 1). We made eight acquisitions, totaling 80 growth rate measurements. We defne the mean growth rate when N pores are saturated (i.e. clog size has reached saturation) as ⁎ vN. We have N ∈ [0, Ntot − 1] where Ntot is the total number of pores. Note that during an experiment, two or more pores may start to clog nearly simultaneously. Consequently, for a given experiment, all values of N are not necessarily observed. As an example, if the two frst pore-clogging events are simultaneous, the data for the third one will count as an N = 2 event and such an experiment lacks a N = 1 event. Figure 3 displays the ratio ⁎ ⁎ v v / N 0 as a function of N. Ǥ Figure 2 shows an example of the time evolution of the area of aggregated particles at each of the pore entrances in a single microfuidic chip. Te curves all display the same characteristic shape. Afer an initial time period where the curves are quite uneven, each shows a rapid quasi-linear growth up to a saturation level. In the present experiment, pore clogging is mainly initiated by the capture of particle SCIENTIFIC REPORTS \| (2018) 8:12460 ȁǣͷͶǤͷͶ͹;ȀͺͷͻͿ;ǦͶͷ;Ǧ͹Ͷ͹;ͿǦͽ 2 www.nature.com/scientificreports/ Figure 3. ⁎ ⁎ v v / N 0 ratio versus N. Te error bars are related to statistical uncertainty (standard deviation over root square of the number of events); dashed points are low statistic points (less than three events). Te diferent lines show the predictions of Eq. 3, with diferent ways of computing 〈k〉N, see legend for details. Figure 3. ⁎ ⁎ v v / N 0 ratio versus N. Te error bars are related to statistical uncertainty (standard deviation over root square of the number of events); dashed points are low statistic points (less than three events). Te diferent lines show the predictions of Eq. 3, with diferent ways of computing 〈k〉N, see legend for details. aggregates. Tis is not surprising as the ratio nanoslit height/particle diameter is only 3.3. Also, the presence of small aggregates in the suspension cannot be ruled out even if care is taken to prevent the aggregate presence (by sonicating the suspension prior to its use). Te larger of these aggregates can sometimes be identifed on the images, at the nanoslit entrance, once they have been captured. Aggregates partially obstruct the pore and then initiate the slow clog growth sometimes visible at the beginning of the clogging, see e.g. the black and blue data points in Fig. 2. When the pore is fully blocked, all the particles are sieved from the fow and most are captured on the aggregate (some may move laterally) so that the clog begins its fast-growth phase. Te saturation of the clog is apparently due to a balance between drag (note that the fow rate through a pore decreases when the clog grows, leading to a decrease of the drag force exerted on the particles) and the combination of double layer repulsion and Brownian difusion, resulting in a zero particle fux surface, similar to the situation described by Bacchin et al.12. Despite some large error bars, a clear increase of ⁎ ⁎ v v / N 0 is observed when the number of saturated pores increases, from = ⁎ ⁎ v v / 1 N 0 for N = 0 to ≈ ⁎ ⁎ v v / 3 N 0 for N = 9. As already mentioned, explaining this increase of ⁎ ⁎ v v / N 0 with N is the main goal of this paper and we now propose a phenomenological model. Ǥ Clog growth rate is proportional to the fow rate through a free pore Qf and the local concentration of particles10. Two mechanisms can explain the growth rate rise: particle concentration increase and/or fow rate increase. In the present fxed pressure-drop confguration, we assume that the fow rate through a free pore, Qf, remains constant as long as clogging did not begin and that a saturated clog acts as a flter, with fuid permeating through it at a fow rate Qs < Qf. SCIENTIFIC REPORTS \| (2018) 8:12460 ȁǣͷͶǤͷͶ͹;ȀͺͷͻͿ;ǦͶͷ;Ǧ͹Ͷ͹;ͿǦͽ 3 www.nature.com/scientificreports/ Figure 4. Lef: image of the development of four adjacent polymersome clogs at the entrance of pores. Red lines delimit the nanoslits. For (a) to (f) the corresponding times are t = 0, 14, 28, 42, 56, 70 s. Right: Sliding average of instantaneous longitudinal velocities of multiple polymersomes fowing through each nanoslit versus time. Nanoslits numbers correspond to numbers written on the lef fgure (f). Figure 4. Lef: image of the development of four adjacent polymersome clogs at the entrance of pores. Red lines delimit the nanoslits. For (a) to (f) the corresponding times are t = 0, 14, 28, 42, 56, 70 s. Right: Sliding average of instantaneous longitudinal velocities of multiple polymersomes fowing through each nanoslit versus time. Nanoslits numbers correspond to numbers written on the lef fgure (f). In the present experiment, an estimate of the fow rate within a free pore cannot be obtained easily (see Experimental methods). However, we now present some data from another experiment supporting the above assumptions. Tese experiments are performed in micro-system similar to those used in the present study (only two dimensions of the nanoslits change: 1650 nm in height and 10 µm in width), where a dilute suspension of self-assembled objects made of polymers (polymersomes) is fowed through the nanoslits (for more details see Liot et al.28). Tese polymersomes can be tracked and their instantaneous longitudinal velocities recorded. Because of the polydispersity of such objects, some of them are larger than the nanoslits height and can clog them quickly, as presented in Fig. 4 (lef). Figure 4 (right) shows a sliding average of the instantaneous longitudinal velocities vy of tracked polymersomes fowing through each nanoslit. Two diferent behaviors are observed: a strong decrease of the velocity (and thus of the fow rate) for nanoslits 2 and 3 and a less pronounced decrease for nanoslit 2 and 4, for which clogging is not as rapid. Performed with a similar fow control than the present experiments, these data show that an increase of the fow rate can be ruled out in the present experiments with colloids. Furthermore the nanoslit 4 reveals constant polymersomes longitudinal velocity up to t = 35 s, when clogging becomes dramatic. Whereas the neighbouring pore (nanoslit 3) is blocked, no decrease of the fow rate in nanoslit 4 can be observed. We conclude that Qf remains constant in a free pore during the fltration process. Since an increase of fow rate is excluded, the main idea of the present model is that the particles driven by the permeation fow close to a clogged pore will be “redistributed” along the membrane surface to fow through open pores, leading to a particle concentration increase close to a free pore. p g p p A possible mechanism to explain this redistribution is Brownian difusion. Te typical time scale t* between two successive pore clogging events is about 100 s (see Fig. 2). Te difusion coefcient of the particles at ambient temperature is29 D = 1.7 × 10−12 m2.s−1. Teir typical displacement during this time interval in one direction is δ µ = = ⁎ x Dt 2 18 m. Tis typical length scale rises to about 60 µm when considering a typical experiment duration, '(1000s). Terefore, particle difusive redistribution is expected to be a relevant mechanism in the present experiments. Note these estimates are obtained by using the difusion coefcient for an isolated particle. Close to a clog (∼100 nm), one may certainly expect difusive transport to be afected by the particle-particle interactions, but this is not the case regarding the difusive transport along the membrane, from a saturated to a free pore, where the particle concentration is expected to be close to the bulk concentration φ0. Also, convective efects are expected to play a role. For instance, the fow rate decrease in pores with a saturated clog will lead to thinner stream tubes “feeding” these pores, and to larger ones for free pores. And it is enough for the particles to difuse until they are “caught by” a stream tube fowing into a free pore. Secondary fows induced by clog spatial extension over the membrane could also have a similar efect, providing a convective component to the redistri- bution mechanism. However, as made clear below, it is crucial to understand that the model described later is not afected by the details of the redistribution mechanism. y Tis redistribution of particles is limited: all the particles redistributed from a saturated pore will be “sucked down” by the frst free pores (on both sides of the saturated pore). In fact, in the case of a difusive redistribution mechanism, advection of a particle through an open pore is much stronger than the difusive transport away from it. Tis can be appreciated by building a Péclet number using the particle diameter and a typical fow velocity U: πη = Pe d U k T 3 / p B 2 . A typical velocity within the nanoslit can be taken as Uf = Qf/(hw), where Qf is computed from velocity feld through a channel with rectangular cross-section with ∆P = 20 mbar30. Away from the nano- slit, a typical velocity in the flow stream that will end up flowing through the nanoslit can be estimated as Uf × (h × w)/(δ × 23 µm), which is ≈Uf/50. Te Péclet is fnally in the range 6–300, taking the typical velocity U in the range Uf/50 − Uf, showing that a particle will not be able to difuse across a free pore, but will be captured by the fow into it. To strengthen this point, we compute the probability, when a pore saturates, that the next one to clog is at a distance ∆x and observe a deviation from a stochastic process only for one inter-pore distance (see Fig. 2 in Supplementary materials). We now propose a stationary phenomenological model to estimate the infuence of the redistributed particles on clog growth rate. Assume that a free pore has k successive neighbouring saturated pores, including lef and right directions. SCIENTIFIC REPORTS \| (2018) 8:12460 ȁǣͷͶǤͷͶ͹;ȀͺͷͻͿ;ǦͶͷ;Ǧ͹Ͷ͹;ͿǦͽ Figure 5 sketches the physical confguration for a k = 2 case, with the two clogs saturated on the SCIENTIFIC REPORTS \| (2018) 8:12460 ȁǣͷͶǤͷͶ͹;ȀͺͷͻͿ;ǦͶͷ;Ǧ͹Ͷ͹;ͿǦͽ 4 www.nature.com/scientificreports/ Figure 5. Sketch of a situation with two saturated pores neighbouring a free pore (therefore, k = 2). Figure 5. Sketch of a situation with two saturated pores neighbouring a free pore (therefore, k = 2). lef side of the considered free pore. Each saturated pore acts as a source of particles difusing in both directions away from the clog, toward the closest free pores. Only the growth rate of these frst pores will be infuenced by the saturated ones. Te efective concentration of the suspension fowing through a free pore will be simply φ φ =    +    k Q Q 1 2 , (1) k s f 0 (1) each neighbouring clogged pore contributing equally to φk in the present model. However, the variable k is not easily available: it is specifc to each free pore, depending on its environment which changes with time, and it depends on the confguration of free and saturated pores. Tereby, this problem has a statistical facet. When N pores out of Ntot are saturated, the efective concentration of the suspension fowing in a given opened pore, aver- aged over all opened-clogged pores confgurations is defned as: φ φ 〈 〉 =    + 〈〉    k Q Q 1 2 , (2) k N N s f 0 (2) where 〈k〉N is the average number of neighbouring saturated pores adjacent to free pores. Te clog growth rate is linked to the efective concentration of the suspension fowing through the pore: φ ∝〈 〉 ⁎ v Q N k N f 10. Finally, the ratio of clog growth rate with N clogged pores to clog growth rate with no clogged pore can be written as: where 〈k〉N is the average number of neighbouring saturated pores adjacent to free pores. Te clog growth rate is linked to the efective concentration of the suspension fowing through the pore: φ ∝〈 〉 ⁎ v Q N k N f 10. Finally, the ratio of clog growth rate with N clogged pores to clog growth rate with no clogged pore can be written as: = + 〈〉 ⁎ ⁎ v v k Q Q 1 2 , (3) N N s f 0 (3) with 〈k〉N computed using a tree diagram approach (see Supplementary materials 3). with 〈k〉N computed using a tree diagram approach (see Supplementary materials 3). ing a tree diagram approach (see Supplementary materials 3 with 〈k〉N computed using a tree diagram approach (see Supplementary materials 3). Te fow rate Qf is determined as already explained. Te fow rate Qs through a porous clog depends on its hydraulic resistance Rh clog, which can be estimated using the Blake-Kozeny equation31: η ε ε = − A R whd 150 (1 ) , (4) h clog p 2 3 η ε ε = − A R whd 150 (1 ) , h clog p 2 3 (4) where ε represents the clog porosity, η the fuid dynamic viscosity, and A a typical length of the clog. To estimate A, we consider that, because they form in a corner, the clogs are roughly quarter-spheres, and assume that the radius of the clog corresponds to A. Using the projected area at saturation, we obtain µ ∼ A 7 m. Te porosity can be estimated from a previous study24, which shows that for pH = 6 (close to that in the present study), and for a small fltration cake of colloids, the porosity can reach ε = 0.83. Tis value is in good agreement with macroscopic measurements made by Brenner20. With these parameters, the hydraulic resistance estimate is ≈ × R 2 10 h clog 17 kg. m−4.s−1. Te hydraulic resistance of the rectangular pore is very similar: = . × R 2 3 10 h slit 17 kg.m−4.s−130. Te resulting fow rate Qs through a saturated pore will be about one-half of that in a free pore: Qs/Qf ≈ 0.5. Tis value is of the same order of magnitude as the velocity decrease before clogging observed in the case of polymersome clogs (Fig. 4). Te result of the model given by Eq. 3 is plotted as a solid line in Fig. 3. Tis model contains no free parameter: 〈k〉N is computed numerically and the ratio Qs/Qf is estimated on solid grounds. Te model is in relatively good agreement with the experimental data. A key point, observed experimentally and well predicted by the model, is that the cross-talk between pores becomes more and more important as N increases. Te clogging of a free pore will be infuenced by a distant, saturated pore, if there are only saturated pores between them. To support this point, Fig. 3 also shows the results of the model obtained considering only the frst one, two, three and four adjacent neighbour(s) in each direction to compute 〈k〉N. We observe the convergence of these curves towards the full model (solid line), where the infuence of all successive saturated neighbours is taken into account, which highlights the “long-range” cross-talk between pores captured in the model. Note that because of large error bars at large N, this cannot be decreed as the only valid model. g y To summarize and conclude, we have directly imaged a fxed-pressure-drop fltration process of Brownian particles through nanoslits, leading to the formation of clogs at the nanoslits entrance. The observation of cross-talk between pores, with an increase of clog growth rate with the number of saturated pores, is the central SCIENTIFIC REPORTS \| (2018) 8:12460 ȁǣͷͶǤͷͶ͹;ȀͺͷͻͿ;ǦͶͷ;Ǧ͹Ͷ͹;ͿǦͽ 5 5 www.nature.com/scientificreports/ point of this paper. To the authors’ knowledge, this is the frst time such an experimental observation is reported. Such a result will certainly impact the current understanding of membrane fouling dynamics. Te direct access to phenomena at this scale provided by the microfabrication, imaging, and fow control, together with a systematic data analysis method thus enables us to demonstrate an original basic phenomenon, i.e. the cross-talk between pores in colloidal fltration. p To better explain the cross-talk physics, several experimental parameters should be varied. First, tuning the “strength” of Brownian difusion will assess the role of this mechanism on the redistribution of the particles along the membrane. Using less viscous fuids or smaller colloidal particles would allow access to the smaller Péclet numbers needed to explore this issue. Tuning the chip geometry is a second one. References 1. Tavakkoli, M. et al. Indirect Method: A Novel Technique for Experimental Determination of Asphaltene Precipitation. Energy & Fuels 29, 2890–2900, https://doi.org/10.1021/ef502188u (2015). 2. Fuller, S. B., Wilhelm, E. J. & Jacobson, J. M. Ink-jet printed nanoparticle microelectromechanical systems. Journal of Microelectromechanical systems 11, 54–60, http://ieeexplore.ieee.org/abstract/document/982863/ (2002). 3. Rothberg, M. B. Coronary Artery Disease as Clogged Pipes: A Misconceptual Model. Circulation: Cardiovascular Quality and Outcomes 6, 129–132, https://doi.org/10.1161/CIRCOUTCOMES.112.967778 (2013). p g 4. Pang, L. et al. Deformability and size-based cancer cell separation using an integrated microfluidic device. The Analyst 140, 7335–7346, https://doi.org/10.1039/C5AN00799B (2015). p g 5. Zhang, W., Tang, X., Weisbrod, N. & Guan, Z. A review of colloid transport in fractured rocks. Journal of Mountain Science 9, 770–787, https://doi.org/10.1007/s11629-012-2443-1 (2012). g 6. Hilgers, C. & Urai, J. L. Experimental study of syntaxial vein growth during lateral fuid fow in transmitted light: frst results. Journal of Structural Geology 24, 1029–1043 (2002). f gy ( ) 7. Dressaire, E. & Sauret, A. Clogging of microfuidic systems. Sof Matter 13, 37–48, https://doi.org/10.1039/C6SM01879C (2017). 8. Sauret, A. et al. Clogging by sieving in microchannels: Application to the detection of contaminants in colloidal suspensions. Applied Physics Letters 105, 074101, https://doi.org/10.1063/1.4893459 (2014). y p g 9. Zuriguel, I. et al. Clogging transition of many-particle systems fowing through bottlenecks. Scientifc Reports 4, 7324, https://doi. org/10.1038/srep07324 (2014). g p 10. Wyss, H. M., Blair, D. L., Morris, J. F., Stone, H. A. & Weitz, D. A. Mechanism for clogging of microchannels. Physical Review E 74 https://doi.org/10.1103/PhysRevE.74.061402 (2006). p g y 11. Dersoir, B., de Saint Vincent, M. R., Abkarian, M. & Tabuteau, H. Clogging of a single pore by colloidal particles. Microfuidics and Nanofuidics 19, 953–961, https://doi.org/10.1007/s10404-015-1624-y (2015). f p g y ( ) 12. Bacchin, P., Marty, A., Duru, P., Meireles, M. & Aimar, P. Colloidal surface interactions and membrane fouling: Investigations at pore scale. Advances in Colloid and Interface Science 164, 2–11, http://linkinghub.elsevier.com/retrieve/pii/S0001868610001922 https://doi.org/10.1016/j.cis.2010.10.005 (2011). p g j ( ) 13. Duru, P. & Hallez, Y. A Tree-Step Scenario Involved in Particle Capture on a Pore Edge. Langmuir 31, 8310–8317, https://doi. org/10.1021/acs.langmuir.5b0l1298 (2015). g g 14. Robert de Saint Vincent, M., Abkarian, M. & Tabuteau, H. Dynamics of colloid accumulation under fow over porous obstacles. Sof Matter 12, 1041–1050, https://doi.org/10.1039/C5SM01952D (2016). p g ( ) 15. Dersoir, B., Schofeld, A. B. & Tabuteau, H. Clogging transition induced by self fltration in a slit pore. For instance, an increase of the pore period δ should have a direct infuence on the cross-talk: at a given Brownian difusion magnitude, the difusion time scale between pores may become too large to allow for cross-talk. Another important quantity for the model is the fow rate Qs through a saturated pore. It depends on the clog saturation size and internal struc- ture, which both result from a balance between drag forces and repulsive interactions between the accumulated Brownian particles. Tuning the internal clog structure is then an interesting perspective. Tis may be achieved by changing the suspension salinity. For instance, at low ionic force, the repulsive interactions between particles, and thus the “efective volume” occupied by each of them, will be higher. It could lead to a less dense and more permeable clog which should increase the cross-talk efect. Work along these directions is currently in progress, even if made tricky by the challenges that are inherent to such experiments, as discussed in the experimental method section. Another interesting study could be to increase the particle volume fraction to observe possible collective difusion infuence on the cross-talk phenomenon. Finally, the model presented in this paper could be extended to diferent geometries, particularly 2-D membranes. Tis confguration could reduce the cross-talk efect because of the more important number of neighbours, limited to two in our 1-D experiment (so the factor 1/2 in the right-hand side term of Eq. 4). It will also impact the computation of 〈k〉N. Nevertheless, it would be interesting to make similar measurements on 2-D membranes, such as microsieves, with diferent pore patterns and compare with the predictions of the phenomenological model. We acknowledge the Fédération FERMaT and University of Toulouse (Project NEMESIS) for funding these researches. O. Liot warmly acknowledges M. Socol for his chip microfabrication and experimental help. Tis work was partly supported by LAAS-CNRS micro and nanotechnologies platform member of the French RENATECH network. researches. O. Liot warmly acknowledges M. Socol for his chip microfabrication and experimental help. Tis work was partly supported by LAAS-CNRS micro and nanotechnologies platform member of the French RENATECH network. References p p g ( ) ( ) 27. Shin, S., Ault, J. T., Warren, P. B. & Stone, H. A. Accumulation of Colloidal Particles in Flow Junctions Induced by Fluid Flow and Difusiophoresis. Physical Review X 7, https://doi.org/10.1103/PhysRevX.7.041038 (2017). p p g 27. Shin, S., Ault, J. T., Warren, P. B. & Stone, H. A. Accumulation of Colloidal Particles in Flow Junctions Induced by Fluid Flow and Difusiophoresis. Physical Review X 7, https://doi.org/10.1103/PhysRevX.7.041038 (2017). y g y 28. Liot, O. et al. Transport of nano-objects in narrow channels: infuence of Brownian difusion, confnement and particle nature. Journal of Physics: Condensed Matter 30, 234001, https://doi.org/10.1088/1361-648X/aac0af (2018). f y p g 29. Cussler, E. L. Difusion: Mass Transfer in Fluid Systems Google-Books-ID: dq6LdJyN8ScC.(Cambridge University Press, 2009). 30 B H T ti l i f idi (O f d i it O f d 2007) htt // b fl it dt dk/b /TMF/ bli ti / 29. Cussler, E. L. Difusion: Mass Transfer in Fluid Systems Google-Books-ID: dq6LdJyN8ScC.(Cambridge University Press, 2009). 30. Bruus, H. Teoretical microfuidics (Oxford university press Oxford, 2007). http://web-fles.ait.dtu.dk/bruus/TMF/publications/ books/Bruus_TMFbook_Sample_Chapter.pdf. 29. Cussler, E. L. Difusion: Mass Transfer in Fluid Systems Google-Books-ID: dq6LdJyN8ScC.(Cambridge University Press, 2009). 30. Bruus, H. Teoretical microfuidics (Oxford university press Oxford, 2007). http://web-fles.ait.dtu.dk/bruus/TMF/publications/ books/Bruus_TMFbook_Sample_Chapter.pdf. 29. Cussler, E. L. Difusion: Mass Transfer in Fluid Systems Google-Books-ID: dq6LdJyN8ScC.(Cambridge University Press, 2009). 30. Bruus, H. Teoretical microfuidics (Oxford university press Oxford, 2007). http://web-fles.ait.dtu.dk/bruus/TMF/publications/ books/Bruus TMFbook Sample Chapter pdf p p p 31. Bird, R. B. Transport phenomena. Applied Mechanics Reviews 55, R1–R4, https://doi.org/10.1115/1.1424298 (2002). 31. Bird, R. B. Transport phenomena. Applied Mecha O.L. and A.S. performed experiments and analysis. O.L. developed the model with P.D. P.J. and O.L. fabricated the microfuidic chips and assembled the experimental device. J.F.M. led the scientifc project development within the context of the NEMESIS Chaire d’Attractivité at University of Toulouse. O.L. led the manuscript writing with inputs from P.D. and in which P.B., J.F.M. and P.J. took part. © Te Author(s) 2018 SCIENTIFIC REPORTS \| (2018) 8:12460 ȁǣͷͶǤͷͶ͹;ȀͺͷͻͿ;ǦͶͷ;Ǧ͹Ͷ͹;ͿǦͽ References Sof Matter 13, 2054–2066, https://doi.org/10.1039/C6SM02605B (2017). p g 16. Cejas, C. M., Monti, F., Truchet, M., Burnouf, J.-P. & Tabeling, P. Particle Deposition Kinetics of Colloidal Suspensions in Microchannels at High Ionic Strength. Langmuir 33, 6471–6480, https://doi.org/10.1021/acs.langmuir.7b01394 (2017). g g g p g g 17. Kim, Y., Ahn, K. H. & Lee, S. J. Clogging mechanism of poly(styrene) particles in the fow through a single micro-pore. Journal of Membrane Science 534, 25–32, http://linkinghub.elsevier.com/retrieve/pii/S0376738817303757 https://doi.org/10.1016/j. memsci.2017.04.010 (2017). 18. Laar, T. v. d., Klooster, S. t., Schroën, K. & Sprakel, J. Transition-state theory predicts clogging at the microscale. Scientifc Reports 6 https://doi.org/10.1038/srep28450 (2016). p g p 19. Sendekie, Z. B. & Bacchin, P. Colloidal Jamming Dynamics in Microchannel Bottlenecks. Langmuir 32, 1478–1488, https://doi. org/10.1021/acs.langmuir.5b04218 (2016). g g ( ) 20. Brenner, H. Tree-dimensional fltration on a circular leaf. AIChE Journal 7, 666–671 (1961). g g ( ) 20. Brenner, H. Tree-dimensional fltration on a circular leaf. AIChE Journal 7, 666–671 (1961). É g g 20. Brenner, H. Tree-dimensional fltration on a circular leaf. AIChE dimensional fltration on a circular leaf. AIChE Journal 7, 666–671 (196 É 21. Ghidaglia, C., de Arcangelis, L., Hinch, J. & Guazzelli, É. Transition in particle capture in deep bed fltration. Physical Review E 53, R3028, https://doi.org/10.1103/PhysRevE.53.R3028. (1996). p g y 22. Narayan, R., Coury, J. R., Masliyah, J. H. & Gray, M. R. Particle p g y 22. Narayan, R., Coury, J. R., Masliyah, J. H. & Gray, M. R. Particle capture and plugging in packed-bed reactors. Industrial & engineering chemistry research 36, 4620–4627, https://doi.org/10.1021/ie970101e. (1997). chemistry research 36, 4620–4627, https://doi.org/10.1021/ie970101e y p g 23. Hong, S., Faibish, R. S. & Elimielech, M. Kinetics of Permeate Flux Decline in Crossflow Membrane Filtration of Colloidal Suspensions. Journal of Colloid ans Interface Science 196, 267–277 (1997). p J f f , ( ) 24. Hieke, M., Ruland, J., Anlauf, H. & Nirschl, H. Analysis of the Porosity of Filter Cakes Obtained by Filtration of Colloidal Suspensions. Chemical Engineering & Technology 32, 1095–1101, https://doi.org/10.1002/ceat.200800609 (2009). SCIENTIFIC REPORTS \| (2018) 8:12460 ȁǣͷͶǤͷͶ͹;ȀͺͷͻͿ;ǦͶͷ;Ǧ͹Ͷ͹;ͿǦͽ 6 www.nature.com/scientificreports/ 25. Song, L. & Elimelech, M. Teory of concentration polarization in crossfow fltration. Journal of the Chemical Society, Faraday Transactions 91, 3389–3398 (1995). 26. Bacchin, P., Meireles, M. & Aimar, P. Modelling of fltration: from the polarised layer to deposit formation and compaction. Desalination 145, 139–146, https://hal.archives-ouvertes.fr/hal-00201115 https://doi.org/10.1016/S0011-9164(02)00399-5 (2002). Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-30389-7. Competing Interests: Te authors declare no competing interests. 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https://openalex.org/W2118314032	https://scholarcommons.sc.edu/cgi/viewcontent.cgi?article=1561&context=emec_facpub	English	null	Nanopillar array with a λ/11 diameter fabricated by a kind of visible CW laser direct lithography system	Nanoscale research letters	2,013	cc-by	6,863	Nanopillar Array with a λ/11 Diameter Fabricated by a Kind of Nanopillar Array with a /11 Diameter Fabricated by a Kind of Visible CW Laser Direct Lithography System Visible CW Laser Direct Lithography System Chen Zhang University of South Carolina - Columbia, zhang294@mailbox.sc.edu Follow this and additional works at: https://scholarcommons.sc.edu/emec_facpub Part of the Nanoscience and Nanotechnology Commons University of South Carolina University of South Carolina Scholar Commons Scholar Commons Faculty Publications Mechanical Engineering, Department of 2013 Nanopillar Array with a λ/11 Diameter Fabricated by a Kind of Nanopillar Array with a /11 Diameter Fabricated by a Kind of Visible CW Laser Direct Lithography System Visible CW Laser Direct Lithography System Chen Zhang University of South Carolina - Columbia, zhang294@mailbox.sc.edu K. Wang J. Bai S. Wang W. Zhao See next page for additional authors Follow this and additional works at: https://scholarcommons.sc.edu/emec_facpub Part of the Nanoscience and Nanotechnology Commons Publication Info Publication Info Published in Nanoscale Research Letters, Volume 8, Issue 280, 2013, pages 2-9. This Article is brought to you by the Mechanical Engineering, Department of at Scholar Commons. It has been accepted for inclusion in Faculty Publications by an authorized administrator of Scholar Commons. For more i f i l di @ ilb d University of South Carolina University of South Carolina Scholar Commons Scholar Commons Mechanical Engineering, Department of Faculty Publications Faculty Publications Author(s) Author(s) Chen Zhang, K. Wang, J. Bai, S. Wang, W. Zhao, F. Yang, C. Gu, and Guiren Wang This article is available at Scholar Commons: https://scholarcommons.sc.edu/emec_facpub/559 Publication Info Publication Info Published in Nanoscale Research Letters, Volume 8, Issue 280, 2013, pages 2-9. This Article is brought to you by the Mechanical Engineering, Department of at Scholar Commons. It has been accepted for inclusion in Faculty Publications by an authorized administrator of Scholar Commons. For more information, please contact digres@mailbox.sc.edu. © 2013 Zhang et al.; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Author(s) Author(s) This article is available at Scholar Commons: https://scholarcommons.sc.edu/emec_facpub/559 Zhang et al. Nanoscale Research Letters 2013, 8:280 http://www.nanoscalereslett.com/content/8/1/280 Open Access Open Access Nanopillar array with a λ/11 diameter fabricated by a kind of visible CW laser direct lithography system Chen Zhang1,2, Kaige Wang1, Jintao Bai1, Shuang Wang1, Wei Zhao2, Fang Yang2, Changzhi Gu3 and Guiren Wang2,4,5 Abstract Nanoscale functional structures are indispensable elements in many fields of modern science. In this paper, nanopillar array with a pillar diameter far smaller than Abbe's diffraction limit is realized by a new kind of continuous wave (CW) laser direct lithography technology. With atomic force microscopy technology, the average diameter of nanopillars on thin OIR906 photoresist film is about 65 nm and the smallest diameter is 48 nm, which is about 1/11 of the incident laser wavelength. Also, the influences of coma and astigmatism effects to the shape and size of nanopillar are numerically simulated by utilizing vector integral. As far as we know, it is the first time that nanopillar array is implemented by a donut-shaped 532-nm visible CW laser. The study presents a new, simple, inexpensive, and effective approach for nanopillar/pore array fabrication. Keywords: Nanopillar, Diffraction limit, Lithography, Coma, Astigmatism beam lithography [14,15], nanoimprint technology [ and laser lithography [17]. * Correspondence: wangkg@nwu.edu.cn; wanggu@cec.sc.edu 1Institute of Photonics and Photo-technology, International Scientific and Technological Cooperation Base of Photoelectric Technology and Functional Materials and Application, Northwest University, 229 North Taibai Rd, Xi'an 710069, People's Republic of China 2Department of Mechanical Engineering, University of South Carolina, 300 Main St., Columbia, SC 29208, USA Full list of author information is available at the end of the article Background d Laser was triggered by a digital pulse generator (DG535, Stanford Research System, Inc., Sunnyvale, CA, USA), and pulse lasting time is 120 ms. A high- performance digital charge-coupled device (CCD) camera (QICAM, QImaging Co., Ltd, Surrey, Canada) is applied for alignment and imaging. Figure 1b is the laser spot imaged in the focal plane by the CCD. This structure of laser beam has been utilized during the following nanopillar array fabrication. Positive tone photoresist (OIR906, Fujifilm Electronic Materials USA, Inc., Valhalla, NY, USA) is adopted through the whole experiment. This resist is coated on a glass wafer by a spinner, and its thick- ness is approximately 800 nm. Even, in a continuous wave (CW) laser two-photon ab- sorption method, photoresist is tailored and the whole system is costly. Furthermore, two laser sources are re- quired in both photo-inhibiting and absorbance modula- tion methods, and the photoresist materials should have particular properties that result in restrictions in choos- ing light sources and resist materials. In the paper, we will report a kind of nanopillar array with a pillar diameter much smaller than Abbe's diffrac- tion limitation by visible CW laser direct lithography technology. A 532-nm CW laser beam, which is modified by a phase mask to generate a nanolevel dark core in the focus space, is proposed and applied in nanopillar/pore fabricating. The nanopillar array is obtained when the laser beam is irradiated to the positive tone photoresist, while nanopore will be generated with a negative tone photo- resist. To the best of our knowledge, this is the first time that nanopillar arrays are fabricated with a spatial donut shape, structured visible CW laser. Experimental results are measured by AFM, and the distortion and the incon- sistency of nanopatterns are analyzed with theoretical simulation. This preliminary work explores a novel, easy, and effective method of maskless CW laser direct writing technology to carry out functional nanopillar/pore arrays. In principle, with the modulation of the vortex phase- shifting plate, the circularly polarized Gaussian beam is generated as a donut-shaped pattern on the focal plane. The dimension of the dark core of the donut-shaped pattern is smaller than the diffraction limitation [31]. During the experiment, the photoresist at the center of the pattern will not be exposed because of the null in- tensity point. Background d Since the positive tone photoresist was ap- plied in this work, a kind of nanopillar structure, whose diameter is far below diffraction limitation, could be obtained in the center of the donut-shaped pattern with appropriate input laser power. Background d [16], Nowadays, nanoscale structures such as nanopillar and nanopore arrays are considered essential functional nano- texturizations for modern scientific research and applica- tion. Nanopillar arrays have been employed in the study of field emission [1], solar cell industry [2], biological sens- ing [3], micro-/nanoscale fluidics, near-field optics, and the lab-on-a-chip technology [4]. Nanopore arrays have also been recognized as valuable structures in many advanced fields such as photovoltaic [5] and photonic crystal research [6], gas detection [7], and especially in biological molecules detection and separation [8]. Fitting with foregoing scien- tific advancements, the nanoscale fabricating methods and technologies have been made good progress. Nano- pillar and nanopore arrays can be fabricated with direct growth approaches (metal-organic chemical vapor depo- sition, hydride vapor phase epitaxy, molecular beam epi- taxy) [9-11], nanosphere-assist etching [12,13], electronic Since the merits of fabricating speediness and cleanli- ness, maskless process, controllable pattern shape and size, and capability of lithograph in three dimensions [18,19], laser direct lithography technology is one of the most attractive approaches to fabricate nanoscale func- tional structures as compared with the disadvantages such as expensive, heavy, or low precision of other methods. Choi's group has reported implementing 100- nm-level nanostructure arrays over a large scale by means of laser interference lithography [20-23]. Scott and Li have respectively fabricated sub-100-nm isotropic voxel [24] and voxel with a 40-nm axial size [25] by photo-initiation inhibiting technology. Cao has obtained a nanoline with a width of 130 nm and nanodots with a diameter of 40 nm [26] by polymerization inhibiting, too. In Andrew's work, the nanolines with an average width of 36 nm were drawn employing absorbance modulation lithography [27]. Tanaka and Thiel have shown fabricating spatial voxel to sub-120 nm with the two-photo-absorption technology [28,29]. Qi got a single polymerized tip with a diameter of 120 nm with the same technical route [30]. However, the utilization of femtosecond laser systems makes the lithography system complex and expensive. Page 2 of 9 Zhang et al. Nanoscale Research Letters 2013, 8:280 http://www.nanoscalereslett.com/content/8/1/280 Zhang et al. Nanoscale Research Letters 2013, 8:280 http://www.nanoscalereslett.com/content/8/1/280 During laser lithography, the photoresist-coated glass wafer is mounted onto a three-dimensional (3D) piezo- electric scanning stage (P-611.3SF along with the E-664.S3 Amplifier/Controller, Physik Instrument, Auburn, MA, USA). The rapid motion of PI stage is controlled by a PC program. Methods The laser direct writing system in our experiments is sche- matically shown in Figure 1a. The light source is a CW laser with its center wavelength at 532 nm (DHOM-VL- 532-2000, Suzhou Daheng Optics and Fine Mechanics Co., Ltd, Suzhou, China). A spatial filter is placed behind the laser head to achieve a high-quality beam mode. A λ/4 wave plate (WP) is used to transfer the linearly polarized 532-nm laser into a right-handed circularly polarized beam. A vortex phase plate (PP) changes phase from 0 to 2π in anticlockwise direction. Here, a high numerical aperture (NA) (1.4) oil-immersed objective (Apoplan 100×/1.4, Olympus Optical Co., Ltd, Tokyo, Japan) is employed to focus the laser beam. Laser power at the input pupil of the objective is approximately 16 μW. The procedure of experiment is composed of the steps of spin coating, preexposure baking, exposing, post-exposure baking, developing, and hard baking in sequence. The ob- tained nanostructures are measured, characterized, and an- alyzed with an atomic force microscopy (AFM, Veeco Dimension 3100 AFM system, Veeco Instruments Inc., Plainview, NY, USA). To obtain the nanopatterns with high precision and consistency, the focal sphere should be accur- ately focused onto the surface of the photoresist. Further- more, the motion of the scanning stage is required to be Figure 1 Schematic diagram of experimental setup (a) and laser focal spot (b). Figure 1 Schematic diagram of experimental setup (a) and laser focal spot (b). Page 3 of 9 Zhang et al. Nanoscale Research Letters 2013, 8:280 http://www.nanoscalereslett.com/content/8/1/280 Zhang et al. Nanoscale Research Letters 2013, 8:280 http://www.nanoscalereslett.com/content/8/1/280 Figure 3 shows the typical nanopillars fabricated in our experiments. The AFM images of Figure 3a, b, c show the three different nanopillars which are fabricated with the same laser power. Figure 3d,e,f is the corre- sponding cross-sectional information along the black lines in Figure 3a, b, c, respectively. These black lines are drawn as symmetry axis of the patterns in Figure 3a, b, c. It is noted that in Figure 3a, the pattern is semilunar, and the bright part, which is embedded in the semilunar pattern, has a width of about 241 nm along the dark line. The pattern in Figure 3b becomes donut-shaped, and in the pattern is the nanopillar with a pillar width of 71 nm. Experimental results Figure 2 is a typical image of a nanopillar array fabri- cated in the experiments. The top surface pattern of the overall topography is displayed as Figure 2a. The scan range is about 10 μm × 10 μm. Each nanopillar is lo- cated in a circular pit whose external diameter is around 950 nm. The average diameter of the nanopillar is 65 nm, which is much smaller than the size of Abbe's limit. Figure 2b is an AFM 3D image of the nanopillar array. Figure 2c represents the cross-sectional topog- raphy along the dark line which is shown in Figure 2a, and it illustrates the flatness of the coating surface. Figure 2d, e shows more details about the typical nanopillar in the array. Figure 2d is the top view of the nanopillar which is marked by the arrow in the nanopillar array of Figure 2a. A dark line in Figure 2d acts as the symmetry axis of the pattern. It passes through the apex of the nanopillar, and its corresponding cross-sectional image is illustrated in Figure 2e. With careful calibration and ana- lysis, it is found that the diameter of the pillar is around 48 nm, which is about λ/11, much smaller than the diffrac- tion limit λ/2, where λ is the incident laser wavelength at about 532 nm. Figure 2 demonstrates that the nanopillar array can be manufactured to sub-diffraction limit size with our donut-shaped CW visible laser system. Comparing the experimental pillars in Figure 2 with the laser spot shown in Figure 1b, as well as in Figure 3, it seems that the nanopillars' location deviated a little from the center of the donut-shaped beam. Meanwhile, the en- tire donut-shaped pattern seems changed to an elliptical shape rather than a cylindrical donut shape. In order to fabricate large area-distributed nanopillar/pore array with Figure 2 Typical image of a nanopillar array fabricated in the experiments. (a) AFM image of nanopillar array fabricated with 532-nm CW laser and (b) its corresponding 3D image. (c) Roughness of coating along the dark line in (a). (d) Enlargement of one unit and (e) its cross section marked in (a). Figure 2 Typical image of a nanopillar array fabricated in the experiments. (a) AFM image of nanopillar array fabricated with 532-nm CW laser and (b) its corresponding 3D image. (c) Roughness of coating along the dark line in (a). Methods In Figure 3c, the nanopillar is almost located at the center of the pattern, and its pillar diameter is around 58 nm. The cross-sectional drawing (Figure 3d,e,f) reflect the asymmetry of depth in the patterns as well as the nonuniformly distributed light intensity. The depth of the left-side pit in Figure 3f is larger than that in Figure 3e, d, while the depth of the two pits in Figure 3a is the smallest. This result indicates that the focal spot has a concentrated and better symmetry of intensity distribution in the case of Figure 3c. synchronized with laser exposure for fast fabricating nanopatterns. synchronized with laser exposure for fast fabricating nanopatterns. Experimental results (d) Enlargement of one unit and (e) its cross section marked in (a). Zhang et al. Nanoscale Research Letters 2013, 8:280 http://www.nanoscalereslett.com/content/8/1/280 Page 4 of 9 Figure 3 AFM images of typical nanopillars. (a) Near the rim of the pit. (b) Close to the center of the pit. (c) At the center of the pit. (d) Cross section of pattern in (a). (e) Cross section of pattern in (b). (f) Cross section of pattern (c). Figure 3 AFM images of typical nanopillars. (a) Near the rim of the pit. (b) Close to the center of the pit. (c) At the center of the pit. (d) Cross section of pattern in (a). (e) Cross section of pattern in (b). (f) Cross section of pattern (c). high consistency with the system, the reasons of the nano- scale patterns transformed are systematical analyzed. Figure 4a, b, c; corresponding coefficient Ac values are 0.5, 0.25 and 0.1, respectively. Figure 4d, e, f stands for the calculated simulations of optical intensity on the yz plane with Ac values equal to 0.5, 0.25 and 0.1 in se- quence. Figure 4g, h, i shows the corresponding cross- sectional profiles of light intensity distribution on the y axis as Ac is 0.5, 0.25 and 0.1, respectively. These figures in Figure 4 clearly illustrate the gradual transformation of light distribution induced by coma effect. The dark core of the donut-shaped pattern is stretched along one direction with the increase of Ac. Meanwhile, light intensity changes and becomes a monosymmetric distribution. It can be clearly observed that the dark spot at the core of the laser beam turns into an elliptical shape as Ac increases. It is well known that the transformation of donut-shaped patterns might be caused by the laser quality, the photo- resist surface roughness, the optical system errors, or la- boratory personnel operational interferences. However, this phenomenon should not be caused by the laser beam qual- ity because the laser focal spot has a symmetric donut shape on the focal plane which is shown in Figure 1b. Otherwise, the surface roughness should not be the issue that can be clarified in Figure 2c in which the coating photoresist surface is flat. During lithography, the laser beam is well aligned to expose the resist vertically; thus, shape deformation is not caused by a tilt photoresist wafer. Experimental results Besides the factors mentioned above, optical system er- rors can affect laser distribution. Spherical aberration, coma, and astigmatism are three primary factors of optical system errors. In general, the focal spot cannot be transformed to an irregular shape under the influence of spherical aberration. On the contrary, coma may cause one-directional deformation of the focal spot, while astig- matism can split the laser spot into two parts. There are two more factors: one is that this kind of laser lithography system is not sensitive to the influence of the spherical ab- erration; another is that the objective is designed as an ap- lanatic lens which eliminates the spherical aberration of the objective. Taking these factors into account, theoretical ana- lysis and numerical calculation will be focused on the influ- ences of coma and astigmatism effect. Aberration influence theory of the focal donut spot is described in the Appendix. It makes sense to compare the results of the experiments and simulations. Their resemblances are easily found out. First, the calculated results shown in Figure 4a, b, c have similar patterns with those experimental patterns imaged in Figure 4a, b, c, respectively. The donut-shaped focal spot is a semilunar appearance in both experiment and simula- tion. Next, the gradual transformation of nanopillars in the experiment has the same variation tendency with the dark spots in the numerical simulation. Figure 4d, e, f illus- trates the asymmetric intensity distribution on the yz plane; they explain the reasons why the two sides of the nanopillars are ruptured with different depths. Further- more, Figure 4g, h, i has shown that the depletion of light intensity increased with the increased Ac, which correctly reflects the variation of depths at the two sides of the nanopillars in Figure 4d, e, f. Thus, coma effect is the main influence factor which results in nonideal nanopillar pat- terns in Figures 2 and 3. Astigmatism influence It is also meaningful to compare the experimental re- sults shown in Figure 6 with the simulation results in Figure 7; the pattern of the marked experimental result in Figure 6a is found very similar with the simulation result in Figure 7b with Aa = 0.1. It can be seen from Figure 7f that the distribution is symmetric with the origin, and the light intensity is different along x = y and x = −y. These calculated results explain the laser lithog- raphy symmetric depth on the two sides of the nano- pillar shown in Figure 7d, e. The widths of the longer axis and the shorter axis of the pillar top are 83 and 47 nm, respectively, which is illustrated in Figure 7d, e. Figure 6a presents images of the other kind of nanopillar with distorted pattern caused by astigmatism besides the situations shown in Figure 3 (the noise of background in Figure 6a is due to AFM software processing). We take the typical pattern marked by the arrow in Figure 6a. Figure 6b, c presents the zoomed-in images of the marked nanopillar in Figure 6a. In Figure 6b, c, dark lines pass through the top of the nanopillar, and they are drawn as the symmetry axes for the nanostructure in two perpendicular directions. Figure 6d, e presents the cross sections along the dark lines in Figure 6b, c, re- spectively. In Figure 6, it is obvious that the nanostruc- tures fabricated by laser lithography are almost located at the center of the patterns; however, they are an elliptic cylinder. It is also evident that the patterns in Figure 6b, c, d,e are symmetric to the two dark lines, but not com- pletely the same as that in Figure 5a. As has been explained earlier, spherical aberration influence is negli- gible since an aplanatic lens is employed as the objective lens. Therefore, this kind of experimental phenomenon could only be induced by astigmatism effect. In conclusion, combining the experimental work and the numerical simulation, it can be illustrated that the nanopillar structure could be transformed by both coma and astigmatism effects. The diameter of the nanopillar is increased and the height of the nanopillar is decreased with enhanced coma value. The shape of the nanopillar is likely to be compressed into a belt form as the astig- matism influence enhanced. Coma influence Figure 4 is the simulation result of coma effect for the structured laser beam as coefficient Ac which is assigned with different values. The intensity distribution of the donut-shaped laser spot on the xy plane is revealed in It should be noted that because of the conical shape of AFM probe tip, the height of the nanopillars is not exactly available with AFM observation. However, the spatial characters of the donut-shaped focal spot can be Zhang et al. Nanoscale Research Letters 2013, 8:280 http://www.nanoscalereslett.com/content/8/1/280 Page 5 of 9 Figure 4 Simulation result of coma effect. The simulated donut-shaped focal spot intensity vs coma effect on the xy plane: (a) Ac = 0.5, (b) Ac = 0.25 and (c) Ac = 0.1. The corresponding intensity on the yz plane: (d) Ac = 0.5, (e) Ac = 0.25, and (f) Ac = 0.1. Intensity along the y axis: (g) Ac = 0.5, (h) Ac = 0.25, and (i) Ac = 0.1. Figure 4 Simulation result of coma effect. The simulated donut-shaped focal spot intensity vs coma effect on the xy plane: (a) Ac = 0.5, (b) Ac = 0.25 and (c) Ac = 0.1. The corresponding intensity on the yz plane: (d) Ac = 0.5, (e) Ac = 0.25, and (f) Ac = 0.1. Intensity along the y axis: (g) Ac = 0.5, (h) Ac = 0.25, and (i) Ac = 0.1. We deem that the influence of coma effect caused by the ×100/1.4 objective lens is insignificant since this type of objective is aplanatic which dispels coma influence of the objective. Also, the focal spot has a well-defined symmetric shape before patterning the photoresist as is correctly reflected, and the height of the nanopillar can be relatively revealed. Figure 5 is the simulation about the donut-shaped laser distributing on the focal plane and the axial plane. It indicates that the height of the nanopillar can be as large as one λ or more. Figure 5 Intensity distribution of the ideally donut-shaped laser spot. (a) The lateral plane and (b) the vertical plane. igure 5 Intensity distribution of the ideally donut-shaped laser spot. (a) The lateral plane and (b) the vertical plane. Page 6 of 9 Zhang et al. Nanoscale Research Letters 2013, 8:280 http://www.nanoscalereslett.com/content/8/1/280 displayed in Figure 1b. In addition, the extents of coma effect, which is shown in Figure 3a, b, c, are different under the same experimental conditions. Coma influence Therefore, we consider that coma effect of the laser lithography system should be caused by mechanical disturbance. In fact, the mechanical vibration during the system working may disturb the laser beam and then induce an angle of devi- ation between the laser beam and objective lens. corresponding profiles of intensity distribution along x = y and x = −y are shown in Figure 7e, f, g, h. In Figure 7a, b, c,d, some clues about the gradual transformation of the donut-shaped laser spot could be found. As Aa is in- creasing, the laser pattern is pulled into two opposite di- rections and finally separated into two parts while the center shape varies from a circular to a belt-like struc- ture. In addition, the light intensity in the center of the focal spot is no longer zero when Aa is bigger, which is apparent in Figure 7g, h. Astigmatism influence In the subsequent work, the effects of coma and astigmatism of the donut-shaped laser direct writing system should be carefully dealt. Figure 7 is the numerical simulation of astigmatism in- fluence on the donut-shaped focal spot. Figure 7a,b,c,d shows the intensity distribution calculated with different Aa values which are 0.05, 0.1, 0.2, and 0.3, respectively. The Theoretically, the resolution of this laser lithography system increases when laser intensity enhances; thus, the resolution would be extremely small. However, it cannot Figure 6 Images of the other kind of nanopillar. (a) AFM image of the other kind of nanopillar. (b, c) Enlarged image of the marked pattern in (a) along different directions. (d, e) The corresponding cross sections of (b) and (c). Figure 6 Images of the other kind of nanopillar. (a) AFM image of the other kind of nanopillar. (b, c) Enlarged image of the marked pattern in (a) along different directions. (d, e) The corresponding cross sections of (b) and (c). Zhang et al. Nanoscale Research Letters 2013, 8:280 http://www.nanoscalereslett.com/content/8/1/280 Page 7 of 9 Figure 7 Numerical simulation of astigmatism influence on the donut-shaped focal spot. Simulated light intensity distribution vs astigmatism coefficient. (a) Aa = 0.05, (b) Aa = 0.1, (c) Aa = 0.2, and (d) Aa = 0.3. Intensity along x = y and x = −y (e) Aa = 0.05, (f) Aa = 0.1, (g) Aa = 0.2, and (h) Aa = 0.3. Figure 7 Numerical simulation of astigmatism influence on the donut-shaped focal spot. Simulated light intensity distribution vs astigmatism coefficient. (a) Aa = 0.05, (b) Aa = 0.1, (c) Aa = 0.2, and (d) Aa = 0.3. Intensity along x = y and x = −y (e) Aa = 0.05, (f) Aa = 0.1, (g) Aa = 0.2, and (h) Aa = 0.3. Competing interests Competing interests The authors declare that they have no competing interests. g The authors declare that they have no competing interests. Abbreviations where f is the focal length of the lens and l0 represents the amplitude factor in the image space; E0 is the ampli- tude of input Gaussian beam; A1(θ, ϕ) is the wavefront aberration function, θ is the angle between the optical axis and given ray; ϕ is the azimuthal coordinate at the input plane and φs(θ, ϕ) is the phase delay generated by the phase mask; x, y, and z indicate the Cartesian coordi- nates of the point p in the focal region; i is the plural; k = 2πn/λ stands for the wave number, where λ is the wavelength of the incident light and n is the refractive index of the focal space medium. Abbreviations AFM: Atomic force microscopy; CCD: Charge-coupled device; CW: Continuous wave; NA: Numerical aperture; PP: Phase plate; WP: Wave plate. Abbreviations AFM: Atomic force microscopy; CCD: Charge-coupled device; CW: Continuous wave; NA: Numerical aperture; PP: Phase plate; WP: Wave plate. Aberration theory about high NA objective ð4Þ Figure 8 is a schematic for laser spot distribution on a focal plane. The Gaussian beam is converted clockwise, is polarized by WP, and then passes through the PP and incident into the high NA objective lens. The compo- nents of the diffracted electric field at point P, which is near to the focal spot, can be expressed by the vectorial Debye theory as in Equation 1 [32]: Ac and Aa are coefficients for coma and astigmatism, respectively. Both Ac and Aa multiply λ, representing the departure of the wavefront at the periphery of the exit pupil. The values for λ, n, NA and θmax adopted in simulation correspond to the practical values in the ex- periment. Refractive index of oil n = 1.52; γ is supposed to be 1, which means that the objective is fulfilled by the Gaussian beam. E p ð Þ ¼ Ex Ey Ez 0 @ 1 A¼ −if l0 λ ∫α 0∫2π 0 E0 ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ cosθ p A1 θ; ϕ ð Þ exp ik x sinθ cosϕ þ y sinθ sinϕ þ z cosθ ð Þ ½ Therefore, the intensity distribution at point P is writ- ten as in Equation 5: A ik x sinθ cosϕ þ y sinθ sinϕ þ z cosθ ð Þ ½ I p ð Þ ¼ Ex j j2 þ Ey 2 þ Ez j j2 ð5Þ ð5Þ The electrical distributions for the donut-shaped pat- tern affected by aberrations are carried out using Matlab software. The electrical distributions for the donut-shaped pat- tern affected by aberrations are carried out using Matlab software. ð1Þ Coma: efficient, and low-cost method of fabricating nanopillars. It could pave a new way to fabricate nanopillars/pore arrays of large area distribution for optical nanoelements and biophotonic sensors while integrated with high-speed scan- ning system. A1 θ; ϕ ð Þ ¼ exp ikAcλ sinθ sinθmax 3 cosϕ " # ð3Þ ð3Þ Astigmatism: Appendix A1 θ; ϕ ð Þ ¼ exp ikAaλ sinθ sinα 2 cos2ϕ " # ð4Þ Authors' contributions CZ carried out specimen preparation, data acquisition and analysis of measurement and simulation and drafted the manuscript. KW, JB, GW and CG conceived the experiment, designed the plan and directed the drafting of the manuscript. SW, WZ and FY contributed to the simulation program improvement and participated in drafting the manuscript. All authors read and approved the final manuscript. Conclusions be that small due to optical aberration effects in the sys- tem and the material utilized in the experiment. In this work, the smallest resolution that was obtained with the photoresist OIR906 is 48 nm, which is 1/11 of the inci- dent wavelength. It is expected that the resolution should be finer with a smaller aberration influence. Nanopillar array has been successfully obtained on a spin-coated thin film of OIR906 photoresist, employing a kind of novel visible CW laser direct lithography sys- tem. The diameter of the fabricated nanopillar was able to be as small as 48 nm, which is 1/11 of the wavelength of the incident laser. The lithographic nanopatterns were calibrated and analyzed with AFM. Shape influences of the coma effect and astigmatism effect were simultaneously analyzed using vector integral. The simulation results ex- plain the distortion and inconsistency of the fabricated nanopatterns well. The work has demonstrated a simple, The patterning speed of the lithography system is mainly determined by factors that include the scanning speed of position stage, exposure time, and pattern com- plexity. In this report, it takes approximately 4 min to pat- tern a nanopillar array within the area of 100 × 100 μm2. Furthermore, an improved lithography system, which is be- ing built in our laboratory, is capable to reduce the fabrica- tion time to 1 min on the same pattern. Figure 8 Schematic drawing of light intensity distribution on the focal plane. In addition, the size of the donut-shaped pattern is re- lated to the wavelength of the incident beam. The beam with a shorter wavelength will generate a smaller donut- shaped pattern on the focal plane. Feature sizes can be tuned by shifting the wavelength of the laser with a fixed input power. In fact, we have quantitatively simulated how the donut-shaped patterns changed with the differ- ent wavelengths such as λ = 800 and 400 nm. The results showed that the radius of the pattern is 468 nm (at 800 nm) and 234 nm (at 400 nm). Figure 8 Schematic drawing of light intensity distribution on the focal plane. Figure 8 Schematic drawing of light intensity distribution on the focal plane. Zhang et al. Nanoscale Research Letters 2013, 8:280 http://www.nanoscalereslett.com/content/8/1/280 Zhang et al. Nanoscale Research Letters 2013, 8:280 http://www.nanoscalereslett.com/content/8/1/280 Page 8 of 9 Page 8 of 9 Authors' information h d d The amplitude of the Gaussian beam at the input plane is expressed as in Equation 2: CZ is a Ph.D. candidate of the Institute of Photonics and Photo-technology, Northwest University, Xi'an, China, with a research direction that is concerned on laser technology and application. KW is a professor of the Institute of Photonics and Photo-technology, Northwest University, Xi'an, China. His research direction focuses on nanotechnology, nanobiophotonics, and soft matter physics. JB is a professor of the Institute of Photonics and Photo-technology, Northwest University, Xi'an, China. His main research areas are all-solid-state laser, laser devices and laser technology. SW is a lecturer of the Institute of Photonics and Photo-technology, Northwest University, Xi'an, China. His study concentrates on biophotonics and biomedical optics. WZ is a Ph.D. candidate of the Department of Mechanical Engineering, University of South Carolina, Columbia, USA. His research topics are related to applied optics and fluid dynamics. FY is a postdoc in the Department of Mechanical Engineering, University of South Carolina, Columbia, USA. He works on high resolution microscopy system and MEMS. CG is a researcher of Institute of Physics, Chinese Academy of Sciences, Beijing, China. He works in the fields of nanostructure and nanodevices. GW is an associate professor at the Department of Mechanical Engineering and is interested in nanotechnology, bioMEMS, and lab-on-chip. E0 γ; θ ð Þ ¼ A0 exp −γ2ρ2 ð2Þ ð2Þ where A0 is the amplitude, γ is the truncation parameter and expressed as γ = a/ω (a is the aperture radius and ω is the beam size at the waist), while ρ stands for the ra- dial distance of a point from its center normalized by the aperture radius of the focusing system and ρ = sinθ/ sinθmax, where θmax is the maximal semi-aperture angle of the objective lens, and in our system, θmax = 67.07°. A1(θ, ϕ) represents wavefront aberration as expressed as in Equations 3 and 4: Page 9 of 9 Page 9 of 9 Zhang et al. Nanoscale Research Letters 2013, 8:280 http://www.nanoscalereslett.com/content/8/1/280 Acknowledgments Thi k 19. Liao Y, Song JX, Li E, Luo Y, Shen YL, Chen DP, Cheng Y, Xu ZZ, Sugioka K, Midorikawa K: Rapid prototyping of three-dimensional microfluidic mixers in glass by femtosecond laser direct writing. Lab Chip 2012, 12:746–749. 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https://openalex.org/W4250545299	https://pure.rug.nl/ws/files/177428059/1_s2.0_S0920563204001604_main.pdf	English	null	A Broad Iron Line in the Chandra/HETG Spectrum of 4U 1705-44	International Astronomical Union colloquium	2,004	cc-by	3,076	University of Groningen University of Groningen A broad iron line in the chandra/hetg spectrum of 4U 1705-44 Published in: Nuclear Physics B DOI: 10.1016/j.nuclphysbps.2004.04.102 IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF also known as Version of record Document Version Publisher's PDF, also known as Version of record Link to publication in University of Groningen/UMCG research database Citation for published version (APA): di Salvo, T., Iaria, R., Méndez, M., Burderi, L., Robba, N. R., Stella, L., & van der Klis, M. (2004). A broad iron line in the chandra/hetg spectrum of 4U 1705-44. Nuclear Physics B, 132, 600-603. Article 600-603. https://doi.org/10.1016/j.nuclphysbps.2004.04.102 Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). A broad iron line in the Chandra/HETG spectrum of 4U 1705–44 T. Di Salvoa∗, R. Iariab, M. M´endezc, L. Burderid, N.R. Robbab, L. Stellad and M. van der Klisa aAstronomical Institute ”Anton Pannekoek,” University of Amsterdam and Center for High-Energy Astrophysics, Kruislaan 403, NL 1098 SJ Amsterdam, the Netherlands A broad iron line in the Chandra/HETG spectrum of 4U 1705–44 T. Di Salvoa∗, R. Iariab, M. M´endezc, L. Burderid, N.R. Robbab, L. Stellad and M. van der Klisa aAstronomical Institute ”Anton Pannekoek,” University of Amsterdam and Center for High-Energy Astrophysics, Kruislaan 403, NL 1098 SJ Amsterdam, the Netherlands T. Di Salvoa∗, R. Iariab, M. M´endezc, L. Burderid, N.R. Robbab, L. Stellad and M. van der Klisa aAstronomical Institute ”Anton Pannekoek,” University of Amsterdam and Center for High-Energy Astrophysics, Kruislaan 403, NL 1098 SJ Amsterdam, the Netherlands bDipartimento di Scienze Fisiche ed Astronomiche, Universit`a di Palermo, via Archiraﬁn.36, 90123 Palermo, Italy cSRON National Institute for Space Research, Sorbonnelaan 2, 3584 CA Utrecht, th cSRON National Institute for Space Research, Sorbonnelaan 2, 3584 CA Utrecht, the Netherlands dOsservatorio Astronomico di Roma, via Frascati 33, 00040 Roma, Italy cSRON National Institute for Space Research, Sorbonnelaan 2, 3584 CA Utrecht, the Netherlands dOsservatorio Astronomico di Roma, via Frascati 33, 00040 Roma, Italy dOsservatorio Astronomico di Roma, via Frascati 33, 00040 Roma, Italy dOsservatorio Astronomico di Roma, via Frascati 33, 00040 Roma, Italy dOsservatorio Astronomico di Roma, via Frascati 33, 00040 Roma, Italy We present preliminary results from a Chandra 30 ks observation of the atoll source 4U 1705–44. In particular we concentrate on the study of discrete features in the energy spectrum at energies below ∼3 keV, as well as the iron Kα line, using the HEG spectrometer on board of the Chandra satellite. The iron Kα line at ∼6.6 keV is found to be intrinsically broad (FW HM ∼1.7 keV); its width can be explained by reﬂection from a cold accretion disk extending down to ∼17 km from the neutron star center or by broadening in the hotter (∼3 −4 keV) Comptonizing corona. ∗This work was partially supported by the Netherlands Organization for Scientiﬁc Research (NWO). 0920-5632/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.nuclphysbps.2004.04.102 Copyright strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of th or copyright holder(s), unless the work is under an open content license (like Creative Commons). The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license. More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne- amendment. Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 24-10-2024 3. SPECTRAL ANALYSIS We selected the ﬁrst-order spectra from the HEG and MEG. Data were extracted from re- gions around the grating arms; to avoid over- lapping between HEG and MEG data we used a region size of 20. The background spectra were computed by extracting data above and below the dispersed ﬂux, again with the same total width in cross-dispersion. The contribution from the background is ∼0.4% of the total count rate. We used the standard CIAO tools to create detector response ﬁles (see [4]) for the HEG +1 and −1 order (background-subtracted) spectra, which we ﬁt simultaneously using the XSPEC v11.2 data analysis package [1]. In some intervals (especially at those wavelength corresponding to the junc- tion between two CCDs) the HEG +1 and −1 orders give diﬀerent residuals with respect to the same continuum model. The reason of these dif- ferences is not clear yet and is under investiga- tion. In the present work we exclude those inter- vals corresponding to the junction between two CCDs and in which the two orders give diﬀerent results. The energy ranges used in the following spectral analysis are: 1.4–5.8 keV and 6.3–11 keV for −1 order, and 1.4–3.1 keV and 3.4–11 keV for +1 order, respectively. 1. INTRODUCTION Suppl.) 132 (2004) 600–603 601 XMM-Newton, can also contribute to broaden the line. we utilize the HEG ﬁrst-order spectrum, which is less aﬀected by pile-up (less than 10%) with respect to the MEG ﬁrst-order spectrum. 4U 1705–44 is an atoll source (see [6]) which also shows type-I X-ray bursts. We selected this source for a Chandra observation because a broad (1.1 keV FWHM) iron emission line at 6.5 keV has been reported [12], [2]. One of the goals of our Chandra observation was the study the iron line proﬁle to discriminate among the various models that have been proposed to explain the large line width. 1. INTRODUCTION fects arising from the strong gravity in the vicin- ity of the neutron star smears the reﬂected fea- tures, so that the line will have a characteristi- cally broad proﬁle, the detailed shape of which depends on the inclination and on how deep the accretion disk extends into the neutron star po- tential [5], [9]. Broad emission lines (FWHM up to ∼1 keV) at energies in the range 6.4 – 6.7 keV are often ob- served in the spectra of low-mass X-ray binaries (LMXBs) hosting an old accreting neutron star. These lines are identiﬁed with the Kα radiative transitions of iron at diﬀerent ionization states. Sometimes an iron absorption edge at energies around 8 keV has been detected. These features are powerful tools to investigate the structure of the accretion ﬂow close to the central source; in particular, important information can be ob- tained from the detailed spectroscopy of the iron Kα emission line and absorption edge, since they are determined by the ionization state, geometry and velocity ﬁeld of the reprocessing plasma. An alternative location of the line emitting region is the so-called Accretion Disk Corona (ADC), probably formed by evaporation of the outer layers of the disk illuminated by the emis- sion of the central object (e.g. [11]). In this case the width of the line is explained by ther- mal Comptonization of the line photons in the ADC. This produces a genuinely broad Gaus- sian distribution of line photons, with σ ≥ EFe(kTe/mec2)1/2, where EFe is the centroid en- ergy of the iron line and kTe is the electron tem- perature in the ADC (see [7], [3] for more de- tailed calculations). This mechanism can explain the width of the iron line for temperatures of the emitting region of few keV. To explain the large width of these lines it has been proposed that they originate from emission reprocessed by the accretion disc surface illumi- nated by the primary Comptonized spectrum [5]. In this model, the combination of Doppler eﬀects from the high orbital velocities and relativistic ef- The presence of several unresolved components, which can eventually be resolved by the high res- olution X-ray instruments on board Chandra and ∗This work was partially supported by the Netherlands Organization for Scientiﬁc Research (NWO). 0920-5632/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.nuclphysbps.2004.04.102 T. Di Salvo et al. / Nuclear Physics B (Proc. 2. OBSERVATIONS Residuals in units of σ when the iron line is not included in the model. To ﬁt the discrete residuals with respect to the previous model we add to the model several Gaus- sian lines. The best ﬁt model is reported in Table 1 together with the most signiﬁcant of these dis- crete features. energy range between 4 and 11 keV, ﬁx the con- tinuum parameters to the best ﬁt values, and sub- stitute the Gaussian line at 6.6 keV with the laor model [8]. This model gives a slightly better ﬁt (χ2(d.o.f.) = 224.7(214) for the Gaussian line and χ2(d.o.f.) = 207.5(211) for the Laor line). The line best ﬁt parameters are shown in Table 2. 2. OBSERVATIONS Our observation of 4U 1705–44 was performed using the High-Energy Transmission Grating Spectrometer (HETGS) on board of Chandra starting on 2001 July 1. The total integration time was 24.4 ks, and the observation was contin- uous. The HETGS consists of two transmission gratings, the Medium-Energy Grating (MEG) and the High-Energy Grating (HEG). It allows high-resolution spectroscopy from 1.2 to 31 ˚A (0.4–10 keV) with a peak spectral resolution at 12 ˚A of ∆λ/λ ∼1000 for HEG ﬁrst order. The dispersed spectra were recorded with an array of six charged coupled devices (CCDs) that are part of the Advanced CCD Imaging Spectrome- ter (ACIS-S; see http://chandra.harvard.edu for more details). ) The brightness of the source required addi- tional eﬀorts to mitigate the “photon pile-up” ef- fects. We applied a “subarray” during the obser- vation that reduced the CCD frame time to 1.4 s. The zeroth-order image is aﬀected by heavy pile- up; the event rate is so high that two or more events are detected in the CCD during the 1.4-s frame exposure. Pile-up distorts the spectrum be- cause detected events overlap and their deposited charges are collected into single, apparently more energetic events. Moreover, many events (∼90%) are lost as the grades of the piled-up events over- lap those of highly energetic background parti- cles, and are rejected by the on board software. We therefore will ignore the zeroth-order events in all subsequent analyses. On the other hand, the grating spectra are not, or only moderately (less than 20%), aﬀected by pile-up. In our analysis, We ﬁt the HEG ﬁrst order spectra of 4U 1705– 44 to a continuum model. The best ﬁt model consists of a blackbody plus the Comptoniza- tion model comptt [10], modiﬁed by absorption from neutral matter, which gives a χ2 red(d.o.f.) of 1.64(846). The addition of a broad (σ ∼0.7 keV) Gaussian line centered at 6.57 keV proves necessary, giving χ2 red(d.o.f.) = 1.25(843). Data and residuals in units of σ with respect to this model are shown in Figure 1; several features are still visible in the residuals with respect to the best ﬁt continuum model. In Figure 2 we show the residuals in units of σ when the iron line is not included in the model. From this ﬁgure it is apparent that the iron line is intrinsically broad and shows a complex proﬁle. T. Di Salvo et al. 2. OBSERVATIONS / Nuclear Physics B (Proc. Suppl.) 132 (2004) 600–603 602 Figure 1. HEG ﬁrst order spectrum of 4U 1705– 44 (top panel) and residuals in units of σ with re- spect to the best ﬁt continuum model (blackbody, Comptonization, and a broad Gaussian line, bot- tom panel). Table 1 Results of the ﬁtting of the 4U 1705–44 HEG ﬁrst order spectrum in the 1–11 keV energy band. Parameter Value NH (×1022 cm−2) 3.12 ± 0.13 kTBB (keV) 0.131 ± 0.017 NormBB 1.03 ± 0.74 kT0 (keV) 0.313 ± 0.074 kTe (keV) 3.85 ± 0.99 τ 10.6 ± 1.8 Normcomp 1.22+0.40 −0.10 E1 (keV) 1.475 ± 0.008 σ1 (keV) 0.0154 ± 0.0066 I1 (10−3 cm−2 s−1) 6.6+3.2 −2.0 EQW1 (eV) 3.07 E2 (keV) 2.163 ± 0.025 σ2 (keV) 0.087 ± 0.029 I2 (10−2 cm−2 s−1) 1.11+0.66 −0.31 EQW2 (eV) 15.0 E3 (keV) 2.647 ± 0.021 σ3 (keV) 0.065+0.052 −0.019 I3 (10−3 cm−2 s−1) 5.5+4.0 −1.6 EQW3 (eV) 10.9 E4 (keV) 3.058 ± 0.007 σ4 (keV) 0.0181 ± 0.0081 I4 (10−3 cm−2 s−1) 1.65+0.67 −0.53 EQW4 (eV) 4.38 EFe (keV) 6.571 ± 0.068 σFe (keV) 0.73 ± 0.11 IFe (10−2 cm−2 s−1) 2.79+0.80 −0.44 EQWFe (eV) 336 Figure 1. HEG ﬁrst order spectrum of 4U 1705– 44 ( l) d id l i i f i h Figure 1. HEG ﬁrst order spectrum of 4U 1705– 44 (top panel) and residuals in units of σ with re- spect to the best ﬁt continuum model (blackbody, Comptonization, and a broad Gaussian line, bot- tom panel). Figure 2. Residuals in units of σ when the iron line is not included in the model. The model consists of a blackbody, a Comptonized spec- trum modeled by comptt, and ﬁve Gaussian emission lines. The blackbody normalization, NormBB, is in units of L39/D2 10, where L39 is the source luminosity in units of 1039 ergs/s and D10 is the distance to the source in units of 10 kpc. kT0 is the temperature of the seed photon (Wien) spectrum and Normcomp is the normalization of the comptt model in XSPEC v.11 units. For the discrete features, I is the intensity of the line and EQW is the corresponding equivalent width. Uncertainties are 90% conﬁdence level for a single parameter of interest. Figure 2. Residuals in units of σ when the iron line is not included in the model. Figure 2. 3.1. The K-shell iron line proﬁle The residuals in the K-shell iron line range are quite complex (see Fig. 2), showing a broad fea- ture centered at ∼6.6 keV. We therefore try to ﬁt this feature with the line proﬁle expected from a thin Keplerian accretion disk; we consider the T. Di Salvo et al. / Nuclear Physics B (Proc. Suppl.) 132 (2004) 600–603 603 Table 2 Iron line parameters from the Laor model. Parameter Value Energy (keV) 6.32 ± 0.04 Rin (Rg) 8+2 −1 Rout (Rg) 390+10 −80 Inclination (deg) 82+5 −4 Index 2.36+0.06 −0.13 I (10−2 cm−2 s−1) 3.6 ± 0.5 Index is the index of the power law dependence of emissivity which scales as r−Index. Uncertainties are 90% conﬁdence level for a single parameter of interest. Table 2 Iron line parameters from the Laor model. Parameter Value Energy (keV) 6.32 ± 0.04 Rin (Rg) 8+2 −1 Rout (Rg) 390+10 −80 Inclination (deg) 82+5 −4 Index 2.36+0.06 −0.13 I (10−2 cm−2 s−1) 3.6 ± 0.5 see also [3]). Assuming an electron temperature kTe = 3.8 keV, as derived from the ﬁt of our data to the Comptonization model, we can explain the width of the iron line for a Thomson optical depth of τT ∼1.4. Therefore, it is possible that the line is produced in the outer region of the Comptoniz- ing corona, where the optical depth decreases (as- suming that the temperature remains constant). This is not unreasonable given that any contribu- tion to the line produced inside the Comptonizing region, where the optical depth can be as high as 10 (see Table 1), would be completely smeared by Comptonization. Therefore, we expect to see only that part of the line that is produced in the outer Comptonizing region. Index is the index of the power law dependence of emissivity which scales as r−Index. Uncertainties are 90% conﬁdence level for a single parameter of interest. Index is the index of the power law dependence of emissivity which scales as r−Index. Uncertainties are 90% conﬁdence level for a single parameter of interest. 4. DISCUSSION AND CONCLUSIONS 1. K.A. Arnaud, in Astronomical Data Analysis Software and Systems V, A.S.P. Conference Series, Vol. 101 (1996), G.H. Jacoby and J. Barnes, eds., p. 17. We have analysed the HEG spectrum from a Chandra 25 ks observation of the X-ray burster and atoll source 4U 1705–44, and we detect an emission line at 6.6 keV, which we interpret as K-shell ﬂuorescent emission of Fe. The high en- ergy resolution of the HEG shows that the line is intrinsically broad (FWHM ∼1.7 keV), in agreement with previous measurements (see e.g. [2]). Therefore, the most probable origin of this line is an accretion disk (in this case the large width of the line would be due to Doppler and relativistic smearing eﬀects) or from a hot corona (in this case the large width of the line would be due to Compton broadening). 2. D. Barret and J.F. Olive, ApJ 576 391 (2002). 3. W.M. Brandt and G. Matt, MNRAS 268 1051 (1994). 4. J.E. Davis, ApJ 562 575 (2001). 5. A.C. Fabian, et al. MNRAS 238 729 (1989). 6. G. Hasinger and M. van der Klis, A&A 225 79 (1989). 7. T. Kallman and N.E. White, ApJ 341 955 (1989). 8. A. Laor, ApJ 376 90 (1991). 9. L. Stella, Nature 344 747 (1990). 10. L. Titarchuk, ApJ 434 570 (1994). In the case the line is produced by reﬂection in an accretion disk we estimate that the re- quired inner radius of the disk is 8 Rg or ∼17 km for a 1.4 M⊙neutron star. This would be in disagreement with the low temperature and high normalization of the blackbody com- ponent (see Tab. 1), which would imply, as- suming that the blackbody is produced by the optically thick disk emission, a large inner disk radius (note, however, that the blackbody nor- malization is poorly constrained). Alternatively, Comptonization in the corona could explain the large width of the line. Detailed calculations give σFe = 0.019EFeτT (1 + 0.78kTe), where τT is the Thompson optical depth and kTe is in keV ([7], 11. N.E. White and S.S. Holt, ApJ 257 318 (1982). 12. N.E. White, et al., MNRAS 218 129 (1986).
https://openalex.org/W2729880573	https://europepmc.org/articles/pmc5599677?pdf=render	English	null	Angle-resolved stochastic photon emission in the quantum radiation-dominated regime	Scientific reports	2,017	cc-by	9,356	Angle-resolved stochastic photon emission in the quantum radiation- dominated regime Received: 9 May 2017 Accepted: 22 August 2017 Published: xx xx xxxx Received: 9 May 2017 Accepted: 22 August 2017 Published: xx xx xxxx Jian-Xing Li , Yue-Yue Chen, Karen Z. Hatsagortsyan & Christoph H. Keitel Signatures of stochastic effects in the radiation of a relativistic electron beam interacting with a counterpropagating superstrong short focused laser pulse are investigated in a quantum regime when the electron’s radiation dominates its dynamics. We consider the electron-laser interaction at near-reflection conditions when pronounced high-energy gamma-ray bursts arise in the backward- emission direction with respect to the initial motion of the electrons. The quantum stochastic nature of the gamma-photon emission is exhibited in the angular distributions of the radiation and explained in an intuitive picture. Although, the visibility of the stochasticity signatures depends on the laser and electron beam parameters, the signatures are of a qualitative nature and robust. The stochasticity, a fundamental quantum property of photon emission, should thus be measurable rather straightforwardly with laser technology available in near future. The next generation petawatt laser systems1, 2 will open a door not only to novel regimes of laser-matter interac- tion3, 4, but also to new perspectives for the investigation of fundamental problems5–9. In ultrastrong laser fields the quantum properties of electron radiation, the discrete and probabilistic character of photon emission, can be conspicuous. While the discreteness of the radiation photon energy is known to be observed straightforwardly, e.g., in the Compton scattering as a shift of the emission frequency10–12, the signatures of the stochastic char- acter of photon emission are more subtle and elaborate for observation. The latter has impact on the radiation back-action to the electron dynamics and should be more apparent in the so-called radiation dominated regime (RDR) of interaction8, 13, 14, when multiple emission of photons by an electron becomes probable. One of the conceptual consequences of stochasticity effects (SE) in photon emissions, i.e., the probabilistic nature of photon emission, is the broadening of the energy spread of an electron beam in a plane laser field15, 16, while similar effect can cause electron stochastic heating in a standing laser field17. However, in an experiment in a focused laser beam, competing effects may arise, e.g., an additional energy spreading of the electron beam due to the difference of radiative losses of electrons in the electron-beam cross section. Max-Planck-Institut für Kernphysik, Saupfercheckweg 1, 69117, Heidelberg, Germany. Correspondence and requests for materials should be addressed to J.-X.L. (email: Jian-Xing.Li@mpi-hd.mpg.de) or K.Z.H. (email: k.hatsagortsyan@mpi-k.de) www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Received: 9 May 2017 Accepted: 22 August 2017 Published: xx xx xxxx Scientific REPOrts \| 7: 11556 \| DOI:10.1038/s41598-017-11871-0 The setup and radiation spectrah without recoil). The simulation methods including and excluding the SE are described in detail in the section “Methods”.i We employ a linearly polarized tightly focused laser pulse with a Gaussian temporal profile, which propagates along +z-direction and polarizes in x-direction. The spatial distribution of the electromagnetic fields takes into account up to the ε3-order of the nonparaxial solution46, 47, where ε = w0/zr, w0 is the laser focal radius, = z k w /2 r 0 0 2 the Rayleigh length with the laser wave vector k0 = 2π/λ0, and λ0 the laser wavelength. The expressions of the laser fields are represented in the section “Methods”. i p A typical angular distribution of radiation which carries the signature of the stochastic nature of photon emis- sion, is illustrated in Fig. 1; φ = 0° and ±180° correspond to the positive and negative directions of the laser polarization, respectively. The peak intensity of the 6-cycle (FWHM) laser pulse is I ≈ 4.9 × 1023 W/cm2 (ξ = 600), λ0 = 1 μm, and w0 = 2 μm. The electron beam, with radius we = λ0, length Le = 6λ0, and density ne ≈ 1015 cm−3, initially counterpropagates with the laser pulse, i.e. θ = ° 180 e i( ) . The initial mean kinetic energy of the electron beam is ε0 = 180 MeV (γ0 ≈ 353, the maximum value of χ during interaction 1 max χ < ), and the energy and angu- lar spread are Δε/ε0 = Δθ = 0.02. The electron-beam parameters are typical for current laser-plasma acceleration setups5–7.hi Figures 1(a,b) demonstrate RAD including and excluding SE, respectively. The radiation is most significant along the strongest field component Ex of the linearly polarized laser field. However, in a tightly focused laser beam, other components of the electric field, Ey and Ez, are not negligible and play a significant role in the electron dynamics and the photon emission. Consequently, the electrons radiate continuously over the azimuthal angle φ, with a Gaussian radiation distribution with respect to φ corresponding to the laser transverse profile. Moreover, the radiation sweeps from the polar angle θ θ = ° = 180 e i( ) down to θ ≈ 11°. The electrons initially counterpropa- gate with the laser pulse, emit forwards, and create a high-intensity spectral region around θ ≈ 180°. Angle-resolved stochastic photon emission in the quantum radiation- dominated regime Another SE signature is the so-called electron straggling effect during radiation in strong fields18–21, when the electrons propagate a long distance without radia- tion due to SE, resulting in the increase of the yield of high-energy photons. However, we will show below that the straggling effect is rather weak in the considered regime. Different signatures of quantum radiation reaction have been discussed22–35 which include all quantum effects, such as photon recoil, stochasticity, and interferences35. However, they do not isolate information on the specific role of stochasticity. Unfortunately, so far unequivocal SE have not been observed in an experiment, which would also be important as a clear test for the underlying theories. We underline that in this paper SE refers to the quantum probabilistic nature of photon emission, but not to the chaotic classical dynamics as, e.g., in ref. 36. The quantum effects in strong laser fields are determined by the invariant parameter χ ≡ µν ν e F p m ( ) / 2 3 37, 38, where Fμν is the field tensor, pν = (ε, p) the incoming electron 4-momentum, and e and m are the electron charge and mass, respectively (Planck units ħ = c = 1 are used throughout). The RDR, when the radiation losses during a laser period are comparable with the electron initial energy, is characterized by the parameter αξχ ≡ >∼ R 18, where α is the fine structure constant, ξ ≡ \|e\|E0/(mω0) the invariant laser field parameter, while E0 and ω0 are the laser field amplitude and frequency, respectively. In the quantum RDR the stochastic nature of photon emission is a fundamental quantum property and has to be taken into account during the high-energy photon radiation39–42, that significantly affects the electron dynamics and the high-energy photon emission. Scientific REPOrts \| 7: 11556 \| DOI:10.1038/s41598-017-11871-0 1 www.nature.com/scientificreports/ In this paper, we investigate signatures of the stochastic nature of photon emission in the nonlinear Compton scattering in the quantum RDR during the interaction of a superstrong short focused laser pulse with a counter- propagating relativistic electron beam. We consider the interaction in the electron near-reflection regime14, where the front electrons are reflected and emit gamma-rays in the near-backward direction29: due to the combined effects of the laser focusing and radiation reaction, the front electrons of the electron beam are reflected and emit ultrashort gamma-rays in the near-backward direction with respect to the initial electron motion29. The setup and radiation spectrah The considered quantum RDR requires the invariant parameters  m ( / ) (1 cos ) 10 1 0 6 χ γ ω ξ β θ γξ ≡ − ≈ < − and αξχ ≡ >∼ R 1, while the electron near-reflection regime does /2 γ ξ ∼ , where γ is the Lorenz factor of the elec- tron, and β the electron velocity scaled by the light speed in vacuum. The two conditions above demand γ ξ ∼ ∼103, i.e., an electron beam of GeV energies and laser intensities of 1023–1024 W/cm2 anticipated in next generation facilities1, 2.h g The calculation of the radiation is based on Monte-Carlo simulations employing QED theory for the electron radiation and classical equations of motion for the propagation of electrons between photon emissions40–42. In superstrong laser fields 1  ξ , the coherence length of the photon emission is much smaller than the laser wave- length and the typical size of the electron trajectory38, 43. Then, the photon emission probability is determined by the local electron trajectory, consequently, by the local value of the parameter χ44. The radiation in the quantum regime ignoring the SE are calculated by employing the Sokolov equation22–24, when the radiation is emitted con- tinuously along the electron trajectory and modifies accordingly the classical equations of motion. In this work we consider the regime when the quantum invariant field parameter χ is not small, χ <1. Therefore, the quan- tum recoil in photon emission is significant even in the case when the probabilistic character (stochasticity) of photon emission is neglected. Therefore radiation intensity and the radiation reaction should be calculated using quantum corrections. The Sokolov equation is a phenomenologically derived equation of motion for an electron in the  ξ 1 limit, which is based on the energy-momentum conservation within the system of the electron and emitted photons at each formation length of radiation [however, it is argued in ref. 45 that this equation cannot be derived from QED by taking the classical limit]. The Sokolov equation adapts the electron energy-momentum at each step according to the radiated energy-momentum including quantum corrections. It provides a natural way to treat the electron dynamics in the external field classically but to take into account in the radiation-reaction quantum-recoil corrections (but not stochasticity). In contrast, the conceptually more systematic Lorentz-Abraham-Dirac or Landau-Lifshitz equations account for the radiation reaction only classically (i.e. Angle-resolved stochastic photon emission in the quantum radiation- dominated regime In the consid- ered case with 1 χ < , the straggling effect is rather weak, however, the electron near-reflection regime offers a possibility to observe the SE in the angular distribution of the radiation. In fact, the photon emissions in different laser cycles are essentially modified due to the SE in the RDR, and the latter is mapped into the broad backward-emission angles when the electron is at the near-reflection condition. We calculate angle-resolved radi- ation intensity and photon numbers and show that, due to the stochastic nature of photon emission, the radia- tion’s angular distribution (RAD) has a single prominent peak in the backward direction, which is broad and easily observable in an experiment. In contrast, when the SE are ignored, the backward radiation yields an angular distribution with several peaks. Furthermore, we investigate the influences of the laser and electron-beam param- eters (the laser focal radius, the laser pulse duration and the electron initial energy) on the visibility of those SE signatures, to optimize parameters for a future experimental setup. Scientific REPOrts \| 7: 11556 \| DOI:10.1038/s41598-017-11871-0 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 1. Angle-resolved radiation energy εR in units of the electron rest energy m vs the emission polar angle θ and the azimuthal angle φ: (a) including and (b) excluding SE, in a 6-cycle focused laser pulse. Color coded is log10[dεR/dΩ] rad−2, with the emission solid angle Ω. The laser and electron beam counterpropagate, with the initial propagation polar angles θ = ° 0 L i( ) and θ = ° 180 e i( ) , respectively. All other parameters are given in the text. Figure 1. Angle-resolved radiation energy εR in units of the electron rest energy m vs the emission polar angle θ and the azimuthal angle φ: (a) including and (b) excluding SE, in a 6-cycle focused laser pulse. Color coded is log10[dεR/dΩ] rad−2, with the emission solid angle Ω. The laser and electron beam counterpropagate, with the initial propagation polar angles θ = ° 0 L i( ) and θ = ° 180 e i( ) , respectively. All other parameters are given in the text. The radiation distributions with respect to the polar angle θ with and without SE are essentially different in Fig. 1. While with SE the RAD is smoothly peaking at θ = 180° and at small angles, without SE it shows a band structure corresponding to the radiation emerging from different laser cycles. A similar behaviour is seen in RAD for photon numbers (See the Supplemental Materials for the details, which includes the impacts of different parameters on the radiation angular distributions). The radiation distributions with respect to the polar angle θ with and without SE are essentially different in Fig. 1. While with SE the RAD is smoothly peaking at θ = 180° and at small angles, without SE it shows a band structure corresponding to the radiation emerging from different laser cycles. A similar behaviour is seen in RAD for photon numbers (See the Supplemental Materials for the details, which includes the impacts of different parameters on the radiation angular distributions). p g A quantitative comparison between RADs including and excluding SE is represented in Fig. 2. www.nature.com/scientificreports/ We focus on the strongest radiation domain along the polarization plane in the region of −15° ≤ φ ≤ +15°, analysing the radiation energy d Rε /[dθ sin(θ)] =   ∫− + 15 15 dφ dεR/dΩ and the photon number d∼ NR/[dθ sin(θ)] =   ∫− + 15 15 dφ dNR/dΩ in this domain. The stochastic nature of photon emission is clearly discernible in RAD: a single broad high-intensity gamma-photon peak is formed in the near-reflection direction when SE is included, while in the case without SE multiple radiation peaks emerge corresponding to the emission from different laser cycles. Therefore, the main detectable difference of RADs with and without SE is the peak number of the radiation: the previous has only one radiation peak, and the latter has several peaks corresponding to the laser-cycle structure. The setup and radiation spectrah Due to radi- ative losses, the electron energy decreases, facilitating the reflection condition γ ≈ ξ/2 in the strong-field region and inducing electron reflection, during which the radiation flash sweeps down to θ ≈ 11°. The emission angle after the reflection with respect to the electron average motion / δθ ξ γ ∼ is determined by the values of ξ and γ in situ after the reflection. In the region of θ ≈ 0°, the radiation intensity is vanishing, because of the very low χ value for the co-propagating electron: the radiation energy εR ∝ χ ∝ γξ(1−β cos θ) ≈ 0 at θ ≈ 0°. Scientific REPOrts \| 7: 11556 \| DOI:10.1038/s41598-017-11871-0 2 Analysis of the radiation properties We proceed discussing the role of SE in shaping RAD. The dynamics and radiation of a sample electron is ana- lysed in Fig. 3 (an electron at the beam center, z = Le/2 and x = y = 0, is considered). We follow the mean longitu- dinal momentum pz and the mean motion direction θe of the electron during interaction with the laser pulse in dependence on the laser phase t k z ( )/(2 ) 0 0 η ω π = − , see Fig. 3(a,b), when SE are included or excluded, respec- tively. To reproduce SE we repeat the simulation 200 times for the same initial conditions of the electron. The single-electron radiation angle is m p arctan( / ) e z θ ξ ∼ (0 eθ π ≤ ≤ ). The point = p 0 z corresponds to the electron reflection, when the emission angle sharply changes from the forward into the backward direction.i l g p y g In the case without SE, the single-electron radiation angle is well defined at each moment during interaction. In contrast, in the case with SE one observes spreading of the average longitudinal momenta and the emission angles at a certain laser phase η , see Fig. 3(a,b), which stems from the probabilistic character of the photon-emission process. In each possible trajectory, the electron emits photons at different moments and pho- tons of different energies. Consequently, the electron has different energy and emission angle at the same laser phase η (same ξ).h The dynamics of the photon emission in the SE case described above is illustrated in Fig. 3(c,d) for two sample trajectories, having a small and a large energy after the reflection in Fig. 3(a). Although both of the trajectories begin with the same initial condition, in the first case the number of photon emissions before the reflection ( 7 5 η < . ) is larger. In the second case the electron “straggles” (does not emit large number of photons) most of the time before the reflection, only emitting a large photon near the reflection η ≈ .5 5 (See the Supplemental Scientific REPOrts \| 7: 11556 \| DOI:10.1038/s41598-017-11871-0 3 www.nature.com/scientificreports/ Figure 2. RAD: (a) radiation energy dεR/[dθ sin(θ)] (rad−1), and (b) photon number d∼ NR/[dθ sin(θ)] (rad−1), emitted in the region of −15° ≤ φ ≤ +15° of the azimuthal angle, see Fig. 1. Analysis of the radiation properties The emitted photon number d∼ NR/ [dθ sin(θ)] in the whole 2π-region of the azimuthal angle: (c) all photon energies, and (d) the photon energies above 50 MeV. Blue-solid curves are with SE, and red-dotted curves are without SE. The employed laser and electron beam parameters are the same as in Fig. 1. Figure 2. RAD: (a) radiation energy dεR/[dθ sin(θ)] (rad−1), and (b) photon number d∼ NR/[dθ sin(θ)] (rad−1), emitted in the region of −15° ≤ φ ≤ +15° of the azimuthal angle, see Fig. 1. The emitted photon number d∼ NR/ [dθ sin(θ)] in the whole 2π-region of the azimuthal angle: (c) all photon energies, and (d) the photon energies above 50 MeV. Blue-solid curves are with SE, and red-dotted curves are without SE. The employed laser and electron beam parameters are the same as in Fig. 1. Materials for the details, which includes the impacts of different parameters on the radiation angular distribu- tions). Because of the latter, the parameter χ after the reflection is larger for the first trajectory with respect to the second, yielding significantly more radiative loss for the first trajectory than for the second one, and correspond- ingly, smaller energy (larger emissison angle) for the first trajectory than for the second one after the reflection (See the Supplemental Materials for the details, which includes the impacts of different parameters on the radia- tion angular distributions). Thus, different probabilistic dynamics of the photon emission, i.e. SE, induces spread- ing of the photon emission angle at each laser phase.h Materials for the details, which includes the impacts of different parameters on the radiation angular distribu- tions). Because of the latter, the parameter χ after the reflection is larger for the first trajectory with respect to the second, yielding significantly more radiative loss for the first trajectory than for the second one, and correspond- ingly, smaller energy (larger emissison angle) for the first trajectory than for the second one after the reflection (See the Supplemental Materials for the details, which includes the impacts of different parameters on the radia- tion angular distributions). Thus, different probabilistic dynamics of the photon emission, i.e. SE, induces spread- ing of the photon emission angle at each laser phase.h g p g p The angle-resolved radiation intensity and radiation energy are shown in Fig. 3(e,f), respectively, for the case without SE. Analysis of the radiation properties In each cycle strongest radiation arises near the peaks of the cycles at a certain angle. Between adja- cent radiation peaks, there is a gap in emission angle corresponding to the weak-field part of the laser cycle. Therefore, the RAD reveals the laser-cycle structure when SE are neglected.h h y g The RAD with SE for a single electron is shown in Fig. 3(g,h). Since with SE the radiation angle in each laser cycle has a very broad spread shown in Fig. 3(b,g), the gaps in emission angle between adjacent radiation peaks of each cycle are filled out. Consequently, the radiation intensity in this case shows a single gamma-radiation peak corresponding to the peak of the laser pulse. Note that the discussed qualitative features of the RAD for a sample electron do not depend on the initial position of the electron in the electron beam. The variation of the initial position introduces only a slight modification of a quantitative character (See the Supplemental Materials for the details, which includes the impacts of different parameters on the radiation angular distributions).hf f g The electron straggling effect in our setup is estimated in Fig. 2(c,d) for the emitted photon number integrated over the whole 2π-region of the azimuthal angle. The straggling effect is observed as an enhancement of the emission of high-energy photons when the SE are included, see Fig. 2(d) for the emitted photon number above 50 MeV. However, the electron straggling effect in the considered regime is insignificant and not relevant for the observation of SE. Optimal conditions for observation of stochasticity effects h f h l f l d l d d The two thin lines are with SE and show the boundaries at η >∼10 (note that the same color lines in (a,b) do not correspond to the same electron). (c,d) The variation of the parameter χ with respect to η of a sample electron with SE. The blue marks indicate the photon emissions. The single electron radiation, integrated over the azimuthal angle of −15° ≤ φ ≤ +15°, without SE: (e) radiation intensity vs emission phase η, log10[d R 2ε/[dηdθ sin(θ)]], and (f) radiation energy d R ε/ [dθ sin(θ)]. (g,h) Correspond to (e,f), respectively, for the case including SE. The sample electron parameters are given in the text, and the laser parameters are the same as in Fig. 1. Figure 3. (a) The mean longitudinal momentum pz and (b) the mean motion direction (photon-emission hh case cannot be observed any more. Therefore, a tightly focused laser pulse with a focal radius of roughly 2 times of the electron-beam radius applies well for the observation of SE.h The dependence of RAD on the laser-pulse length is shown in Fig. 5. As the laser-pulse length increases from 4λ0 to 10λ0, the main (highest) radiation peak in the region of 11 45   θ ° < < ° gradually decreases, and the pla- teaus between the peaks gradually increase. In principle, for a longer laser pulse there are more peaks correspond- ing to the laser cycles in the “without SE” case, which is helpful to distinguish SE. However, for a longer laser pulse, e.g., LL = 10λ0, the laser-field gradient is smaller than for shorter laser pulses, consequently, the ratio between the peaks and the plateaus becomes smaller, and the visibility of the signatures becomes worse. Then, a short laser pulse of LL ≈ 6λ0 is optimal: it has a more-cycle structure than for ultrashort pulses, and the visibility of the laser-cycle structure is better than longer pulses.h y g p The role of the initial kinetic energy of the electron beam on the radiation angular distribution is analysed in Fig. 6. As the electron kinetic energy ε0 increases from 120 MeV to 240 MeV, the radiation energy εR ∝ χ ∝ ξγ increases as well since 0 γ ε ∼ and ξ being constant is assumed. Optimal conditions for observation of stochasticity effects h f h l f l d l d d We investigate the optimization of the laser (focal radius, pulse duration) and electron (energy) parameters for the best identification of SE. The influence of the laser focusing effect on RAD is studied in Fig. 4. The case of w0 = 2λ0 is optimal, being the same as in Figs 1 and 2, when the SE are exhibited by a broad smooth peak of the gamma radiation in backward direction. For w0 = λ0 = we, the near-reflection radiation is rather weak, and there is no laser-cycle structure in the “without SE” case. For w0 = 3λ0, the main (highest) gamma-photon peaks in the region of 11 45   θ ° < < ° are much stronger apparently than those for w0 = 2λ0, since all electrons are much closer to the laser-intensity center, which also weakens the shift of the main peaks in polar angle when the SE are excluded. Consequently, higher angular resolution is required to distinguish the “without SE” case from the one with SE. In the extreme case of a plane-wave laser pulse the structure of multiple gamma-photon peaks in the “without SE” Scientific REPOrts \| 7: 11556 \| DOI:10.1038/s41598-017-11871-0 4 www.nature.com/scientificreports/ Figure 3. (a) The mean longitudinal momentum pz and (b) the mean motion direction (photon-emission direction) θe (polar angle) of a sample electron. The thick line is without SE. The two thin lines are with SE and show the boundaries at η >∼10 (note that the same color lines in (a,b) do not correspond to the same electron). (c,d) The variation of the parameter χ with respect to η of a sample electron with SE. The blue marks indicate the photon emissions. The single electron radiation, integrated over the azimuthal angle of −15° ≤ φ ≤ +15°, without SE: (e) radiation intensity vs emission phase η, log10[d R 2ε/[dηdθ sin(θ)]], and (f) radiation energy d R ε/ [dθ sin(θ)]. (g,h) Correspond to (e,f), respectively, for the case including SE. The sample electron parameters are given in the text, and the laser parameters are the same as in Fig. 1. Figure 3. (a) The mean longitudinal momentum pz and (b) the mean motion direction (photon-emission direction) θe (polar angle) of a sample electron. The thick line is without SE. Scientific REPOrts \| 7: 11556 \| DOI:10.1038/s41598-017-11871-0 Optimal conditions for observation of stochasticity effects h f h l f l d l d d The peaks in RAD shift right to a larger polar angle since the emission angle after reflection satisfies ( ) / e i e ( ) δθ θ θ ξ γ = − ∼ . Therefore, larger initial kinetic energies are favourable for generation of more clear SE signatures, but one has to keep in mind that for the discussed SE features the reflection condition γ ξ ∼ /2 should be held in advance. l Finally, the role of the colliding angle between the laser pulse and the electron beam is investigated, as shown in Fig. 7. The figure shows that the variation of the colliding angle of 5° does not change the qualitative SE Scientific REPOrts \| 7: 11556 \| DOI:10.1038/s41598-017-11871-0 5 www.nature.com/scientificreports/ Figure 4. RAD dεR/[dθ sin(θ)]: (a) with SE, and (b) without SE, respectively. The laser focal radius w0 equals: (red, dotted) λ0, (blue, solid) 2λ0 and (green, dash-dotted) 3λ0. The black-dash curves show the case of a 6-cycle plane-wave laser pulse, scaled by a factor of 10−2. ξ = 600 for all cases. All other laser and electron parameters are the same as in Fig. 1. Figure 4. RAD dεR/[dθ sin(θ)]: (a) with SE, and (b) without SE, respectively. The laser focal radius w0 equals: (red, dotted) λ0, (blue, solid) 2λ0 and (green, dash-dotted) 3λ0. The black-dash curves show the case of a 6-cycle plane-wave laser pulse, scaled by a factor of 10−2. ξ = 600 for all cases. All other laser and electron parameters are the same as in Fig. 1. Figure 5. RAD dR ε /[dθ sin(θ)]: (a) with SE, and (b) without SE, respectively. The laser-pulse length LL is: (red- solid) 4λ0, (blue-dash) 6λ0, (black-dash-dotted) 8λ0 and (green-dotted) 10λ0. The electron-beam length Le is chosen to be equal to the corresponding laser-pulse length. ξ = 600 for all cases. All other laser and electron parameters are the same as in Fig. 1. Figure 5. RAD dR ε /[dθ sin(θ)]: (a) with SE, and (b) without SE, respectively. The laser-pulse length LL is: (red- solid) 4λ0, (blue-dash) 6λ0, (black-dash-dotted) 8λ0 and (green-dotted) 10λ0. The electron-beam length Le is chosen to be equal to the corresponding laser-pulse length. ξ = 600 for all cases. All other laser and electron parameters are the same as in Fig. 1. Figure 6. RAD dεR/[dθ sin(θ)]: (a) with SE, and (b) without SE, respectively. signatures. This is the strength of the qualitative SE signatures that they are robust with respect to the variation of the process parameters. signatures. This is the strength of the qualitative SE signatures that they are robust with respect to the variation of the process parameters. Conclusion We have revealed signatures of the stochastic nature of gamma-photon emission during nonlinear Compton scattering of a superstrong short focused laser pulse by a counterpropagating electron beam in the quantum radiation-dominated regime. The signatures are manifested in the qualitative features of the angular distribution of the radiation in the near-backward direction with respect to the initial electron motion, which arises when the electron energy is at the reflection condition. When the stochasticity effects are included, a single broad gamma-photon peak is formed in the near-reflection radiation angular distribution, while several gamma-photon peaks would arise if there were no stochasticity in the photon emission. The signatures are enhanced with tightly focused and short laser pulses. They are robust with respect to variation of the laser and electron parameters and can be observed in near-future laser facilities, such as ELI and XCELS. Optimal conditions for observation of stochasticity effects h f h l f l d l d d The mean kinetic energy of the electron beam ε0 is: (red-solid) 120 MeV, (blue-dashed) 180 MeV and (green-dotted) 240 MeV, respectively. All other laser and electron parameters are the same as in Fig. 1. Figure 6. RAD dεR/[dθ sin(θ)]: (a) with SE, and (b) without SE, respectively. The mean kinetic energy of the electron beam ε0 is: (red-solid) 120 MeV, (blue-dashed) 180 MeV and (green-dotted) 240 MeV, respectively. All other laser and electron parameters are the same as in Fig. 1. Scientific REPOrts \| 7: 11556 \| DOI:10.1038/s41598-017-11871-0 6 www.nature.com/scientificreports/ Figure 7. RAD dεR/[dθ sin(θ)]: (a) with SE, and (b) without SE, respectively. The blue-solid and red-dash curves present the results with θcolliding = 180° and 175°, respectively. All other laser and electron parameters are the same as in Fig. 1. Figure 7. RAD dεR/[dθ sin(θ)]: (a) with SE, and (b) without SE, respectively. The blue-solid and red-dash curves present the results with θcolliding = 180° and 175°, respectively. All other laser and electron parameters are the same as in Fig. 1. Methods h k In this work, we employ a linearly polarized focused Gaussian laser pulse propagating along +z direction and polarized in x direction. Scientific REPOrts \| 7: 11556 \| DOI:10.1038/s41598-017-11871-0 www.nature.com/scientificreports/ www.nature.com/scientificreports/ E0 is the amplitude of the laser fields with normalization factor Fn = i to keep + + = E E E E x y z 2 2 2 0 at the focus, yielding the scaled coordinates      x x w y y w z z z x y , , , , (8) r 0 0 2 2 2 ρ = = = = + (8) where = zr k w 2 0 0 2 is the Rayleigh length, f i z i  = + , η = ω0t − k0z, and φCEP the carrier-envelop phase. Note that φCEP = 0 is employed throughout.i where = zr k w 2 0 0 2 is the Rayleigh length, f i z i  = + , η = ω0t − k0z, and φCEP the carrier-envelop phase. Note that φCEP = 0 is employed throughout.i p y g In superstrong laser fields 1 ξ , the photon emission probability W is determined by the local electron tra- ectory, consequently, by the local value of the parameter χ38, 44: ∫ η ω αχ ωω χ ω π =   +   ⋅ ω ∞ d W d d m K x dx K k p ( ) ( ) 3 ( ) , (9) r r 2 2 5/3 2 2/3 0 r (9) where  k k k p ( )/ ( ) 0 0 ω χ = ⋅ ⋅ is the normalized emitted photon energy, k0, k and p are the four-vectors of the driving laser photon, the emitted photon and the electron, respectively, and ωr = 2ω/3(1 − χω). The applicability of the constant crossed-field approximation for the emission probability in Eq. (9) related to the laser intensity has been estimated and justified (See the Supplemental Materials for the details, which includes the impacts of differ- ent parameters on the radiation angular distributions). The photon emission of electrons is considered to be a Monte-Carlo stochastic process40–42. During the electron-laser interaction, for each propagation coherent length Δη, the photon emission will take place if the condition (dW/dη)Δη ≥ Nr is fulfilled, where Nr is a uniformly distributed random number in [0, 1]. Herein, the coherent length Δη is inversely proportional to the invariant laser field parameter ξ, i.e., 1/ η ξ ∆ ∼ . www.nature.com/scientificreports/ However, to keep the total photon emission energy consistent, i.e., to exclude numerical error of the simulation of photon emission, we choose 1/ η ξ ∆ ∼ . The photon emission probability W dW d d W d d d , 2 min max ∫ η η η η ω ω = ∆ = ∆ ω ω where ωmin is the driving laser photon energy, and ωmax equals the electron instantaneously kinetic energy. In addition, the emitted photon energy ωR is determined by the relation: where ωmin is the driving laser photon energy, and ωmax equals the electron instantaneously kinetic energy. In addition, the emitted photon energy ωR is determined by the relation: ∫ ∫ ω ω ω η ω η ω ω = ∆ = ∼ ω ω ω ω W dW d d W d W d d d N 1 ( ) ( ) ,r 2 min R min R ∫ ∫ ω ω ω η ω η ω ω = ∆ = ∼ ω ω ω ω W dW d d W d W d d d N 1 ( ) ( ) ,r 2 min R min R where, Nr ∼ is an another independent uniformly distributed random number in [0, 1]. Between the photon emis- sions, the electron dynamics in the laser field is governed by classical equations of motion: where, Nr ∼ is an another independent uniformly distributed random number in [0, 1]. Between the photon emis- sions, the electron dynamics in the laser field is governed by classical equations of motion: β = − + × . p E B d dt e( ) (10) (10) Given the smallness of the emission angle ~1/γ for an ultrarelativistic electron, the photon emission is assumed to be along the electron velocity8, 38. The photon emission induces the electron momentum change pf ≈ (1 − ωR/cp)pi, where pi,f are the electron momentum before and after the emission, respectively. In this simulation, the interfer- ence effects between emissions in adjacent coherent lengths are negligible since the laser fields employed are superstrong, i.e., 1  ξ . Therefore, the photon emissions happening in each coherent length are independent of each other. However, as the interference effects are significant, i.e. Methods h k The expressions of the electromagnetic fields are presented in the following refs 46 and 47: E iE f x f 1 4 , (1) x 2 2 2 3 4  ε ρ = −    +    −       E iE f x f 1 4 , (1) x 2 2 2 3 4  ε ρ = −    +    −       ε = − E iE f xy , (2) y 2 2  E E f x x f f f 2 4 , (3) z 3 2 3 2 4 4   ε ε ρ ρ =    +    − + −       = B 0, (4) x ε ρ ρ = −    +    −       B iE f f 1 2 4 , (5) y 2 2 2 3 4 ε ε ρ ρ =    +    + −       B E f y y f f f 2 2 4 , (6) z 3 2 3 2 4 4   ( ) E E F fe e e , (7) n f i T 0 2 CEP 2 0 2 = ρ η φ τ − + − E iE f x f 1 4 , (1) x 2 2 2 3 4  ε ρ = −    +    −       ε = − E iE f xy , (2) y 2 2  E E f x x f f f 2 4 , (3) z 3 2 3 2 4 4   ε ε ρ ρ =    +    − + −       = B 0, (4) x ε ρ ρ = −    +    −       B iE f f 1 2 4 , (5) y 2 2 2 3 4 ε ε ρ ρ =    +    + −       B E f y y f f f 2 2 4 , (6) z 3 2 3 2 4 4   where, ( ) E E F fe e e , (7) n f i T 0 2 CEP 2 0 2 = ρ η φ τ − + − E iE f x f 1 4 , (1) x 2 2 2 3 4  ε ρ = −    +    −       ε = − E iE f xy , (2) y 2 2  E E f x x f f f 2 4 , (3) z 3 2 3 2 4 4   ε ε ρ ρ =    +    − + −       = B 0, (4) x ε ρ ρ = −    +    −       B iE f f 1 2 4 , (5) y 2 2 2 3 4 ε ε ρ ρ =    +    + −       B E f y y f f f 2 2 4 , (6) z 3 2 3 2 4 4   where, ( ) E E F fe e e , (7) n f i T 0 2 CEP 2 0 2 = ρ η φ τ − + − (1) ε = − E iE f xy , (2) y 2 2  ε = − E iE f xy , (2) y 2 2  ε = − E iE f xy , y 2 2  (2) (3) (4) (5) (6) (7) Scientific REPOrts \| 7: 11556 \| DOI:10.1038/s41598-017-11871-0 7 Scientific REPOrts \| 7: 11556 \| DOI:10.1038/s41598-017-11871-0 www.nature.com/scientificreports/ 1  ξ < , the photon emission probabilities in adjacent coherent lengths are summed up to compare with the random number Nr until the photon emission happens.f As the stochastic effects are not taken into account, the rate of the electron radiation loss  can be considered s as d k k d W d d ( ) (11) 0 2 min max ∫ ω η ω = ⋅ . ω ω  (11) Implementing the radiation losses due to quantum radiation reaction into the electron classical dynamics leads to the following equation of motion22–24: Implementing the radiation losses due to quantum radiation reaction into the electron classical dynamics leads to the following equation of motion22–24: dp d e m F p m p F F p , (12) c c τ τ = − + α αβ β α αβ βγ γ    (12) where τ is the proper time, τ ξ ξ = ⋅ = ⋅     d d k p d d k x ( ) , ( ) (13) 0 0 (13) x the four-vector of the coordinate, τc ≡ 2e2/(3m), and  2 c 2 2 αω ξ = the classical radiation loss rate. The radiation energy and photon number at each step from the initial phase ηi to the final phase ηf read: x the four-vector of the coordinate, τc ≡ 2e2/(3m), and  2 c 2 2 αω ξ = the classical radiation loss rate. The radiation energy and photon number at each step from the initial phase ηi to the final phase ηf read: Scientific REPOrts \| 7: 11556 \| DOI:10.1038/s41598-017-11871-0 8 www.nature.com/scientificreports/ Figure 8. RAD dεR/[dθ sin(θ)]: (a) with SE, and (b) without SE, respectively. The blue-solid and red-dash curves present the results with the simulation step number Nstep = 2.7 × 104 and 5.4 × 104. All other laser and electron parameters are the same as in Fig. 1. Figure 8. RAD dεR/[dθ sin(θ)]: (a) with SE, and (b) without SE, respectively. The blue-solid and red-dash curves present the results with the simulation step number Nstep = 2.7 × 104 and 5.4 × 104. All other laser and electron parameters are the same as in Fig. 1. ∫ ε η = = . References Th 84, 1177 (2012). 8. Di Piazza, A., Müller, C., Hatsagortsyan, K. Z. & Keitel, C. H. Extremely high-intensity laser interactions with fundamental quantum systems. Rev. Mod. Phys. 84, 1177 (2012). 9. Mourou, G. & Tajima, T. Summary of the IZEST science and aspiration. Eur. Phys. J. Spec. Topics 223, 979–984 (2014). 10 Goldman I I Intensity effects in compton scattering Sov Phys JETP 19 954 (1964); Zh Eksp Teor Fiz 46 1412 (1964 9. Mourou, G. & Tajima, T. Summary of the IZEST science and aspiration. Eur. Phys. J. Spec. Topics 223, 979–984 (2014). 10. Goldman, I. I. Intensity effects in compton scattering. Sov. Phys. JETP 19, 954 (1964); Zh. Eksp. Teor. Fiz. 46, 1412 (1964 j y p y p p 10. Goldman, I. I. Intensity effects in compton scattering. Sov. Phys. JETP 19, 954 (1964); Zh. Eksp. Teor. Fiz. 46, 1 j y y p p 10. Goldman, I. I. Intensity effects in compton scattering. Sov. Phys. JETP 19, 954 (1964); Zh. Eksp. Teor. Fiz. 46, 1412 (1964). yf p g y p 11. Brown, L. S. & Kibble, T. W. Interaction of intense laser beams with electrons. Phys. Rev. 133, A705 (1964). 12. Berestetskii, V. B., Lifshitz, E. M. & Pitevskii, L. P. Quantum e 3. Koga, J., Esirkepov, T. Zh & Bulanov, S. V. Nonlinear thomson scattering in the strong radiation damping regime. Phys. Plasmas 12 093106 (2005). 14. Di Piazza, A., Hatsagortsyan, K. Z. & Keitel, C. H. Strong signatures of radiation reaction below the radiation-dominated reg Phys. Rev. Lett. 102, 254802 (2009).f y 15. Neitz, N. & Di Piazza, A. Stochasticity effects in quantum radiation reaction. Phys. Rev. Lett. 111, 054802 (2013).i f 16. Neitz, N. & Di Piazza, A. Electron-beam dynamics in a strong laser field including quantum radiation reaction. Phys. Rev. A 022102 (2014).f 022102 (2014). 17. Bashinov, A. V., Kim, A. V. & Sergeev, A. M. Impact of quantum effects on relativistic electron motion in a chaotic regime. Phys. Rev. E 92, 043105 (2015). 7. Bashinov, A. V., Kim, A. V. & Sergeev, A. M. Impact of quantum effects on relativistic electron motion in a chaotic regime. Phys. Rev E 92, 043105 (2015). , ( ) 18. Shen, C. S. & White, D. Energy straggling and radiation reaction for magnetic bremsstrahlung. Phys. Rev. Lett. 28, 455–459 (1972). 19. Duclous, R., Kirk, J. G. & Bell, A. R. where ωarb = εarb. l arb arb To prove explicitly the convergence of our Monte-Carlo simulations, we re-simulate the radiation of Fig. 2 via employing a smaller step size, as shown in Fig. 8. The blue-solid curves present the same radiation as in Fig. 2 with the simulation step number Nstep = 2.7 × 104, and the red-dash curves show the radiation with a smaller simula- tion step size, i.e., larger step number of Nstep = 5.4 × 104. The simulations clearly show that the behaviour of the photon emission angular distribution does not change with decreasing the simulation step size, i.e., increasing the simulation step number, which indicates the convergence of our results. Besides, the simulations based on the “Sokolov” equation have been compared with those based on a modified “Landau-Lifshitz” equation (rescaled radiation but no stochasticity effects)48, and they demonstrate good coincidences (See the Supplemental Materials for the details, which includes the impacts of different parameters on the radiation angular distributions). References Th 1. The Extreme Light Infrastructure (ELI). http://www.eli-laser.eu/. 1. The Extreme Light Infrastructure (ELI). http://www.eli-laser.eu/. h g p . Exawatt Center for Extreme Light Stidies (XCELS). http://www.x h g 2. Exawatt Center for Extreme Light Stidies (XCELS). http://www.xcels.iapras.ru/. g p p 3. Sarri, G. et al. Ultrahigh brilliance multi-mev gamma-ray beams from nonlinear relativistic thomson scattering. Phys. Rev. Lett. 224801 (2014). 4 S i G t l G ti f t l d hi h d it l t it i l i th l b t N t C 6 (2015 g p p 3. Sarri, G. et al. 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Nonlinear collective effects in photon-photon and photon-plasma interactions. Rev. Mod. Phy 591 (2006). ( ) 6. Mourou, G. A., Tajima, T. & Bulanov, S. V. Optics in the relativistic regime. Rev. Mod. Phys. 78, 309–371 (2006). ( ) 6. Mourou, G. A., Tajima, T. & Bulanov, S. V. Optics in the relativistic regime. Rev. Mod. Phys. 78, 309–371 (2006 ourou, G. A., Tajima, T. & Bulanov, S. V. Optics in the relativistic reg 7. Esarey, E., Schroeder, C. B. & Leemans, W. P. Physics of laser-driven plasma-based electron accelerators. Rev. Mod. Phys. 81, (2009). 8. Di Piazza, A., Müller, C., Hatsagortsyan, K. Z. & Keitel, C. H. Extremely high-intensity laser interactions with fundamental quantum systems. Rev. Mod. Phys. www.nature.com/scientificreports/ η η d N W , (14) R R i f  (14) Then, the photon number of the radiation energy εR above an arbitrary energy εarb at each step yields: W d d d W d d , (15) arb 2 arb max i f ∫ ∫ ω η η ω = ω ω η η (15) where ωarb = εarb. T li Scientific REPOrts \| 7: 11556 \| DOI:10.1038/s41598-017-11871-0 Author Contributions J.-X.L., K.Z.H. and C.H.K. conceived the project, J.-X.L. and Y.-Y.C. developed the simulation model and carried out the calculations. All contributed to the development of ideas, discussion of the results, and preparation of the manuscript. J.-X.L., K.Z.H. and C.H.K. conceived the project, J.-X.L. and Y.-Y.C. developed the simulation model and carried out the calculations. All contributed to the development of ideas, discussion of the results, and preparation of the manuscript. www.nature.com/scientificreports/ 192, 313 (20 q y p y 3. Khokonov, M. K. & Bekulova, I. Length of formation of processes in a constant external field at high energies. Techn. Phys. 55, 728 (2010). 4. Baier, V. N., Katkov, V. M. & Strakhovenko, V. M. Electromagnetic Processes at High Energies in Oriented Single Crystals (World Scientific, Singapore, 1994). Scientific, Singapore, 1994). 45. Ilderton, A. & Torgrimsson, G. 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https://openalex.org/W4283390154	https://zenodo.org/record/6697165/files/73.pdf	English	null	Listening Difficulties of Non-English Majored Freshmen at Tay Do University, In Vietnam	International journal of social science and human research	2,022	cc-by	9,651	1.1 Rationale Nowadays, English is considered as the most popular language used to exchange information and ideas among different nations. Thanks to English, countries are able to have common understanding and a sense of global citizenship in this multilingual world. It is an effective means to learn how to appreciate different cultures, communities and people. Moreover, English makes people understand each other more in the process of economic cooperation and other fields in today’s era when countries tend to integrate for the whole development. English has gradually become an indispensable tool in the modern time. When society is more developed, it is essential for people to have expert English skills to meet the demands of integration into the world economy and exchange of culture with other countries. As a result, mastering English gives people many greater opportunities of career advancement and success in life. According to Vandergrift (1997) listening skill requires hypothesizing, prediction, generalizing, revising and checking for the listening inputs. Students have to prepare carefully before listening. Most of students face some problems in listening because they cannot understand what the speaker says. English learners need to pay attention when learning listening and attempt to comprehend the content. The learners shall concern to the related factors to improve their skill. Unlike reading, while the readers can go back to the text any time they want, the listeners have no chance of going back to the part they may pass. Hence, learners demand to improve listening skill and it is un-denied about the important of listening. Listening plays an important role in communication, according to Mendelsohn (1994) in the total time spent on communicating, listening takes up 40-50%; speaking is 25-30%; reading is 11-16%; and writing is about 9%. This shows that, listening takes a half of time in communication. Moreover, that is a main factor, without listening, communication cannot operate effectively. But English learners have crucial problems in listening because universities pay attention to grammar, and vocabulary. Listening and speaking skills are not significant parts of books and teachers do not consider these skills in their classes, Hamouda (2013). There are many reasons why it is difficult for learners to listen. International Journal of Social Science And Human Research ISSN(print): 2644-0679, ISSN(online): 2644-0695 Volume 05 Issue 06 June 2022 DOI: 10.47191/ijsshr/v5-i6-73, Impact factor- 5.871 Page No: 2475-2485 International Journal of Social Science And Human Research ISSN(print): 2644-0679, ISSN(online): 2644-0695 Volume 05 Issue 06 June 2022 DOI: 10.47191/ijsshr/v5-i6-73, Impact factor- 5.871 Page No: 2475-2485 Listening Difficulties of Non-English Majored Freshmen at Tay Do University, In Vietnam Nga H.T. NGUYEN Tay Do University ABSTRACT: Listening skill plays an important role in studying a foreign language, especially English. However, students face a lot of difficulties in English listening. This study aims to find out the status and difficulties in listening comprehension of 149 non- English majored freshmen majoring in Pharmacy, Accounting, Business Administration, Food Technology, and Information Technology at Tay Do University. A listening achievement test was offered when the participants were studying TOEIC-based course 1. The research results found out some listening problems due to (1) lack of vocabulary knowledge, (2) inability to recognize and distinguish sounds, (3) limited reasoning ability and limited ability to use listening strategies such as judgment, taking notes, or memorizing, and (4) lack of concentration. The above results also showed some issues to which teachers need to pay attention to help learners improve their listening skill. KEYWORDS: Listening skill, non-English majored students, Tay Do University 1.2 Research aim The study was conducted with a view to find out the difficulties that non-English majored freshmen faced in listening comprehension so that students can recognize their own obstacles in learning listening and find out appropriate ways to listen in English effectively. 1 3 Research question The study was conducted with a view to find out the difficulties that non-English majored freshmen faced in listening comprehension so that students can recognize their own obstacles in learning listening and find out appropriate ways to listen in English effectively. Listening Difficulties of Non-English Majored Freshmen at Tay Do University, In Vietnam language proficiency, memory, concentration, age, gender, ability to use first language, background knowledge and (5) Characteristics of information processing like using listening strategies. In addition, sometimes learners make mistakes when listening text has much specialized terminology vocabulary. Specialized terminology vocabulary is some words specialized for particular areas. If learners do not have rich and plentiful vocabulary, they will be confused when listening. In reality, the speakers may use synonyms, antonyms to explain the word. Underwood (1989) points that, for students listening to a foreign language, it is certain that they cannot know all words in the speech like mother tongue. Therefore, when they hear an unknown word which can be like a suddenly dropped barrier, they may stop and think about the meaning of word. With this reason, a good listener is a person who can understand total of the listening text. Learning listening skills is the most difficult part in learning a foreign language. Listening is assuming greater and greater importance in foreign language classrooms. Rost (1994) emphasizes that listening can be regarded as a necessary skill in the preparation of foreign language and can even be considered a good predictor of language achievement. It is expected that this research would provide an overview of challenges in listening comprehension process so that both teachers and students can find out suitable and effective solutions. 2.1 Participants The participants of this study are 149 non-English majored freshmen majoring in Pharmacy (36), Accounting (24), Business Administration (27), Food Technology (33), and Information Technology (29) at Tay Do University. Students have studied 30/60 periods in TOEIC-based course 1 with Very Easy TOEIC book (2nd Ed.) by Anne Taylor and Garett Byrne (2006), Ho Chi Minh Publishing House. So these students have been familiarized with the questions of TOEIC test format, but at a basic level, the number of sentences for each listening part is less than the amount the student has to take the TOEIC test according to the international standards. 1.1 Rationale According to Ur (1996), English learners often face difficulties while listening, such as: (1) Not recognize the sounds, (2) Have the habit of having to understand all the words in a sentence, (3) Cannot understand when native speakers speak quickly and naturally, (4) Need to listen many times to understand, (5) Find it hard to grasp information and cannot predict what the speaker is about to say, and (6) feel tired and lack of concentration if listening for a long time. Also, Rubin (1994) points out five influential factors to listening. These are (1) characteristics of the listening such as speaking speed, pauses, stress and rhyme, the difference between first and second languages, etc.; (2) characteristics of the speaker such as gender and language proficiency; (3) Assignment characteristics such as exercise type; (4) Listener characteristics such as IJSSHR, Volume 05 Issue 06 June 2022 Page 2475 www.ijsshr.in 1.3 Research question q he research was conducted to answer these following questions: The research was conducted to answer these following questions: 1. How is the listening comprehension of non-English majored freshmen at Tay Do University? 2. What are some problems that non-English majored freshmen at Tay Do University face in liste 2. What are some problems that non-English majored freshmen at Tay Do University face in listening comprehension? 3. RESULT AND DISCUSSION 3. RESULT AND DISCUSSION 3. RESULT AND DISCUSSION 3.1 Students’ listening ability Figure 1: Students’ listening ability in different classes 55.80% 55.87% 55.55% 52.76% 46.24% 0.00% 20.00% 40.00% 60.00% Pharmacy Accounting Business Administration Food Technology Information Technology 3.1 Students’ listening ability 3.1 Students’ listening ability Figure 1: Students’ listening ability in different classes The results from figure 1 showed that students’ ability to do the TOEIC test of different departments at Tay Do University were at the average level. In which Accounting, Pharmacy and Business Administration were nearly at the same points 55.87%, 55.80% and 55.55%. Food Technology was a little bit lower 52.76%. And Information Technology was the lowest, at below the average 46.24%. Difficulties in each part were shown in detail in the following figures. 55.80% 55.87% 55.55% 52.76% 46.24% 0.00% 20.00% 40.00% 60.00% Pharmacy Accounting Business Administration Food Technology Information Technology Figure 1: Students’ listening ability in different classes The results from figure 1 showed that students’ ability to do the TOEIC test of different departments at Tay Do University were at the average level. In which Accounting, Pharmacy and Business Administration were nearly at the same points 55.87%, 55.80% and 55.55%. Food Technology was a little bit lower 52.76%. And Information Technology was the lowest, at below the average 46.24%. Difficulties in each part were shown in detail in the following figures. The results from figure 1 showed that students’ ability to do the TOEIC test of different departments at Tay Do University were at the average level. In which Accounting, Pharmacy and Business Administration were nearly at the same points 55.87%, 55.80% and 55.55%. Food Technology was a little bit lower 52.76%. And Information Technology was the lowest, at below the average 46.24%. Difficulties in each part were shown in detail in the following figures. Figure 2: Students’ listening ability in each part 57.11% 60.61% 39.20% 57.92% 0.00% 20.00% 40.00% 60.00% 80.00% Part 1 Part 2 Part 3 Part 4 Figure 2: Students’ listening ability in each part According to the results in Figure 2, the average points for four parts were 53.71%. In which the highest points were part 2 (questions & responses) with 60.61%, students listened to the short questions with only three option responses better than the others. It may be due to lack of attention, or poor listening technique. 2.2 Instruments Listening Difficulties of Non-English Majored Freshmen at Tay Do University, In Vietnam Test the ability to listen in combination with inference, listen to detailed ideas but need to be able to memorize or take notes because the information appears in long sentences, there are many ideas, there are no words expressing the same idea appearing in the talk. Listening test is done with the efforts to limit the special influence of external factors. Specifically, the format of the listening test is similar to the form students have been exposed to in the lesson. The content revolves around the common topics of the TOEIC test with the same duration. The speaker does not have a very strange or very special tone. The listening environment is not mixed with noise. Listening test is done with the efforts to limit the special influence of external factors. Specifically, the format of the listening test is similar to the form students have been exposed to in the lesson. The content revolves around the common topics of the TOEIC test with the same duration. The speaker does not have a very strange or very special tone. The listening environment is not mixed with noise. 3. RESULT AND DISCUSSION The second one was for part 4 (short talks) with 57.92%, students listening to the talk with familiar to daily life were better than what they had not got experience. It may cause by lack of reasoning ability. The third one was part 1 (picture description) with and 57.11%. Students got higher points for distinguishing prepositions of position than distinguishing nouns indicating places and objects. This showed that students were lacking in vocabulary or this lexical knowledge is still passive, and the ability to recognize sounds was still poor. The lowest scores were part 3 (short conversations) with only 39.20 %. This may be due to students' lack of deductive skills or poor listening strategies. The above results show that the students' listening ability was only slightly higher than the average level, although the test was similar to the learning content in terms of format and difficulty. In which, the ability of students to listen to single sentences is better than listening to conversations or talks, especially when listening to short conversations. Difficulties in each conversation were shown in detail in the following tables and figures. 2.2 Instruments The research was carried out to find out the difficulties of non-English majored freshmen when listening to English. The data collection tool was a listening achievement test offered when the participants were studying TOEIC-based course 1. The test was designed according to TOEIC-based test, selected from the book Developing Skills for the TOEIC Test by Paul Edmunds and Anne Taylor (2012), close to the type of listening students are studying in class with equivalent difficulty. The test is nearly 10 minutes long, includes 22 multiple-choice questions (only part 2 has 3 choices, while the rest ones have 4 choices), divided into 4 parts described in detail in the following table. Table 1: Listening test description Part Number of questions Content Purpose Part 1 Picture Description 5 Listen four statements, select the one statement that describes the picture. Test the ability to distinguish the places, things, positions, and actions. Part 2 Questions and Responses 5 Listen a question or a statement followed by three responses. Select the best response. Test the ability to distinguish the sounds almost the same, and distinguish how to answer Yes/No questions and Wh-questions Part 3 Short Conversations 6 Listen to two short talks given by a single speaker, then answer three questions about what is said in each talk. Test the ability to listen to details, listen to inferences, and distinguish words that are easily confused because they have similar pronunciations IJSSHR, Volume 05 Issue 06 June 2022 Page 2476 Page 2476 www.ijsshr.in Listening Difficulties of Non-English Majored Freshmen at Tay Do University, In Vietnam Part 4 Short Talk 6 Test the ability to listen in combination with inference, listen to detailed ideas but need to be able to memorize or take notes because the information appears in long sentences, there are many ideas, there are no words expressing the same idea appearing in the talk. Listening test is done with the efforts to limit the special influence of external factors. Specifically, the format of the listening test is similar to the form students have been exposed to in the lesson. The content revolves around the common topics of the TOEIC test with the same duration. The speaker does not have a very strange or very special tone. The listening environment is not mixed with noise. Picture 1-Distinguish nouns of place Figure 3: Picture 1-Distinguish nouns of place Picture 1. (A) This is a meeting room. (B) This is a church. (C) This is a garden. (D) This is a hospital. This was an easy question with four different nouns of place. 49.79% chose the correct answer. Look at figure 3, the column in the middle indicated the highest column, with 59.26% for Business Administration. The Pharmacy was in the second position, with a share of 55.56 %, only 3.7% lower than the Business Administration. With a particular value of 54.55%, the Food Technology was ranked the third. The Accounting ranked the fourth with a percentage of 41.67%. Finally, the Information Technology had the lowest figure of 37.93% compared to the others. With this question, there could not have difficulties in pronunciation, but only half of students could get points. This could come to the conclusion that students were lack of vocabulary. Part 1-Picture description In part 1 students listened to 4 simple sentences to choose 1 sentence with describing the given pictures. IJSSHR, Volume 05 Issue 06 June 2022 Page 2477 Page 2477 www.ijsshr.in Listening Difficulties of Non-English Majored Freshmen at Tay Do University, In Vietnam Table 2: Part 1-Picture description Statement Picture 1 Picture 2 Picture 3 Picture 4 P Listening Difficulties of Non-English Majored Freshmen at Tay Do University, In Vietnam Table 2: Part 1-Picture description Statement Picture 1 Picture 2 Picture 3 Picture 4 Picture 5 Pharmacy 55.56% 44.45% 61.11% 52.78% 80.56% Accounting 41.67% 45.83% 70.83% 58.33% 83.33% Business Administration 59.26% 48.15% 74.07% 51.85% 74.07% Food Technology 54.55% 57.58% 63.64% 45.45% 66.67% Information Technology 37.93% 48.27% 55.17% 34.48% 62.07% Average (57.11%) 49.79% 48.86% 64.96% 48.58% 73.34% Listening Difficulties of Non-English Majored Freshmen at Tay Do University, In Vietnam According to the results in table 2, the percentage of students who got good answer was about 57.11%. In which, distinguishing action words that were usually used was the highest with 73.34%, while distinguishing action words that were rarely used was the lowest with 48.58%. In addition, distinguish prepositions of position, 64.96% was better than distinguishing nouns indicating places (49.79%) and objects (48.86%). This showed that students were lacking in vocabulary or this lexical knowledge is still passive, and the ability to recognize sounds was still poor, so students could recognize these words when reading, but when listening to the sounds, they could not. Difficulties in each picture were shown in detail in the following figures. Picture 1-Distinguish nouns of place Figure 3: Picture 1-Distinguish nouns of place Picture 1. (A) This is a meeting room. (B) This is a church. (C) This is a garden. (D) This is a hospital. This was an easy question with four different nouns of place. 49.79% chose the correct answer. 55.56% 41.67% 59.26% 54.55% 37.93% 0.00% 20.00% 40.00% 60.00% 80.00% Pharmacy Accounting Business Administration Food Technology Information Technology Picture 1-Distinguish nouns of place Picture 2-Distinguish nouns for objects Picture 2-Distinguish nouns for objects Figure 4: Picture 2-Distinguish nouns for objects Picture 2. (A) They have a kite. (B) They have a balloon. (C) They have an airplane. (D) They have a book. This was also an easy question with differentiating nouns for objects. The four things were easily identified. But only 48.86% chose the correct answer. Let’s look at figure 4, the highest column with 57.58% was for Food Technology. With 48.27%, Information Technology came in the second. Business Administration was 48.15%. Accounting was 45.83%. And Pharmacy was the lowest with 44.45%. This may know that students were lack of knowledge about vocabulary; they did not have enough vocabulary although these were simple things usually seen in daily life. 44.45% 45.83% 48.15% 57.58% 48.27% 0.00% 20.00% 40.00% 60.00% 80.00% Pharmacy Accounting Business Administration Food Technology Information Technology Picture 2-Distinguish nouns for objects Figure 4: Picture 2-Distinguish nouns for objects 44.45% 45.83% 48.15% 57.58% 48.27% 0.00% 20.00% 40.00% 60.00% 80.00% Pharmacy Accounting Business Administration Food Technology Information Technology Figure 4: Picture 2-Distinguish nouns for objects Picture 2. (A) They have a kite. (B) They have a balloon. (C) They have an airplane. (D) They have a book. This was also an easy question with differentiating nouns for objects. The four things were easily identified. But only 48.86% chose the correct answer. question with differentiating nouns for objects. The four things were easily identified. But only 48.86% chose the correct answer. Let’s look at figure 4, the highest column with 57.58% was for Food Technology. With 48.27%, Information Technology came in the second. Business Administration was 48.15%. Accounting was 45.83%. And Pharmacy was the lowest with 44.45%. This may know that students were lack of knowledge about vocabulary; they did not have enough vocabulary although these were simple things usually seen in daily life. Let’s look at figure 4, the highest column with 57.58% was for Food Technology. With 48.27%, Information Technology came in the second. Business Administration was 48.15%. Accounting was 45.83%. And Pharmacy was the lowest with 44.45%. This may know that students were lack of knowledge about vocabulary; they did not have enough vocabulary although these were simple things usually seen in daily life. Let’s look at figure 4, the highest column with 57.58% was for Food Technology. With 48.27%, Information Technology came in the second. Business Administration was 48.15%. Accounting was 45.83%. Picture 4. Distinguish verbs of action Picture 4. Distinguish verbs of action Figure 6: Picture 4-Distinguish verbs of action 52.78% 58.33% 51.85% 45.45% 34.48% 0.00% 20.00% 40.00% 60.00% 80.00% Pharmacy Accounting Business Administration Food Technology Information Technology Figure 6: Picture 4-Distinguish verbs of action Picture 4. (A) The man is singing. (B) The man is dancing. (C) The man is angry. (D) The man is rowing. This is a rather difficult question differentiating action verbs, the picture was about the rarely used vocabulary, students had to choose basing on excluding known answers. 48.58% chose the correct answer. Picture 4. (A) The man is singing. (B) The man is dancing. (C) The man is angry. (D) The man is rowing. This is a rather difficult question differentiating action verbs, the picture was about the rarely used vocabulary, students had to choose basing on excluding known answers. 48.58% chose the correct answer. Figure 6 showed that 58.33% for Accounting. Pharmacy was in the second with 52.78%. Business Administration was 51.85% and 45.45% for Food Technology. Finally, Information Technology had the lowest figure of 34.48%. This was a difficult question about distinguishing verbs of action, only about half of students could overcome this exercise, that was acceptable. Picture 5. Distinguish verbs of action Picture 5. Distinguish verbs of action Figure 7: Picture 5-Distinguish verbs of action 80.56% 83.33% 74.07% 66.67% 62.07% 0.00% 50.00% 100.00% Pharmacy Accounting Business Administration Food Technology Information Technology Figure 7: Picture 5-Distinguish verbs of action Picture 5. (A) The person is writing. (B) The person is cutting. (C) The person is painting. (D) The person is laughing. This picture was also about distinguishing verbs of action but it was easier than the previous one due to simple vocabulary that was familiar to students. 73.34% chose the correct answer. Picture 5. (A) The person is writing. (B) The person is cutting. (C) The person is painting. (D) The person is laughing. This picture was also about distinguishing verbs of action but it was easier than the previous one due to simple vocabulary that was familiar to students. 73.34% chose the correct answer. Figure 7 showed that the second column had the highest percentage of 83.33% for Accounting. Pharmacy was with 80.56%. Business Administration was rated the third with 74.07%, and 66.67% was for Food Technology. Finally, Information Technology had the lowest 62.07%. It is easier for students to distinguish action verbs for this picture because most of the vocabulary they knew and often encountered. Picture 2-Distinguish nouns for objects And Pharmacy was the lowest with 44.45%. This may know that students were lack of knowledge about vocabulary; they did not have enough vocabulary although these were simple things usually seen in daily life. IJSSHR, Volume 05 Issue 06 June 2022 www.ijsshr.in IJSSHR, Volume 05 Issue 06 June 2022 Page 2478 Page 2478 www.ijsshr.in Listening Difficulties of Non-English Majored Freshmen at Tay Do University, In Vietnam Picture 3. Distinguish prepositions of position Figure 5: Picture 3-Distinguish prepositions of position 61.11% 70.83% 74.07% 63.64% 55.17% 0.00% 20.00% 40.00% 60.00% 80.00% Pharmacy Accounting Business Administration Food Technology Information Technology Listening Difficulties of Non-English Majored Freshmen at Tay Do University, In Vietnam Picture 3. Distinguish prepositions of position Figure 5: Picture 3-Distinguish prepositions of position Picture 3. (A) The girl is stretching. (B) The girl is next to the dog. (C) The girl is dancing. (D) The girl is under the dog. There were four sentences, but the picture given was about the prepositions of position. Students easily only focused on B and D. 64.96% chose the correct answer, much higher than the result of picture 1 and 2. From figure 5, the middle column revealed the highest column with 74.07% for Business Administration. Accounting was in the second with 70.83%. Food Technology was 63.64%, and 61.11% for Pharmacy. Information Technology was the lowest with 55.17%. Students appeared to place a high value on identifying prepositions of position. They maybe do well for this question. Part 2-Questions & Responses Q p In part 2 students listened to the question and chose a corresponding answer out of the 3 options they heard. In part 2 students listened to the question and chose a corresponding answer out of the 3 options they h IJSSHR, Volume 05 Issue 06 June 2022 www.ijsshr.in IJSSHR, Volume 05 Issue 06 June 2022 Page 2479 www.ijsshr.in Listening Difficulties of Non-English Majored Freshmen at Tay Do University, In Vietnam Table 3: Part 2-Questions & Responses Statement Question 1 Question 2 Question 3 Question 4 Question 5 Pharmacy 47.22% 77.77% 86.11% 61.11% 69.44% Accounting 45.83% 70.83% 83.33% 58.33% 62.50% Business Administration 44.44% 74.07% 70.37% 48.15% 62.96% Food Technology 36.36% 75.76% 81.82% 54.55% 42.42% Information Technology 31.03% 68.96% 82.75% 41.38% 37.93% Average (60.61%) 40.97% 73.48% 80.87% 52.70% 55.05% ening Difficulties of Non-English Majored Freshmen at Tay Do University, In Vietnam According to the results in table 3, the percentage of students who chose correctly was 60.61%. In which, question 1 – “What time” only got 40.97%, the lowest out of 5 questions. Thus, students were not good at distinguishing between similar sounds "at" and "It's" maybe due to lack of attention, or poor listening technique. Question 2 asked students to distinguish two similar-sounding sentences “How old are you?” and “How are you?”. The result of this sentence is better with 73.48% of students answering correctly, maybe because the number of rhymes in the two sentences was different, students could easily recognize it. Question 3 was a yes/no question which had the highest score of 80.87%, the reason could be because the answer to the question Yes – No was quite short and simple, so most students recognized it and chose the right one. With the questions “where” and “why”, the answer was at the average 52.70%, and 55.05%, respectively. Difficulties in each question were shown in detail in the following figures. Question 1 – What time Figure 8: Question 1 – What time 47.22% 45.83% 44.44% 36.36% 31.03% 0.00% 10.00% 20.00% 30.00% 40.00% 50.00% Pharmacy Accounting Business Administration Food Technology Information Technology Question 1 – What time Figure 8: Question 1 – What time Question 1. What time does the movie start? (A) At three thirty. (B) It's a good movie. (C) It's three o'clock. “What time” was an easy question that students usually used in daily life, but the result showed that only 40.97% chose the correct answer. Part 2-Questions & Responses Pharmacy, Accounting and Business Administration had nearly the same value as 47.22%, 45.83% and of 44.44% while Food Technology and Information Technology were lower with 36.36% and 31.03%. When being ask “What time”, it was not difficult to give the answer, but students could not choose the correct answer. This revealed that it was difficult for students to distinguish between similar sounds “At and It’s”. 47.22% 45.83% 44.44% 36.36% 31.03% 0.00% 10.00% 20.00% 30.00% 40.00% 50.00% Pharmacy Accounting Business Administration Food Technology Information Technology Figure 8: Question 1 – What time Question 1. What time does the movie start? (A) At three thirty. (B) It's a good movie. (C) It's three o'clock. “What time” was an easy question that students usually used in daily life, but the result showed that only 40.97% chose the correct answer. Pharmacy, Accounting and Business Administration had nearly the same value as 47.22%, 45.83% and of 44.44% while Food Technology and Information Technology were lower with 36.36% and 31.03%. When being ask “What time”, it was not difficult to give the answer, but students could not choose the correct answer. This revealed that it was difficult for students to distinguish between similar sounds “At and It’s”. Question 2 – How Figure 9: Question 2 – How 77.77% 70.83% 74.07% 75.76% 68.96% 60.00% 65.00% 70.00% 75.00% 80.00% Pharmacy Accounting Business Administration Food Technology Information Technology Figure 9: Question 2 – How Question 2. How old are you? (A) I'm fine, thank you. (B) I'm fifteen. (C) I feel sick. 73.48% chose the correct answer. That was good. Students could recognize the different between “How old are you? and How are you?” 114/149 students at five classes got the correct answers, in which Pharmacy had the greatest scale 77.77% while Information Technology was at the lowest 68.96%. When students’ vocabulary was good, it was not difficult for them to distinguish between similar sounds, too. 114/149 students at five classes got the correct answers, in which Pharmacy had the greatest scale 77.77% while Information Technology was at the lowest 68.96%. Part 2-Questions & Responses 61.11% 58.33% 48.15% 54.55% 41.38% 0.00% 20.00% 40.00% 60.00% 80.00% Pharmacy Accounting Business Administration Food Technology Information Technology Figure 11: Question 4 – Where Question 4. Where did she go? (A) Yes, she did. (B) To the supermarket. (C) She's going now. 52.70% got good answer, at the average points. This was students’ lack of attention. The question was so simple but they could not choose the correct answer. Question 4. Where did she go? (A) Yes, she did. (B) To the supermarket. (C) She's going now. 52.70% got good answer, at the average points. This was students’ lack of attention. The question was so simple but they could not choose the correct answer. In figure 11, Pharmacy still was the leaders with 61.11%. Accounting and Food Technology were nearly the same with 58.33% and 54.55%. Business Administration and Information Technology were at the lowest rates with 48.15% and 41.38%. It was not difficult for students to use Wh-question, especially with “Where”, but due to lack of concentration, students were at the average rate only. In figure 11, Pharmacy still was the leaders with 61.11%. Accounting and Food Technology were nearly the same with 58.33% and 54.55%. Business Administration and Information Technology were at the lowest rates with 48.15% and 41.38%. It was not difficult for students to use Wh-question, especially with “Where”, but due to lack of concentration, students were at the average rate only. In figure 11, Pharmacy still was the leaders with 61.11%. Accounting and Food Technology were nearly the same with 58.33% and 54.55%. Business Administration and Information Technology were at the lowest rates with 48.15% and 41.38%. It was not difficult for students to use Wh-question, especially with “Where”, but due to lack of concentration, students were at the average rate only. Question 5 – Why Figure 12: Question 5 – Why 69.44% 62.50% 62.96% 42.42% 37.93% 0.00% 20.00% 40.00% 60.00% 80.00% Pharmacy Accounting Business Administration Food Technology Information Technology Part 2-Questions & Responses When students’ vocabulary was good, it was not difficult for them to distinguish between similar sounds too IJSSHR, Volume 05 Issue 06 June 2022 Page 2480 Page 2480 www.ijsshr.in Listening Difficulties of Non-English Majored Freshmen at Tay Do University, In Vietnam Question 3 – Yes/No Figure 10: Question 3 – Yes/No 86.11% 83.33% 70.37% 81.82% 82.75% 0.00% 50.00% 100.00% Pharmacy Accounting Business Administration Food Technology Information Technology Listening Difficulties of Non-English Majored Freshmen at Tay Do University, In Vietnam Figure 10: Question 3 – Yes/No Question 3. Do you like chocolate ice cream? (A) No, I can't. (B) Yes, I do. (C) Yes, I can. Question 3. Do you like chocolate ice cream? (A) No, I can't. (B) Yes, I do. (C) Yes, I can. 80.87% chose the correct answer, the highest rate compared to the others in part 2 – Questions & Responses. Students can find out the difference in giving the answer for Yes/No question, especially in “Do-Can” one. 80.87% chose the correct answer, the highest rate compared to the others in part 2 – Questions & Responses. Students can find out the difference in giving the answer for Yes/No question, especially in “Do-Can” one. Figure 10 showed that the highest column was Pharmacy with 86.11%. Accounting was only 2.28% behind Pharmacy with 83.83%. Business Administration was rated third, with 82.75%, and 81.82% for Information Technology. Food Technology had the lowest figure of 70.37%. The result revealed that yes/no question was easily understood by students. Question 4 – Where Figure 11: Question 4 – Where 61.11% 58.33% 48.15% 54.55% 41.38% 0.00% 20.00% 40.00% 60.00% 80.00% Pharmacy Accounting Business Administration Food Technology Information Technology Question 4 – Where Figure 11: Question 4 – Where Question 4. Where did she go? (A) Yes, she did. (B) To the supermarket. (C) She's going now. 52.70% got good answer, at the average points. This was students’ lack of attention. The question was so simple but they could not choose the correct answer. In figure 11, Pharmacy still was the leaders with 61.11%. Accounting and Food Technology were nearly the same with 58.33% and 54.55%. Business Administration and Information Technology were at the lowest rates with 48.15% and 41.38%. It was not difficult for students to use Wh-question, especially with “Where”, but due to lack of concentration, students were at the average rate only. Part 3 - Short conversations From table 4, Food Technology had the highest percentage, at 57.58%. Information Technology was with 51.72%. Business Administration with 48.15%. Pharmacy and Accounting were at 38.89% and 33.33%. According to data, students' poor vocabulary made it harder for them to differentiate words during interactions. Question 2. Who does the woman speak to? Key: (A) The operator. (B) The project manager. (C) Her co-worker. (D) The director. Only 20.38% chose the correct answer. Students had to base on the conversation to choose the correct answer, but maybe they did not have enough vocabulary to do this kind of question. From table 4, the highest percentage was 29.17% for According. Pharmacy was at 22.22%. Business Administration and Food Technology were at the same 18.52% and 18.18%. Information Technology had the lowest figure of 13.79%. According to the data, students' poor vocabulary made it difficult for them to listen based on the situation. Question 2. Who does the woman speak to? Key: (A) The operator. (B) The project manager. (C) Her co-worker. (D) The director. Only 20.38% chose the correct answer. Students had to base on the conversation to choose the correct answer, but maybe they did not have enough vocabulary to do this kind of question. From table 4, the highest percentage was 29.17% for According. Pharmacy was at 22.22%. Business Administration and Food Technology were at the same 18.52% and 18.18%. Information Technology had the lowest figure of 13.79%. According to the data, students' poor vocabulary made it difficult for them to listen based on the situation. Question 3. What is the woman going to do? (A) Cancel an appointment. Key: (B) Call again the same day. (C) Call back tomorrow. (D) Wait for a phone call. 39.61% chose the correct answer. The answer was quite similar to the words used in the conversation “This afternoon was same day”. From table 4, Food Technology and Business Administration had the highest percentage, at 45.45% and 44.44%. Accounting was ranked third with a specific value of 41.67%. Pharmacy and Information Technology were at 38.89% and 27.59%. Thus, students' poor listening skill made it harder for them to differentiate words during the interactions. Question 5 – Why Figure 12: Question 5 – Why Question 5. Why did you close the window? (A) The window is closed. (B) It's a window. (C) Because it's cold. 55.05% chose the correct answer, a little bite higher than “Where” question (52.70%), but it was still at the average rate. In figure 12, Pharmacy, Accounting and Business Administration were at the rate 69.44%, 62.69% and 62.50% respectively. Food Technology and Information Technology were only at 42.42% and 37.93%. When having “Why-question”, usually the answer must begin with “Because”. In the answer, there was one statement having “Because” but students could not get good points for this type of question. They limited reasoning ability and limited ability to use listening strategies such as judgment, taking notes, or memorizing. 69.44% 62.50% 62.96% 42.42% 37.93% 0.00% 20.00% 40.00% 60.00% 80.00% Pharmacy Accounting Business Administration Food Technology Information Technology Figure 12: Question 5 – Why Question 5. Why did you close the window? (A) The window is closed. (B) It's a window. (C) Because it's cold. 55.05% chose the correct answer, a little bite higher than “Where” question (52.70%), but it was still at the average rate. Question 5. Why did you close the window? (A) The window is closed. (B) It's a window. (C) Because it's cold. 55.05% chose the correct answer, a little bite higher than “Where” question (52.70%), but it was still at the average rate. Question 5. Why did you close the window? (A) The window is closed. (B) It's a window. (C) Because it's cold. 55.05% chose the correct answer, a little bite higher than “Where” question (52.70%), but it was still at the average rate. In figure 12, Pharmacy, Accounting and Business Administration were at the rate 69.44%, 62.69% and 62.50% respectively. Food Technology and Information Technology were only at 42.42% and 37.93%. When having “Why-question”, usually the answer must begin with “Because”. In the answer, there was one statement having “Because” but students could not get good points for this type of question. They limited reasoning ability and limited ability to use listening strategies such as judgment, taking notes, or memorizing. IJSSHR, Volume 05 Issue 06 June 2022 Page 2481 Page 2481 www.ijsshr.in Part 3 - Short conversations In part 3 students listen to two short conversations to answer questions. The results of table 4 and 5 showed that the students' ability to listen to the conversation was not good, with the average score below 50% - conversation 1 (35.31%), conversation 2 (43.09%). This may be due to students' lack of deductive skills or poor listening strategies. Difficulties in each conversation were shown in detail in the following tables. Table 4: Part 3-Short conversation 1 Statement Question 1 Question 2 Question 3 Pharmacy 38.89% 22.22% 38.89% Accounting 33.33% 29.17% 41.67% Business Administration 48.15% 18.52% 44.44% Food Technology 57.58% 18.18% 45.45% Information Technology 51.72% 13.79% 27.59% Average (35.31%) 45.93% 20.38% 39.61% Script M: Good morning. How may I direct your call? W: I’m calling for the project manager. Is she in? M: Just a moment please. Oh, I’m sorry, she’s out of the office. W: I see. Well, then I’ll try again this afternoon. The average score of conversation 1 was 35.31%. Students listened well to details (45.93% for question 1), students recognized it rather well when the answer was quite similar to the words used in the conversation and was repeated with related words (39.61% answered question 3 correctly). The lowest score was in questions required students to hear information combined with inference (20.38% answered question 2 correctly). Thus, although listening to the conversation was not very good, the students hear the details better when listening to the combination of inference. Let’s find more details in each question. Question 1. Who is the woman calling for? (A) The man. (B) The director. (C) An extension. Key: (D) The project manager. 45.93% chose the correct answer, the highest rate compared to the others in conversation 1. Students got the exactly the answer from what they hear in the conversation “the project manager”. From table 4, Food Technology had the highest percentage, at 57.58%. Information Technology was with 51.72%. Business Administration with 48.15%. Pharmacy and Accounting were at 38.89% and 33.33%. According to data, students' poor vocabulary made it harder for them to differentiate words during interactions. Question 1. Who is the woman calling for? (A) The man. (B) The director. (C) An extension. Key: (D) The project manager. 45.93% chose the correct answer, the highest rate compared to the others in conversation 1. Students got the exactly the answer from what they hear in the conversation “the project manager”. M: Excuse me. I need to check out of my room about 30 minutes late tomorrow. I’m expecting a phone call at 10:30, and I won't be finished by 11:00. Will that be a problem? Part 3 - Short conversations Table 5: Part 3-Short conversation 2 Statement Question 4 Question 5 Question 6 Pharmacy 44.44% 63.64% 22.22% Accounting 33.33% 79.17% 29.17% Business Administration 40.74% 74.07% 29.63% Food Technology 39.39% 75.76% 18.18% Information Technology 27.58% 55.17% 13.79% Average (43.09%) 37.10% 69.56% 22.60% Script IJSSHR, Volume 05 Issue 06 June 2022 IJSSHR, Volume 05 Issue 06 June 2022 Page 2482 www.ijsshr.in Listening Difficulties of Non-English Majored Freshmen at Tay Do University, In Vietnam W N t t ll Y bill h l d b id j t l k i th Key: (B) He is expecting a phone call. (C) He wants to stay longer. (D) He lost his room key. 69.56% chose the correct answer, the highest rate compared to the others in conversation 2. This was an easy question since students got the exactly the answer from what they heard in the conversation “I’m expecting a phone call”. From table 5, Accounting had the greatest rate, at 79.17%. Food Technology and Business Administration were at 75.76% and 74.07% while the industry is ranked third in 63.64% and 55.17% were of Pharmacy and Information Technology. This meant that when students could hear the correct words in the conversation, the score would be high. Question 6. What time will the man probably leave? Key: (A) 11:30 (B) 11:00 (C) 10:30 (D) 10:00. This was a very difficult question, students heard "10:30" and "11:00", but the answer must be “11:30”, thus only 22.60% chose the correct answer. From table 5, Business Administration and Accounting were better than others with 29.63%. and 29.17 %. Pharmacy got 22.22%. Food Technology and Information Technology were 18.18% and 13.79%. In conclusion, students were limited reasoning ability. Part 4-Short talks This talk was about a popular topic – internet that all students were familiar to, the vocabulary was also simple. Thus, students got high points when they had experience of what were talked about. Let’s find more details in each question. q Question 1. What problem was the man having? (A) He couldn’t change his password. (B) The telephone was not working. (C) His computer shut down. Key: (D) He could not connect to the Internet. 79.48% chose the correct answer. The answer was got exactly from the talk “connected to the Internet”. From table 6, Business Administration had the highest proportion of 92.59%. Pharmacy and Accounting came to the next with 88.89% and 83.33%. Information Technology and Food Technology were also good at 68.97% and 63.64%. Question 1. What problem was the man having? (A) He couldn’t change his password. (B) The telephone was not working. (C) His computer shut down. Key: (D) He could not connect to the Internet. 79.48% chose the correct answer. The answer was got exactly from the talk “connected to the Internet”. From table 6, Business Administration had the highest proportion of 92.59%. Pharmacy and Accounting came to the next with 88.89% and 83.33%. Information Technology and Food Technology were also good at 68.97% and 63.64%. Question 2. What did the man forget? (A) His modern. (B) His phone line. (C) His access number. Key: (D) His password. 89.58% chose the correct answer. Students got information from what they heard in the talk without reasoning or guessing ability. From table 6, 94.44% and 92.59% were of Pharmacy and Business Administration. Information Technology, Food Technology and Accounting were at high rates with 89.66%, 87.88% and 83.33%. Question 2. What did the man forget? (A) His modern. (B) His phone line. (C) His access number. Key: (D) His password. 89.58% chose the correct answer. Students got information from what they heard in the talk without reasoning or guessing ability. From table 6, 94.44% and 92.59% were of Pharmacy and Business Administration. Information Technology, Food Technology and Accounting were at high rates with 89.66%, 87.88% and 83.33%. Question 2. What did the man forget? (A) His modern. (B) His phone line. (C) His access number. Key: (D) His password. 89.58% chose the correct answer. Students got information from what they heard in the talk without reasoning or guessing ability. Listening Difficulties of Non-English Majored Freshmen at Tay Do University, In Vietnam W N t t ll Y bill h l d b id j t l k i th W: Not at all. Your bill has already been paid, so just leave your keys in the room. W: Not at all. Your bill has already been paid, so just leave your keys in the room. M: Great. Thank you very much. M: Great. Thank you very much. M: Great. Thank you very much. W: I hope you enjoyed your stay. W: I hope you enjoyed your stay. The average score of conversation 2 was 43.09%. Students listened well to details (69.56% correctly answered question 5). The lowest score was in questions required students to hear information combined with inference (22.60% for question 6 and 37.10% for question 4). Thus, although listening to the conversation was not very good, the students hear the details better when listening to the combination of inference. Let’s find more details in each question. Question 4. Where is this conversation taking place? (A) At a university. Key: (B) At a hotel. (C) On the subway. (D) At home. The answer was based on the situation of "room" and "check-out" to choose the answer “At a hotel”. But 37.10% chose the correct answer, maybe students did not know how to guess what they heard. From table 5, Pharmacy had the highest ratio, at 44.44%. Business Administration and Food Technology were at 40.74% and 39.39%. Accounting and Information Technology were at the lowest rates of 33.33% and 27.58%. Students had difficulty in choosing the right answer based on the situation due to lack of reasoning ability. Question 5. Why will the man leave his room late? (A) He will wake up late. Key: (B) He is expecting a phone call. (C) He wants to stay longer. (D) He lost his room key. 69.56% chose the correct answer, the highest rate compared to the others in conversation 2. This was an easy question since students got the exactly the answer from what they heard in the conversation “I’m expecting a phone call”. From table 5, Accounting had the greatest rate, at 79.17%. Food Technology and Business Administration were at 75.76% and 74.07% while the industry is ranked third in 63.64% and 55.17% were of Pharmacy and Information Technology. This meant that when students could hear the correct words in the conversation, the score would be high. Question 5. Why will the man leave his room late? (A) He will wake up late. Part 4-Short talks In part 4 students listen to two short talks to answer questions. The results of table 6 and 7 showed that the students' ability to listen to the talks was completely different, in which talk 1 got 77.10% while talk 2 only had 38.75%. This may be due to students' background knowledge. Difficulties in each talk were shown in detail in the following tables. Table 6: Part 4-Short talk 1 Statement Question 1 Question 2 Question 3 Pharmacy 88.89% 94.44% 61.11% Accounting 83.33% 83.33% 66.67% Business Administration 92.59% 92.59% 74.07% Food Technology 63.64% 87.88% 57.58% Information Technology 68.97% 89.66% 51.72% Average (77.10%) 79.48% 89.58% 62.23% Script of short talk 1 Table 6: Part 4-Short talk 1 M: Yesterday, I was trying to get my computer connected to the Internet. It was a complete disaster! First, I couldn’t figure out the access number for the modem. Then, I forgot my password. When I finally remembered my password, the telephone line was busy. I waited for twenty minutes, but it was no use. I gave up because I had to go to work. I'll have to try again today. access number for the modem. Then, I forgot my password. When I finally remembered my password, the telephone line was busy. I waited for twenty minutes, but it was no use. I gave up because I had to go to work. I'll have to try again today. All three questions of talk 1 were about listen to details. The average score was very good at 77.10%. Students listened well to details (89.58%, 79.48% and 62.23%). This talk was about a popular topic – internet that all students were familiar to, the vocabulary was also simple. Thus, students got high points when they had experience of what were talked about. Let’s find more details in each question. All three questions of talk 1 were about listen to details. The average score was very good at 77.10%. Students listened well to details (89.58%, 79.48% and 62.23%). This talk was about a popular topic – internet that all students were familiar to, the vocabulary was also simple. Thus, students got high points when they had experience of what were talked about. Let’s find more details in each question. All three questions of talk 1 were about listen to details. The average score was very good at 77.10%. Students listened well to details (89.58%, 79.48% and 62.23%). Listening Difficulties of Non-English Majored Freshmen at Tay Do University, In Vietnam Question 3. Why did the man give up trying yesterday? Key: (A) He had to leave for work. (B) His phone didn’t work. (C) He didn’t have change for a phone call. (D) He hates waiting. 62.23% chose the correct answer, the lowest when compared with the others in talk 1. This maybe because in the multiple choices there were two words “work” that made students confused. From table 6, Business Administration had the highest score of 74.07%. Accounting and Pharmacy were at 66.67% and 61.11%. And 57.58% and 51.72% were of Food Technology and Information Technology. Students had a little difficulty due to incorrect judgment during the listening process. Table 7: Part 4-Short talk 2 Statement Question 4 Question 5 Question 6 Pharmacy 30.56% 47.22% 38.89% Accounting 37.50% 50.00% 41.67% Business Administration 22.22% 55.56% 22.22% Food Technology 21.21% 63.64% 33.33% Information Technology 41.38% 55.17% 20.69% Average (38.75%) 30.57% 54.32% 31.36% Script of short talk 2 M: Welcome to scenic Durango. Our resort offers many activities that you can enjoy in the beautiful sunshine each day. For example, we offer river rafting and rock climbing. We also offer mountain biking trips, golf lessons and tournaments, and many other sports. If you need further information or want to sign up for any of these activities, please ask for our activities coordinator, Roxie Lane. She will be pleased to help. p p This talk was about the information of a resort with many details. Most students only stayed in hotels when travelling. So if based on actual experience, it would not be true. And this was proved that the average score of talk 2 was only 38.75%. Students listened well to details (54.32% correctly answered question 5). The lowest scores were in questions required students to hear information combined with inference (31.36% for question 6 and 30.57% for question 4). Thus, although listening to the conversation was not very good, the students hear the details better when listening to the combination of inference. Let’s find more details in each question. Question 4. What kinds of activities are offered at this resort? (A) Indoor activities. Key: (B) Outdoor activities. (C) Both indoor and outdoor activities. (D) Only children’s activities. This was a difficult question. 30.57% chose the correct answer. Usually based on the experience that at a resort there were many activities, so students chose (C) Both indoor and outdoor activities. Listening Difficulties of Non-English Majored Freshmen at Tay Do University, In Vietnam In addition, there was no phrase as outdoor activities but students had to base on “sunshine” to choose the correct answer. From table 7, Information Technology had the largest share of 41.38%. Accounting and Pharmacy were with 37.50% and 30.56%. Business Administration and Food Technology were with a rate of 22.22% and 21.21%. Question 5. Which water activity is offered? (A) Swimming. (B) Scuba diving. (C) Water polo. Key: (D) River rafting. This was rather easy question since students could get the answer from what they heard, but only 54.32% chose the correct answer. Maybe there were too many details appearing at the same time made students confused. From table 7, Food Technology had the highest percentage at 63.64%. Business Administration, Information Technology and Accounting were 55.56%, 55.17% and 50.00%. Pharmacy had the lowest figure of 47.22%. Question 6. Who should guests contact with questions? (A) The hotel manager. Key: (B) The activities coordinator. (C) The front desk. (D) The clerk. This question was easy because the answer key was mentioned in the talk, but this was also difficult since it was hard to get the “activities coordinator” in a series of information. Then maybe students chose the answer based on the experience that when staying in a hotel, if they needed something, they usually asked the “front desk”. This experience did not help them in choosing the answer. And only 31.36% got good points. From table 7, Accounting had the greatest rate at 41.67%. Pharmacy and Food Technology were 38.89% and 33.33%. Business Administration and Information Technology were ranked 22.22%, and 20.69%. Part 4-Short talks From table 6, 94.44% and 92.59% were of Pharmacy and Business Administration. Information Technology, Food Technology and Accounting were at high rates with 89.66%, 87.88% and 83.33%. IJSSHR, Volume 05 Issue 06 June 2022 Page 2483 www.ijsshr.in Listening Difficulties of Non-English Majored Freshmen at Tay Do University, In Vietnam listening to details was better than listening with inference. It meant that students did not use their listening. listening to details was better than listening with inference. It meant that students did not use their reasoning ability well when listening. The research results found out that students faced some listening problems due to (1) lack of vocabulary knowledge, (2) inability to recognize and distinguish sounds, (3) limited reasoning ability and limited ability to use listening strategies such as judgment, taking notes, or memorizing, and (4) lack of concentration. To help learners improve their listening skills, teachers should pay attention to the following issues: Learners need to regularly supplement their knowledge of vocabulary. Practicing the ability to distinguish homonyms is also considered a necessary activity. Learners need to practice thinking, reasoning, interpreting, and judgment before, during and after listening. Listening and taking notes are also important skills that improve concentration and getting information. 4. CONCLUSION Research results showed that the listening ability of non-English freshmen at Tay Do university was only at average, although the difficulty of the listening test and the content were similar to the lesson content. The above results also contributed to pointing out some difficulties of students in listening and the causes of these difficulties. Specifically, (1) students distinguished action words and prepositions of position better than nouns of places and objects, possibly due to lack of lexical knowledge. (2) the ability to distinguish some words was still limited, distinguishing similar sounds was not good, possibly due to poor vocabulary knowledge, or poor listening technique. (3) students listened to separated sentences better than to conversations or talks, or listening to single sentences with less information was better than to long sentences with many ideas. This maybe show slow speed of sound recognition, ability to concentrate, take notes and memorize limited. (4) listening to information mentioned only once was not good. This showed that the ability to concentrate, take notes and memorize was still limited, perhaps students expected to hear it many times. And (5) IJSSHR, Volume 05 Issue 06 June 2022 Page 2484 www.ijsshr.in Listening Difficulties of Non-English Majored Freshmen at Tay Do University, In Vietnam 6) Ur, P., 1996. A course in Language Teaching Practice and Theory. Cambridge University Press, Cambridge. 7) Vandergrift, L. (1997). The Comprehension Strategies of Second Language (French) Listeners: A Descriptive Study. ACTFL. 30(3), 387-409. 996. A course in Language Teaching Practice and Theory. Cambridge University Press, Cambridge. REFERENCES 1) Hamouda, A. (2013). An Investigation of Listening Comprehension Problems Encountered by Saudi Students in the EL Listening Classroom. International Journal of Academic Research in Progressive Education and Development, 2(2): 113- 155. 1) Hamouda, A. (2013). An Investigation of Listening Comprehension Problems Encountered by Saudi Students in the EL Listening Classroom. International Journal of Academic Research in Progressive Education and Development, 2(2): 113- 155. 2) Mendelsohn, D. J. (1994). Learning to Listen: A Strategy-based Approach for Second Langua 2) Mendelsohn, D. J. (1994). Learning to Listen: A Strategy-based Approach for Second Language Learner. Dominie Press 3) R M (1994) I d i li i L d P i 2) Mendelsohn, D. J. (1994). Learning to Listen: A Strategy-based Approach for Second Language Learner. Dominie Press 3) Rost, M. (1994). Introducing listening. London: Penguin. 4) Rubin, J. (1994). A View of Second Language Listening Comprehension Research. Modern Language Journal, 78(2): 199- 217. 5) Underwood, M. (1989). Teaching listening. Longman. g g g 6) Ur, P., 1996. A course in Language Teaching Practice and Theory. Cambridge University Press, Cambridge. 6) Ur, P., 1996. A course in Language Teaching Practice and Theory. Cambridge University Press, Cam 7) Vandergrift, L. (1997). The Comprehension Strategies of Second Language (French) Listeners: A Descriptive Study. ACTFL. 30(3), 387-409. There is an Open Access article, distributed under the term of the Creative Commons Attribution – Non Commercial 4.0 International (CC BY-NC 4.0) (https://creativecommons.org/licenses/by-nc/4.0/), which permits remixing, adapting and building upon the work for non-commercial use, provided the original work is properly cited. There is an Open Access article, distributed under the term of the Creative Commons Attribution – Non Commercial 4.0 International (CC BY-NC 4.0) (https://creativecommons.org/licenses/by-nc/4.0/), which permits remixing, adapting and building upon the work for non-commercial use, provided the original work is properly cited. IJSSHR, Volume 05 Issue 06 June 2022 www.ijsshr.in Page 2485 There is an Open Access article, distributed under the term of the Creative Commons Attribution – Non Commercial 4.0 International (CC BY-NC 4.0) (https://creativecommons.org/licenses/by-nc/4.0/), which permits remixing, adapting and building upon the work for non-commercial use, provided the original work is properly cited. IJSSHR, Volume 05 Issue 06 June 2022 IJSSHR, Volume 05 Issue 06 June 2022 There is an Open Access article, distributed under the term of the Creative Commons Attribution – Non Commercial 4.0 International (CC BY-NC 4.0) (https://creativecommons.org/licenses/by-nc/4.0/), which permits remixing, adapting and building upon the work for non-commercial use, provided the original work is properly cited. IJSSHR, Volume 05 Issue 06 June 2022 Page 2485 www.ijsshr.in
https://openalex.org/W4376320838	https://zenodo.org/records/7931078/files/4.40.pdf	Zulu	null	UMUMTA'LIM MAKTABLARINING ALGEBRA DARSLARI ORQALI O'QUVCHILARNING MANTIQIY MADANIYATINI SHAKLLANTIRISH USULLARI VA BUNDA MANTIQIY MASALALARNING O'RNI	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	1,653	UMUMTA'LIM MAKTABLARINING ALGEBRA DARSLARI ORQALI O'QUVCHILARNING MANTIQIY MADANIYATINI SHAKLLANTIRISH USULLARI VA BUNDA MANTIQIY MASALALARNING O'RNI Q Sattorova Gulnoza Rustam qizi TDPU matematika o'qitish metodikasi yo'nalishi 401-guruh talabasi https://doi.org/10.5281/zenodo.7931078 Annotatsiya. Bu maqolada, umumta'lim maktablarining algebra darslarida o'quvchilarning mantiqiy madaniyatini shakllantirish usullari va mantiqiy masalalarning o'rni haqida fikr yoritiladi. Maqola, mantiqiy madaniyatni rivojlantirishga qaratilgan savollar va ularni yechish usullarini ko'rsatadi. Mantiqiy masalalar yechish o'quvchilarga turli xil muammolarni yechishga, savollarni tahlil qilishga va ularga javob topishga yordam beradi. Bu esa, ularni turli xil fanlarda muvaffaqiyatga erishish va ularga yangi tajribalar olishga yordam beradi. https://doi.org/10.5281/zenodo.7931078 Annotatsiya. Bu maqolada, umumta'lim maktablarining algebra darslarida o'quvchilarning mantiqiy madaniyatini shakllantirish usullari va mantiqiy masalalarning o'rni haqida fikr yoritiladi. Maqola, mantiqiy madaniyatni rivojlantirishga qaratilgan savollar va ularni yechish usullarini ko'rsatadi. Mantiqiy masalalar yechish o'quvchilarga turli xil muammolarni yechishga, savollarni tahlil qilishga va ularga javob topishga yordam beradi. Bu esa, ularni turli xil fanlarda muvaffaqiyatga erishish va ularga yangi tajribalar olishga yordam beradi. Kalit so’zlar: mantiqiy madaniyat, shakllantirish, mantiqiy masalalar yechish, umumta'lim maktabi, algebra Umumta'lim maktablarida algebra darslari o'quvchilarning mantiqiy madaniyatini shakllantirishga yordam beradigan muhim usullar hisoblanadi. Bu usullar orqali o'quvchilar mantiqiy masalalarni tahlil qilish va yechishda kuchliroq bo'lishadi. Mantiqiy madaniyat, insoniyat tarixining bir qismi hisoblanadi. Bu madaniyat insonning mantiqiy o'ylash, fikrlash va tushunish ko'nikmalariga asoslanadi. Mantiqiy madaniyatning shakllanishi esa insonning o'zida tushunish, ko'rgan va bilgan narsalarning manosini anglashiga bog'liqdir. Bu esa o'z navbatida insonning yaxshi mantiqiy ko'nikmalarga ega bo'lishiga qulaylik keltiradi. Algebra darslari mantiqiy madaniyatni shakllantirish usullari qatorini taqdim etadi. Bu usullar orqali o'quvchilar mantiqiy savollar va masalalarni ochiq va tahlil qilishni o'rganishadi. Mantiqiy savollarga mantiqiy amallar yordam beradi. Masalan, yana bir narsa bilish va boshqa narsalardan ajratish amalini bajarish. Bu esa o'quvchilar uchun mantiqiy o'ylashni oshiradi. Mantiqiy masalalarning o'rni esa o'quvchilar uchun juda muhimdir. Bu masalalar orqali o'quvchilar mantiqiy amallar bilan tanishadi va ularni tahlil qilishni o'rganadi. Mantiqiy masalalar o'quvchilarning tushunish qobiliyatini oshiradi va ularning mantiqiy ko'nikmalariga ega bo'lishiga yordam beradi. Shu bilan birga, algebra darslari orqali o'quvchilarning mantiqiy madaniyatini shakllantirish usullari juda muhim hisoblanadi. Bu usullar orqali o'quvchilar mantiqiy savollarni tahlil qilish va yechishda kuchliroq bo'lishadi. Bu esa ularning mantiqiy ko'nikmalariga ega bo'lishiga yordam beradi va ularga mantiqiy madaniyatni oshirishga yordam beradi. Mantiqiy masalalar o'quvchilar uchun juda muhim hisoblanadi, chunki ular o'quvchilarning mantiqiy o'ylash va muhokama qobiliyatlarini oshiradi. Bu masalalar bir necha turdagi bo'lishi mumkin, masalan, tasavvur amallari, soniy tasavvur masalalari, haqiqiy yoki jinoyatli isbot masalalari, va boshqalar. Mantiqiy masalalarni yechish usullari ham juda ko'pdir. Ular o'quvchilarning kuchliroq bo'lishi, ularning mantiqiy ko'nikmalarini kuchaytirishi va tasavvur qobiliyatlarini oshirishi mumkin. “UMUMIY O’RTA TA’LIM MAKTABLARIDA MADANIY MUHITNI YARATISHNING PEDAGOGIK TEXNOLOGIYALARI: MUAMMO VA YECHIMLAR” MAVZUSIDAGI RESPUBLIKA ILMIY-AMALIY ANJUMANI, MAY 15-16, 2023 Algebra darslarida o'quvchilar mantiqiy masalalarni tahlil qilishni o'rganish orqali, ular mantiqiy o'ylash va muhokama qobiliyatlarini oshirishadi. Bu esa ularning har bir sohada muvaffaqiyatga erishishiga yordam beradi. Shuningdek, mantiqiy masalalar orqali o'quvchilar mantiqiy madaniyatning qolgan aspektlarini o'rganishiga ham imkon beradi. Bu masalalar o'quvchilarga muhim tajriba beradi va ularni mantiqiy tushunchalarini oshirishga, ularning o'zini boshqalar bilan soliqqa solish va tahlil qilish qobiliyatlarini rivojlantirishga yordam beradi. Algebra darslari orqali o'quvchilar mantiqiy madaniyatni shakllantirish usullari va mantiqiy masalalarining o'rni juda katta e'tiborga sazovor. Bu usullar o'quvchilarning mantiqiy o'ylash va muhokama qobiliyatlarini oshirishga, ularning har bir sohada muvaffaqiyatga erishishiga, shuningdek, ularni hayotda muhim mantiqiy muhokama va qarashlarga hazil qilishiga yordam beradi. Bu esa ularning mustaqil fikrlash, tushunish va aniqlash qobiliyatlarini rivojlantirishiga yordam beradi. Umumta'lim maktablarining algebra darslari, o'quvchilarning mantiqiy madaniyatini shakllantirish usullarini o'rganishda muhim ahamiyatga ega. Bu usullar o'quvchilarning akademik hayotlarida, shuningdek, kundalik hayotlarida ham muhim ahamiyatga ega bo'lib, ularni mantiqiy va tahliliy fikrlash uchun tayyorlaydi. Mantiqiy masalalar yechish va o'rganish, o'quvchilarni ta'lim jarayonida mantiqiy madaniyatni rivojlantirish uchun yana bir vosita hisoblanadi. Ular o'quvchilarning tahlil qilish, yechim topish va isbotlash qobiliyatlarini rivojlantiradi. Bu esa ularni mustaqil va o'zlashtirilgan fikrlashni o'rganishga yordam beradi. Algebra darslarida o'quvchilar mantiqiy masalalar orqali tahlil qilish, yechim topish va isbotlash qobiliyatlarini oshirish orqali, boshqa fanlarda ham muvaffaqiyatga erishishga yordam beradi. Bu esa ularning o'zlarining qarashlarni tahlil qilish va muhokama qilish, hayotda o'z vazifalarini bajarish uchun katta ahamiyatga ega bo'ladi. Shunday qilib, umumta'lim maktablarida o'qituvchilar algebra darslari orqali o'quvchilarning mantiqiy madaniyatini shakllantirish usullari va mantiqiy masalalarining o'rni haqida qiziqarli o'quvchilar tayyorlashga intiladi. Bu esa ularni har bir sohada muvaffaqiyatga erishish va hayotda mantiqiy muhokama va qarashlarni tahlil qilishga tayyorlaydi. Jamiyatimizning mantiqiy madaniyatni rivojlantirishga va mukammal jamiyatga aylanishiga qaratilgan maqsadlariga erishishda, umumta'lim maktablarida o'quvchilarni mantiqiy madaniyatni rivojlantirishda juda muhim ahamiyatga ega bo'lgan algebra darslari katta rol o'ynaydi. Bu esa mustaqil va o'zlashtirilgan fikrlash va tahlil qilishni o'rganishda o'quvchilarga yordam beradi va ularni muvaffaqiyatga erishishga yordam beradi. Bundan tashqari, umumta'lim maktablarida o'quvchilar mantiqiy madaniyatni shakllantirish usullari orqali, turli xil qarashlarni tahlil qilish va javob topishni o'rganishadi. Bu esa ularni turli masalalar yechish va yechim topishda o'zining xususiyatlarini va qobiliyatlarini oshiradi. Shu bilan birga, mantiqiy masalalar tahlil qilish uchun katta ahamiyatga ega bo'lib, bu esa o'quvchilarning mantiqiy madaniyatini rivojlantirish uchun ham muhimdir. Mantiqiy masalalar, o'quvchilarning qarashlarini tahlil qilish va o'z fikrlarini yoritishda ularga yordam beradi. Shu bilan birga, ularni isbotlash qobiliyatlarini oshiradi va mantiqiy jarayonni yaxshi tahlil qilishga yordam beradi. UMUMTA'LIM MAKTABLARINING ALGEBRA DARSLARI ORQALI O'QUVCHILARNING MANTIQIY MADANIYATINI SHAKLLANTIRISH USULLARI VA BUNDA MANTIQIY MASALALARNING O'RNI Masalan, masalani bo'lgan tahlilga ajratish, masalaga bir yoki bir nechta yechimlarni ko'rsatish, yechimlarni tartiblash va shu kabi usullar masalani yechishda yordam beradi. 331 “UMUMIY O’RTA TA’LIM MAKTABLARIDA MADANIY MUHITNI YARATISHNING PEDAGOGIK TEXNOLOGIYALARI: MUAMMO VA YECHIMLAR” MAVZUSIDAGI RESPUBLIKA ILMIY-AMALIY ANJUMANI, MAY 15-16, 2023 Shuningdek, o'quvchilarning yechim topish qobiliyatlarini oshiradi va ularga turli xil masalalar yechish uchun qo'shimcha qobiliyatlar beradi. 332 “UMUMIY O’RTA TA’LIM MAKTABLARIDA MADANIY MUHITNI YARATISHNING PEDAGOGIK TEXNOLOGIYALARI: MUAMMO VA YECHIMLAR” MAVZUSIDAGI RESPUBLIKA ILMIY-AMALIY ANJUMANI, MAY 15-16, 2023 3-masala. 1, 2, 3, ..., 8 raqamlaridan ularni takrorlamay tuzilgan 8 xonali sonlar ichida 1 va 8 raqamlari yonma – yon turadiganlari nechta? Quyidagi hollar bo‘lishi mumkin: 1 birinchi o‘rinda, 8 ikkinchi o‘rinda, ..., 1 yettinchi o‘rinda, 8 sakkizinchi o‘rinda, bunday hollar soni 7 ta. Bundan tashqari, 1 va 8 larning yuqoridagi 7 holda o‘rinlarini almashtirib, yana 7 ta (ular yonma – yon turadigan) holni topamiz. Demak, 1 va 8 ni yonma – yon qilib, 14 usul bilan qo‘yish mumkin. Bu usullarning har biriga boshqa qolgan raqamlarning 6! ta o‘rin almashtirishlari mos keladi. Shunday qilib, 1 va 8 raqamlari yonmayon turadigan o‘rin almashtirishlar soni 2 · 7 · 6! = 2 · 7! ga teng. 4-masala. Gul sotuvchida 5 ta qizil va 10 ta oq chinnigul qolibdi. A’zamxon singlisi Mubinabonuga 2 ta qizil va 3 ta oq chinniguldan iborat guldasta sovg‘a qilmoqchi. Buni u necha xil usul bilan amalga oshirishi mumkin? O'quvchilarning mantiqiy madaniyatini shakllantirish usullari va mantiqiy masalalar yechish, jamiyatimizni o'sishiga va rivojlantirishiga ham yordam beradi. Bu esa ularning turli xil fanlarda muvaffaqiyatga erishishiga va ularga yangi tajribalar olishiga yordam beradi. Shuningdek, bu mantiqiy madaniyatni shakllantirish usullari va mantiqiy masalalar yechish, o'quvchilarning hayotda mantiqiy muhokama va qarashlarni tahlil qilish uchun tayyorlashda muhim ahamiyatga ega. Jamiyatning rivojlanishi, har doim mantiqiy madaniyatni rivojlantirishga bog'liqdir. Bu esa, o'quvchilarni mantiqiy madaniyatni shakllantirish usullari va mantiqiy masalalar yechish orqali tayyorlash, ularni turli xil fikrlash tarzlarini rivojlantirish va o'zlarini mustaqil o'rganishga yordam beradi. Mantiqiy madaniyatni shakllantirish usullari va mantiqiy masalalar yechish, o'quvchilarni turli xil masalalarni yechishga, savollarni tahlil qilishga va ularga javob topishga yordam beradi. Bu esa, ularni turli xil fanlarda muvaffaqiyatga erishish va ularga yangi tajribalar olishga yordam beradi. Shu bilan birga, mantiqiy madaniyatni shakllantirish usullari va mantiqiy masalalar yechish, o'quvchilarning hayotda mantiqiy muhokama va qarashlarni tahlil qilish uchun tayyorlashda muhim ahamiyatga ega. Shuningdek, mantiqiy madaniyatni shakllantirish usullari va mantiqiy masalalar yechish, jamiyatimizning o'sishiga va rivojlantirishiga ham yordam beradi. “UMUMIY O’RTA TA’LIM MAKTABLARIDA MADANIY MUHITNI YARATISHNING PEDAGOGIK TEXNOLOGIYALARI: MUAMMO VA YECHIMLAR” MAVZUSIDAGI RESPUBLIKA ILMIY-AMALIY ANJUMANI, MAY 15-16, 2023 Algebra darslarida o'quvchilar, mantiqiy madaniyatni shakllantirish uchun bir qadam ko'rsatadi, bu esa ularni o'zlarini o'rganishga, mustaqil fikrlashga va o'zlashtirilgan fikrlashni tahlil qilishga yordam beradi. Bu esa ularni boshqa fanlarda ham muvaffaqiyatga erishishga yordam beradi, chunki turli xil fanlarda mantiqiy fikrlash va tahlil qilish uchun tayyorlik zarur. Jamiyatimizda, mantiqiy madaniyatni rivojlantirish va jamiyatni mukammal jamiyatga aylanish maqsadida, umumta'lim maktablarida o'quvchilarni mantiqiy madaniyatni shakllantirish usullari va mantiqiy masalalar yechishning o'rni hamda ahamiyati juda katta. Bu esa ularni har bir sohada muvaffaqiyatga erishish va hayotda mantiqiy muhokama va qarashlarni tahlil qilishga tayyorlaydi. Bundan tashqari, mantiqiy madaniyatni rivojlantirish, jamiyatimizni har qanday sohada rivojlantirish uchun ham muhimdir. Bu esa yangi tajribalar olish, muhim vaqtlar bo'ylab yaxshi qarashlarni topish va jamiyatimizni o'sishiga yordam beradi. Shu bilan birga, bu mantiqiy madaniyatni shakllantirish usullari va mantiqiy masalalar o'rni, o'quvchilarga mustaqil o'rganish va o'zlarining fikrlash tarzlarini rivojlantirishda ham muhim o'ziga xosdir. O'quvchilarimiz, mantiqiy madaniyatni rivojlantirish uchun turli xil usullardan foydalanishadi. Ba'zilari, ko'proq amaliy usulni iste'mol qilishni yaxshi ko'radilar, boshqalar esa nazariy yondashuvni qo'llab-quvvatlashni ko'proq yoqtirishadi. Mantiqiy madaniyatni rivojlantirish uchun yaxshi usullardan biri, o'quvchilarni tahlil qilishni o'rganishdir. Bu esa ularni turli xil savollarni yechishga, savollar tahlil qilishga va javob topishga yordam beradi. Mantiqiy masalalar yechish esa o'quvchilarga ko'proq amaliy tajriba va jarayonni tahlil qilishga yordam beradi. Mantiqiy madaniyatni shakllantirish usullari va mantiqiy masalalar yechish orqali, o'quvchilarimiz mustaqil fikrlashni va o'zini mustaqil fikrlashni rivojlantirishadi. Bu esa ularning turli xil masalalarni yechish va ularga javob topishda o'zining fikrlash tarzini rivojlantirishga yordam beradi. Bundan tashqari, mantiqiy madaniyatni shakllantirish usullari va mantiqiy masalalar yechish, o'quvchilarning o'zlarini tahlil qilish va yechim topish qobiliyatlarini oshiradi. Shu bilan birga, ularni turli xil fanlarda ham muvaffaqiyatga erishish uchun tayyorlaydi. Fikrimizning dalili uchun 7-sinf darsligidan bir necha misol keltiramiz. 1-masala. Idishda 1, 2, 3, ..., 10 sonlari yozilgan sharlar bor. Idishdan uchta shar olamiz. Nechta holda ularda yozilgan sonlar yig‘indisi 9 ga teng bo‘ladi? Nechta holda 9 dan katta bo‘ladi? 2-masala. 3 ta tovuq, 4 ta o‘rdak va 2 ta g‘oz bor. Bir nechta parrandani shunday tanlab olingki, ular ichida tovuq, o‘rdak va g‘oz bo‘lsin. Shunday tanlashlar soni nechta bo‘ladi? 333 REFERENCES 1. Jumayev M.E. va boshqalar. Matematika o’qitish metodikasi (kasb-hunar kollejlari o’quvchilari uchun o’quv qo’llanma) – T.: Ilm-Ziyo, 2003, 240-bet o quvchilari uchun o quv qo llanma) – T.: Ilm-Ziyo, 2003, 240-bet 2. Jumayev M.E. Matematika o’qitish metodikasidan praktikum - Toshkent.: O’qituvchi, 2004, 328 bet. 2. Jumayev M.E. Matematika o’qitish metodikasidan praktikum - Toshkent.: O’qituvchi, 2004, 328 bet. 334
https://openalex.org/W2558947371	https://digitalcommons.unl.edu/cgi/viewcontent.cgi?article=1523&context=entomologyfacpub	English	null	Integration of Plant Defense Traits with Biological Control of Arthropod Pests: Challenges and Opportunities	Frontiers in plant science	2,016	cc-by	25,743	University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Entomology, Department of Entomology, Department of Faculty Publications: Department of Entomology This Article is brought to you for free and open access by the Entomology, Department of at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Faculty Publications: Department of Entomology by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Peterson, Julie A.; Ode, Paul J.; Oliveira-Hofman, Camila; and Harwood, James D., "Integration of Plant Defense Traits with Biological Control of Arthropod Pests: Challenges and Opportunities" (2016). Faculty Publications: Department of Entomology. 495. http://digitalcommons.unl.edu/entomologyfacpub/495 Citation: Peterson JA, Ode PJ, Oliveira-Hofman C and Harwood JD (2016) Integration of Plant Defense Traits with Biological Control of Arthropod Pests: Challenges and Opportunities. Front. Plant Sci. 7:1794. doi: 10.3389/fpls.2016.01794 Integration of Plant Defense Traits with Biological Control of Arthropod Pests: Challenges and Opportunities James D. Harwood University of Kentucky Follow this and additional works at: http://digitalcommons.unl.edu/entomologyfac ollow this and additional works at: http://digitalcommons.unl.edu/entomologyfacpub Part of the Entomology Commons, and the Plant Sciences Commons Part of the Entomology Commons, and the Plant Sciences Commons Peterson, Julie A.; Ode, Paul J.; Oliveira-Hofman, Camila; and Harwood, James D., "Integration of Plant Defense Traits with Biological Control of Arthropod Pests: Challenges and Opportunities" (2016). Faculty Publications: Department of Entomology. 495. http://digitalcommons.unl.edu/entomologyfacpub/495 This Article is brought to you for free and open access by the Entomology, Department of at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Faculty Publications: Department of Entomology by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. REVIEW published: 30 November 2016 doi: 10.3389/fpls.2016.01794 REVIEW published: 30 November 2016 doi: 10.3389/fpls.2016.01794 Integration of Plant Defense Traits with Biological Control of Arthropod Pests: Challenges and Opportunities Julie A. Peterson1, Paul J. Ode2, Camila Oliveira-Hofman3 and James D. Harwood4 1 Department of Entomology, West Central Research and Extension Center, University of Nebraska–Lincoln, North Platte, NE, USA, 2 Department of Bioagricultural Sciences and Pest Management, Colorado State University, Fort Collins, CO, USA, 3 Department of Entomology, University of Nebraska–Lincoln, Lincoln, NE, USA, 4 Department of Entomology, University of Kentucky, Lexington, KY, USA Integration of Plant Defense Traits with Biological Control of Arthropod Pests: Challenges and Opportunities Julie A. Peterson1, Paul J. Ode2, Camila Oliveira-Hofman3 and James D. Harwood4 1 Department of Entomology, West Central Research and Extension Center, University of Nebraska–Lincoln, North Platte, NE, USA, 2 Department of Bioagricultural Sciences and Pest Management, Colorado State University, Fort Collins, CO, USA, 3 Department of Entomology, University of Nebraska–Lincoln, Lincoln, NE, USA, 4 Department of Entomology, University of Kentucky, Lexington, KY, USA 1 Department of Entomology, West Central Research and Extension Center, University of Nebraska–Lincoln, North Platte, NE, USA, 2 Department of Bioagricultural Sciences and Pest Management, Colorado State University, Fort Collins, CO, USA, 3 Department of Entomology, University of Nebraska–Lincoln, Lincoln, NE, USA, 4 Department of Entomology, University of Kentucky, Lexington, KY, USA Crop plants exhibit a wide diversity of defensive traits and strategies to protect themselves from damage by herbivorous pests and disease. These defensive traits may be naturally occurring or artiﬁcially selected through crop breeding, including introduction via genetic engineering. While these traits can have obvious and direct impacts on herbivorous pests, many have profound effects on higher trophic levels, including the natural enemies of herbivores. Multi-trophic effects of host plant resistance have the potential to inﬂuence, both positively and negatively, biological control. Plant defense traits can inﬂuence both the numerical and functional responses of natural enemies; these interactions can be semiochemically, plant toxin-, plant nutrient-, and/or physically mediated. Case studies involving predators, parasitoids, and pathogens of crop pests will be presented and discussed. These diverse groups of natural enemies may respond differently to crop plant traits based on their own unique biology and the ecological niches they ﬁll. Genetically modiﬁed crop plants that have been engineered to express transgenic products affecting herbivorous pests are an additional consideration. For the most part, transgenic plant incorporated protectant (PIP) traits are compatible with biological control due to their selective toxicity to targeted pests and relatively low non- target impacts, although transgenic crops may have indirect effects on higher trophic levels and arthropod communities mediated by lower host or prey number and/or quality. Host plant resistance and biological control are two of the key pillars of integrated pest management; their potential interactions, whether they are synergistic, complementary, or disruptive, are key in understanding and achieving sustainable and effective pest management. Edited by: Rex Brennan, The James Hutton Institute, UK Reviewed by: Andrew Nick Birch, The James Hutton Institute, UK Julie Graham, The James Hutton Institute, UK Michelle Fountain, NIAB East Malling Research, UK *Correspondence: Julie A. Peterson julie.peterson@unl.edu Specialty section: This article was submitted to Crop Science and Horticulture, a section of the journal Frontiers in Plant Science Received: 18 March 2016 Accepted: 15 November 2016 Published: 30 November 2016 Keywords: host plant resistance, tritrophic interactions, transgenic crops, biological control, herbivore-induced plant volatiles (HIPVs) Keywords: host plant resistance, tritrophic interactions, transgenic crops, biological control, herbivore-induced plant volatiles (HIPVs) Edited by: Rex Brennan, The James Hutton Institute, UK Peterson JA, Ode PJ, Oliveira-Hofman C and Harwood JD (2016) Integration of Plant Defense Traits with Biological Control of Arthropod Pests: Challenges and Opportunities. Front. Plant Sci. 7:1794. doi: 10.3389/fpls.2016.01794 INTRODUCTION TO KEY CONCEPTS Interestingly, some studies also describe variable responses of predators on diﬀerent plants using plant- based defenses such as glandular trichomes and allelochemical production (De Clercq et al., 2000). These variable responses therefore highlight the need for careful consideration of the eﬀects of diﬀerent plant traits on pest suppression. The interactions between plants, herbivores, and their natural enemies are referred to as tritrophic interactions and this multi-trophic exchange is key to understanding the interactions between host plant resistance and biological control. Natural enemies can be considered an extension of plant defense if plant traits, such as release of herbivore-induced plant volatiles (HIPVs), draw in these natural enemies. The literature is replete with examples of natural enemies acting in a top-down fashion, reducing herbivore populations, thereby providing plant defense. The concept of breeding plants to select for heritable traits that reduce pest impacts has been a part of agricultural production for over 100 years (Painter, 1951; Smith, 2005) and can be separated into tolerance and resistance mechanisms (Stout, 2013). Tolerance allows plants to withstand pest injury while resistance is conferred by plant traits that reduce the extent of pest injury and can be divided into constitutive or inducible and direct or indirect plant defenses (Stout, 2013). A constitutive defense is expressed in a plant regardless of whether it has been attacked by an herbivore, whereas an inducible defense is only expressed (or expressed to a greater degree) after attack. Direct defenses aﬀect the herbivore without a mediating factor, whereas indirect defenses act via the actions of natural enemies. While indirect resistance may have the most obvious implications for biological control, other forms of resistance and tolerance also impact pest control by natural enemies. Holistic consideration of all these mechanisms is critical for their successful integration into pest control schemes. The intention of this section is to provide a general introduction to the key concepts that provide context for the remainder of this review article. For more in-depth discussion of these topics, please refer to the many texts that review these topics (i.e., Painter, 1951; Panda and Khush, 1995; Kogan, 1998; Bellows et al., 1999; Agrawal, 2000a; Landis et al., 2000; Hajek, 2004; Smith, 2005; Heil, 2008; Radcliﬀe et al., 2009; van Lenteren, 2012; Stout, 2013; Pedigo and Rice, 2014). INTRODUCTION TO KEY CONCEPTS The worldwide population is growing, with projections of 9–10 billion people living on Earth by 2050 (United Nations, 2004; Lutz and Samir, 2010). Global food demands are increasing concomitantly, with a need for heightened food security, increased agricultural productivity and improved water use eﬃciency of crops. In a global review of factors contributing to losses for eight November 2016 \| Volume 7 \| Article 1794 Frontiers in Plant Science \| www.frontiersin.org 1 Host Plant Resistance and Biocontrol Peterson et al. major food and cash crops, animal pests came in second only to weeds, causing potential yield losses of 17.6% (Oerke and Dehne, 2004). Clearly, crop pests are responsible for signiﬁcant losses to agricultural commodities worldwide despite profound eﬀorts at management. Identiﬁcation and promotion of sustainable solutions to these agricultural threats are essential for meeting future needs. The concepts of Integrated Pest Management (IPM), ﬁrst championed by Stern et al. (1959), support practical eﬀorts to achieve sustainable pest management. IPM has been described as “the harmonious use of multiple methods to control” pests, using “a set of decision rules based on ecological principles and economic and social considerations” (Kogan, 1998). Ideally, IPM incorporates the use of economic thresholds (Higley and Peterson, 2009) and a variety of control tactics (mechanical, physical, cultural, chemical, biological, and host plant resistance) making it essential to understand the interactions between diﬀerent control tactics. Two key approaches for sustainable pest management have been (1) host plant resistance, the selection or development (via traditional breeding or genetic modiﬁcation) and use of crop plants that possess defensive traits against herbivores and disease, and (2) biological control, the use of living organisms that are natural enemies of crop pests. natural enemy abundance due to reproduction or aggregation) or functional (changes in natural enemy behavior) (Hajek, 2004). Seminal work on functional responses of predators to their prey items by Holling (1966) demonstrated that rate of prey discovery, search time, handling time, and predator hunger were important factors in determining functional response. In the years since Holling’s research, studies in pest management have frequently examined how predators respond to prey, documenting the existence of functional responses in the context of biological control (e.g., De Clercq et al., 2000; Lee and Kang, 2004; Rutledge and O’Neil, 2005). INTRODUCTION TO KEY CONCEPTS This review will focus on the interactions between biological control and host plant resistance, addressing the mechanisms and potential outcomes of interactions, with special attention to genetically modiﬁed insect- resistant crops and case studies for application of host plant resistance and biological control in cropping systems. IMPACT OF PLANT TRAITS ON BIOLOGICAL CONTROL Biological control programs use natural enemies (predators, parasitoids, and pathogens) of targeted pests to keep populations below the economic threshold. Classical biological control is the importation and establishment of natural enemies to control exotic pests while augmentation biological control incorporates the supplemental release of natural enemies. Conservation biological control involves modiﬁcation of the environment or existing agronomic practices to protect and enhance speciﬁc natural enemies already present in the ecosystem (e.g., Landis et al., 2000; Eilenberg et al., 2001). The maintenance of natural enemy populations via conservation biological control can be a practical and sustainable option for low-value and high- acreage commodities, such as maize and other annual ﬁeld crops (Thorbek et al., 2004; Naranjo et al., 2015). The responses of natural enemies to pest population changes are critically important and these can be classiﬁed as numerical (changes in The mechanisms by which plant defensive traits can aﬀect biological control can be divided into four major categories: semiochemically, plant toxin-, plant nutrient-, and physically mediated interactions. These have been widely recognized as the major mechanisms by which the three trophic levels interact (Price, 1986; Thomas and Waage, 1996; Agrawal, 2000a) and will be reviewed in detail here. Their integration (see Discussion) into biological control programs is critical as we develop sustainable solutions for pest management. Frontiers in Plant Science \| www.frontiersin.org Semiochemically Mediated Interactions Such information conveyed to natural enemies has profound consequences for the biological control services aﬀorded by them and maximizes the top-down eﬀect of such species on herbivorous pests. The quantity of HIPVs released may reﬂect the level of herbivory and determine the level of attractiveness to parasitoids. In studies of Cotesia glomerata (L.) (Hymenoptera: Braconidae) attacking Pieris rapae (L.) (Lepidoptera: Pieridae), plants attacked by more herbivores or induced with higher concentrations of JA (simulating higher levels of herbivory) were more attractive to C. glomerata (Geervliet et al., 1998; Bruinsma et al., 2009). Yet, HIPV production may also inﬂuence the plant’s attractiveness to herbivores. In an interesting study of two chrysomelid beetles (Gynandrobrotica guerreroensis (Jacoby) and Cerotoma ruﬁcornis Olivier) attacking wild lima beans [Phaseolus lunatus L. (Fabales: Fabaceae)], female beetles were repelled by HIPVs produced by induced plants regardless of level of induction (possibly reﬂecting competition and a lack of enemy-free space) whereas males were attracted by weakly induced plants (possibly indicating the presence of a mate) but repelled by strongly induced plants (Ballhorn et al., 2013). The eﬀect of such changes in herbivore densities on parasitoid foraging decisions is unexplored. Furthermore, parasitoid species identity may also inﬂuence plant volatile production. Cabbage [Brassica oleracea L. (Brassicales: Brassicaceae)] produced similar HIPV proﬁles when attacked by imported cabbageworm Pieris rapae (L.) or large cabbage white P. brassicae (L.) (Lepidoptera: Pieridae) (Poelman et al., 2011). Yet, intriguingly, herbivore regurgitant characteristics were strongly inﬂuenced by the species of parasitoid developing within the herbivore, which diﬀerentially expressed genes within the plant’s JA-signaling pathway. Even hyperparasitoids use HIPVs to locate their parasitoid hosts; the hyperparasitoid Lysibia nana Gravenhorst (Hymenoptera: Ichneumonidae) was more attracted to P. rapae hosts attacked by C. glomerata than those attacked by C. rubecula or unparasitized hosts. Field surveys showed hosts parasitized by C. glomerata are more likely to be hyperparasitized than C. rubecula-parasitized hosts and this preference was due to diﬀerences in HIPV proﬁles elicited by Yet, plant responses to herbivory are more complex than simple wound responses to mechanical damage, which cannot explain the speciﬁcity of some plant responses to herbivores. In addition to physical damage, herbivores secrete substances that may modify plant responses. Semiochemically Mediated Interactions Plants produce a wide range of volatile compounds that are the predominant signals used by arthropod herbivores to November 2016 \| Volume 7 \| Article 1794 2 Host Plant Resistance and Biocontrol Peterson et al. locate suitable host plants (Schoonhoven et al., 2005). These volatile proﬁles can change both quantitatively and qualitatively following herbivory (Dicke, 1999; Páre and Tumlinson, 1999; Heil and Ton, 2008), dramatically altering their attractiveness (or repellency) to herbivores and their natural enemies (Heil, 2014). Feeding, especially by chewing herbivores, results in mechanical damage to plant tissues eliciting a wound response thereby creating electrical, hydraulic, and chemical signals (e.g., systemin; Kessler and Baldwin, 2002). This action results in local and systemic release of linolenic acid from plant cell membranes and is converted by the enzyme lipoxygenase (LOX) to 13-hydroperoxide, which enters one of two pathways (Walling, 2000; Kessler and Baldwin, 2002). In one pathway, 13-hydroperoxide may be hydrolyzed by hydroperoxide lyase to yield ‘green leaf volatiles’ (GLVs; e.g., C6 alcohols and aldehydes) and these, and other volatiles such as terpenoids, are often considered indirect defenses because they attract natural enemies. Alternatively, 13-hydroperoxide can enter the octadecanoid pathway, resulting in the production of jasmonic acid (JA), ultimately producing an array of anti- herbivore defenses including proteinase inhibitors (anti-digestive proteins), polyphenol oxidases (anti-nutritive enzymes), and a bewildering diversity of plant-speciﬁc toxins (Walling, 2000; Kessler, 2015; see Plant Toxin-Mediated Interactions). These inducible defensive chemicals are generally termed direct defenses in that they directly deter or inhibit feeding by herbivores. Wäschke et al., 2013). These GLVs, and others produced via diﬀerent pathways such as volatile terpenoids (Kessler and Baldwin, 2002; Dudareva et al., 2013; Kessler, 2015), play a crucial role in signaling speciﬁc information for parasitoids regarding the status of herbivores and their natural enemies. The information conveyed in HIPVs can provide information on the species of herbivore present, the level of herbivory damage sustained, the developmental stage of the host, and even whether the herbivore has been previously parasitized. For instance, tomato plants attacked by tobacco budworm Heliothis virescens (F.) (Lepidoptera: Noctuidae), but not the closely related tomato fruitworm Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae), emit a volatile proﬁle that is highly attractive to the specialist parasitoid of the tobacco budworm, Cardiochiles nigriceps Viereck (Hymenoptera: Braconidae) (De Moraes et al., 1998). Frontiers in Plant Science \| www.frontiersin.org Plant Toxin-Mediated Interactions While some evidence indicates that diﬀerent herbivores can diﬀerentially induce plant defenses (e.g., Stout et al., 1998; Agrawal, 2000b; Poelman et al., 2008), the eﬀects on higher trophic levels are poorly studied. The most challenging belowground pest of maize production in North America and Europe is the western corn rootworm (WCR) Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae). Upon injury to the roots, European maize hybrids induce a strong production of EβC locally and a weak systemic response throughout root tissues (Hiltpold et al., 2011). EβC released into the rhizosphere recruits the entomopathogenic nematode (EPN) Heterorhabditis megidis Poinar, Jackson and Klein (Rhabditida: Heterorhabditidae). In ﬁeld studies, maize hybrids producing EβC had signiﬁcantly higher rates of H. megidis infection in WCR larvae and reduced rootworm adult emergence than non-EβC-emitting hybrids; non-EβC-emitting maize varieties do not recruit H. megidis when attacked by the WCR (Rasmann et al., 2005). g p p y Unlike indirect defenses (see Semiochemically Mediated Interactions), direct plant defenses typically have negative eﬀects on parasitoid ﬁtness (Ode, 2006, 2013) and occur through one of three, non-mutually exclusive routes. Plant toxins may: (1) reduce host size, having negative consequences for parasitoids feeding on such hosts, (2) pass unmetabolized through the herbivore’s midgut into the hemolymph where they are directly encountered by developing parasitoid larvae (Campbell and Duﬀey, 1979; McGovern et al., 2006; Lampert et al., 2008), or (3) be sequestered for defense against their own natural enemies (Nishida, 2002; Ode, 2006; Lampert et al., 2011a). For example, the catalpa sphinx moth, Ceratomia catalpae (Boisduval) (Lepidoptera: Sphingidae), sequesters the iridoid glycoside catalpol when it feeds on the catalpa plant, Catalpa bignonioides Walter (Lamiales: Bignoniaceae) (Lampert et al., 2010). Interestingly, the parasitoid Cotesia congregata (Say) (Hymenoptera: Braconidae) appears to be little aﬀected by concentrations of catalpol, which also accumulate in the tissues of the parasitoid suggesting the role of this compound as protection against its own hyperparasitoids (Lampert et al., 2011a). Numerous studies have shown the potential of EPNs to suppress WCR populations (Wright et al., 1993; Jackson, 1996; Toepfer et al., 2005, 2008; Kurtz et al., 2009; Hiltpold et al., 2012) but not all EPN species and strains that attack WCR larvae are attracted to EβC (Hiltpold et al., 2010c; Anbesse and Ehlers, 2013; Laznik and Trdan, 2013). Plant Toxin-Mediated Interactions , p g Domestication can inadvertently alter the volatile proﬁles of many crop plants, aﬀecting rates of parasitism. One example is the production of the sesquiterpene (E)-β-caryophyllene (EβC) in maize. EβC is emitted in response to above- (Turlings et al., 1998) and below-ground injury (Rasmann et al., 2005). It serves as an attractant for natural enemies of maize pests (Rasmann et al., 2005; Köllner et al., 2008) and provides protection from herbivores with diﬀerent modes and sites of attack (Köllner et al., 2008). Unfortunately, EβC production has been unintentionally bred out of commercially available North American maize hybrids, but it is still present in European maize lines and teosinte (Zea mays ssp. parviglumis) (Degen et al., 2004; Rasmann et al., 2005). EβC production can be reintroduced by insertion of a gene from oregano, Origanum vulgare L. (Lamiales: Lamiaceae) (Degenhardt et al., 2009), demonstrating the ability to genetically enhance crops to increase natural enemy control of insect pests. Of the more than 100,000 identiﬁed plant secondary metabolites, many play roles in direct defense against herbivorous insects through anti-nutritive, anti-digestive, or toxic compounds. Many of these defensive chemicals are produced constitutively, regardless of whether a plant is attacked by herbivores; others are often inducible via the JA-based signaling pathway described in Semiochemically Mediated Interactions above (Memelink et al., 2001; Agrawal, 2011; De Geyter et al., 2012). While plant anti-herbivore toxins might be expected to exhibit similar responsiveness as semiochemicals to the damage done by speciﬁc herbivores and the presence of their natural enemies, little evidence suggests this is the case. Rather, many secondary compounds are present within only a limited range of plant families (e.g., the glucosinolates are found almost exclusively in plants in the Order Brassicales (Halkier and Gershenzon, 2006), furanocoumarins are primarily associated with the families Apiaceae and Rutaceae (Berenbaum, 1983, 1990)). Speciﬁcity of plant defensive responses to diﬀerent herbivores (‘speciﬁcity of elicitation’ sensu Stout et al., 1998) seems, for the most part, to be quantitative rather than qualitative. For instance, levels of damage caused by diﬀerent herbivores (Van Zandt and Agrawal, 2004) or variable damage by unparasitized vs. parasitized herbivores that results in diﬀerential feeding by herbivores (Ode et al., 2016) may result in the induction of diﬀerent plant defensive compounds. Frontiers in Plant Science \| www.frontiersin.org Semiochemically Mediated Interactions Collectively, these substances are referred to as herbivore-associated molecular patterns (HAMPs; Felton and Tumlinson, 2008; Mithöfer and Boland, 2008) and include substances such as regurgitants and salivary secretions (Alborn et al., 1997; Musser et al., 2002; Schäfer et al., 2011; Tian et al., 2012; Louis et al., 2013), and even frass production (Ray et al., 2015). Behavioral interactions, too, modify plant volatile production with walking on leaf surfaces (Tooker et al., 2010) and oviposition (Hilker and Meiners, 2006; Kim et al., 2012; Hilﬁker et al., 2014) having profound eﬀects. It is therefore unsurprising that plants respond to herbivory in speciﬁc ways that provide informative semiochemical-based information for both herbivores and their natural enemies. Plants emit diﬀerent suites of volatiles, attracting diﬀerent parasitoid complexes, depending on the species of herbivore attacking the plant. Clearly, there is abundant evidence that HAMPs and behavioral interactions of herbivores with host plants alter plant defensive responses beyond that of simple mechanical damage (e.g., Dicke, 1999; Reymond et al., 2000; Kessler and Baldwin, 2002). This highlights a cautionary note when interpreting ﬁndings of the large number of ecological studies using artiﬁcial leaf clippings and hole punches as a proxy for herbivore damage. As discussed above, plant volatiles that attract natural enemies are considered indirect defenses (Vet and Dicke, 1992; Kessler and Baldwin, 2002; Turlings and Wäckers, 2004; November 2016 \| Volume 7 \| Article 1794 3 Host Plant Resistance and Biocontrol Peterson et al. factors inﬂuencing pest control, rather than single elements acting along. and cultural (Levine et al., 2002) management tools. Alternative control strategies, such as recruitment of entomopathogens using plant volatiles, must be explored in order to sustainably manage this critical pest. Case study: Maize Volatiles, Western Corn Rootworm, and Entomopathogenic Nematodes Case study: Maize Volatiles, Western Corn Rootworm, and Entomopathogenic Nematodes Case Study: Cotton, Gossypol and Bt Toxins, Herbivores, and Natural Enemies Cotton, Gossypium hirsutum L. (Malvales: Malvaceae), the most important plant-based ﬁber used by humans worldwide, presents an interesting example of the diﬃculties in breeding for resistance against multiple insect pests. It is consumed by a large number of insect herbivores including the boll weevil, bollworm, pink bollworm, tobacco budworm, armyworms, cotton aphid, whiteﬂies, Lygus bugs, and thrips (Matthews and Tunstall, 1994; Hagenbucher et al., 2013a). Prior to the introduction of Bacillus thuringiensis (Bt) cotton and more eﬀective IPM approaches, insecticides were the primary means of pest control. An array of morphological (e.g., trichomes) and chemical defenses are produced by cotton and of the chemical defenses, terpenoids (especially gossypol and related compounds) are the best studied. Gossypol, present in leaves and seeds, provides resistance to a broad range of lepidopteran pests (Bottger and Patana, 1966). As it is also toxic to humans, breeding eﬀorts have selected for glandless cultivars that produce low gossypol levels, but these cultivars are particularly susceptible to a range of insect pests (Jenkins et al., 1966). Recent eﬀorts using RNAi to produce low gossypol levels in the seeds while maintaining high levels elsewhere have been successful (reviewed in Hagenbucher et al., 2013a), but gossypol also has negative eﬀects on some natural enemies. For instance, Campoletis sonorensis (Cameron) (Hymenoptera: Ichneumonidae) experiences reduced body size, reduced survivorship, and increased development time when developing on H. virescens that had fed on diets high in gossypol (Gunasena et al., 1989), although this negative eﬀect is by no means universal across species (e.g., Sun et al., 2011). Similar to semiochemically induced eﬀects, responses of organisms to diﬀerent compounds are speciﬁc to the exact plant–insect interaction. The recent focus in cotton breeding for insect herbivore resistance has centered on the development of Bt transgenic lines expressing Cry-endotoxins that confer resistance against lepidopteran herbivores. In particular, adoption of Bt cotton has been credited with the eradication of the pink bollworm Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae) in the southwestern United States (Carriére et al., 2003) and substantial declines of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) in China (Wu et al., 2008). The speciﬁcity of Cry toxins against lepidopterans and reduced pesticide use after widespread adoption of Bt cotton has provided an environment favorable to natural enemies, allowing increased control of a wide variety of cotton pests (Naranjo, 2011; Lu et al., 2012). Plant Toxin-Mediated Interactions Heterorhabditis bacteriophora Poinar (Rhabditida: Heterorhabditidae), for instance, is highly eﬀective against WCR larvae (Jackson, 1996; Toepfer et al., 2008; Pilz et al., 2009) but is not attracted to EβC (Hiltpold et al., 2010a,c). Selective breeding of H. bacteriophora, however, can increase the attraction of infective juveniles to EβC-emitting maize roots, thereby increasing WCR mortality (Hiltpold et al., 2010a,b). Maximizing the expression of HIPVs via bioengineering, while increasing EPN responsiveness to volatiles, can help enhance the eﬀectiveness of biological control in crops. However, more studies are needed to assess the costs, viability and potential risks of introducing EβC-emitting maize varieties with EPN releases. The WCR has a high propensity for invasion and adaptation (Gray et al., 2009) and has already developed resistance to multiple chemical (Meinke et al., 1998; Ciosi et al., 2009; Pereira et al., 2015), genetic (Gassmann et al., 2011; Wangila et al., 2015), Whether parasitoids are adversely aﬀected by plant toxins depends in large part on the level of host plant specialization of their herbivorous hosts. The diversity of host plants on November 2016 \| Volume 7 \| Article 1794 4 Host Plant Resistance and Biocontrol Peterson et al. which a given herbivore develops depends, in part, on its ability to metabolize or avoid plant defensive toxins (Schoonhoven et al., 2005). Herbivores feeding on a broader range of host plants typically possess detoxiﬁcation enzyme systems capable of metabolizing a broad array of plant toxins (Krieger et al., 1971; Li et al., 2004; Ali and Agrawal, 2012). Conversely, herbivores with specialized diets tend to have more eﬃcient detoxiﬁcation enzymes that metabolize the narrower range of plant toxins to which they are exposed (Wittstock et al., 2004; Mao et al., 2006). Far less documentation exists regarding the consequences for parasitoids of developing in generalist vs. specialist herbivores because few studies have documented the levels of unmetabolized plant toxins in the hemolymph of herbivores with diﬀerent diet breadths. In one study, signiﬁcantly more xanthotoxin was passed unmetabolized into the hemolymph of the cabbage looper, Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae), a generalist herbivore, than was passed in the hemolymph of the parsnip specialist Depressaria pastinacella (Geeze) (Lepidoptera: Oecophoridae) (Lampert et al., 2011b). In turn, Copidosoma ﬂoridanum Ashmead (Hymenoptera: Encyrtidae) (a parasitoid of T. ni) suﬀered increased mortality and reduced clutch sizes relative to Copidosoma sosares (Walker) (Hymenoptera: Encyrtidae) (a specialist parasitoid of D. Plant Toxin-Mediated Interactions In one study, signiﬁcantly more xanthotoxin was passed unmetabolized into the hemolymph of the cabbage looper, Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae), a generalist herbivore, than was passed in the hemolymph of the parsnip specialist Depressaria pastinacella (Geeze) (Lepidoptera: Oecophoridae) (Lampert et al., 2011b). In turn, Copidosoma ﬂoridanum Ashmead (Hymenoptera: Encyrtidae) (a parasitoid of T. ni) suﬀered increased mortality and reduced clutch sizes relative to Copidosoma sosares (Walker) (Hymenoptera: Encyrtidae) (a specialist parasitoid of D. pastinacella) even though both herbivore-parasitoid combinations were reared on the same artiﬁcial diets (Lampert et al., 2011b). Other studies have documented similar patterns (e.g., Barbosa et al., 1986, 1991). Finally, generalist and specialist herbivores of cruciferous plants are negatively aﬀected by diﬀerent classes of glucosinolates. Generalist herbivores are typically susceptible to both indole and aliphatic glucosinolates, whereas specialist herbivores are susceptible to just indole glucosinolates (Gols et al., 2008a,b; Müller et al., 2010; Harvey and Gols, 2011). However, some specialists are known to sequester glucosinolates, providing protection against their natural enemies [e.g., the turnip sawﬂy Athalia rosae (Hymenoptera: Tenthredinidae) (Müller et al., 2002) and the specialist aphids Brevicoryne brassicae (L.) and Lipaphis erysimi Kaltenbach (Hemiptera: Aphididae)] (Francis et al., 2001; Rossiter et al., 2003; Kazana et al., 2007). Interestingly, survivorship and body size of unparasitized T. ni were negatively correlated with concentrations of aliphatic glucosinolates whereas survivorship and clutch sizes of T. ni parasitized by C. ﬂoridanum were negatively aﬀected by concentrations of indole (and not aliphatic) glucosinolates (Ode et al., 2016). Case Study: Cotton, Gossypol and Bt Toxins, Herbivores, and Natural Enemies Frontiers in Plant Science \| www.frontiersin.org Plant Toxin-Mediated Interactions pastinacella) even though both herbivore-parasitoid combinations were reared on the same artiﬁcial diets (Lampert et al., 2011b). Other studies have documented similar patterns (e.g., Barbosa et al., 1986, 1991). Finally, generalist and specialist herbivores of cruciferous plants are negatively aﬀected by diﬀerent classes of glucosinolates. Generalist herbivores are typically susceptible to both indole and aliphatic glucosinolates, whereas specialist herbivores are susceptible to just indole glucosinolates (Gols et al., 2008a,b; Müller et al., 2010; Harvey and Gols, 2011). However, some specialists are known to sequester glucosinolates, providing protection against their natural enemies [e.g., the turnip sawﬂy Athalia rosae (Hymenoptera: Tenthredinidae) (Müller et al., 2002) and the specialist aphids Brevicoryne brassicae (L.) and Lipaphis erysimi Kaltenbach (Hemiptera: Aphididae)] (Francis et al., 2001; Rossiter et al., 2003; Kazana et al., 2007). Interestingly, survivorship and body size of unparasitized T. ni were negatively correlated with concentrations of aliphatic glucosinolates whereas survivorship and clutch sizes of T. ni parasitized by C. ﬂoridanum were negatively aﬀected by concentrations of indole (and not aliphatic) glucosinolates (Ode et al., 2016). Despite long running discussions about the potential the patterns outlined above will hold. For instance, soybeans, Glycine max (L.) (Fabales: Fabaceae), with the Rag1 gene are resistant to soybean aphid Aphis glycines Matsumura (Hemiptera: Aphididae). Compatibility studies between Rag1 and biological control agents of A. glycines have shown that these agents are less eﬀective (e.g., reduced foraging eﬃciency and survivorship) on soybean varieties containing the resistant Rag1 gene (Lundgren et al., 2009b; Ghising et al., 2012; Ode and Crompton, 2013). which a given herbivore develops depends, in part, on its ability to metabolize or avoid plant defensive toxins (Schoonhoven et al., 2005). Herbivores feeding on a broader range of host plants typically possess detoxiﬁcation enzyme systems capable of metabolizing a broad array of plant toxins (Krieger et al., 1971; Li et al., 2004; Ali and Agrawal, 2012). Conversely, herbivores with specialized diets tend to have more eﬃcient detoxiﬁcation enzymes that metabolize the narrower range of plant toxins to which they are exposed (Wittstock et al., 2004; Mao et al., 2006). Far less documentation exists regarding the consequences for parasitoids of developing in generalist vs. specialist herbivores because few studies have documented the levels of unmetabolized plant toxins in the hemolymph of herbivores with diﬀerent diet breadths. November 2016 \| Volume 7 \| Article 1794 Case Study: Cotton, Gossypol and Bt Toxins, Herbivores, and Natural Enemies However, Bt has not been without its downsides as damage by some pests, for example, mirid bugs (Lu et al., 2010), have been Despite long-running discussions about the potential (in)compatibilities of biological control and breeding programs for plant resistance (e.g., Bergman and Tingey, 1979; van Emden, 1991; Bottrell et al., 1998; Cortesero et al., 2000; Poppy and Sutherland, 2004), surprisingly little is known about the severity of these incompatibilities. This is primarily a reﬂection of the independent paths that host plant resistance and biological control programs have taken; i.e., IPM is rarely practiced in reality. Part of the diﬃculty lies in the fact that when crop varieties are bred for insect resistance, rarely do we know the exact mechanism involved. Nonetheless, breeding programs likely select for plant defensive toxins in many cases, which likely mediate resistance. When true, we expect that many of November 2016 \| Volume 7 \| Article 1794 5 Host Plant Resistance and Biocontrol Peterson et al. documented to increase with the widespread use of Bt cotton, presumably because of competitive release from lepidopterans. Another complication involves improved success of the cotton aphid Aphis gossypii Glover (Hemiptera: Aphididae) on Bt cotton. Suppression of feeding by lepidopteran herbivores on Bt cotton reduces induction of key defensive terpenoids, such as gossypol, making these plants much more susceptible to aphids, which do not induce terpenoids (Hagenbucher et al., 2013b). Furthermore, induced terpenoids from non-Bt cotton end up in the hemolymph of the aphids, reducing success of attack by the parasitoid Lysiphlebus testaceipes (Cression) (Hymenoptera: Braconidae) (Hagenbucher et al., 2014b). Reduced parasitism was most likely due to reduced parasitoid acceptance of aphids feeding on lepidopteran-infested non-Bt cotton. Finally, as honeydew is an important source of nutrition for foraging parasitoids, the eﬀect of honeydew from lepidopteran-infested Bt and non-Bt cotton on two important parasitoids of cotton pests, L. testaceipes and the whiteﬂy parasitoid Eretmocerus eremicus Rose and Zolnerowich (Hymenoptera: Aphelinidae) was compared. While gossypol and other terpenoids were signiﬁcantly higher in the honeydew produced on lepidopteran- infested non-Bt cotton, this did not aﬀect the quality of the honeydew in terms of its eﬀects on parasitoid longevity or fecundity (Hagenbucher et al., 2014a). more susceptible to infection with nuclear polyhedrosis virus (NPV) (Hamm and Wiseman, 1986). However, the slow-growth– high-mortality hypothesis does not hold true for all tritrophic interactions. Plant Nutrient-Mediated Interactions The proteins, sugars, lipids, nucleic acids, vitamins, and minerals contained within plant tissue provide the nutrition necessary for growth, development, and survival of many insects. In turn, the nutrients provided by plants to herbivores aﬀect the nutrients subsequently available to their natural enemies. The presence, quantity, quality, and availability of these nutrients varies signiﬁcantly between plant species and varieties, and can be aﬀected by season, plant phenology, and other biotic and abiotic conditions (Fox et al., 1990; Roth and Lindroth, 1995; Walde, 1995; Stadler and Mackauer, 1996). g g Some insects are truly omnivorous, having a ﬂexible trophic strategy that allows them to utilize either plant or prey resources, with the potential to inﬂict crop damage if engaging in phytophagy. For example, the western ﬂower thrips Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae) feeds on plant material and arthropod prey, leading to its role as both a serious pest (Grazia-Tommasini, 1995; Kirk and Terry, 2003) and a biological control agent (Trichilo and Leigh, 1986; Wilson et al., 1996; Agrawal and Karban, 1997; Milne and Walter, 1997). Furthermore, Agrawal et al. (1999) revealed that the presence of prey [eggs of the Paciﬁc spider mite Tetranychus paciﬁcus McGregor (Thysanoptera: Tetranychidae)] reduced feeding by F. occidentalis on cotton by nearly 50%. However, when cotton plants were ﬁrst exposed to feeding pressure by spider mites, eliciting systemically induced plant defenses that lower host plant quality, herbivory by F. occidentalis was reduced (Agrawal et al., 1999). When both induced host plant defenses and T. paciﬁcus egg prey were available, feeding preference shifted to consume half the amount of cotton tissue and twice the number of prey (Agrawal et al., 1999). Thus, host plant quality and prey availability are important factors for arthropods with omnivorous trophic tendencies. A key indirect interaction between host plant nutrition and natural enemies occurs when herbivore growth and development is delayed by suboptimal plant quality, extending the period of time when herbivores are vulnerable to attack (Moran and Hamilton, 1980; Price et al., 1980; Price, 1986; Loader and Damman, 1991; reviewed in Benrey and Denno, 1997). An example of this “slow-growth–high-mortality” hypothesis was reported for the Mexican bean beetle Epilachna varivestis Mulsant (Coleoptera: Coccinellidae) feeding on soybean. The spined soldier bug, Podisus maculiventris (Say) (Hemiptera: Pentatomidae), was better able to control E. Case Study: Cotton, Gossypol and Bt Toxins, Herbivores, and Natural Enemies For example, Leather and Walsh (1993) found that pine beauty moth Panolis ﬂammea Denis and Schiﬀermüller (Lepidoptera: Noctuidae) larvae were not more vulnerable to natural enemies when development was delayed by host plant quality. Some natural enemies, such as parasitoids, may actually be at a disadvantage when their hosts are smaller and/or of lower quality, and smaller hosts may also aﬀect the sex ratio and fecundity of parasitoid populations (Kuo, 1986). It is therefore important to examine whether the presence of smaller and lower quality hosts due to suboptimal plant nutrition has a large enough impact on parasitoids as to aﬀect their ability to suppress pest populations. Many natural enemies also engage in omnivory, supplementing their prey-based diet with plant-provided resources (reviewed in Lundgren, 2009), particularly during periods when prey abundance is low. This can allow for more stable interactions between predators and prey (Agrawal, 2000a) and may facilitate early season colonization of crop ﬁelds and better pest suppression due to this “lying in wait” of natural enemies prior to arrival of the pest species (Settle et al., 1996; Eubanks and Denno, 1999; Athey et al., 2016). Therefore, good quality plant hosts in the case of omnivorous natural enemies is essential for a positive relationship between plant and biocontrol. Plants expressing herbivore defense traits can have direct impacts on facultatively phytophagous predators but the literature is lacking in how these interactions will impact the compatibility of host plant resistance with biological control (Lundgren, 2009). Frontiers in Plant Science \| www.frontiersin.org Plant Nutrient-Mediated Interactions varivestis on crop varieties that lowered the herbivore’s growth rate (Price et al., 1980), although the exact resistance mechanism was not known. In addition to a longer period of vulnerability, a slow herbivore growth rate can be advantageous if the natural enemy’s functional response is stronger when consuming smaller prey, as tends to be the case with predators (Price, 1986). Insect pathogens, in particular, are positively associated with the slow-growth– high-mortality hypothesis (Schuster et al., 1983; Hamm and Wiseman, 1986). In one case, S. frugiperda feeding on resistant maize plants had reduced growth and vigor, making them Extraﬂoral nectaries (EFN) are a plant-provided resource that deserve additional attention because of their role in natural enemy nutrition. It is hypothesized that the main function of extraﬂoral nectar is to recruit predators and parasitoids for the protection of the plant against herbivores, an example of November 2016 \| Volume 7 \| Article 1794 Frontiers in Plant Science \| www.frontiersin.org 6 Host Plant Resistance and Biocontrol Peterson et al. indirect host plant resistance (Bentley, 1977; Koptur, 1992; Turlings and Wäckers, 2004). Some EFN emit olfactory signals that are attractive to natural enemies, such as parasitoids (Lewis and Takasu, 1990; Stapel et al., 1997). By providing nutritional resources, the presence of EFN can lead to enhanced herbivore suppression by arthropod natural enemies, such as ants (Bentley, 1977; Smiley, 1986), spiders (Ruhren and Handel, 1999), predatory mites (Bakker and Klein, 1992), coccinellids (Stephenson, 1982) and parasitoids (Lindgren and Lukefahr, 1977). Interestingly, some plants produce a consistent low level of EFN, but increase production in response to herbivory; in this manner, extraﬂoral nectaries can be considered both constitutive and inducible indirect host plant resistance (Wäckers et al., 2001; Wäckers and Bonifay, 2004; Lundgren, 2009; Heil, 2015). The applied implications of EFN production by crop plants is examined in the case study with cotton below. survival are evident when provided EFN in the diet (Taylor and Pfannenstiel, 2009; Pfannenstiel and Patt, 2012). The majority of modern cotton varieties now produce EFN, but past breeding eﬀorts illustrate the diﬃculty in managing plant traits aﬀecting both pests and natural enemies. Rogers (1985) recommended that for the case of nectar-producing cotton, varieties should be developed that produce nectar that is palatable to beneﬁcial species, but not pests. However, the feasibility of this suggestion has not been explored. Plant Nutrient-Mediated Interactions Recommendations to improve the recruitment of natural enemies to cotton ﬁelds include selecting for varieties with enhanced nectar production. For example, most cotton leaves bear a single nectary, but some have three (Cortesero et al., 2000) and a breeding challenge is whether cotton varieties can be developed with a greater number of nectaries. It is evident that plant nutrients are critically important to a diverse array of natural enemies across multiple functional groups. Integration of this resource into biological control programs through selective enhancement or provisioning of additional nectar sources can assist when developing sustainable solutions to pest management. Clearly, challenges exist when selectively breeding for plant defense traits (described here and in other sections), but careful consideration of their integration with biological control can provide synergistic levels of pest control. Case Study: Extraﬂoral Nectar-Producing Cotton, Its Herbivores, and Natural Enemies , The ability of extraﬂoral nectar to attract natural enemies for biological control of cotton pests has long been exploited. Cook (1904, 1905) reported on the practice of indigenous farmers in Guatemala, who purposely cultivated cotton near nests of the tropical ant Ectatomma tuberculatum (Olivier) (Hymenoptera: Formicidae). In addition to feeding on EFN, these ants attacked boll weevil Anthonomus grandis Boheman (Coleoptera: Curculionidae) adults. Subsequently, plant breeding eﬀorts in the mid 1900’s attempted to develop cotton varieties that lacked EFN, due to the observation that both natural enemies and some lepidopteran pests, such as P. gossypiella, beneﬁtted from cotton nectaries (Lukefahr and Griﬃn, 1956; Lukefahr and Rhyne, 1960; Bentley, 1983). However, the beneﬁt of a modest reduction in lepidopteran pests was outweighed by the disadvantage of reduced natural enemy populations, although this conclusion was doubted at the time (Rogers, 1985; Schuster and Calderon, 1986). The population of natural enemies in “nectarless” cotton varieties was up to 35% lower than EFN-producing cotton and the presence of EFN in cotton had positive impacts on the attraction, retention, and eﬃciency of many predators, including chrysopids, anthocorids, and coccinellids (Schuster et al., 1976). Similarly, the parasitoid Microplitis croceipes (Cresson) (Hymenoptera: Braconidae), which attacks larvae of the bollworm H. zea, is stimulated to stay longer and attack a greater number of hosts in the presence of nectar (Stapel et al., 1997). Many other examples exist in the literature, providing clear evidence for widespread beneﬁts of EFN to parasitoids (e.g., Treacy et al., 1987). Another functional group of natural enemies, cursorial wandering spiders such as Cheiracanthium inclusum (Hentz) (Araneae: Miturgidae) and Hibana futilis (Banks) (Araneae: Anyphaenidae), are important nocturnal predators of lepidopterous pest eggs in cotton (Pfannenstiel, 2008) and consume EFN in the ﬁeld (Taylor and Pfannenstiel, 2008). Furthermore, Hibana futilis responds to olfactory cues from extraﬂoral nectar and engages in restricted area searching following contact with nectar (Patt and Pfannenstiel, 2008, 2009) and profound improvements of Frontiers in Plant Science \| www.frontiersin.org Case Study: Plant Epicuticular Waxes, the Diamondback Moth, and Its Predators Plant epicuticular waxes primarily serve to control water, gas and solute exchange (Riederer and Müller, 2006). In addition, these waxes mediate other ecological functions including host plant resistance against pathogens (Reina-Pinto and Yephremov, 2009) and herbivores (Eigenbrode et al., 1991b; Müller, 2008). The interactions between B. oleracea (cabbage, broccoli, cauliﬂower, kale, and others), Plutella xylostella (L.) (Lepidoptera: Plutellidae), and its predators highlight the interface between plant waxes and herbivore resistance. Gene mutations yield B. oleracea cultivars with altered chemical structures and diﬀerent crystallization patterns of epicuticular lipids (Macey and Barber, 1970; Netting et al., 1972; Baker, 1974). As a consequence, mutants usually have decreased epicuticular waxes and produce a “glossy” phenotype instead of their normal wax “glaucous” phenotype (Eigenbrode and Espelie, 1995). Although information is limited (Verkerk and Wright, 1996), evidence suggests that glossy plants exhibit resistance against neonate P. xylostella larvae (Lin et al., 1983; Eigenbrode and Shelton, 1990; Eigenbrode et al., 1991a) and that physical and chemical diﬀerences inﬂuence neonate behavior (Eigenbrode et al., 1991b). Neonates on glossy varieties disperse further and faster, spending less time palpating, biting, mining, and spinning silk (Eigenbrode and Shelton, 1990; Eigenbrode et al., 1991a). This non-preference behavior causes a lack of establishment, reduced feeding and increased larval mortality (Eigenbrode and Shelton, 1990; Eigenbrode et al., 1991a). The plant surface is a complex microenvironment playing a critical role in insect–plant interactions, impacting insect behavior (such as attraction, retention, and host choice), feeding (such as attachment and accessibility of nutrients), and dispersal (by impeding insect movement) (Chapman, 1977; Southwood, 1986). Leaf surface structures that defend the plant from herbivores, such as leaf toughness, cuticle thickness, epicuticular waxes, trichomes and spines, can have direct and indirect eﬀects on natural enemies. An indirect eﬀect can occur if physical defense traits, such as leaf toughness, delay the development of herbivores. The extended period of vulnerability to natural enemies can thereby enhance biological control (slow-growth–high-mortality hypothesis, see Plant Nutrient- Mediated Interactions). A common example of direct eﬀects is when trichomes are physically disruptive to natural enemy movement. In general, trichomes have more harmful than beneﬁcial eﬀects on predators, although most of these eﬀects are sublethal (Riddick and Simmons, 2014a,b). Case Study: Plant Epicuticular Waxes, the Diamondback Moth, and Its Predators The functional response or attack rate of predators and parasitoids is typically lower when their prey or hosts are found on plants with greater trichome density (e.g., Krips et al., 1999; Kumar et al., 1999; De Clercq et al., 2000; Stavrinides and Skirvin, 2003; Madadi et al., 2007; Jalalizand et al., 2012), although the opposite has been found as well (Koveos and Broufas, 2000). These interactions have signiﬁcant implications for pest management; for example, biological control is possible on glabrous cucumber varieties, but is seriously hindered on those with dense trichomes due to the reduction in searching eﬃciency by the parasitoid Encarsia formosa Gahan (Hymenoptera: Aphelinidae) attacking greenhouse whiteﬂies Trialeurodes vaporariorum Westwood (Hemiptera: Aleyrodidae) (Hulspas-Jordaan and van Lenteren, 1978). Clusters of trichomes on the underside of plant leaves can form domatia, commonly used by predatory arthropods for shelter (O’Dowd and Willson, 1991; Walter, 1996; Agrawal and Karban, 1997); the positive impact of domatia on biological control has been well-documented for predatory phytoseiid mites (reviewed in Schmidt, 2014). In general, arthropods need to be either quite large (Rabb and Bradley, 1968; Obrycki and Tauber, 1984) or very small (Krips et al., 1999) to move along a leaf surface unimpeded by physical plant defense structures. The eﬀect of trichome density on natural enemy movement can be a function of the relationship between natural enemy size and trichome spacing (Buitenhuis et al., 2014). This myriad of physical plant traits clearly has an important g Host plant resistance conferred by the glossy phenotype is also enhanced by predators. Field studies revealed that green lacewing Chrysoperla carnea (Stephens) (Neuroptera: Chrysopidae), insidious ﬂower bug Orius insidiosus (Say) (Hemiptera: Anthocoridae), and convergent lady beetle Hippodamia convergens Guérin-Méneville (Coleoptera: Coccinellidae), all generalist predators, signiﬁcantly increased P. xylostella larval mortality in glossy, but not normal wax, varieties (Eigenbrode et al., 1995). The reduction in mining behavior renders the larvae more exposed to predators (Eigenbrode et al., 1995). Predators also walked faster, spent more time walking, and covered more leaf area on glossy leaves compared to normal wax varieties (Eigenbrode et al., 1996). Increased mobility was attributed to increased traction/adhesion of predators on glossy vs. normal wax plants. The crystallization and composition of natural waxes have an impact on how natural enemies, such as H. Physically Mediated Interactions Just as some tritrophic interactions involve both semiochemicals and toxins, physically mediated interactions do not always function alone. For example, substances such as resin or latex physically limit herbivores by trapping or immobilizing them, while simultaneously delivering various toxins (Konno, 2011), and glandular trichomes release sticky and toxic compounds serving as a physical and chemical defense against herbivores (Levin, 1973; Southwood, 1986; Cortesero et al., 2000). Plant architecture aﬀects the dispersion of herbivores on a host plant, which may in turn aﬀect searching behavior and host- ﬁnding abilities of natural enemies. For example, the leaves of winter wheat varieties developed for resistance to Russian wheat aphid Diuraphis noxia (Kurdjumov) (Hemiptera: Aphididae) remain ﬂat, compared to susceptible varieties whose leaves furl in response to aphid feeding (Hawley et al., 2003), exposing aphids to disturbances such as wind, rain, and predators inducing them to fall from the plant (von Berg et al., 2008). Characteristics that aﬀect falling behavior of herbivores can aﬀect predation rates as they experience vulnerability to ground-dwelling predators and may also face additional challenges from natural enemies as they attempt to recolonize the plant (Sunderland et al., 1986; Winder, 1990; Winder et al., 1994). The size and morphology of certain plant structures that confer resistance to herbivores can aﬀect biological control by altering where pests feed, how long they are exposed and how apparent or accessible the pests are to natural enemies, particularly if plant morphology can delay internally feeding pests from entering the plant’s tissues. An example would be husk tightness and length in sweet corn plants conferring resistance to H. zea larvae attempting to enter the ear and feed on developing kernels (Cameron and Anderson, 1966; Wiseman and Davis, 1990). Plant structures may also act to hide the herbivore from its November 2016 \| Volume 7 \| Article 1794 Frontiers in Plant Science \| www.frontiersin.org 7 Host Plant Resistance and Biocontrol Peterson et al. natural enemies. For example, open-leaf brassica varieties, such as Brussels sprouts, have higher parasitism on P. rapae compared to heading varieties, such as cabbage, due to larvae being able to feed in leaf folds protected from parasitoids (Pimentel, 1961). Physically Mediated Interactions Furthermore, the size of plant structures impacts the ability of parasitoids to oviposit in pests, particularly if larger fruits allow pests to feed deeper than the parasitoid’s ovipositor can reach, creating “enemy-free space” and potentially facilitating host switching by pests (Bush, 1974; Price et al., 1980; Jeﬀries and Lawton, 1984; Bernays and Graham, 1988). also aﬀecting (positively and negatively) the ability of natural enemies to attack pest species. This trade-oﬀis evident in many examples of physically mediated interactions. In addition to trichomes, another plant surface characteristic that can impact natural enemies is the presence and composition of epicuticular waxes, which will be discussed in the following section. Frontiers in Plant Science \| www.frontiersin.org Mechanisms of Plant Trait-Mediated Interactions: Summary y Plant traits have a profound (and often complex) array of impacts on herbivores and natural enemies. The examples cited within each section above for semiochemically, plant toxin-, plant nutrient-, and physically mediated interactions show the diversity and gradient of interactions occurring between natural enemies and HPR and how these can interact synergistically or antagonistically to suppress the target pest. For instance, semiochemically mediated traits serve as indirect plant defenses by impacting signaling pathways and attraction/repellency between the members of tritrophic interactions. Conversely, plant toxins act as direct defense against herbivores and this in turn can alter host suitability for natural enemies. Insect host/prey vulnerability via the slow-growth–high-mortality hypothesis can be mediated by plant nutrition. Plant-provided nutritional resources can also be linked to the success of natural enemies due to omnivory by predators and/or parasitoids. Moreover, physically mediated traits are known to function together with other traits to deter herbivory, but physical plant defenses are also responsible for increasing or decreasing herbivores’ vulnerability to natural enemies and trichomes can have direct negative impacts on biological control by decreasing natural enemy search eﬃciency. Manipulation of plant traits through plant breeding or bioengineering, as well as knowledge of the ecology and biology of herbivores and natural enemies, can work together to aid crop protection. In the last two decades, another control tactic, Bt, has become a staple of the agricultural landscape throughout much of the world (although notably less so in Europe). This technology will be discussed below given its importance in pest control programs throughout the world. Many natural enemies consume plant-provided non-prey foods (see Plant Nutrient-Mediated Interactions) and when these plant-provided resources are GM crops, they are likely to contain Bt toxins. The expression of transgenic proteins is inﬂuenced by many biotic and abiotic factors, including environment, geography, crop phenology and genetics, and the speciﬁc transgenic event and protein expressed (Fearing et al., 1997; Duan et al., 2002; Grossi-de-Sa et al., 2006; Obrist et al., 2006a; Lundgren et al., 2009a). Most Bt crops employ a constitutive promoter that expresses Bt proteins throughout the life of the plant in nearly all tissues. Natural enemies that engage in facultative phytophagy of these plants are therefore likely to be exposed to the Bt toxins. Mechanisms of Plant Trait-Mediated Interactions: Summary Despite this exposure, laboratory feeding assays and ﬁeld studies do not report negative impacts (Pilcher et al., 1997; Armer et al., 2000; Lundgren and Wiedenmann, 2002; Geng et al., 2006; Ludy and Lang, 2006; Obrist et al., 2006b; Torres et al., 2006; Li et al., 2008), most likely due to the high speciﬁcity of Bt proteins against target pests and the lack of necessary physiological conditions in non-target arthropods. It is therefore unlikely this pathway has a signiﬁcant impact on biological control in transgenic crops. Case Study: Plant Epicuticular Waxes, the Diamondback Moth, and Its Predators convergens and Chrysoperla plorabunda (Fitch) (Neuroptera: Chrysopidae) attach to the leaf surface, thereby aﬀecting their ability to exert biological control (Eigenbrode et al., 1999; Eigenbrode and Jetter, 2002). This myriad of physical plant traits clearly has an important eﬀect on the feeding eﬃciency of herbivores. However, integration of plant physical traits with biological control is a complex issue with characteristics hindering herbivore damage In summary, this system has multiple pest suppression factors working together. Plutella xylostella neonates are less likely November 2016 \| Volume 7 \| Article 1794 8 Host Plant Resistance and Biocontrol Peterson et al. lysis of epithelial cells on the gut wall, perforations in the midgut lining, cessation of feeding, and death by septicemia. Bt toxins target a narrow spectrum of pest insects that possess speciﬁc physiological traits (i.e., gut pH and toxin receptor sites in the midgut), and thus pose less direct toxicity risk to non-target species than broad-spectrum insecticides (Marvier et al., 2007; Wolfenbarger et al., 2008; Naranjo, 2009; Duan et al., 2010; Peterson et al., 2011). Commercialized Bt crops include maize, cotton, and soybeans that are protected against a suite of coleopteran and lepidopteran pests. The planting of Bt crops has increased dramatically since their introduction in the mid-1990’s; for example, in the United States, the percentage of Bt maize was only 1% of the total crop grown in 1996 but 81% of all maize grown in 2015 (United States Department of Agriculture National Agricultural Statistics Service, 2015). The ecological interactions between insect-resistant GM crops and biological control are complex and have been addressed in numerous comprehensive reviews (e.g., Obrycki et al., 2004; Lundgren et al., 2009a; Hilbeck and Otto, 2015). Two major categories for how GM crops inﬂuence biological control, proposed by Lundgren et al. (2009a), are discussed below: (1) toxicity-based pathways, including natural enemy consumption of toxic plant or prey foods; and (2) crop-induced changes to the environment, including unintended alterations to the crop plant and a decrease in prey quality and/or density that alter functional and numerical responses as well as the community ecology of natural enemies. to accept glossy varieties, which increases their mortality and vulnerability to predation (via decreased mining behavior). Predators on glossy varieties have a greater ability to walk and hence, locate and attack prey, due to increased adhesion to the surface of leaves. Altogether, host plant resistance for P. Case Study: Plant Epicuticular Waxes, the Diamondback Moth, and Its Predators xylostella in glossy varieties increases biological control by natural enemies, and hence overall suppression of this key pest of Brassica plants. November 2016 \| Volume 7 \| Article 1794 Frontiers in Plant Science \| www.frontiersin.org GENETICALLY MODIFIED CROPS AND INTERACTIONS WITH BIOLOGICAL CONTROL In addition to development time, natural enemy size can be reduced if feeding on lower quality prey or hosts; smaller size in insects can result in reduced fecundity and dispersal capacity (Honˇek, 1993; Kazmer and Luck, 1995), further delaying natural enemy population growth (Lundgren et al., 2009a). The majority of interactions discussed above operate at the scale of a single crop ﬁeld or smaller. However, some eﬀects of the proliferation of GM crops are observed at the landscape or community scale. For example, Bt maize has been associated with area wide suppression of ECB in the midwestern United States (Hutchison et al., 2010). Despite reduced ECB populations that confer economic beneﬁts to growers planting non-Bt maize, management of this pest is still critical for seed corn, popcorn, and other crops not protected by Bt toxins. Therefore, suppression of ECB due to biological control by natural enemies such as the specialist parasitoid Macrocentrus grandii (Goidanich) (Hymenoptera: Braconidae) and the entomopathogenic microsporidian Nosema pyrausta (Paillot) (Microsporidia: Nosematidae) is a valuable service. Despite the large reduction in ECB populations, infection dynamics of N. pyrausta have not signiﬁcantly changed (Lewis et al., 2009), although parasitism rates by M. grandii were lowest when ECB hosts were found in small aggregations (White and Andow, 2005). Therefore, the area wide suppression of Bt- targeted prey or hosts does not always aﬀect the interactions of pests with their natural enemies. The most likely action by which GM crops could inﬂuence natural enemy ﬁtness and fecundity is through a reduction in prey quality and/or prey density. Numerous studies have shown that consumption of Bt-containing plant tissue negatively aﬀects the growth and development of herbivorous species, thereby impacting their natural enemies (e.g., Lövei and Arpaia, 2005; Hilbeck and Schmidt, 2006; Romeis et al., 2006; Lawo et al., 2010; Garcia et al., 2012; Tian et al., 2014; Han et al., 2015). For example, Hilbeck et al. (1998a) reported that the generalist predator C. carnea experienced reduced larval survival and longer development time when fed a diet of European corn borer (ECB), Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae), that had consumed Bt corn. However, generalist predators are capable of preferential feeding on healthy prey (Ferry et al., 2006) and are able to shift their dietary preferences to consume the mixture of nutrients required for optimal ﬁtness (Mayntz et al., 2005; Raubenheimer et al., 2007; Marques et al., 2015). GENETICALLY MODIFIED CROPS AND INTERACTIONS WITH BIOLOGICAL CONTROL Therefore, generalist predators may be able to compensate for reduced quality of select prey due to Bt toxin consumption, having a negligible impact on biological control. For entomopathogens, species that are specialists of Bt-targeted pests are likely to see population reductions, whereas generalists will continue to persist in Bt crop ﬁelds (Obrycki et al., 2004). Parasitoids often do not have the ﬂexibility to select hosts unaﬀected by Bt toxins and are therefore more likely to be adversely aﬀected (Bernal et al., 2004; Marvier et al., 2007; Wolfenbarger et al., 2008; Bernal, 2010). Specialist parasitoid populations are reduced due to a lack of suitable hosts and may also suﬀer direct mortality if they are developing inside of a host that suﬀers mortality due to ingestion of Bt toxins (Agrawal, 2000a). For hosts that are only partially susceptible to Bt toxins, reduced host quality can result in sublethal eﬀects on parasitoids (e.g., Bernal et al., 2002; Baur and Boethel, 2003; Vojtech et al., 2005; Ramirez- Romero et al., 2007; Walker et al., 2007), but host-mediated impacts of Bt crops on parasitoids are not universal and vary depending on the plant, host, and parasitoid. For example, the soybean looper Chrysodeixis includens (Walker) (Lepidoptera: Noctuidae) is moderately susceptible to the Bt toxins expressed in transgenic cotton and exhibits slower development time and lower prepupal weight (Baur and Boethel, 2003). Parasitism by Cotesia marginiventris (Cresson) (Hymenoptera: Braconidae) on these hosts results in longer larval development time, reduced adult longevity, and reduced egg production. However, when In addition to transgenic Bt crops, other herbicide-resistant and insecticidal GM crops are commercially available or under review by governmental agencies. The adoption of herbicide- tolerant crops that confer resistance to herbicides such as glyphosate, glufosinate, and 2,4-Dichlorophenoxyacetic acid (2,4- D) has been rapid. In the United States, 89% of corn and upland cotton and 94% of soybeans planted in 2015 had GM herbicide- tolerance traits (United States Department of Agriculture National Agricultural Statistics Service, 2015). Furthermore, herbicide-tolerant canola, alfalfa, and sugar beets are currently being grown in the United States, albeit in reduced frequency. This adoption has led to changes in the agricultural landscape, including reduced within-ﬁeld plant diversity (Heard et al., 2005; Culpepper, 2006; Pleasants and Oberhauser, 2013), potentially aﬀecting natural enemies and conservation biological control. The potential consequence of GM herbicide-tolerant crops on biological control is addressed in detail by Lundgren et al. (2009a). GENETICALLY MODIFIED CROPS AND INTERACTIONS WITH BIOLOGICAL CONTROL Transgenic genetically modiﬁed (GM) crops have been engineered to incorporate genes derived from another species that confer nutritional or agronomic beneﬁts, such as resistance to insect pests, viruses, herbicides, or protection from environmental conditions (e.g., low water availability). Among insect-resistant GM crops, Bacillus thuringiensis (Bt) crops are the most common and express insecticidal proteins derived from a naturally occurring soil bacterium. The insecticidal mode of action occurs when Bt toxins bind to receptors on the midgut lining of susceptible insects, causing Natural enemies may be exposed to Bt toxins by consuming or parasitizing prey/hosts that have fed on GM crops, a pathway similar to plant toxin-mediated interactions (see Plant Toxin- Mediated Interactions). One factor mitigating the exposure of natural enemies is that for crop pests that are highly susceptible to Bt toxins, ingestion of a very small amount of toxin elicits lethal eﬀects. Exposure to natural enemies can be greater if the herbivore consuming a GM crop plant is only partially susceptible to the toxin and therefore consumes a greater quantity of plant tissue. Many herbivores do contain transgenic toxins November 2016 \| Volume 7 \| Article 1794 Frontiers in Plant Science \| www.frontiersin.org 9 Host Plant Resistance and Biocontrol Peterson et al. (e.g., Harwood et al., 2005; Meissle et al., 2005; Obrist et al., 2005, 2006b; Peterson et al., 2016), but accumulation in higher trophic levels is uncommon (Dutton et al., 2002; Obrist et al., 2006a; Paula and Andow, 2016). While tritrophic transfer of Bt proteins has been documented, it is at low levels (e.g., Harwood et al., 2005, 2007; Meissle et al., 2005; Zwahlen and Andow, 2005; Obrist et al., 2006a; Wei et al., 2008; Chen et al., 2009; Meissle and Romeis, 2009; Peterson et al., 2009, 2016; Tian et al., 2010; Han et al., 2015). Early studies reported that some predators had negative sub-lethal eﬀects from exposure to Bt-containing prey (Hilbeck et al., 1998a,b; Ponsard et al., 2002) but it was subsequently revealed that this was the result of reduced prey quality rather than direct exposure to Bt toxins (Romeis et al., 2004; Torres and Ruberson, 2006). C. ﬂoridanum parasitizes loopers that have fed on Bt cotton, wasp pupal development time and adult longevity are unaﬀected, but fewer adults are produced per host (Baur and Boethel, 2003), revealing the diﬀerence in eﬀects between species. Frontiers in Plant Science \| www.frontiersin.org GENETICALLY MODIFIED CROPS AND INTERACTIONS WITH BIOLOGICAL CONTROL Transgenic insecticidal traits other than Bt have been studied; for example, potatoes, rice, maize, sugarcane, wheat, and other crops have been engineered to express snowdrop lectin GNA, a protein produced by the common snowdrop plant Galanthus nivalis (Asparagales: Amaryllidaceae) that expresses anti-hemipteran properties (Gatehouse et al., 1996; Sudhakar et al., 1998; Wang et al., 2005; Zhangsun et al., 2007; Duan et al., 2015). However, negative impacts of snowdrop lectin on natural enemies have been reported (Birch et al., 1999; Sétamou et al., 2002a,b,c; Horgervorst et al., 2006; Li and Romeis, 2009). The November 2016 \| Volume 7 \| Article 1794 Frontiers in Plant Science \| www.frontiersin.org 10 Host Plant Resistance and Biocontrol Peterson et al. next generation of transgenic insecticidal crops in the commercial pipeline utilizes RNA interference (RNAi), where small double stranded RNA molecules expressed in the plant selectively silence targeted genes in herbivores that feed on the plant (Siomi and Siomi, 2009). For the western corn rootworm (WCR), silencing the DvSnf7 gene using genetically modiﬁed RNAi maize induces mortality of this pest (Baum et al., 2007; Bolognesi et al., 2012) but the interactions between RNAi crops and biological control are not fully understood. While the reported spectrum of insecticidal activity of DvSnf7 RNAi is limited to a subset of species related to the WCR (Bachman et al., 2013), further risk-assessment is clearly required. The potential hazards of GM RNAi crops to natural enemies include oﬀ-target gene silencing, silencing of the targeted gene in non-target organisms, immune stimulation, and saturation of the RNAi machinery; however, these interactions may be highly complex and diﬃcult to predict (see reviews by Lundgren and Duan, 2013; Casacuberta et al., 2015; Roberts et al., 2015). Consequently, understanding the potential eﬀect that GM crops have on natural enemy-pest dynamics will allow for better integration of this technology with biological control services. Genetically engineered biotech crops undoubtedly aﬀord signiﬁcant levels of pest suppression; research on the compatibility of this approach with biological control is critical to address the long-term integration of both approaches. attractive to biological control agents are clearly compatible with biological control approaches. These interactions have been discussed in the Semiochemically Mediated Interactions and Case study: Maize Volatiles, Western Corn Rootworm, and Entomopathogenic Nematodes sections. Too often, however, little is known about the mechanisms underlying plant resistance to herbivory. In turn, parasitoids can reduce herbivore pressure allowing for increased plant yields. GENETICALLY MODIFIED CROPS AND INTERACTIONS WITH BIOLOGICAL CONTROL Parasitoids, especially solitary species, can reduce damage done by herbivores, resulting in direct yield beneﬁts to the plant; even gregarious parasitoids, which often induce increased feeding by individual herbivores, can reduce long-term population sizes of herbivores. Indeed, the widespread success of many insect biological control programs speaks to the ability of parasitoids (and predators) to have positive eﬀects on plant production and yield. An underappreciated facet of this interaction between parasitoids and plant ﬁtness/yield is the potential for parasitoids to reduce the likelihood of evolution of herbivore resistance to plant resistance traits. This is discussed further in section “Biological Control Can Reduce the Likelihood of Resistance Evolution.” Considerations for the Use of Volatiles to Recruit Biological Control Agents Most studies involving HIPVs are undertaken in laboratory and greenhouse settings, with fewer studies conducted on the eﬃcacy of HIPVs as host–plant resistance mechanisms in cropping systems at the ﬁeld scale (Orre et al., 2010; Simpson et al., 2011a,b). Our understanding of arthropod responses to chemical compounds is still evolving, but eﬀorts in developing HIPV strategies for crops are already in place via baiting/lures (Kaplan, 2012) or via bioengineering (Degenhardt et al., 2003, 2009). However, eﬀorts to increase natural enemy eﬃcacy by increasing plant attractiveness via HIPVs cannot ignore potential side eﬀects. Extensive reviews of the challenges and the future of HIPV use in pest management have been published (Dicke, 2009, 2015; Alba et al., 2012; Kaplan, 2012; Heil, 2014) and there are many unknown factors and risks associated with the use of HIPV-based pest management tactics. Cropping systems are often considered low-diversity environments because of monocultural practices but in reality there are a multitude of organisms in any given ﬁeld emitting and receiving chemical cues. We know that HIPVs targeted to attract natural enemies also attract herbivores, plant parasites, and members of the fourth trophic level. Releasing HIPV technology without examining the ecological factors present may render the technology ineﬀective. Several studies have shown that application of synthetic elicitors such as methyl jasmonate (MeJA) to induce elevated plant volatile production can also attract herbivores (Ballhorn et al., 2013) as well as hyperparasitoids (Kaplan, 2012; Heil, 2014), both outcomes that would be counterproductive to the potential for increased rates of parasitism by primary parasitoids. Additional spatio-temporal considerations must be understood to apply this technology in a large ﬁeld setting. Moreover, it is unclear how the intentional use of HIPV technology impacts the net-eﬃciency of the HIPV-emitting crop. For example, the use of synthetic green Frontiers in Plant Science \| www.frontiersin.org November 2016 \| Volume 7 \| Article 1794 Biological Control Can Reduce the Likelihood of Resistance Evolution Pesticide resistance is listed as the third most serious threat to global agriculture (behind soil erosion and water pollution) (Pimentel, 2005). Resistance is a pest population’s decreased response to a pesticide or control agent (including plant defense traits) as a result of previous exposure (McKenzie, 1996) and over 540 arthropod species have developed resistance to at least one pesticide (Arthropod Pesticide Resistance Database, 2016). The evolution of resistance to GM crops is of particular concern. For example, the WCR developed resistance to Cry3Bb1 Bt proteins with cross-resistance to mCry3A within 8 years of commercial release in the U.S. (Gassmann et al., 2011; Wangila et al., 2015). The impacts of resistance are often severe and far-reaching: they can lead to economic losses and increased pesticide usage. Delaying or preventing adaptation to pesticides, insecticidal GM crops and host plant defense traits can be achieved through the adoption of an integrated resistance management plan, and biological control can play a large role in these eﬀorts. The impact of biological control on the rate of evolution of pest resistance is dependent upon whether natural enemies disproportionately attack resistant prey/hosts (thereby slowing resistance evolution) or susceptible prey/hosts (thereby accelerating resistance evolution) (Gould et al., 1991). In a high-dose/refuge strategy, such as that used for Bt crops, susceptible pests developing in refuges are frequently found at higher densities than resistant pests feeding on high- dose plants. Therefore, if natural enemies preferentially attack hosts found at higher densities (positive density-dependent mortality), the rate of resistance evolution will be faster than if natural enemies prefer less dense hosts (inverse density- dependent mortality) or are unaﬀected by host density (density- independent mortality) (Heimpel et al., 2005). For example, Coleomegilla maculata De Geer (Coleoptera: Coccinellidae) exhibits inverse density-dependent predation on the egg masses of the Colorado potato beetle Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae), decreasing the rate at which this pest develops resistance to Bt potatoes (Arpaia et al., 1997). However, the introduction of alternative prey can alter feeding patterns of this generalist predator, thereby aﬀecting its inﬂuence on resistance evolution (Mallampalli et al., 2005). As described, biological control can inﬂuence the rate of resistance evolution via top-down inﬂuence. However, the manner in which host plant resistance traits are implemented can also have an eﬀect on the evolution of resistance through bottom-up selection. Top-Down vs. Bottom-Up Control of Herbivorous Populations Top-Down vs. Bottom-Up Control of Herbivorous Populations As emphasized throughout this review, IPM ideally integrates a range of approaches to reduce damage caused by insect pests. Two of these approaches, HPR and biological control, are essentially forms of bottom-up and top-down control of herbivore populations. Whether breeding for increased plant resistance and the use of biological control are compatible and complementary approaches depends, in large part, on the mechanisms involved in HPR and the eﬀects they have on biological control agents. Plant breeding for increased toxicity to herbivores will likely have negative eﬀects on any biological control agents of these herbivores, whether due to direct ingestion of plant toxins or the eﬀects of reduced host or prey size. In this respect, the array of interactions described in the Plant Toxin- Mediated Interactions and Plant Nutrient-Mediated Interactions sections are expected to apply here. An increasing number of studies have demonstrated that HPR has negative consequences for biological control agents through reduced body size or survivorship of individual natural enemies, raising the concern that such approaches are incompatible. Perhaps true in some circumstances, this is not always the case. Even if these control tactics negatively interact, the net eﬀect in suppressing pest populations may be greater than use of either strategy alone. While rarely done, studies evaluating the joint eﬀects of HPR and biological control eﬀorts on pest population dynamics are essential to design eﬀective and sustainable IPM strategies to minimize pest damage. Conversely, eﬀorts to increase HPR by selecting for varieties that increase production of volatiles November 2016 \| Volume 7 \| Article 1794 11 Host Plant Resistance and Biocontrol Peterson et al. Susceptible P. xylostella are killed within 5 days of feeding on Bt plants and consumption of Bt leaves is signiﬁcantly reduced for susceptible larvae than resistant larvae. Consequently, the parasitoid C. plutellae is more attracted to Bt-resistant hosts, as plants with greater feeding damage release more HIPVs, which are attractive to the parasitoid (Schuler et al., 1999). Additionally, natural enemies can slow the evolution of resistance if they increase the ﬁtness costs associated with resistance to crop traits (Raymond et al., 2007) but alternatively may amplify selection for resistance if they attack susceptible prey or hosts more frequently (Gould et al., 1991). For example, susceptible H. virescens feeding on Bt tobacco Nicotiana tabacum L. Top-Down vs. Bottom-Up Control of Herbivorous Populations (Solanales: Solanaceae) took longer to develop, exposing them to greater parasitism by Campoletis sonorensis, and had higher movement rates, increasing risk of infection by the entomopathogenic fungus Nomuraea rileyi (Farlow) Samson (Johnson and Gould, 1992; Johnson et al., 1997a,b). leaf volatiles and MeJA to induce increased HIPV production in ﬁeld grown maize did not result in increased parasitism rates by parasitoids of S. frugiperda (von Mérey et al., 2011, 2012). An essential question that needs additional exploration is whether an increase in biological control due to HIPV-emission will equate to increased crop yields. Biological Control Can Reduce the Likelihood of Resistance Evolution The durability of plant resistance traits is aﬀected by a multitude of factors that inﬂuence selection pressure on herbivorous pests, such as planting of a monoculture of resistant plants vs. mixtures or refuges of non-resistant plants, the mechanism and eﬃcacy of the resistance traits, and the use of pyramiding multiple resistance traits (Stout, 2013). To achieve the greatest durability of plant defense traits, and therefore a more stable and sustainable pest management strategy, both top-down and bottom-up methods for delaying evolution of resistance by arthropod pests should be employed. Frontiers in Plant Science \| www.frontiersin.org How Can We Integrate Host Plant Resistance and Biological Control? To further our understanding of the interactions between plant defense traits and biological control, experts that can conduct research using natural history, molecular and genetic tools, and ﬁeld experimentation must be brought together (Agrawal, 2000a). volatiles, and extraﬂoral nectaries as the most promising plant defense traits for positive synergy with biological control. Plants experience a wide range of biotic associations (both beneﬁcial and antagonistic) above- and belowground that interact in complex ways (Bezemer and van Dam, 2005; van Dam and Heil, 2011). Herbivory and pathogen pressures experienced belowground can inﬂuence above ground interactions between plants, herbivores, and higher trophic levels (e.g., Soler et al., 2007, 2012). Approaches that use beneﬁcial root associates such as arbuscular mycorrhizal fungi and rhizobacteria can not only increase root production and have beneﬁts on yield and aboveground growth, they can stimulate aboveground defensive chemistry providing protection against aboveground herbivores (Gehring and Bennett, 2009; Orrell and Bennett, 2013). Any recommendations that are given to maximize the compatibility of host plant resistance and biological control must also consider other important agronomic and practical factors, such as water availability and water use eﬃciency, fertilization and nutrient availability, weed management, and disease management. However, multiple goals can sometimes be achieved by the adoption of a single practice. For example, indirect host plant resistance, pathogen resistance, and biological control can be simultaneously supported in the case with leaf domatia on grape leaves: both predatory and fungivorous mites use these structures for protection and their presence can decrease incidence of arthropod pests and powdery mildew, a major disease of grapes (Agrawal, 2000a; Norton et al., 2000). For crop producers, agronomic traits other than insect resistance, and ultimately yield, will be the deciding factors for variety or hybrid selection. For crops where the seed market is dominated by transgenics, there may be less choice for the farmer; often only the highest yielding hybrids are chosen for transformation; in order to have the Bt or herbicide resistance traits desired, a smaller pool of varieties are available. Plant breeding often focuses on enhancing agronomic traits, such as drought tolerance, with higher yields as a major driving factor. Therefore, breeding for resistance to arthropod pests may not be the highest priority. How Can We Integrate Host Plant Resistance and Biological Control? Many plant defense traits have been inadvertently lost or weakened through domestication and selective breeding to enhance yields (Brattsen, 1991; Loughrin et al., 1995; Pickett et al., 1997; Rasmann et al., 2005; Chen et al., 2015a,b). Often, indirect defenses that rely upon the attraction or provisioning of natural enemies have also been lost, although eﬀorts have been made to restore these plant traits, such as EβC-production due to an oregano transgene in maize to attract nematodes to attack rootworm larvae (see Case study: Maize Volatiles, Western Corn Rootworm, and Entomopathogenic Nematodes) or artiﬁcial domatia added to commercial cotton plants, which increased the abundance of certain predators (Agrawal et al., 2000). Wild relatives of cotton do have leaf domatia (Fryxell, 1978) and molecular mapping has been used to identify the genes that aﬀect pubescence in cotton (Wright et al., 1999), allowing for the selective expression of pubescence at the leaf vein axils (domatia) that could positively aﬀect natural enemies and biological control in cotton. Looking back to wild relatives of domesticated plant species could be informative for discovering plant defense traits capable of controlling pest species. How Can We Integrate Host Plant Resistance and Biological Control? g Historically, developers of HPR and biological control programs have worked independently, seeking to ﬁnd “single-solution approaches to pest problems” (Thomas and Waage, 1996). Communication between such disparate groups such as plant breeders and natural enemy ecologists may not be inherently high. In reality, there are at least four distinct groups that should come together to better integrate plant defense traits and biological control: (1) HPR researchers (including plant breeders), (2) biological control researchers, (3) ecologists studying community and tritrophic interactions, and (4) extension professionals who are implementing IPM programs and working directly with producers and their advisors (Thomas and Waage, 1996). How can these ﬁelds and groups be brought together? Currently, plant breeding for HPR includes the selection of plant traits with the goal of enhancing direct defenses against herbivorous pests, with little consideration for enhancing plant traits that could improve indirect defenses through the action of natural enemies against pests (Cortesero et al., 2000). Evaluating the impacts of plant resistance characteristics on common natural enemies in the assessment of plant varieties during breeding for HPR would aid in bringing these two methods together. Additionally, fundamental Natural enemies can enhance resistance management for plant defense traits by inﬂicting mortality on those pests that have developed resistance (Liu et al., 2014). In oilseed rape Brassica napus L. (Brassicales: Brassicaceae) expressing Bt toxins, for example, the parasitoid Cotesia vestalis (Halliday) (Hymenoptera: Braconidae) dies with their host if developing inside a Bt- susceptible P. xylostella larva, but does not suﬀer negative eﬀects when parasitizing Bt-resistant caterpillars (Schuler et al., 1999). November 2016 \| Volume 7 \| Article 1794 Frontiers in Plant Science \| www.frontiersin.org 12 Host Plant Resistance and Biocontrol Peterson et al. ecological literature and applied host plant resistance literature have suﬀered from a lack of integration, an observation that has persisted for nearly 30 years (Kogan, 1986; Stout, 2013). An adherence by the host plant resistance community to the three traditional categories of resistance: antibiosis, antixenosis and tolerance (Painter, 1951) may also account for the lack of consideration of the third trophic level (Stout, 2013). Induced, indirect host plant resistance, such as what is seen when herbivore feeding or oviposition on plants triggers the attraction of natural enemies, does not ﬁt into the three traditional categories proposed by Painter (1951). Frontiers in Plant Science \| www.frontiersin.org November 2016 \| Volume 7 \| Article 1794 Practical Implementation of Host Plant Resistance and Biological Control in Integrated Pest Management A successful IPM plan must account for the ecology and biology of the targeted pest(s), environmental factors, and agricultural management. It must be localized; a one size ﬁts all approach will never be eﬀective, yet area wide suppression programs encompassing large regions are sometimes necessary (Schellhorn et al., 2015). This is a signiﬁcant challenge in making prescriptions. An HPR-biological control combination targeting the same pest may work in one region, but not another. Similarly, this combination may work for one type of pest, but not another, even within the same ﬁeld. While HPR and biological control are two of the key pillars of IPM, other essential management tactics include cultural control and chemical control. Another key management tactic is the “stimulo-deterrent diversion” or “push- pull” strategy. Host plant resistance traits can contribute to the “push” component, while biological control by natural enemies may be enhanced by concentration of pests due to the “pull” component (Eigenbrode et al., 2016). Finding a compromise between the strategies of host plant resistance and biological control may prove to be advantageous for selecting management strategies that maximize pest suppression and minimize the likelihood of resistance by reducing selection pressure on pests. For example, glandular pubescence was bred into commercial potato clones for defense against aphids and leafhoppers (Tingey, 1982). In the absence of natural enemies, aphid populations are the lowest on plants with high trichome density; however, when natural enemies are present, biological control is greatest on plants with intermediate trichome density (Obrycki et al., 1983). Therefore, plants with intermediate trichome density were recommended for potato IPM due to their partial resistance to aphids, compatibility with natural enemies, and reduced risk for development of pest resistance (Obrycki et al., 1983). The concept of pairing a partially resistant crop plant with biological control was proposed by van Emden (1988) as two of the three components of a “pest management triad” for aphid control (the third being use of selective insecticides to cause mortality of pests but not natural enemies). Cortesero et al. (2000) identiﬁed leaf domatia, trichomes (in intermediate density), plant signaling via November 2016 \| Volume 7 \| Article 1794 13 Host Plant Resistance and Biocontrol Peterson et al. addressed this important question. However, we will need to continue to explore the dynamic interactions between host plant resistance and biological control as these tritrophic interactions are impacted by changing global conditions, such as climate. REFERENCES Arpaia, S., Gould, F., and Kennedy, G. (1997). Potential impact of Coleomegilla maculata predation on adaptation of Leptinotarsa decemlineata to Bt-transgenic potatoes. Entomol. Exp. Appl. 82, 91–100. doi: 10.1046/j.1570-7458.1997. 00117.x Agrawal, A., Karban, R., and Colfer, R. (2000). How leaf domatia and induced resistance aﬀect herbivores, natural enemies and plant performance. Oikos 89, 70–80. doi: 10.1034/j.1600-0706.2000.890108.x Arthropod Pesticide Resistance Database (2016)). Arthropod Pesticide Resistance Database. 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CONCLUSION In one of the ﬁrst reviews to address the interactions between host plant resistance and biological control for pest management, Bergman and Tingey (1979) stated that “interactions between plant resistance and arthropod predators and parasites remain poorly known.” Since that time, a large body of literature has Practical Implementation of Host Plant Resistance and Biological Control in Integrated Pest Management It is now clear that the mechanisms by which plant defense traits and natural enemies interact are complex and may be synergistic, disruptive, or anywhere on the continuum between. Each is clearly a powerful tool for suppressing herbivore populations and continued eﬀorts to utilize these methods in IPM are essential for environmentally and economically sustainable global crop production. This review provided synthesis for the many facets of these interactions and encompassed the many critical implications these interactions have for agriculture today. Host plant resistance and biological control are both well- suited for adoption in developing countries due to their low cost and lack of need for specialized equipment. The costs of HPR are often built into the price of seed (and may be a one-time expense if farmers can harvest and plant their own seeds subsequently). Biological control may be completely free, if natural control or conservation biological control is used. However, the use of entomopathogens may require application equipment. These biological control methods are in contrast to other types of management, such as chemical control, which may require the use of expensive equipment that is not accessible to farmers in developing countries. A review of these considerations can be found in Thomas and Waage (1996). 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https://openalex.org/W4282572220	https://www.researchsquare.com/article/rs-1693836/latest.pdf	English	null	A Noval Approach towards Neuro Acoustic Loops &amp; their intervention in various Neuro Psychiatric Disorders	Research Square (Research Square)	2,022	cc-by	5,635	A Noval Approach towards Neuro Acoustic Loops & their intervention in various Neuro Psychiatric Disorders Shahzad Aasim (  drshahzadaasim4@gmail.com ) Higher Education https://orcid.org/0000-0001-7310-540X Suhail Ahmad Muheet Butt Hilal Wani Rifat Fatima Abdul Ganie Nizam Ud Din Dar Suhail Ahmad Muheet Butt Hilal Wani Rifat Fatima Abdul Ganie Nizam Ud Din Dar Article License:   This work is licensed under a Creative Commons Attribution 4.0 International License. R d F ll Li Page 1/14 Page 1/14 Abstract The interface of sound in general and music in particular has known and unknown dimensions in human physiology, human psychology and larger well-being of human health. Acoustic therapies and stimulation have been scientifically proven to have a powerful effect on the brain. Recent researches have shown that music can help in many aspects of the brain including pain reduction, stress relief, Seizure, Stroke, The interface of sound in general and music in particular has known and unknown dimensions in human physiology, human psychology and larger well-being of human health. Acoustic therapies and stimulation have been scientifically proven to have a powerful effect on the brain. Recent researches have shown that music can help in many aspects of the brain including pain reduction, stress relief, Seizure, Stroke, memory, and brain injuries. The intervention of sound or music as a type of treatment can be medicine less and mostly concentrates on selective sound frequencies or acoustic loops. In this paper an attempt is made to study the intervention of these sound frequencies in general and their effects on neuro- psychiatric mechanisms and allied phenomenon. The quantification of specific frequencies (Acoustic loops) which will ameliorate the various disorders after their identification can also be correlated with various biochemical parameters that will be analysed with regards to the changes effected by these interventions. The proposed research work might provide a novel approach of treatment for neuro psychiatric disorders using the above stated therapy. The work intends to provide the effect of this therapy on 50 patients/subjects which are taken in isolation wherein the results on selected subjects has been quite demanding. These patients have been already diagnosed with psychiatric disorders at SKIMS and are being re-accessed at NARC using Acoustic Loop Therapy. The proposed work will also provide a scaling or quantitative approach in the intervention of Neuro Acoustic disorders. Introduction Today, the role of sound in science extends beyond the range of audible frequencies: Ultrasonic and other silent acoustic waves have made their way into researchers’ repertoire, helping them push the boundaries of conventional medicine and research. It is known that humans distinguish typically 1400 discrete frequencies and out of these on an equally tempered musical scale there are only 120 discrete frequencies which range from 16 Hz to 16,000 Hz (1) which provide markers for specification of our study. The aim of music is to weave and combine elementary sounds together with combinations and sequences which give pleasure to ear in symphony with notions of harmony and melody. Prior to the study of acoustic loops within the mechanism of sound/music it is also important to know how do we understand the general mechanism of their transfer and cognitive identification of these characteristic frequencies to appreciate how sounds generally may be classed into being musical/unmusical. while we identify conventionally notes being denoted as music and noise as something unmusical, we can say that noise is seen as an irregular disturbance [2] while regular vibrations produce regular musical notes and its further to mention that from variety of notes one obtains approximate noise while no combinations of noise could blend into musical note. The proposed research provides a novel approach for diagnosis of psychiatric disorders using acoustic loop treatment therapy. The proposed work is conducted on 50 patients at NARC who have already be diagnosed for psychiatric ailment by SKIMS. Using various acoustic loop procedures/tests it was observed that only 25 patients/subjects were having psychiatric disorder. This authenticity of diagnosis was also followed by calculating FMTI (Frequency Page 2/14 Modulation Treatment Initial Range) and FMTF (Frequency Modulation Treatment Final Range) followed by biochemical evaluation of the subject. Influence Of Music On Human Brain Through music we can learn much about our human origins and the human brain. Music is a potential method of therapy and a means of accessing and stimulating specific cerebral circuits. There is also an association between musical creativity and psychopathology. Music has been a big part of our species’ development since, well, forever. There are certain parts of the brain that react specifically to music, not just sounds in general. There are regions in the auditory cortex, according to one MIT lab, that show intense activation when listening to music and have little to no activation when you hear, say, a phone ring or someone else’s voice. Our brains are built to listen to music. Music improves brain health and function in many ways. It makes you smarter, happier, and more productive at any age. Listening is good, playing is better that nourishes your moods and vitalizes one’s brain functions. Music has played an important part in every human culture, both past and present. People around the world respond to music in a universal way. And now, advances in neuroscience enable researchers to measure just how music affects the brain. The interest in the effects of music on the brain has produced a new field of research called neuro-musicology which explores how the nervous system reacts to music. Studies have shown that when listening to music, specifically your favourite song, your brain has an increased tolerance for pain and can recover from operations quicker. This implies that the brain stimulates the release of endogenous opioids in the brain. So, if you feel invincible during that part of the song where Gerard Way is yelling about being misunderstood, that’s because you kind of are. Different kinds of music affect our brains in different ways. Tempo, rhythm, instruments, they all change how our bodies and brains react to songs. Role Of Acoustics In The Treatment Of Neuro-psyc Acoustics are a prototypical environmental stressor, has clear health effects in causing hearing loss but other health effects are less evident. Acoustic exposure may lead to minor emotional symptoms but the evidence of elevated levels of aircraft noise leading to psychiatric hospital admissions and psychiatric disorder in the community is contradictory. Despite this there are well documented associations between acoustic treatments and changes in performance, sleep disturbance and emotional reactions such as annoyance. Moreover, annoyance is associated with both environmental acoustic noise level and psychological and physical symptoms, psychiatric disorder and use of health services. It seems likely that existing psychiatric disorder contributes to high levels of annoyance. However, there is also the possibility that tendency to annoyance may be a risk factor for psychiatric morbidity. Although acoustic noise level explains a significant proportion of the variance in annoyance, the other major factor, confirmed in many studies, is subjective sensitivity to acoustic noise. Noise sensitivity is also related to psychiatric disorder. The evidence for noise sensitivity being a risk factor for psychiatric disorder would be greater if it were a stable personality characteristic, and preceded psychiatric morbidity. The stability of Page 3/14 Page 3/14 noise sensitivity and whether it is merely secondary to psychiatric disorder or is a risk factor for psychiatric disorder as well as annoyance is examined in two studies in this monograph: a six-year follow-up of a group of highly noise sensitive and low noise sensitive women; and a longitudinal study of depressed patients and matched control subjects examining changes in noise sensitivity with recovery from depression. A further dimension of noise effects concerns the impact of noise on the autonomic nervous system. Most physiological responses to noise habituate rapidly but in some people physiological responses persist. It is not clear whether this sub-sample is also subjectively sensitive to noise and whether failure to habituate to environmental noise may also represent a biological indicator of vulnerability to psychiatric disorder. In these studies, noise sensitivity was found to be moderately stable and associated with current psychiatric disorder and a disposition to negative affectivity. Noise sensitivity levels did fall with recovery from depression but still remained high, suggesting an underlying high level of noise sensitivity. Noise sensitivity was related to higher tonic skin conductance and heart rate and greater defence/startle responses during noise exposure in the laboratory. Research Mechanism And Methodologies Towards Neuro- Acoustic Research The culmination of different techniques whether PET, MRI, to nail cognitive (7) neuroscience as a discipline with reference to perception of melody and rhythm to track the specific frequencies which effect those changes are very important for us. Surveying the literature, it is seen that cognitive characteristic (11) regarding pitch perception can be traced to neural tissues with certain location for example the auditory cortex, but the conclusively required to pin down the exact mapping of regions which correspond to music only has not been proved with certainty. Although primary areas associated with hearing is superior temporal lobe with many aspects of musical experience associated with cerebellum but the primary auditory cortex (16) descends into many folds in temporal lobes (Hershel’s gyras) and if we compare the quantitative understanding of accessible visible cortex things cannot be properly formulated as the effects of sound waves recess into deeper layers of consciousness. In hindsight the research in music with the brain has been focussed on whether musical pitch processing is also laterized with regards to left hemisphere associated with language (10) and right hemisphere with music, although from certain studies it is not clear whether music and language (13) are independent or not with the caveat that from MRI and PET scans it is observed that non-musicians process musical pitch (13) in right hemisphere while for musicians (10,14) it is left. It has been known that there is correlation of the particular function within human body (physical/psychological) parameters whose disruption after the injury within brain can be traced to inference that the intact region or portion was the necessary component with regards to the neural resources required for the sound functioning of what was disruptively observed. This broad identification of loss of a particular function in general sense to different areas of brain and in particular if there is injury, stroke, neurosurgery or extreme cases of epilepsy with the perception and cognition of music(7) is seen to be affected in non-linear and strikingly unknown ways. It’s seen that persistent loss of music (Amusia) or perception of melodies with the loss of ability to discriminate isolated pitches within melodic context there are clear deficiencies observed in patients with regards to them and there have been difficulties in identifying melodies with more than one or different tone. Research Mechanism And Methodologies Towards Neuro- Acoustic Research It’s also generally known that Sound and music perceived at acoustic level can explain auditory notes with respect to ear and brain, The psychology which is affected by the sound frequencies implores us to ask what disturbances of air molecules are specifically heard as noise and others as music. Subsequent to that what happens to sound wave in the ear and brain when we perform or listen to those frequencies being classified as sound/music. How do these specific frequencies which are identified as music/sound emerge develop or becomes refined from particular expertise and appreciate the deep emotional significance and its power to influence social behaviour varying across cultures (15). The psychology and identification of these frequencies can be properly identified which depends on the understanding of branches of psychology (19) visa viz sensation, perception, neuropsychology, development psychology, social psychology and educational psychology. These studies can also be complemented with the physical properties of sound waves their transmission to ear and neural basis of perception and cognition of sound/music within certain range of specific/unspecified frequencies with reference to subjects identified for the desired study. Pertinent to mention is that the identification of sound/music can be further supplemented with the perception of melody, rhythm and musical structure with meanings implied or communicated visa viz, social and emotional (8) functions which can be properly understood and interpreted from the interplay of various sub disciplines (9) like that of neuroscience, musicology, education, philosophy, ethno-musicology. The frequency of source of sound giving rise to aural sensation called pitch which Pythagoras identified with wavelength and other parameters of sound/music waves include frequency, Amplitude and complexity which correspond with psychological properties of pitch, loudness and Timbre. The regulation of sound waves from different parts of ear towards auditory canal (6,16) with role of cochlea as a device to detect and transduce relevant properties of wave trains into neural impulses with Page 4/14 Page 4/14 the specificity of wavelengths which could be modulated externally in the entire process of hearing is very important. The cognitive task of brain to analyse the incoming sound waves with musically relevant properties and the role of primary auditory cortex (4) to distinguish sounds within relevant frequency ranges or pitch relations associated with the neuro acoustic processes is also significant. Intervention Of Acoustic Loops And Our Study In Relation To The Neuro-psychiatric Disorders Our study parallels with this study (17) when we identify the normal state with one of these ranges in particular alpha and theta which are rejuvenating states commonly associated with relaxation, awareness, intuition creativity and most importantly these states allow body to rest and repair. While the Beta states are anxiety prone states with electric waves or brainwaves associated with them in the mentioned ranges are the states we live with. It is a challenging task of how we can identify these states and with the identification of acoustic loops within certain ranges and then find a way and method to make transition from Beta states to relaxed Alpha and theta states. Although it has to be mentioned the scale varies from different studies available in literature with respect to study in fluctuation of electric fields with regards to the identifiable mental and psychological parameters which are precursors to the changes which can also be affected by sounds/frequencies falling in certain ranges which we observe with regards to subjects/patients under consideration. The Neuro acoustic Audio treatment will primarily be focussing on directing brane wave patterns into calming brain wave states associated with the treatment so that there is a smooth transition into mediative states. The important point to mention is that in our treatment or method in the presence of the intervention of those acoustic loops falling in certain narrow or specific range of frequencies within those mentioned ranges which are loosely classified as alpha, beta, gamma and delta ranges. It is to be mentioned there can be specific/unspecific modes into which patients or subjects are triggered into once they are under the effect of these experimental loops, we induce on the patients which fall into sub frequencies of those general frequencies comparable to the previous scale mentioned. Once the intervention in these specific loops is triggered, they have the potential of decoupling mind from the environment and internally making the patient/subject to go from unspecified mode to specific mode which makes him sensitive or responsive to those corresponding modes only which have been triggered externally by those specific acoustic loops falling within those frequency ranges. Research Mechanism And Methodologies Towards Neuro- Acoustic Research It is also challenging to understand if melodies (3) are simple arrangements of discrete pitches with notable oscillating patterns and how does human mind differentiate and identify (5) as well as remember a distinct arrangement, and if two or more pitches are conjured together and how they are processed in terms of coherent tune. If these questions are addressed in whatever manner, how can we identify sound/music frequencies associated with them to ameliorate various neuropsychiatric disorders with regards to diagnosis or treatment in relation to the intervention of acoustic loops. The use of EEG to measure changes in the electric activity of whole of the brain by placing electrodes on the scalp informs us that there is transmission of impulses with neurons leading to fluctuations in electric Page 5/14 Page 5/14 Page 5/14 field within the neighbourhood of these neurons. A variable scale has been constructed within certain ranges where we observe and mark each pattern associated with certain cognitive and motor activation with regards to the intervention of electric fields. The classification and identification in the literature with respect to different ranges of electric cycles are 1) Delta Ranges- 3Hz 2) Theta-4-7 Hz, 3) Alpha-8-12Hz 4) Beta-12-30Hz. 5) Gamma-30-100Hz . Intervention Of Acoustic Loops And Our Study In Relation To The Neuro-psychiatric Disorders The mode and method of these acoustic loops assumes to induce changes which may be short term to begin with but they can serve also to be diagnostic with regards to how the subject with neuropsychiatric disorders is going to get effected by the intervention of these acoustic loops. These can also serve as a source of identification and stage of disorder regarding patients when the acoustic loops and their corresponding brain wave frequencies which have been mapped and measured with regards to the identified mental states with regards to the neuropsychiatric disorders. The scale identified with regards to intervention of acoustic loops with respect to the different parameters of age, type of identified disorder, nature of mental state, Page 6/14 Page 6/14 environment, case history of patients can be compared with the normal medical treatment which the patient is subjected or treated. The effect of musical instruments in conjunction with the frequencies which is being played apart from the frequency which is characteristic of the instrument has important role in identifying the principal frequency which is identified as part of different acoustic therapies which we are suitable for different therapeutic interventions. Different musical instruments will have different effects when played upon by the same frequency so that the identification of the principal frequency which is useful for the given session regarding particular acoustic loop can be accounted and it is seen there is addition or alteration of given frequency on the same instrument or different instruments with the same frequencies. The resonant frequencies which are produced in a given instrument have potential of identifying the different sub -frequencies which will lie above or below those principal frequencies associated with different loops which we are going to use as part of intervention. One reason of having different effects from the application of different instruments is the nature of matter and properties of the vibrating instruments and knowledge of these properties can be manipulated and be helpful in the identification of the principal frequency as well as the sub frequencies which will help us in understanding and quantifying their effect in ameliorating neuropsychiatric disorders. The identification of the frequencies which correspond to specific Acoustic loops proposes also to give us an indirect identification of scale which is going to give us proposed method of identifying disorders like that of anxiety, depression, OCD, Parkinson, BPD, Psyznopheria, Autism etc. Intervention Of Acoustic Loops And Our Study In Relation To The Neuro-psychiatric Disorders The sensitivity and identification of the scale and also the mechanisms identifying the acoustic loops associated with the scale will in long term help us to make our measurements more precise in long term with respect to the prognosis and diagnostic in relation to the disorders of the subjects which we have identified in the first place. Simultaneously after profiling of subjects /patients with these disorders and after the identification of specific loops which are identified from acoustic scale, various biochemical parameters like serotonin, dopamine, adrenaline, noradrenaline or their metabolites etc. will be analysed with the help of kits and autoanalyzers both in the pre and post intervention state of the subject. The purpose is to develop diagnostic and prognostic tool in terms of biochemical parameters which will not only be helpful in identifying the disorder prior to treatment but also helps us to find the stage of disorder the subject is living with besides evaluating the prognosis. The same would be supplementary to the neuro acoustic loop- based diagnosis and prognosis. In the first phase we have identified 50 patients and worked with acoustic loops previously identified with certain tests which can be loosely phrased in terms like that of frequency modulated tests (FMTS) which synchronise to the Resonance values of those identified frequencies which has been seen in our case to ameliorate the disorders and potentially curing patients which in case of our study has shown promising signs. The parameters or the observables and the identification of frequencies which corresponding to those acoustic loops can be stage wise graphical parameterized in terms of relevant variables which can Page 7/14 Page 7/14 also be compared with the normal medical literature in terms of success or efficacy of treatment. The Preliminary results regarding the intervention of these specific loops regarding diseases in our case has been encouraging which have shown improvements with regards to the identification and treatment of disorders. The bird’s eye analysis of the data regarding for example the patients suffering from anxiety and depression when compared to our treatments in the sessions which were intervened with specific acoustic loops showed positive results of improvements in terms of restoring them to normal behaviour. The various therapies are synonymous with regards to the various loops which have been classified and identified for different patients depending upon varied factors. Treatment Process based on Neuro Acoustic Loops This treatment is carried out under the project on Neuro Acoustic Research having collaboration with Sheri Kashmir Institute of Medical Sciences (SKIMS), J&K, India. The said treatment is done based on various phases of acoustic implementation. The treatment is mainly composed of two phases. i) Registration of Patients This phase involves registering of patients who wish to get checked for any Psychiatric disorder. The information regrinding the patient is kept hidden so that the real identity of the patient is not disclosed. The data is collected solely for research purpose. The complete and accurate patient registration is crucial phase of this research. As the circumstances and policies change often there is a dire need to update patient demographics at each visit. Intervention Of Acoustic Loops And Our Study In Relation To The Neuro-psychiatric Disorders These parameters and factors shall be elaborated in future studies with an broader aim of not only giving qualitative analysis but supplementing the study with the quantitative correspondence with respect to the variables which have been identified and mapped with respect to intervention of acoustic loops in relation to neuropsychiatric and other disorders. ii) Case History Formulation In this phase the case history of the patient taken by the concerned psychiatrist and all the test reading of past are also recorded including various important vitals. The phase provides an environment to ask open-ended questions that require a full response, so as to get the most up-to-date information regarding the patient which could aid in better diagnosis and treatment. iii) Therapies Awareness Based on the case history information the patient/subject is made aware of all the therapies that he/she has to undergo so as to bring clarity in the diagnosis. As we are dealing with patients with psychiatric disorders a proper counselling is being done so that the patients become emotionally and psychologically ready for these acoustic therapies. iv) Loop Selection (in Active Mode) iv) Loop Selection (in Active Mode) Page 8/14 Page 8/14 In this phase a set of acoustic loop stack has already been identified for various ailments. These loops are mostly sound frequencies which are provided to patient in Active or Passive mode. Normally in this phase loop selection for the patient is done in active mode. These loops sensitize the patient’s neuro systems and accordingly provide an adequate output which enables the therapist to depict the level of psychiatric disorder vii) Checking for Psychiatric Disorder & Treatment Provisions It is followed by conclusion of any sort of psychiatric disorder and accordingly acoustic loop based treatment provisions are being determined on patient to patient basis. In the first phase the patient is registered and is made exposed to various acoustic loops which involve therapy awareness, loop selection and based on that perform Frequency Modulation Tests which make patient ready for going to the next phase after determining any neuro psychiatric disorder. The flow diagram is shown in Figure 1. v) Frequency Modulation Test (FMT/EEG) An electroencephalogram (EEG) is a test is performed for the detection of electrical activity in the brain using small, metal discs (electrodes) attached to the patients scalp. The brain cells communicate via electrical impulses and are active all the time, even when asleep. This activity shows up as wavy lines on an EEG recording. This test is provides a key information regarding any neuro psychiatric disorder. vi) Comparing Resonance Value The value of resonance are compared which provides a qualitative mechanism for diagnosing psychiatric disorder. Comparative Data Analysis between NARC & SKIMS Presently these disorder are being purely diagnosed on observation and patient behaviour and a concrete methodology is missing. The proposed research can aid in formulating a standard scaling mechanism which could quantify a neuro psychiatric disorder. Comparative Data Analysis between NARC & SKIMS The data has been gathered and tabulated from around 50 random patients/subjects who have been already diagnosed with psychiatric disorders by Sheri Kashmir Institute of Medical Sciences (SKIMS). These patients have been re-assessed using Acoustic Loop Therapy for any psychiatric disorder. Data has been collaborated on a excel sheet and analysed using python data frames in a standardised template. Duplicate data, any outlier from the same study samples and reported has been identified and removed. As the results are being gathers from two sources (SKIMS & NARC), accordingly two separate collaboration has been made whose confusion matrix is shown as under Figure 2. Confusion matrix for the data shows that around 50% patients who have been diagnosed for an psychiatric disorder at SKIMS have been wrongly diagnosed as per NARC. These tests which accumulated for the diagnosis have been conducted using Acoustic Loops, FMT followed by bio- chemical analysis and are shown in the chart in Figure 3. Page 9/14 Page 9/14 The chart clearly tabulates the value of FMTI and FMTF and the variation of data has been identified wherein 50% of cases have been diagnosed as psychiatric disorder. The neuro acoustic based study variability in prevalence of these psychiatric disorder when these tests on the subjects were conducted. The comparative results between (SKIMS & NARC) like sensitivity, precision, F1 Score etc. are shown in the table below: The chart clearly tabulates the value of FMTI and FMTF and the variation of data has been identified wherein 50% of cases have been diagnosed as psychiatric disorder. The neuro acoustic based study variability in prevalence of these psychiatric disorder when these tests on the subjects were conducted. The comparative results between (SKIMS & NARC) like sensitivity, precision, F1 Score etc. are shown in the table below: S. No. Parameter Value 1. Sensitivity 0.5 2. Precision 1.0 3. False Discovery Rate 0.0 4. False Negative Rate 0.5 5. Accuracy 0.5 6. F1 Score 0.6667 The proposed research could also aid in qualitative scaling of these neuro psychiatric disorders. Presently these disorder are being purely diagnosed on observation and patient behaviour and a concrete methodology is missing. The proposed research can aid in formulating a standard scaling mechanism which could quantify a neuro psychiatric disorder. The proposed research could also aid in qualitative scaling of these neuro psychiatric disorders. Author’s contribution Dr. Shahzad Aasim developed the concept and designed the study did systematic search and helped in carrying out the experimental and helped in preparation of the first draft. All authors participated in the revising of the manuscript before submission. Conclusion And Future Scope The use of specific sound frequencies identified as acoustic loops have been proposed to ameliorate the state of diseases with promising signs within the limited groups and specific range of identified disorders with regards to our study. The paper provides a novel approach towards treatment of Neuro Psychiatric Disorders by using Acoustic Loop therapies on 50 patients where in the results were quite encouraging The novelty of identifying principal frequencies regarding specificity of disease and the type of patient has been also supposed to be validated from other methods with the proposal of indirect scale which will be also verified by known medical and biochemical parameters.. The biochemical profiling could also open new dimension in the prognosis and diagnosis of the neuro psychiatric disorders besides adding to the potential of Neuro Acoustic Loop based diagnosis and therapy. The variation of methods scale and scope of treatment may itself be part of any future study and in this paper the qualitative description has been formulated which can be supplemented or complemented with a future study in that direction. As the data will accumulate over a period of our research an AI & Machine Learning based model can also be evolved in future out of the psychiatric dataset which could also aid is diagnosis and treatment of many neuro psychiatric disorders. Page 10/14 Search Strategy The PubMed database was searched for related work done. Moreover, reference lists of suitable articles were reviewed to find relevant material. Competing interests The authors declare that there were no competing interests. Acknowledgement The authors acknowledge the facilities provided by SKIMS Medical College/JVC hospital, Srinagar, J&K, India and also for providing us with subjects/patients for the study and also keeping space and resources available at our disposal. Funding sources No funding was received for the study. Ethical statement Ethical approval that was required was sought before the start of the work from Sher-i-Kashmir Institute of Medical Sciences (SKIMS-Medical College, Srinagar, J&K, India). Conflict of Interest The authors declare that there was no conflict of interest. 1. The Science and Applications of Acoustics Second Edition Daniel R. Raichel 2. The Theory of Sound John William Strut Baron Rayleigh Vol 1 3. Peretz, I. (1993). Auditory atonalia for melodies. Cognitive Neuropsychology, 10, 21–56. 4. Rauschecker, J. P., & Tian, B. (2000). Mechanisms and streams for processing of ‘what’ and ‘where’ in auditory cortex. Proceedings of the National Academy of Sciences, 97, 11800–11806 References 1. The Science and Applications of Acoustics Second Edition Daniel R. Raichel 2. The Theory of Sound John William Strut Baron Rayleigh Vol 1 3. Peretz, I. (1993). Auditory atonalia for melodies. Cognitive Neuropsychol 4. Rauschecker, J. P., & Tian, B. (2000). Mechanisms and streams for processing of ‘what’ and ‘where’ in auditory cortex. Proceedings of the National Academy of Sciences, 97, 11800–11806 Page 11/14 Page 11/14 5. Lamont, A. (1998). Music, education, and the development of pitch perception: The role of context, age, and musical experience. Psychology of Music, 26, 7–25 6. Lahav, A., Saltzman, E., & Schlaug, G. (2007). Action representation of sound: Audiomotor recognition network while listening to newly acquired actions. Journal of Neuroscience, 27, 308–314 7. Krumhansl, C. (1990). Cognitive foundations of musical pitch. Oxford: Oxford University Press 8. Konecˇni, V. (2008). Does music induce emotion? A theoretical and methodological analysis. Psychology of Aesthetics, Creativity and the Arts, 2, 115–129. 9. Huron, D. (2006). Sweet anticipation: Music and the psychology of expectation. Cambridge, MA: MIT Press. 10. Gaser, C., & Schlaug, G. (2003). Brain structures differ between musicians and non-musicians. Journal of Neuroscience, 23, 9240–9245. Gazzaniga, M. S., Ivry, R. B., & Mangun, G. R. (2002). 11. Cognitive neuroscience: The biology of the mind (2nd ed.). New York: W. W. Norton. 11. Cognitive neuroscience: The biology of the mind (2nd ed.). New York: W. 12. Francès, R. (1958/1988). The perception of music. (W. J. Dowling, Trans.). Hillsdale, NJ: Lawrence Erlbaum Associates, Inc 13. Besson, M., & Schön, D. (2003). Comparison between language and music. In I. Peretz & R. Zatorre (Eds.), The cognitive neuroscience of music (pp. 269–293). New York: Oxford University Press 14. Bever, T., & Chiarello, R. J. (1974, August 9). Cerebral dominance in musicians and non-musicians. Science, 185, 537–539. 14. Bever, T., & Chiarello, R. J. (1974, August 9). Cerebral dominance in musicians and non-musicians. Science, 185, 537–539. 15. Balkwill, L.-L., & Thompson, W. F. (1999). A cross-cultural investigation of the perception of emotion in music: Psychophysical and cultural cues. Music Perception, 17, 43–64 15. Balkwill, L.-L., & Thompson, W. F. (1999). A cross-cultural investigation of the perception of emotion in music: Psychophysical and cultural cues. Music Perception, 17, 43–64 16. Brattico, E., Tervaniemi, M., Näätänen, R., & Peretz, I. (2006). Musical scale properties are automatically processed in the human auditory cortex. Brain Research, 1117, 162–174 16. References Brattico, E., Tervaniemi, M., Näätänen, R., & Peretz, I. (2006). Musical scale properties are automatically processed in the human auditory cortex. Brain Research, 1117, 162–174 17. Clinical Aspects of Music Sound and Acoustics Dr Shahzad Asim KASRC ts of Music Sound and Acoustics Dr Shahzad Asim KASRC Cluster universit 18. Springer Handbook of Acoustics (Medical Acoustics) Editor Rossing Authors Kirk W Beach Barbina Dunmire 18. Springer Handbook of Acoustics (Medical Acoustics) Editor Rossing Authors Kirk W Beach Barbina Dunmire 19. The Psychology of Music Enlarged Edition HP, Krishna Rao 20. Quantum Mechanism Of Music Dr. Shahzad Aasim Global Journal, Physics and Space Science, volume-20, Issue-10, version 1.0 20. Quantum Mechanism Of Music Dr. Shahzad Aasim Global Journal, Physics and Space Science, volume-20, Issue-10, version 1.0 Figures Page 12/14 Page 12/14 igure 1 arious Phases for Acoustic Loop Psychiatric Diagnosis Figure 1 Various Phases for Acoustic Loop Psychiatric Diagnosis Various Phases for Acoustic Loop Psychiatric Diagnosis Page 13/14 Page 13/14 Figure 2 Confusion Matrix Between SKIMS and NARC Data Figure 2 Confusion Matrix Between SKIMS and NARC Data Figure 2 Confusion Matrix Between SKIMS and NARC Data Figure 3 Comparative Analysis of SKIMS and NARC Data based on FMTI and FMTF values Comparative Analysis of SKIMS and NARC Data based on FMTI and FMTF values Page 14/14
https://openalex.org/W4379197498	https://www.nature.com/articles/s41598-023-35764-7.pdf	English	null	Increased plasma lipids in triple-negative breast cancer and impairment in HDL functionality in advanced stages of tumors	Scientific reports	2,023	cc-by	10,501	OPEN Maria Isabela Bloise Alves Caldas Sawada 1,2,3, Monique de Fátima Mello Santana 4, Mozania Reis 1,5, Sayonara Ivana Santos de Assis 4, Lucas Alves Pereira 1, Danielle Ribeiro Santos 4, Valéria Sutti Nunes 4, Maria Lucia Cardillo Correa‑Giannella 6, Luiz Henrique Gebrim 2 & Marisa Passarelli 1,4* The association between plasma lipids and breast cancer (BC) has been extensively explored although results are still conflicting especially regarding the relationship with high-density lipoprotein cholesterol (HDLc) levels. HDL mediates cholesterol and oxysterol removal from cells limiting sterols necessary for tumor growth, inflammation, and metastasis and this may not be reflected by measuring HDLc. We addressed recently diagnosed, treatment-naïve BC women (n = 163), classified according to molecular types of tumors and clinical stages of the disease, in comparison to control women (CTR; n = 150) regarding plasma lipids and lipoproteins, HDL functionality and composition in lipids, oxysterols, and apo A-I. HDL was isolated by plasma discontinuous density gradient ultracentrifugation. Lipids (total cholesterol, TC; triglycerides, TG; and phospholipids, PL) were determined by enzymatic assays, apo A-I by immunoturbidimetry, and oxysterols (27, 25, and 24-hydroxycholesterol), by gas chromatography coupled with mass spectrometry. HDL-mediated cell cholesterol removal was determined in macrophages previously overloaded with cholesterol and 14C-cholesterol. Lipid profile was similar between CTR and BC groups after adjustment per age. In the BC group, lower concentrations of TC (84%), TG (93%), PL (89%), and 27-hydroxicholesterol (61%) were observed in HDL, although the lipoprotein ability in removing cell cholesterol was similar to HDL from CRT. Triple-negative (TN) BC cases presented higher levels of TC, TG, apoB, and non-HDLc when compared to other molecular types. Impaired HDL functionality was observed in more advanced BC cases (stages III and IV), as cholesterol efflux was around 28% lower as compared to stages I and II. The altered lipid profile in TN cases may contribute to channeling lipids to tumor development in a hystotype with a more aggressive clinical history. Moreover, findings reinforce the dissociation between plasma levels of HDLc and HDL functionality in determining BC outcomes. Female breast cancer (BC) is the most commonly diagnosed cancer worldwide, accounting for 11.7% of all cancer cases, and is the fifth leading cause of cancer-related deaths, representing 6.9% of all cancer deaths1. Breast cancer is considered a heterogeneous disease, and its molecular classification is widely used to determine treatment options and prognosis. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports/ www.nature.com/scientificreports/ In recent decades, evidence has emerged linking plasma lipid levels to the development and worse outcomes of BC4–7. This is related to tumor cell reprogramming that enables greater lipid uptake for cell division8. However, clinical and epidemiological data have shown mixed results, with some studies suggesting a positive or negative impact of hyperlipidemia on BC incidence, while others suggest no impact9–13. Similarly, the association between statins and the risk of BC is still controversial14,15. Much attention has been focused on the role of cholesterol in high-density lipoproteins (HDLc) in relation to BC risk and prevention, with most studies suggesting a protective role of plasma HDLc4,10,11. These results may be due to the beneficial activities of HDL in removing excess cholesterol and oxysterols from tumor cells16. Furthermore, HDL has antioxidant and anti-inflammatory activities and acts as a carrier for bioactive lipids, proteins, and microRNAs that may modulate tumor growth and progression17,18. However, there is still contro- versy surrounding this topic, with some studies showing only a weak association between HDLc and BC, while others show a positive or no association at all9,12,13,19. p Similar to the role of HDL in atherosclerosis and other chronic non-communicable diseases, it is possible that the HDLc metric may not be the best predictor variable for determining BC incidence and progression20. Therefore, alterations in HDL functionality should be considered, particularly when taking into account the modulation of this lipoprotein’s composition and function by the tumor microenvironment21. y HDL is well known for its ability to remove cellular lipids, allowing cholesterol for uptake by the liver, secre- tion into bile, and excretion in feces through reverse cholesterol transport. Similarly, in the case of tumor cells, reverse cholesterol transport can be considered a defensive mechanism that prevents cholesterol accumulation that supports cell proliferation and metastasis. The expression of HDL receptors, such as ATP-binding cassette transporter A-1 (ABCA-1) and scavenger receptor class B type 1 (SR-B1), that mediated cholesterol exportation is altered in tumors and is related to epithelial-mesenchymal transition, tumor growth, and metastasis22–25. HDL also mediates the transport of oxysterols produced intracellularly by the oxidation of the cholesterol molecule. Due to its higher hydrophobicity compared to other oxysterols and cholesterol, 27 hydroxycholesterol (27HC) can easily diffuse across the plasma membrane26. www.nature.com/scientificreports/ Additionally, its transfer to HDL is facilitated by the ATP- binding cassette transporters G-1 (ABCG-1) located in the cell membrane of tumor cells and macrophages that infiltrate the tumor area27. Then, 27HC flux outside cells is considered an additional route for reverse cholesterol transport, limiting sterol content that is associated with cell inflammation, oxidative stress, and proliferation18,28. 27 hydroxycholesterol also acts as a selective estrogen receptor modulator (SERM), promoting tumor growth and metastasis in BC. In human BC samples, the expression of the enzymes involved in the production of 27HC (CYP27A1) and metabolism (CYP7B1) is respectively increased and decreased and is associated with poor tumor prognosis29. Furthermore, tumor metastasis is related to 27HC-dependent activation of the liver X receptor (LXR)29–32. p ( ) HDL removes cholesterol and oxysterols from cells, making acceptable the idea that it limits the intracel- lular accumulation of sterols and their negative impact on BC. In this way, it was addressed in newly diagnosed women with BC categorized according to the clinical stage of the disease and molecular classification of the tumor, without pharmacological or surgical intervention, compared to control women (CTR): (1) plasma lipid profile [total cholesterol (TC), HDLc, apolipoprotein B (apoB) and triglycerides (TG)]; (2) the composition of isolated HDL in lipids, main species of oxysterols and apolipoprotein A-I (apoA-I); and (3) the ability of HDL to mediate cholesterol removal from macrophages. Plasma lipids were similar between controls (CTR) and all women with BC, but when categorized according to tumor molecular type, triple-negative (TN) BC cases had higher plasma TC, TG, and apoB values compared to other molecular types. Despite having lower levels of TC, phospholipids (PL), and 27HC, HDL from women with BC maintained its ability to remove cellular cholesterol when compared to HDL from CTR. In advanced BC (covering clinical stages III and IV), despite similar composition in apoA-I and lipids, HDL had an impaired ability to remove cholesterol from macrophages. OPEN This classification is based on the expression of estrogen and progesterone receptors (luminal A; LA, and luminal B; LB), human epidermal growth factor receptor 2 (HER2), or the absence of these receptors (triple-negative; TN)2,3. 1Programa de Pós‑Graduação em Medicina, Universidade Nove de Julho (UNINOVE), São Paulo, Brazil. 2Centro de Referência da Saúde da Mulher (Hospital Pérola Byington), São Paulo, Brazil. 3Hospital da Força Aérea de São Paulo, São Paulo, Brazil. 4Laboratório de Lípides (LIM10), Hospital das Clínicas (HCFMUSP) da Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil. 5Unidade Básica de Saúde Dra. Ilza Weltman Hutzler, São Paulo, Brazil. 6Laboratório de Carboidratos e Radioimunoensaio Lípides (LIM18), Hospital das Clínicas (HCFMUSP) da Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil. email: m.passarelli@ uni9.pro.br \| https://doi.org/10.1038/s41598-023-35764-7 Scientific Reports \| (2023) 13:8998 www.nature.com/scientificreports/ After protein quantification by the Lowry technique36, LDL was incubated with acetic anhy- dride as previously described37. Acetylated LDL was dialyzed and utilized to load macrophages with cholesterol. HDL‑mediated cholesterol efflux from bone marrow‑derived macrophages. The Institutional Animal Care and Research Advisory Committee (Universidade Nove de Julho # 7,070,120,821, 08/23/2021) approved the study according to the U.S. National Institutes of Health Guide for the Care and Use of Labora- tory Animals. All the methods were performed in accordance with ARRIVE guidelines (Supplementary file S1). Thirty male and female C57BL/6 J mice, aged 2–48 weeks, were housed with free access to commercial chow (Nuvilab CR1, São Paulo, Brazil) and drinking water in a conventional animal facility at 22 ± 2 °C under a 12 h light/dark cycle. Intraperitoneal overdose of ketamine hydrochloride (Ketalar) (300 mg/kg of body weight) and xylazine hydrochloride (Rompun) (30 mg/kg of body weight) were used to euthanize the animals, in accord- ance with the norms of the National Council for the Control of Animal Experimentation (CONCEA) of the Ministry of Science, Technology and Innovation (MCTI). Undifferentiated bone marrow cells were obtained from C57BL/6 wild-type mouse´s tibia and femora, as previously described38. Cells were differentiated into macrophages by incubating with L929 cells-conditioned medium (ATCC, American Tissue Culture Collection) and plated in culture dishes for 5 days at 37 °C, under 5% (v/v) CO2. The medium was changed for a new one and after 6 days replaced by DMEM (low glucose, containing 1% penicillin/streptomycin and 10% heat-inactivated fetal calf serum). Macrophages were overloaded with acetylated LDL (50 µg/mL) and 14C-cholesterol (0.3 µCi/ mL) for 48 h, and after washing incubated with DMEM containing fatty acid-free albumin for equilibration of intracellular cholesterol pools. Cells were incubated for 6 h with HDL (50 µg/mL) from BC or control women; control incubations were performed in the absence of HDL (basal efflux). After incubation, the radioactivity in the medium was determined as well as in the cell lipid extract. The cholesterol efflux was determined as 14C-cholesterol in the medium / 14C-cholesterol in the medium + 14C-cholesterol in cells × 100. Values from basal efflux were subtracted from those obtained after incubation with HDL, in order to express the specific ability of HDL in removing cell cholesterol. Oxysterols quantification in isolated HDL. Oxysterols (24-hydroxycholesterol, 25-hydroxycholesterol, and 27-hydroxycholesterol) were measured from 1 mL of extracted HDL as previously described39,40. www.nature.com/scientificreports/ ratio < 2.0 with mean HER2 > 6 signals per cell (greater than 120 signals in 20 nuclei). Tumor samples that did not express either hormonal or HER2 receptors were classified as TN BC34. ratio < 2.0 with mean HER2 > 6 signals per cell (greater than 120 signals in 20 nuclei). Tumor samples that did not express either hormonal or HER2 receptors were classified as TN BC34. i For sample calculation, the number of new cases of BC included for treatment at Hospital Pérola Byington during 2018 (2,985 cases) was taken into account; also the study design, with the comparison of outcome variables between two main groups (CTR vs. BC); the main outcome variables; the effect size of the variables according to the main studies published in the area; and the probability of committing a type 1 error (0.05) and type 2 error (0.20), with 80% power, resulted in 144 patients in each group (pairing 1:1). i For sample calculation, the number of new cases of BC included for treatment at Hospital Pérola Byington during 2018 (2,985 cases) was taken into account; also the study design, with the comparison of outcome variables between two main groups (CTR vs. BC); the main outcome variables; the effect size of the variables according to the main studies published in the area; and the probability of committing a type 1 error (0.05) and type 2 error (0.20), with 80% power, resulted in 144 patients in each group (pairing 1:1). Blood collection. Venous blood was drawn after 12 h fasting and plasma was immediately isolated after centrifugation (3,000 rpm, 4 °C, 15 min). Plasma TC, TG, and HDLc were determined by enzymatic techniques. Low-density lipoprotein cholesterol (LDLc) was determined by the Friedewald formula35, and VLDLc as TG/5. ApoB was quantified by immunoturbidimetry (Randox Lab. Ltd. Crumlin, UK). Isolation of plasma lipoproteins. High-density lipoproteins (HDL; D = 1.063–1.21 g/mL) were isolated from BC and CTR women´s plasma by discontinuous density ultracentrifugation and immediately frozen at − 80 °C in a 5% saccharose solution. HDL composition in lipids (TC, TG, and PL) was determined by enzymatic techniques. ApoA-I was determined by the immunoturbidimetric method (Randox Lab. Ltd. Crumlin, UK). Low‑density lipoprotein isolation and acetylation. LDL (D = 1.019–1.063 g/mL) was obtained by sequential ultracentrifugation of plasma from healthy volunteers and was purified by discontinuous density ultracentrifugation. Material and methods Two hundred and one women newly diagnosed with BC between 18 and 80 years old in any clinical stage and with the molecular classification of the tumor were recruited at Hospital Pérola Byington. A hundred and fifty- seven women without any type of cancer were recruited at Universidade de São Paulo and at Unidade Básica de Saúde Dra. Ilza Weltman Hutzler (control group; CTR). Exclusion criteria were diabetes mellitus, chronic kidney disease (estimated glomerular filtration rate < 60 mL/min/1.73m2), autoimmune diseases, smokers, alcoholics, use of contraceptives, and hormone replacement therapy, pregnancy, previous history of any cancer, and in situ breast disease. After excluding those who did not meet the eligibility criteria, 150 CTR and 163 women with BC remained in the study. All participants were informed about the study and signed an informed written consent previously approved by institutional Ethics Committees, in accordance with the Declaration of Helsinki, includ- ing approval for publication (Universidade Nove de Julho, # 3.139.460; Centro de Referência da Saúde da Mulher, Hospital Pérola Byington, #3.225.220; and Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, #3.317.909).hi The molecular classification of tumors was obtained from medical records being accessed immunohistochemi- cally after percutaneous biopsy, according to the American College of Pathologists33. Samples that were positive for hormone receptors (estrogen and progesterone) were those in which > 1% of the tumor cells showed positive nuclear staining of moderate to strong intensity on immunohistochemistry and were classified as Luminal A and B neoplasia if they presented the Ki67 index below or above 14%, respectively. Samples that exhibited > 10% of invasive tumor cells with strong HER2 staining in the plasma membrane were considered HER2 positive. In case of moderate staining in > 10% of the cells or strong staining in < 10% of the cells, the sample was re-evaluated by in situ hybridization and was considered positive if a HER2/centromere ratio > 2.0; or if a HER2/centromere Scientific Reports \| (2023) 13:8998 \| https://doi.org/10.1038/s41598-023-35764-7 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Briefly, a mixture of 100 ng of oxysterol deuterium-labeled (7α-hydroxycholesterol-d7, 7β-hydroxycholesterol-d7, 25-hydroxycholesterol-d7, 27-hydroxycholesterol-d7) (Avanti Polar Lipids, Alabaster, USA) was added as inter- nal standard. Alkaline saponification was done by adding 10 mL of ethanolic KOH (0.4 M) solution for 2 h, at room temperature with adjustment to pH 7 with phosphoric acid. Then 20 mL of chloroform and 6 mL of water were added to the sample. After strong shaking and centrifugation at 4 °C, the aqueous phase was eliminated and the organic phase evaporated. The lipid extract was dissolved in toluene (1 mL). Oxysterols were separated from cholesterol by solid-phase extraction (Sigma- Aldrich Supelclean LC-Si SPE Tubes SUPELCO, Bellefonte, USA). The sample (1 mL in toluene) was placed in the column previously conditioned with 2 mL of hexane, following washing with 1 mL of hexane. Sterols were eluted with 1.5% isopropanol in hexane (8 mL), and oxysterols were further eluted with 30% isopropanol in hexane (6 mL). Then, the solvent was evaporated and samples were deri- vatized (100 μL of pyridine and 100 μL of N, O-bis (trimethylsilyl) trifluoroacetamide with trimethylchlorosilane (BSTFA; Sigma- Aldrich, St. Louis, USA), for 1 h at 60 °C). One microliter of the derivatized sample (1 µL) was injected into a gas chromatograph coupled to a mass spectrometer (Shimadzu GCMS-QP2010, Kyoto, Japan) by an automatic injector and analyzed in selected ion monitoring. The separation was carried out on a Restek capillary column (100% dimethyl polysiloxane-RxiR -1 ms. Cat. #13,323), 30 m, internal diameter 0.25 mm, for 30 min, using helium as mobile phase, with a constant linear velocity of 44.1 cm/sec. The oven started at 240 °C with an increment of 5◦C/min, for 7 min up to 290 °C. The mass spectrometer operated in impact electron mode at an ionization voltage of 70 eV with the temperature of the ion source at 300 °C40. Comparisons from standard curves peak areas to internal standards were made in order to quantify the oxysterols, as previously described41. https://doi.org/10.1038/s41598-023-35764-7 Scientific Reports \| (2023) 13:8998 www.nature.com/scientificreports/ Statistical analysis. The Shapiro–Wilk test was used to analyze normality; parametric data were repre- sented by the mean and standard deviation of the sample and compared by Student’s t-test, with or without Welch correction, depending on the performance of Levene’s Test regarding sample sphericity. www.nature.com/scientificreports/ When analyzing more than two samples, analysis of variance of non-repeated measures was used with Tukey or Games-Howell post-test, according to the homo or heteroscedasticity, respectively; with bootstrap when necessary. Covariate analyses were performed to adjust the outcome variables with Sidak’s post-test. Non-parametric data were rep- resented by the median, lower and upper quartiles, and compared with each other using the Mann–Whitney test for two samples, and when comparing more than two samples, the Kruskal–Wallis test was used. If normaliza- tion was necessary, variables were log-transformed. Frequencies were compared using the Chi-square test. A value of P < 0.05 was considered statistically significant. IBM® SPSS Statistics (version 27.0), GraphPad Prisma (version 5.04) for Windows, and Microsoft® Excel for Mac (version 16.52) software were used for data tabulation and analysis. Ethical approval and informed consent. The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Ethics Committee of the Universidade Nove de Julho (# 3.139.460; February 2019); Centro de Referência da Saúde da Mulher (Hospital Pérola Byington; #3.225.220; March 2019) and Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (#3.317.909; March 2019). Results In the BC group, the median age and the frequency of postmenopausal status, dyslipidemia and hypertension were higher in comparison to the CTR, although BMI, overweight (BMI ≥ 25 kg/m2) and reported statin use were similar between groups. Among tumors, the frequency (%) of histological types was: ductal (87.7), lobular (7.4), mucinous (4.3), and metaplastic (0.6). As expected, a higher frequency of LA and LB tumors was observed. 70.8% of BC women were categorized in clinical stages I and II, and 29.2% in advanced stages of disease (stages III and IV) (Table 1). The postmenopausal status was similar among clinical stages (stage I = 66.7%; stage II = 59.6%; stage III = 74.2%; and stage IV = 68.8%; χ2 = 2.011; P = 0.570). The luminal types (LA and LB) accounted for the majority of tumors (68%), with most cases classified as stages I and II (70.8%). Women with TN tumors had the Table 1. Age, anthropometric and clinical data of CTR and BC women. Values in median and interquartile ranges 25–75%. CTR, control women; BC, breast cancer women; BMI, body mass index; LA, luminal A; LB, luminal B; TN, triple-negative. Comparisons were done by the χ2 test. CTR BC P n 150 163 Age (years)* 51 (38–59) 55 (49–63) < 0.001 BMI (kg/m2)* 28 (25–31) 27 (24–31) 0.644 n % n % BMI ≥ 25 kg/m2 108 73.5 106 68.4 0.179 Premenopausal 75 50.7 56 34.4 < 0.001 Postmenopausal 73 49.3 107 65.6 < 0.001 Dyslipidemia 18 12 35 21.9 0.021 Statin use 9 6 19 11.8 0.074 Hypertension 33 22.4 61 37.9 0.003 Histological type of BC Ductal 143 87.7 Lobular 12 7.4 Mucinous 7 4.3 Metaplastic 1 0.6 Molecular type of BC LA 46 28.2 LB 65 39.8 HER2 26 16 TN 26 16 Clinical stage of disease I 57 35.4 II 57 35.4 III 31 19.3 IV 16 9.9 Table 1. Age, anthropometric and clinical data of CTR and BC women. Values in median and interquartile ranges 25–75%. CTR, control women; BC, breast cancer women; BMI, body mass index; LA, luminal A; LB, luminal B; TN, triple-negative. Comparisons were done by the χ2 test. Table 1. Age, anthropometric and clinical data of CTR and BC women. Values in median and interquartile ranges 25–75%. CTR, control women; BC, breast cancer women; BMI, body mass index; LA, luminal A; LB, luminal B; TN, triple-negative. Comparisons were done by the χ2 test. www.nature.com/scientificreports/ highest frequency of advanced disease, with 60% being classified as stages III and IV, while those with LA tumors had the lowest percentage of advanced disease (8.9%) (Supplementary file S2).i highest frequency of advanced disease, with 60% being classified as stages III and IV, while those with LA tumors had the lowest percentage of advanced disease (8.9%) (Supplementary file S2).i i Plasma lipid profile adjusted per age and the ratios TC/apoB and TG/HDLc were similar between CTR and BC groups (Table 2). In isolated HDL, the concentrations of TC, TG, and PL were lower in the BC group, while apoA-I was similar in both groups. The 27HC content in HDL was lower in BC as compared to CTR even after adjustment for age, but the concentrations of 24HC and 25HC in HDL were similar between groups. Despite some alterations in composition, HDL particles from BC and CTR presented similar abilities in mediating macrophage cholesterol removal (Table 2). g When comparing the molecular types of BC, age and BMI were similar, but TC was higher in TN as compared to LA, LB, and HER2 tumors. Besides, plasma levels of TG, apoB, and non-HDLc were greater in TN as compared to LB and HER2 (Table 3). Although plasma lipids were different in TN tumors, the ratios TC/apoB (P = 0.065) and TG/HDLc (P = 0.091) did not reach statistical difference. Oxysterols in HDL were similar among LA, LB, HER2, and TN as well as the ability of cell cholesterol removal (Table 3).l When subjects were categorized according to the stage of the disease reflected by increased levels of Ki67, it was observed that plasma lipids and their ratios were similar among groups. The composition of HDL in TC, PL, apoA-I, and oxysterols was similar among stages. Only HDL-TG was increased on stage III as compared to I, II, and IV. Reduced cholesterol efflux was observed with HDL isolated from BC women in more advanced stages of the disease (III and IV) as compared to stages I and II. (Table 4). Discussionh The present study investigated plasma lipid levels, HDL composition, and functionality in newly diagnosed women with BC compared to CTR women, taking into account the molecular classification of the tumor and the clinical stage of the disease. The results showed that (1) BC cases (including all molecular types) had similar concentrations of plasma lipids adjusted for age but HDL particles less enriched in TC, PL, TG, and 27HC com- pared to CTR; (2) TN cases had a higher concentration of plasma lipids as compared to other molecular types of tumor; (3) HDL functionality along the first step of reverse cholesterol transport was impaired in advanced stages of BC, meaning less cholesterol removal from macrophages. Table 2. Plasma lipids, HDL composition, and HDL-mediated cholesterol efflux in women with BC and CTR. Plasma lipids were determined by enzymatic colorimetric methods and apoB by immunoturbidimetry. HDLc was determined after precipitation of lipoproteins containing apoB; non-HDLc was calculated as TC-HDLc. Oxysterols were quantified in isolated HDL by gas chromatography-mass spectrometry (GC–MS). The cholesterol efflux was determined in BMDM overloaded with 14C-cholesterol and acetylated LDL, utilizing 50 µg/mL of HDL as a cholesterol acceptor. BMI, body mass index; TC, total cholesterol; TG, triglycerides; apoB, apolipoprotein B; HDLc, high-density lipoprotein cholesterol; LDLc, low density lipoprotein cholesterol; VLDLc, very low density lipoprotein cholesterol; PL, phospholipids; apoA-I, apolipoprotein A-I; 24HC, 24-hydroxycholesterol (CTR, n = 82; BC, n = 82); 25HC, 25-hydroxycholesterol (CTR, n = 82; BC, n = 82); 27HC, 27-hydroxycholesterol (CTR, n = 81; BC, n = 80). Comparisons were made using the Mann–Whitney test; data shown in median and interquartile ranges (25–75%). P values were age-adjusted. Results CTR BC P n 150 163 Age (years)* 51 (38–59) 55 (49–63) < 0.001 BMI (kg/m2)* 28 (25–31) 27 (24–31) 0.644 n % n % BMI ≥ 25 kg/m2 108 73.5 106 68.4 0.179 Premenopausal 75 50.7 56 34.4 < 0.001 Postmenopausal 73 49.3 107 65.6 < 0.001 Dyslipidemia 18 12 35 21.9 0.021 Statin use 9 6 19 11.8 0.074 Hypertension 33 22.4 61 37.9 0.003 Histological type of BC Ductal 143 87.7 Lobular 12 7.4 Mucinous 7 4.3 Metaplastic 1 0.6 Molecular type of BC LA 46 28.2 LB 65 39.8 HER2 26 16 TN 26 16 Clinical stage of disease I 57 35.4 II 57 35.4 III 31 19.3 IV 16 9.9 Table 1. Age, anthropometric and clinical data of CTR and BC women. Values in median and interquartile ranges 25–75%. CTR, control women; BC, breast cancer women; BMI, body mass index; LA, luminal A; LB, luminal B; TN, triple-negative. Comparisons were done by the χ2 test. Table 1. Age, anthropometric and clinical data of CTR and BC women. Values in median and interquartile ranges 25–75%. CTR, control women; BC, breast cancer women; BMI, body mass index; LA, luminal A; LB, luminal B; TN, triple-negative. Comparisons were done by the χ2 test. https://doi.org/10.1038/s41598-023-35764-7 Scientific Reports \| (2023) 13:8998 \| www.nature.com/scientificreports/ Discussionh CTR BC P n* 132 126 Age (years) 48 (37–58) 55 (49–63) < 0.001 BMI (kg/m2) 28 (25–31) 27 (24–31) 0.391 TC (mg/dL) 172 (151–199) 178 (155–206) 0.423 TG (mg/dL) 84 (59–122) 92 (68–116) 0.224 apoB (mg/dL) 111 (89–139) 107 (82–141) 0.428 HDLc (mg/dL) 41 (35–51) 40 (32–48) 0.109 LDLc (mg/dL) 111 (95–131) 115 (95–142) 0.522 VLDLc (mg/dL) 17 (12–24) 18 (14–23) 0.224 non-HDLc (mg/dL) 127 (109–156) 136 (113–163) 0.252 TC/apoB (mg/dL) 1,5 (1,3–1,9) 1,7 (1,4–2,0) 0.065 TG/HDL (mg/dL) 1,9 (1,3–3,1) 2,3 (1,5–3,5) 0.091 HDL-TC (mg/dL) 44 (34–55) 37 (30–46) 0.001 HDL-TG (mg/dL) 14 (10–19) 13 (10–16) 0.039 HDL-PL (mg/dL) 98 (80–131) 87 (73–116) 0.006 HDL-apoA-I (mg/dL) 104 (82–141) 112 (82–142) 0.700 24HC (ng/mL) 38.9 (25.9–99.0) 34.9 (24.1–65.4) 0.203 25HC (ng/mL) 8.1 (5.1–10.7) 6.9 (5.4–9.3) 0.111 27HC (ng/mL) 12.6 (7.4–19.4) 7.7 (4.6–13.1) 0.002 14C-cholesterol efflux (%) 12 (9–18) 13 (9–19) 0.501 CTR BC P n* 132 126 Age (years) 48 (37–58) 55 (49–63) < 0.001 BMI (kg/m2) 28 (25–31) 27 (24–31) 0.391 TC (mg/dL) 172 (151–199) 178 (155–206) 0.423 TG (mg/dL) 84 (59–122) 92 (68–116) 0.224 apoB (mg/dL) 111 (89–139) 107 (82–141) 0.428 HDLc (mg/dL) 41 (35–51) 40 (32–48) 0.109 LDLc (mg/dL) 111 (95–131) 115 (95–142) 0.522 VLDLc (mg/dL) 17 (12–24) 18 (14–23) 0.224 non-HDLc (mg/dL) 127 (109–156) 136 (113–163) 0.252 TC/apoB (mg/dL) 1,5 (1,3–1,9) 1,7 (1,4–2,0) 0.065 TG/HDL (mg/dL) 1,9 (1,3–3,1) 2,3 (1,5–3,5) 0.091 HDL-TC (mg/dL) 44 (34–55) 37 (30–46) 0.001 HDL-TG (mg/dL) 14 (10–19) 13 (10–16) 0.039 HDL-PL (mg/dL) 98 (80–131) 87 (73–116) 0.006 HDL-apoA-I (mg/dL) 104 (82–141) 112 (82–142) 0.700 24HC (ng/mL) 38.9 (25.9–99.0) 34.9 (24.1–65.4) 0.203 25HC (ng/mL) 8.1 (5.1–10.7) 6.9 (5.4–9.3) 0.111 27HC (ng/mL) 12.6 (7.4–19.4) 7.7 (4.6–13.1) 0.002 14C-cholesterol efflux (%) 12 (9–18) 13 (9–19) 0.501 Table 2. Plasma lipids, HDL composition, and HDL-mediated cholesterol efflux in women with BC and CTR. Plasma lipids were determined by enzymatic colorimetric methods and apoB by immunoturbidimetry. HDLc was determined after precipitation of lipoproteins containing apoB; non-HDLc was calculated as TC-HDLc. Oxysterols were quantified in isolated HDL by gas chromatography-mass spectrometry (GC–MS). The cholesterol efflux was determined in BMDM overloaded with 14C-cholesterol and acetylated LDL, utilizing 50 µg/mL of HDL as a cholesterol acceptor. Table 2. Plasma lipids, HDL composition, and HDL-mediated cholesterol efflux in women with BC and CTR. Plasma lipids were determined by enzymatic colorimetric methods and apoB by immunoturbidimetry. HDLc was determined after precipitation of lipoproteins containing apoB; non-HDLc was calculated as TC-HDLc. Oxysterols were quantified in isolated HDL by gas chromatography-mass spectrometry (GC–MS). The cholesterol efflux was determined in BMDM overloaded with 14C-cholesterol and acetylated LDL, utilizing 50 µg/mL of HDL as a cholesterol acceptor. BMI, body mass index; TC, total cholesterol; TG, triglycerides; apoB, apolipoprotein B; HDLc, high-density lipoprotein cholesterol; LDLc, low density lipoprotein cholesterol; VLDLc, very low density lipoprotein cholesterol; PL, phospholipids; apoA-I, apolipoprotein A-I; 24HC, 24-hydroxycholesterol (CTR, n = 82; BC, n = 82); 25HC, 25-hydroxycholesterol (CTR, n = 82; BC, n = 82); 27HC, 27-hydroxycholesterol (CTR, n = 81; BC, n = 80). Comparisons were made using the Mann–Whitney test; data shown in median and interquartile ranges (25–75%). P values were age-adjusted. Discussionh BMI, body mass index; TC, total cholesterol; TG, triglycerides; apoB, apolipoprotein B; HDLc, high-density lipoprotein cholesterol; LDLc, low density lipoprotein cholesterol; VLDLc, very low density lipoprotein cholesterol; PL, phospholipids; apoA-I, apolipoprotein A-I; 24HC, 24-hydroxycholesterol (CTR, n = 82; BC, n = 82); 25HC, 25-hydroxycholesterol (CTR, n = 82; BC, n = 82); 27HC, 27-hydroxycholesterol (CTR, n = 81; BC, n = 80). Comparisons were made using the Mann–Whitney test; data shown in median and interquartile ranges (25–75%). P values were age-adjusted. Table 2. Plasma lipids, HDL composition, and HDL-mediated cholesterol efflux in women with BC and CTR. Plasma lipids were determined by enzymatic colorimetric methods and apoB by immunoturbidimetry. HDLc was determined after precipitation of lipoproteins containing apoB; non-HDLc was calculated as TC-HDLc. Oxysterols were quantified in isolated HDL by gas chromatography-mass spectrometry (GC–MS). The cholesterol efflux was determined in BMDM overloaded with 14C-cholesterol and acetylated LDL, utilizing 50 µg/mL of HDL as a cholesterol acceptor. BMI, body mass index; TC, total cholesterol; TG, triglycerides; apoB, apolipoprotein B; HDLc, high-density lipoprotein cholesterol; LDLc, low density lipoprotein cholesterol; VLDLc, very low density lipoprotein cholesterol; PL, phospholipids; apoA-I, apolipoprotein A-I; 24HC, 24-hydroxycholesterol (CTR, n = 82; BC, n = 82); 25HC, 25-hydroxycholesterol (CTR, n = 82; BC, n = 82); 27HC, 27-hydroxycholesterol (CTR, n = 81; BC, n = 80). Comparisons were made using the Mann–Whitney test; data shown in median and interquartile ranges (25–75%). P values were age-adjusted. Discussionh https://doi.org/10.1038/s41598-023-35764-7 Scientific Reports \| (2023) 13:8998 \| www.nature.com/scientificreports/ LA LB HER2 TN P n 46 65 26 26 Age (years) 60 (52–64) 54 (48–63) 55 (49–61) 53 (41–61) 0.107 BMI (kg/m2) 27 (24–31) 28 (25–31) 27 (24–31) 27 (24–30) 0.793 TC (mg/dL) 193 (162–228) 173 (155–205) 173 (151–193) 198 (173–214) 0.022# TG (mg/dL) 103 (66–139) 84 (67–110) 82 (63–105) 102 (91–134) 0.026$ apoB (mg/dL) 129 (90–151) 98 (81–123) 100 (81–127) 134 (102–158) 0.005$ HDLc (mg/dL) 41 (34–49) 42 (33–53) 42 (32–52) 42 (36–50) 0.997 LDLc (mg/dL) 123 (99––155) 111 (90–138) 117 (85–134) 127 (98–148) 0.116 VLDLc (mg/dL) 21 (13–28) 17 (13–22) 16 (13–21) 20 (18–27) 0.026$ non-HDLc (mg/dL) 147 (121–184) 129 (104–162) 135 (105–151) 155 (130–174) 0.013$ TC/apoB 1.7 (1.4–2.0) 1.8 (1.5–2.1) 1.8 (1.5–2.0) 1.5 (1.3–2.0) 0.313 TG/HDLc 2.3 (1.3–3.4) 1.9 (1.3–3.1) 1.7 (1.3–3.2) 2.5 (2.0–3.4) 0.358 HDL-TC (mg/dL) 39 (30–49) 40 (27–52) 41 (29–49) 45 (23–58) 0.886 HDL-TG (mg/dL) 15 (12–34) 17 (11–62) 13 (11–18) 24 (14–91) 0.055 HDL-PL (mg/dL) 90 (71–119) 90 (77–121) 104 (82–124) 96 (77–117) 0.435 HDL-apoA-I (mg/dL) 114 (83–142) 108 (76–150) 108 (86–139) 107 (90–123) 0.894 24HC (ng/mL) 28 (22–49) 36 (23–78) 35 (28–58) 35 (24–88) 0.681 25HC (ng/mL) 7.3 (5.6–9.2) 8.3 (6.4–9.8) 6.6 (4.6–9.4) 6.0 (4.5–7.6) 0.110 27HC (ng/mL) 5.8 (4.5–10.6) 10.9 (4.4–13.8) 7.2 (4.7–14.6) 5.7 (4.1–14.2) 0.580 14C-cholesterol efflux (%) 14 (10–17) 12 (9–19) 13 (8–15) 11 (8–18) 0.921 Table 3. Plasma lipids, HDL composition, and HDL-mediated cholesterol efflux according to the molecular type of BC. Plasma lipids were determined by enzymatic colorimetric methods and apoB by immunoturbidimetry. HDLc was determined after precipitation of lipoproteins containing apoB; non-HDLc was calculated as TC-HDLc. Oxysterols were quantified in isolated HDL by gas chromatography-mass spectrometry (GC–MS). The cholesterol efflux was determined in BMDM overloaded with 14C-cholesterol and acetylated LDL, utilizing 50 µg/mL of HDL as a cholesterol acceptor. CTR, control women; BC, breast cancer; BMI, body mass index; TC, total cholesterol; TG, triglycerides; apoB, apolipoprotein B; HDLc, high- density lipoprotein cholesterol; LDLc, low density lipoprotein cholesterol; VLDLc, very low density lipoprotein cholesterol; PL, phospholipids; apoA-I, apolipoprotein A-I; 24HC, 24-hydroxycholesterol (CTR, n = 82; BC, n = 82); 25HC, 25-hydroxycholesterol (CTR, n = 82; BC, n = 82); 27HC, 27-hydroxycholesterol (CTR, n = 81; BC, n = 80). Comparisons were made using the Kruskall-Wallis test with a significance of 0.05. Data shown in median and interquartile ranges (25–75%). LA, luminal A; LB, luminal B; TN, triple negative. Table 3. Plasma lipids, HDL composition, and HDL-mediated cholesterol efflux according to the Discussionh # in comparison to LA, LB, and HER2; $ in comparison to LB and HER2. Plasma lipids are considered contributing factors for many types of cancer, including BC42–46, although the interpretation and comparison of studies are limited by the heterogeneity of BC, disease duration and staging, ethnic diversity, age, menopausal status, lifestyle, and oncological treatments, which are confounding factors. Furthermore, diabetes mellitus, insulin resistance, and obesity, which can alter lipoprotein profiles, are also present in many cases of BC as potential contributors to the disease development and progression. The current study exclusively included newly diagnosed, treatment-naïve participants, excluding smoking habit and women with diabetes mellitus and comorbidities that affect lipids. Moreover, the data were adjusted for age, which was higher in the BC group. As a result, many factors contributing to dyslipidemia were minimized, and the strict matching between the CTR and BC groups likely accounted for the absence of variation in the plasma lipid profile between all BC cases and CTR. Even with the exclusion data of women informing dylipidemia and/or statin use, results were similar (data not shown). https://doi.org/10.1038/s41598-023-35764-7 Scientific Reports \| (2023) 13:8998 \| www.nature.com/scientificreports/ Table 4. Discussionh Plasma lipids, HDL composition, and HDL-mediated cholesterol efflux of BC Plasma lipids were determined by enzymatic colorimetric methods and ap I II III IV P n 57 57 31 16 Age (years) 60 (49–64) 53 (46–61) 56 (51–61) 55 (48–66) 0.287 BMI (kg/m2) 27 (24–31) 28 (25–31) 27 (25–31) 25 (23–30) 0.291 Ki67 20 (10–30) 20 (10–30) 30 (20–70) 35 (30–72) < 0.001 I–III < 0.001 I–IV < 0.001 II–III 0.015 II–IV 0.005 III–IV 0.416 I–II 0.122 TC (mg/dL) 187 (162–218) 179 (155–203) 189 (167–208) 183 (154–241) 0.600 TG (mg/dL) 94 (64–117) 92 (73–122) 92 (73–115) 84 (60–111) 0.760 apoB (mg/dL) 105 (81–138) 107 (87–137) 109 (84–141) 105 (84–150) 0.972 HDLc (mg/dL) 42 (33–51) 42 (33–50) 46 (39–55) 41 (33–45) 0.376 LDLc (mg/dL) 121 (98–146) 109 (92–137) 118 (97–139) 117 (98–172) 0.476 VLDLc (mg/dL) 21 (13–28) 17 (13–22) 16 (13–21) 20 (18–27) 0.760 non-HDLc (mg/dL) 140 (118–169) 132 (105–159) 139 (116–163) 132 (108–196) 0.640 TC/apoB 1.8 (1.4–2.1) 1.7 (1.4–2.0) 1.7 (1.4–2.0) 1.6 (1.2–2.0) 0.685 TG/HDLc 1.9 (1.5–3.2) 2.3 (1.4–3.3) 2.1 (1.0–3.3) 2.4 (1.6–2.7) 0.875 HDL-TC (mg/dL) 44 (30–51) 38 (28–49) 45 (26–67) 38 (31–51) 0.336 HDL-TG (mg/dL) 15 (12–44) 15 (11–39) 38 (14–92) 18 (10–56) 0.032 I–IV 0.831 II–IV 0.809 III–IV 0.035 I–II0.968 –III 0.009 II–III 0.009 HDL-PL (mg/dL) 90 (75–119) 89 (76–114) 117 (87–124) 87 (79–113) 0.116 HDL-apoA-I (mg/dL) 116 (83–144) 104 (78–135) 114 (85–141) 109 (94–149) 0.733 24HC (ng/mL) 31 (19–48) 35 (26–75) 28 (25–50) 56 (34–128) 0.276 25HC (ng/mL) 6.5 (5.1–8.5) 7.1 (5.3–8.7) 9.7 (6.6–10.6) 6.0 (4.3–8.9) 0.247 27HC (ng/mL) 8.8 (4.6–15.1) 9.7 (4.6–14.4) 5.0 (3.9–9.1) 5.7 (3.4–10.1) 0.267 14C-cholesterol efflux (%) 14 (10–20) 13 (10–18) 9 (7–15) 9 (7–12) 0.011 III–IV 0.424 II–IV 0.014 I–IV 0.004 II–III 0.088 I–III 0.033 I–II 0.578 Table 4. Plasma lipids, HDL composition, and HDL-mediated cholesterol efflux according to clinical stages of BC. Plasma lipids were determined by enzymatic colorimetric methods and apoB by immunoturbidimetry. HDLc was determined after precipitation of lipoproteins containing apoB; non-HDLc was calculated as TC-HDLc. Oxysterols were quantified in isolated HDL by gas chromatography-mass spectrometry (GC– MS). The cholesterol efflux was determined in BMDM overloaded with 14C-cholesterol and acetylated LDL, utilizing 50 µg/mL of HDL as a cholesterol acceptor. www.nature.com/scientificreports/ Although the ability of HDL to remove cell cholesterol was preserved in all women with BC when compared to CTR women, the reduced content of TC, PL, and 27HC in the HDL particles from BC cases may be a conse- quence of alterations in the tumor cells that hinder sterol exportation to HDL. In addition to the intrinsic ability of HDL to receive cholesterol, it is important to consider that lipid efflux is modulated by cellular components, such as the bioavailability of free or unesterified cholesterol and the content and functionality of HDL recep- tors sch as ABCA-1, ABCG-1, and SR-B157. ABCA-1, which is expressed in the mammary gland, is indicated to be reduced in BC and associated with positive lymph nodes58. Its expression negatively affects the therapeutic efficiency of chemotherapy59 and is considered by some authors as a marker of TN tumors60. Conversely, its deficiency contributes to an increase in cellular and mitochondrial cholesterol content, which hinders cell death processes mediated by this organelle, thereby favoring tumor cell survival24. 6 6 y g y g Solid tumors are known to accumulate large amounts of cholesterol48,61,62, which is mainly due to the increased synthesis and uptake of lipoproteins48,63. The scavenger receptor class B type 1 (SR-B1) mediates the efflux of free cholesterol from cells and promotes its transfer to HDL. However, SR-B1 can also facilitate the uptake of modified lipoproteins, leading to the supply of cholesterol to cells and tumor progression64. Higher expression of SR-B1 is associated with increased aggressiveness and a worse prognosis of tumors23,65,66, while mutations in the Scarb1 gene have been shown to inhibit tumor proliferation67. g Moreover, the reduced lipid content in HDL could be associated with the decreased detachment of surface components of TG-rich lipoproteins during lipolysis mediated by lipoprotein lipase, which is now recognized as a process of reverse transport of remnant cholesterol68. However, it is difficult to determine which mechanisms are involved in shaping the composition of HDL based on the present findings. These events cannot be captured by a simple determination of HDLc in plasma, which may explain some of the controversies surrounding the association between HDLc and BC. According to the clinical stage of the disease, a diminished intrinsic capacity of the HDL particle to remove cell cholesterol was observed in stages III and IV in comparison to stages I and II. www.nature.com/scientificreports/ www.nature.com/scientificreports/ In the casuistic of the present investigation, as anticipated, the majority of tumors were of the luminal types in in stages I and II. Triple-negative cases had the highest frequency of advanced disease and higher levels of TC, apoB, nonHDLc, VLDLc, and TG compared to LA, LB, and HER-2. However, HDLc, composition, and functionality of the HDL particle were similar among all types. These alterations were independent of BMI and may represent a distinct characteristic of TN tumors, directing lipids as an energy source and structural com- ponents for tumor growth in a histotype with a more aggressive clinical course, lower survival rate, and a higher metastasis rate. These factors should be considered when exploring the association between plasma lipids and BC and deserves further investigation.i g Also, findings are consistent with previous studies that have reported increased levels of VLDL-TG in TN BC. However, changes in CT/apoB and TG/HDLc did not reach statistical significance. A retrospective cohort study demonstrated that a high pretreatment TG/HDLc ratio was an independent predictor of the overall survival rate in TN tumors47. The enhanced expression of the LDL receptor and LDLR-related proteins 5 and 6 were found in TN tumors and associated with the higher ability of tumor growth and invasion; while the knockdown of LDLR-5 and 6 decreased tumorigenesis48–51. A syngeneic tumor graft model demonstrated that tumor growth leads to changes in host lipid metabolism, promoting the synthesis of very low-density lipoproteins while inhibiting their metabolism, ultimately providing more energy to the tumor52. Furthermore, a targeted plasma liquid chromatography-tandem mass spectrometry (LC–MS/MS) approach identified potential lipid biomarkers in TN BC cases, such as ceramides, phosphatidyl- choline, lysophosphatidylcholine, and diacylglycerol, thus confirming the alteration of the lipid profile in this molecular subtype53. Unlike other studies, it was not found in the present investigation any changes in plasma lipids in cases of HER2-positive BC45,54,55. However, in individuals with HER2-positive BC, the analysis of the lipoprotein pro- file using nuclear magnetic resonance demonstrated an enhanced level of specific VLDL subfractions, which served as a marker of plasma lipid alteration compared to control women, even with similar BMI between the groups. Reductions in HDL subfractions were surrogate markers for the response to neoadjuvant chemotherapy follow-up56. Discussionh CTR, control women; BC, breast cancer; BMI, body mass index; TC, total cholesterol; TG, triglycerides; apoB, apolipoprotein B; HDLc, high-density lipoprotein cholesterol; LDLc, low density lipoprotein cholesterol; VLDLc, very low density lipoprotein cholesterol; PL, phospholipids; apoA-I, apolipoprotein A-I; 24HC, 24-hydroxycholesterol (CTR, n = 82; BC, n = 82); 25HC, 25-hydroxycholesterol (CTR, n = 82; BC, n = 82); 27HC, 27-hydroxycholesterol (CTR, n = 81; BC, n = 80). Comparisons were made using the Kruskall-Wallis test with a significance of 0.05. Data shown in median and interquartile ranges (25–75%). Table 4. Plasma lipids, HDL composition, and HDL-mediated cholesterol efflux according to clinical stages of BC. Plasma lipids were determined by enzymatic colorimetric methods and apoB by immunoturbidimetry. HDLc was determined after precipitation of lipoproteins containing apoB; non-HDLc was calculated as TC-HDLc. Oxysterols were quantified in isolated HDL by gas chromatography-mass spectrometry (GC– MS). The cholesterol efflux was determined in BMDM overloaded with 14C-cholesterol and acetylated LDL, utilizing 50 µg/mL of HDL as a cholesterol acceptor. CTR, control women; BC, breast cancer; BMI, body mass index; TC, total cholesterol; TG, triglycerides; apoB, apolipoprotein B; HDLc, high-density lipoprotein cholesterol; LDLc, low density lipoprotein cholesterol; VLDLc, very low density lipoprotein cholesterol; PL, phospholipids; apoA-I, apolipoprotein A-I; 24HC, 24-hydroxycholesterol (CTR, n = 82; BC, n = 82); 25HC, 25-hydroxycholesterol (CTR, n = 82; BC, n = 82); 27HC, 27-hydroxycholesterol (CTR, n = 81; BC, n = 80). Comparisons were made using the Kruskall-Wallis test with a significance of 0.05. Data shown in median and interquartile ranges (25–75%). Scientific Reports \| (2023) 13:8998 \| https://doi.org/10.1038/s41598-023-35764-7 www.nature.com/scientificreports/ www.nature.com/scientificreports/ To our knowledge, this is the first study demonstrating the loss of HDL function along the first step of reverse cholesterol transport according to disease burden, independent of plasma lipids. However, the study has certain limitations, such as the absence of detailed clinical data, including components of metabolic syndrome and visceral adiposity, as well as lifestyle information. Nevertheless, BMI was similar between the groups, and comorbidities that were associated with changes in lipid metabolism were excluded. Moreover, it was not iden- tified the specific HDL structure components that were responsible for the impairment of cholesterol efflux, independently of lipids and apo A-I. A more detailed analysis of HDL proteomics, lipidomics, and microRNAs may provide additional information. A subanalysis of TN cases from this casuistic (n = 27) showed an enhanced antioxidant role of the HDL particle—reflected by retardation in LDL oxidation—that was positively associated to the apoA-I but independent of HDLc70.hi The results of this investigation confirmed that TN tumors have increased levels of plasma lipids, potentially driving the more aggressive type of BC. This study also contributes to a better understanding of the role of HDL in BC, particularly in patients with a worse prognosis, where HDL’s ability to remove excess cellular cholesterol is impaired. In comparison to the CTR group, only the composition of HDL was distinctive in subjects with BC. A prospective cohort study will aid in determining the prognostic value of HDL composition and functionality in predicting BC outcomes. p g Finally, results contribute to the growing understanding of HDL’s role in breast cancer pathophysiology. Since routine laboratory metrics cannot directly indicate this, it is essential to recognize the impact of HDL functional- ity on BC and develop new metrics accordingly. Data availability y All data reported are included in the manuscript and raw data can be kindly shared upon personal request to the corresponding author MP (m.passarelli@uni9.pro.br). Received: 8 November 2022; Accepted: 23 May 2023 Received: 8 November 2022; Accepted: 23 May 2023 References Oncogenesis 5(1), e189 (2016) k Ä l d l d d f h ff f bl d l d b k ( ) jl p p g g g 9. Nowak, C. & Ärnlöv, J. A Mendelian randomization study of the effects of blood lipids on breast cancer risk. Nat. Commun. 9(1) 3957 (2018).hf ( ) 10. Li, X. et al. The effect of preoperative serum triglycerides and high-density lipoprotein-cholesterol levels on the prognosis of breast cancer. Breast 32, 1–6 (2017). 1. Fan, Y. et al. Decreased serum HDL at initial diagnosis correlates with worse outcomes for triple-negative breast cancer but not non-TNBCs. Int. J. Biol. Markers 30(2), e200–e207 (2015). 2. His, M. et al. Associations between serum lipids and breast cancer incidence and survival in the E3N prospective cohort study Cancer Causes Control. 28, 77–88 (2017). 3. Chandler, P. D. et al. Lipid biomarkers and long-term risk of cancer in the Women’s Health Study. Am. J. Clin. 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Beloribi-Djefaflia, S., Vasseur, S. & Guillaumond, F. Lipid metabolic reprogramming in cancer cells. www.nature.com/scientificreports/ This observation was inde- pendent of changes in HDL composition and plasma lipids, highlighting the dissociation between plasma levels of HDLc and the overall functionality of HDL as a particle. Increasing levels of intracellular cholesterol promote the formation of oxysterols, which suggests an increase in the concentration of 27HC in the tumor microenvi- ronment that negatively influences BC progression. The underlying mechanisms of the reduced HDL ability in removing cell cholesterol in advanced stages of BC were not investigated in the present study. HDL functionality may be impaired by its chemical modification resulting from glycoxidative and inflammatory stress that often accompanies cancer69. Consequently, a vicious circle may be established, which compromises tumor choles- terol homeostasis, exacerbating cancer progression and impairing HDL function. In this sense, HDL may even promote tumor growth, as experimental studies have previously shown that modified HDL was oncogenic42,69. Once again, these findings support the idea that the tumor’s energy demand adapts systemic metabolism to its advantage.h The concept of reverse causation suggests that the tumor may modulate HDL, which may disassociate HDL from being a predictor of tumor risk. Therefore, HDL would serve more as a marker of tumor evolution rather than a protector or inducer of tumor genesis. Bone marrow-derived macrophages were used in this investigation to minimize cellular effects on HDL functionality measurement. However, it is crucial to consider that in vivo, the interplay between lipoprotein subsets and tumor cell biology determines HDL functionality and cell cholesterol content. 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Acknowledgementsh g The authors are thankful to Rafaela V Coutinho, Karina CLB Lima, Jessica AV Siqueira, Julia HC Dezotti, Raiana C Novaes, Jessica Mosello, Karina Cezar, and Jacira X Carvalho for helping in blood collection and obtaining clinical data; to Fernanda Faccioli Schober and Nurya Bustamante de Araújo, from Universidade Nove de Julho Animal Facility Unity for animal caring. Author contributions Conceptualization, M.P., L.H.G.; casuistic selection: M.I.B.A.C.S., M.R.; methodology, M.R., M.F.M.S., S.I.S.A., D.S.P., L.A.P., V.S.N.; formal analysis, M.I.B.A.C.S., M.L.C.C.G., M.P.; investigation; data curation, M.I.B.A.C.S., M.P.; writing—original draft preparation, M.I.B.A.C.S., M.P.; writing—review and editing, M.P.; resources, M.P.; project administration, M.P.; funding acquisition, M.P. All authors have read and agreed to the published ver- sion of the manuscript. www.nature.com/scientificreports/ 91(1), 54–69 (2004). 61. Freeman, M. R. & Solomon, K. R. 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Cancer Res. 64, 1515–1521 (2004). 6. Llaverias, G. et al. Role of cholesterol in the development and progression of breast cancer. Am. J. Pathol. 178, 402–412 (2011). 7. Cao, W. M. et al. A mutant high-density lipoprotein receptor inhibits proliferation of human breast cancer cells. Cancer Res. 64 1515–1521 (2004). 67. Cao, W. M. et al. A mutant high-density lipoprotein receptor inhibits proliferation of human breast cancer cells. Cancer Res. 64, 1515–1521 (2004). Scientific Reports \| (2023) 13:8998 \| https://doi.org/10.1038/s41598-023-35764-7 Fundingh g This research was funded by Fundação de Amparo à Pesquisa do Estado de São Paulo, FAPESP (Grants # 2019/18431-4 to MP; #2022/11186-7 to DSP; #2021/04989-3 to SISA; 2021/02401-9 to MFMS). MP is a recipi- ent of a research award from Conselho Nacional de Desenvolvimento Científico e Tecnológico, CNPq, Brazil. © The Author(s) 2023 Competing interests h p g The authors declare no competing interests. Additional informationh Additional information Supplementary Information The online version contains supplementary material available at https://doi.org/ 10.1038/s41598-023-35764-7. Correspondence and requests for materials should be addressed to M.P. Correspondence and requests for materials should be addressed to M.P. Reprints and permissions information is available at www.nature.com/reprints. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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https://openalex.org/W3102591873	https://www.nature.com/articles/s41598-017-03264-0.pdf	English	null	Abrupt p-n junction using ionic gating at zero-bias in bilayer graphene	Scientific reports	2,017	cc-by	6,753	Abrupt p-n junction using ionic gating at zero-bias in bilayer graphene OPEN Received: 21 March 2017 Accepted: 25 April 2017 Published: xx xx xxxx Received: 21 March 2017 Accepted: 25 April 2017 Published: xx xx xxxx Sameer Grover1, Anupama Joshi1,2, Ashwin Tulapurkar2 & Mandar M. Deshmukh1 Graphene is a promising candidate for optoelectronic applications. In this report, a double gated bilayer graphene FET has been made using a combination of electrostatic and electrolytic gating in order to form an abrupt p-n junction. The presence of two Dirac peaks in the gating curve of the fabricated device confirms the formation of a p-n junction. At low temperatures, when the electrolyte is frozen intentionally, the photovoltage exhibits a six-fold pattern indicative of the hot electron induced photothermoelectric effect that has also been seen in graphene p-n junctions made using metallic gates. We have observed that the photovoltage increases with decreasing temperature indicating a dominant role of supercollision scattering. Our technique can also be extended to other 2D materials and to finer features that will lead to p-n junctions which span a large area, like a superlattice, that can generate a larger photoresponse. Our work creating abrupt p-n junctions is distinct from previous works that use a source–drain bias voltage with a single ionic gate creating a spatially graded p-n junction. Graphene1 has unique optical2–4 and electronic5 properties which has made it a promising material for opto- electronic devices such as photodetectors. The creation of p-n junctions with tunable chemical potentials allows the transduction of light into electrical signals. In conventional electrostatic gating, a metallic gate separated by a dielectric is used. The maximum carrier density, typically 5 × 1012 cm−2 for silicon dioxide, is limited by the breakdown strength of the dielectric. g Electrolytic gating6 is an alternate technique that has the advantage of large achievable carrier densities, ~4 × 1013 cm−2. This is limited by the leakage current through the electrolyte7. The large capacitance is a result of the formation of an interfacial electrical double layer8 with a thickness of ~1 nm. Electrolytic gating has been used for tuning the carrier density in various semiconductors such as organic polymers9, carbon nanotubes10, and superconductors11, 12. Other non-tunable techniques for large doping density include chemical doping13, 14, expo- sure to electron beams15 or light16, 17, incorporating dopant atoms in the lattice18, 19 and other growth techniques20. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Received: 21 March 2017 Accepted: 25 April 2017 Published: xx xx xxxx Abrupt p-n junction using ionic gating at zero-bias in bilayer graphene OPEN Our Work Bilayer graphene Hybrid gating (combination of electrostatic and electrolytic gating) using ionic liquid, formation of abrupt junction and photoresponse study. Table 1. Comparison of reports of formation of p-n junction in graphene and related 2-D materials. Reference Material Used Study Zhang et al.28 MoS2 Spatially graded p-n junction with bias dependent barrier height using ionic liquid Chakraborty et al.27 Bilayer Graphene Spatially graded p-n junction with bias dependent barrier height using solid polymer electrolyte He et al.26 Graphene (CVD) p-n-p junction using ionic liquid and electrostatic back gate. Junction is abrupt but each region is a few millimetres wide and spatial control of the geometry is not possible. Our Work Bilayer graphene Hybrid gating (combination of electrostatic and electrolytic gating) using ionic liquid, formation of abrupt junction and photoresponse study. Table 1. Comparison of reports of formation of p-n junction in graphene and related 2-D materials. Table 1. Comparison of reports of formation of p-n junction in graphene and related 2-D materials. Figure 1. Comparison of the gating scheme discussed in this work with that used previously. (a) Schematic of our gating scheme and the corresponding band diagram with abrupt p-n junction and independent control of VDS. (b) Electrolytic gating scheme used in previous experiments27, 28 where a combination of VDS and VGS is used to create a junction which is not abrupt and does not allow independent control of VDS and the barrier height. Figure 1. Comparison of the gating scheme discussed in this work with that used previously. (a) Schematic of our gating scheme and the corresponding band diagram with abrupt p-n junction and independent control of VDS. (b) Electrolytic gating scheme used in previous experiments27, 28 where a combination of VDS and VGS is used to create a junction which is not abrupt and does not allow independent control of VDS and the barrier height. Figure 1 shows a comparison of the abrupt p-n junction profile that we have formed and compared it to previous reports that use a source–drain bias voltage to create a gradual junction using electrolytic gating. To demonstrate the p-n junction we measure the gating curve and observe two Dirac peaks. We also study the electrical properties of the p-n junction and the photoresponse as a function of the junction barrier height and temperature. Abrupt p-n junction using ionic gating at zero-bias in bilayer graphene OPEN We find that the photoresponse is dominated by the photothermoelectric effect, characterized by a sixfold pattern in the photovoltage, similar to the results obtained with electrostatic dual gates29. The photovolt- age increases as the temperature decreases which is indicative of hot electron thermalization by disorder assisted supercollisions30. Abrupt p-n junction using ionic gating at zero-bias in bilayer graphene OPEN Graphene can be electrolytically gated using different types of electrolytes such as aqueous solutions of salts21, solid polymer electrolytes of lithium or potassium salts in a polymer matrix6, ionic liquids22 and their gels23. Electrolytic gating has been used previously in order to explore properties such as superconductivity in MoS2 24. The achievement of large carrier density is important for the study of properties of graphene below the Bloch-Grüneisen temperature25. The added advantage of this technique is that the ionic liquids have larger optical transmission compared to a metallic top gate. In this article, we demonstrate the formation of a p-n junction in graphene using a combination of electrostatic and electrolytic gating at zero bias. Part of the graphene is covered with a protective layer of hydrogen silsesqui- oxane (HSQ) resist which prevents the electrolytic top gate from influencing the entire graphene region. Creating p-n junctions at zero bias using electrolyte gates is scalable and has not been done before. Graphene p-n junctions have previously been created using metallic gates and with electrolytic gating by putting ionic liquid drop on mil- limetre sized CVD graphene26 and by applying a drain-source bias comparable with the gate voltage27. Using the existing non-zero bias technique, an abrupt p-n junction is difficult to realize. With our technique, we can create an abrupt profile which is important for optoelectronic applications. Further, we have independent control of the source drain bias voltage and p-n junction barrier height and using a zero bias is possible. Table 1 summarizes work related to the formation of p-n junction by various techniques and establishes novel aspect of our work. 1Department of Condensed Matter Physics and Materials Science, Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai, 400005, India. 2Department of Electrical Engineering, Indian Institute of Technology Bombay, Mumbai, 400076, India. Correspondence and requests for materials should be addressed to M.M.D. (email: deshmukh@tifr.res.in) 1 www.nature.com/scientificreports/ Reference Material Used Study Zhang et al.28 MoS2 Spatially graded p-n junction with bias dependent barrier height using ionic liquid Chakraborty et al.27 Bilayer Graphene Spatially graded p-n junction with bias dependent barrier height using solid polymer electrolyte He et al.26 Graphene (CVD) p-n-p junction using ionic liquid and electrostatic back gate. Junction is abrupt but each region is a few millimetres wide and spatial control of the geometry is not possible. Experimentl p Graphene flakes identified with visual contrast and Raman spectroscopy (Supplementary Section I) were mechan- ically exfoliated on Si/SiO2 (300 nm) chips and metallization with titanium (7 nm) and gold (85 nm) is done using standard electron beam lithography. The ionic liquid reacts with electrodes made from chromium and gold and this limits the metals that can be used. A large in-plane electrode is made in the vicinity of graphene to serve as the top gate electrode. p g In order to mask the graphene to protect it from the ionic liquid, we have tried partial coverage with polym- ethylmethacrelate (PMMA) resist, with overexposed PMMA, and HSQ resist. We found that PMMA tended to dissolve in the ionic liquid over time and LiClO4 in a PEO matrix was not sufficiently optically transparent (Supplementary Section II). Multiple electrodes are present on each of the regions so that the the electrical prop- erties can be separately measured and we can verify that the resist protects graphene from ionic gating. We have used a thick (500 nm) HSQ protective layer patterned with a dose of 350 μC/cm2. We have also fabricated devices completely covered with HSQ in order to verify that they are not affected by the ionic top gate (Supplementary Section III). ) An optical image of the device used and the measurement scheme is shown in Fig. 2. Measurements are formed in an optical cryostat with simultaneous measurement of the resistance and photovoltage. p y p g Ionic liquids31 are salts that are liquid at room temperature, are transparent and stable. We have used the ionic liquid EMI-Im (also called EMI-TFSI). Its glass transition temperature32 is 175 K and melting point33 is 258 K. Below the freezing point, the ions in ionic liquid are immobilized and do not respond to externally applied fields. Scientific Reports \| 7: 3336 \| DOI:10.1038/s41598-017-03264-0 2 www.nature.com/scientificreports/ Figure 2. Optical image and resistance measurements. (a) Schematic of a graphene device which is gated both electrostatically and electochemcically and the measurement circuit with simultaneous measurement of resistance and photovoltage. (Supplementary Section VIII). (b) Optical image of the graphene device on which measurements have been performed. The substrate is silicon with 300 nm of silicon dioxide. Source and drain electrodes, and a top gate electrode which is large in comparison with graphene, are made from titanium and gold. Experimentl Source and drain electrodes, and a top gate electrode which is large in comparison with graphene, are made from titanium and gold. The graphene flake is partially masked with 500 nm thick HSQ. The ionic liquid drop is small and does not touch the aluminium wire bonds. (c,d,e,f) Resistance measurements as a function of top gate and back gate voltages at 280 K measured separately for the exposed and covered regions. (c) and (d) Resistance variation with the top gate and back gate in the region exposed to the ionic liquid. This region is influenced by both the gates. (e) and (f) Resistance variation with the top and back gates in the region covered with HSQ. The absence of gating with the top gate shows that HSQ is able to shield part of the graphene flake. Our measurements are conducted above (273 K) and below (30–150 K) the freezing point. Changing the voltage applied to the ionic gate at T ≤ 150 K is done by cycling the temperature. Ionic liquids are hygroscopic34 and their electrical properties are degraded by water absorption. Dehydration is usually done by heating in vacuum or by freeze drying35. Our measurements are performed in an optical cryostat, and the device is initially cooled to around 30 K in vacuum, leading to removal of water. Our measurements are conducted above (273 K) and below (30–150 K) the freezing point. Changing the voltage applied to the ionic gate at T ≤ 150 K is done by cycling the temperature. Ionic liquids are hygroscopic34 and their electrical properties are degraded by water absorption. Dehydration is usually done by heating in vacuum or by freeze drying35. Our measurements are performed in an optical cryostat, and the device is initially cooled to around 30 K in vacuum, leading to removal of water. We have noticed that it is necessary to put a very small amount of ionic liquid so that the size of the droplet is small. When the ionic liquid drop covers a large area, the back gate capacitance increases, similar to the effect seen by Xia et al.36. We have estimated that the effective increase in back gate capacitance is by factor of ~7 for a drop size of ~1 mm2 (Supplementary Section IV). Besides this, a large ionic liquid drop can touch the aluminium wire bonds with which it reacts. Experimentl The graphene flake is partially masked with 500 nm thick HSQ. The ionic liquid drop is small and does not touch the aluminium wire bonds. (c,d,e,f) Resistance measurements as a function of top gate and back gate voltages at 280 K measured separately for the exposed and covered regions. (c) and (d) Resistance variation with the top gate and back gate in the region exposed to the ionic liquid. This region is influenced by both the gates. (e) and (f) Resistance variation with the top and back gates in the region covered with HSQ. The absence of gating with the top gate shows that HSQ is able to shield part of the graphene flake. Figure 2. Optical image and resistance measurements. (a) Schematic of a graphene device which is gated Figure 2. Optical image and resistance measurements. (a) Schematic of a graphene device which is gated both electrostatically and electochemcically and the measurement circuit with simultaneous measurement of resistance and photovoltage. (Supplementary Section VIII). (b) Optical image of the graphene device on which measurements have been performed. The substrate is silicon with 300 nm of silicon dioxide. Source and drain electrodes, and a top gate electrode which is large in comparison with graphene, are made from titanium and gold. The graphene flake is partially masked with 500 nm thick HSQ. The ionic liquid drop is small and does not touch the aluminium wire bonds. (c,d,e,f) Resistance measurements as a function of top gate and back gate voltages at 280 K measured separately for the exposed and covered regions. (c) and (d) Resistance variation with the top gate and back gate in the region exposed to the ionic liquid. This region is influenced by both the gates. (e) and (f) Resistance variation with the top and back gates in the region covered with HSQ. The absence of gating with the top gate shows that HSQ is able to shield part of the graphene flake. Figure 2. Optical image and resistance measurements. (a) Schematic of a graphene device which is gated both electrostatically and electochemcically and the measurement circuit with simultaneous measurement of resistance and photovoltage. (Supplementary Section VIII). (b) Optical image of the graphene device on which measurements have been performed. The substrate is silicon with 300 nm of silicon dioxide. Experimentl We use a 25 μm wire-bonder wire to wick a small amount of the ionic liquid and put in on the graphene device. The HSQ surface, in contrast with overexposed PMMA tends to repel the ionic liquid and this can be an added factor in the ionic liquid preferentially covering only the exposed parts of graphene. The refractive index of the ionic liquid and HSQ are nearly identical (~1.5) so that a surface of uniform refractive index is presented to the incident light (Supplementary Figure S5).i g y g We have also monitored the current through the top gate using a sourcemeter to find the usable electrical limits of the top gate voltage (Supplementary Section V). A top gate voltage of ±2.5 V results in a current less than 1 nA. This current does not change if we turn on the laser illumination, indicating an absence of photochemical reactions. After applying a top gate voltage of 4 V, we have observed that the Ti/Au electrodes directly under the ionic liquid were corroded and the ones protected by HSQ were intact, further indicating that HSQ is able to effectively shield the underlying material from the ionic liquid (Supplementary Figure S5). f y y g q pp y g Figure 2 shows resistance measurements at 280 K as a function of the two gates for each of the exposed and masked region separately. The region which is exposed to the ionic liquid shows gating with both the top and back gates. We have calculated the ionic gate capacitance using the back gate capacitance of 11.5 nF/cm2 and the capacitance ratio η = CTG/CBG ~ 90; this gives the ionic gate capacitance CTG = 1 μF/cm2. We observe that the Scientific Reports \| 7: 3336 \| DOI:10.1038/s41598-017-03264-0 3 www.nature.com/scientificreports/ Figure 3. Photovoltage and resistance measurements at 120 K. To change the top gate voltage, the device is warmed to 273 K and the top gate voltage is adjusted and allowed to stabilize for half an hour before the device is cooled down to 120 K. (a) The measured resistance as a function of both gates. The dotted lines indicate the charge neutrality peaks of the two regions and the polarity of the exposed and covered regions is indicated. (b) The corresponding photovoltage at the junction measured simultaneously with the resistance. There is a clear signature of photothermoelectric effect indicated by the sixfold pattern29. Experimentl Six regions with alternating positive and negative signs are indicated by plus and minus signs and dotted lines that separate the six regions have been drawn. (c) Slices of the resistance data in Fig. 3(a) exhibit two Dirac peaks. (d) Slices of the photovoltage with multiple zero crossings. Figure 3. Photovoltage and resistance measurements at 120 K. To change the top gate voltage, the device is warmed to 273 K and the top gate voltage is adjusted and allowed to stabilize for half an hour before the device is cooled down to 120 K. (a) The measured resistance as a function of both gates. The dotted lines indicate the charge neutrality peaks of the two regions and the polarity of the exposed and covered regions is indicated. (b) The corresponding photovoltage at the junction measured simultaneously with the resistance. There is a clear signature of photothermoelectric effect indicated by the sixfold pattern29. Six regions with alternating positive and negative signs are indicated by plus and minus signs and dotted lines that separate the six regions have been drawn. (c) Slices of the resistance data in Fig. 3(a) exhibit two Dirac peaks. (d) Slices of the photovoltage with multiple zero crossings. resistance of the region of the flake which is masked by HSQ remains unaffected by the top gate. All subsequent measurements are done across the p-n junction.h p j The variation of the resistance across the p-n junction at 273 K for all values of \|VTG\| ≤ 0.8 V and VBG ≤ 25 V is shown in Supplementary Section VI and indicates the formation of p-n junction at room temperature. Similar electrical measurements performed at 120 K are shown in Fig. 3. These consist of four regions corresponding to p-n, n-p, n-n and p-p doping. The dotted lines indicate the charge neutrality peaks of the two regions and the polarity of the exposed and covered regions is indicated. The maximum carrier density was determined by using the known value of the ionic gate capacitance and the maximum gate voltage applied and it is found to be 7 × 1012 cm−2.i To change the top gate voltage, the device was first warmed to 273 K and the top gate voltage is adjusted and allowed to stabilize for 30 minutes before the device is cooled down to 120 K. Experimentl The existence of two distinct values of ntg where Stg = Sbg leads to the two photoresponse zeros seen in Fig. 3(d). liquid and covered with HSQ respectively). This manifests itself as two intersections of the photoresponse curve with zero when measuring the photovoltage as a function of the each gate, a shown in Fig. 3(c). The conventional mechanism of photoresponse generation in semiconductors is the generation of an electron hole pair due to the the absorption of a photon and their separation under the influence of a built-in electric field such as that arising at a p-n junction. This is referred to as the photovoltaic effect and the net photoresponse depends on the magni- tude and direction of band bending.f g We schematically illustrate in Fig. 4 that the photovoltaic effect cannot explain the multiple polarity reversals that are exhibited by the p-n junction. Along the highlighted line shown in Fig. 4 with a constant value of VBG, the charge density in the region covered with HSQ remains constant. Along this line, only the Fermi level of the exposed region changes. This change is monotonic and the photoresponse will only change sign once by going through zero at the flat band condition at point 2. However, the experimentally measured photoresponse data goes through zero twice, at point 2 and point 4, and therefore cannot be explained by the photovoltaic effect.hff g g yf The photothermoelectric effect is consistent with our observations of a six-fold pattern. Under the effect of light illumination, the temperature at the p-n junction increases and the temperature at the source and drain contacts remains at the bath temperature. The difference in carrier densities on either side of the junction leads to different Seebeck coefficients in the two regions. This results in a net photo-induced voltage developing across the device.hfi The multiple polarity reversals arise because of the functional form of the Seebeck coefficient’s dependence on the charge density, illustrated in Fig. 4. The Seebeck coefficient is positive for hole doping and negative for electron doping. It is an odd function and goes through a maximum before decreasing at large carrier densities. Experimentl The photovoltage generated at 120 K at the p-n junction is given in Fig. 3 along with line slices of the data for given values of the top gate voltage. The data has been acquired by repeated warming and cooling for each value of the top gate voltage. The junction is robust and does not show any thermal cycling effects (Supplementary Section IX).h y y gf pp y The plot of the photovoltage at the junction shown in Fig. 3(b) as a function of the top and back gate voltages exhibits a six-fold pattern. The photovoltage is zero along the lines nTG = 0, nBG = 0 and nTG = nBG and alterna- tively positive and negative on either side (nTG and nBG are the carrier densities in the region exposed to the ionic Scientific Reports \| 7: 3336 \| DOI:10.1038/s41598-017-03264-0 4 www.nature.com/scientificreports/ Figure 4. The magnitude and signs of the Seebeck coefficient that leads to the six-fold pattern seen in the photovoltage data. Along the constant VBG line, the charge density in the covered region is constant and the exposed regions changes. The photovoltage is zero at point 2 and 4 where Seebeck coefficients are equal. The photovoltaic response is zero at point 2, which is the flat band condition where the Fermi levels in both regions are equal. At point 4, the photoresponse is zero despite unequal Fermi levels and provides evidence of the photothermoelectric effect. The plot on the right schematically shows the variation of Stg with ntg at fixed values of Sbg and nbg. The existence of two distinct values of ntg where Stg = Sbg leads to the two photoresponse zeros seen in Fig. 3(d). Figure 4. The magnitude and signs of the Seebeck coefficient that leads to the six-fold pattern seen in the photovoltage data. Along the constant VBG line, the charge density in the covered region is constant and the exposed regions changes. The photovoltage is zero at point 2 and 4 where Seebeck coefficients are equal. The photovoltaic response is zero at point 2, which is the flat band condition where the Fermi levels in both regions are equal. At point 4, the photoresponse is zero despite unequal Fermi levels and provides evidence of the photothermoelectric effect. The plot on the right schematically shows the variation of Stg with ntg at fixed values of Sbg and nbg. Experimentl For a given value of the Seebeck coefficient of the covered region influenced by the back gate, there are two distinct carrier densities where Seebeck coefficient of the exposed top-gated region will be equal to that of back gated region, resulting in a zero photovoltage. Along the highlighted line in Fig. 4, the sign and magnitude of the Seebeck coefficient and the photovoltage at five distinct points is highlighted.hffi fi p gi p g g The photovoltage is given by difference in the Seebeck coefficients in the two regions: gi p g g difference in the Seebeck coefficients in the two regions: = − ∆ PV V V S n S n T ( , ) [ ( ) ( )] (1) BG TG BG TG = − ∆ PV V V S n S n T ( , ) [ ( ) ( )] BG TG BG TG (1) and the Mott relation relates the Seebeck coefficient and the charge density: and the Mott relation relates the Seebeck coefficient and the charge density: π = S k T e d lnR dn dn dE 3 ( ) (2) B 2 2 (2) For bilayer graphene27, α γ = − + n sign E E E ( ) [ ] F F F 1 2 and α γ α = − + \| dn dE n / 4 2 2 , where n < 0(n > 0) for electron (hole) doping, α π = − v ( ( ) ) f 2 1  , and the inter-layer hopping energy γ1~390 meV. The resistance data is fitted to the form µ = − + R L W e / ( ) n n 1 1 2 0 2 , from which we get = − + d lnR dn ( )/ n n n 2 0 2 .fih + n n0 0 We can estimate the value of the Seebeck coefficient from the experimental data. The photovoltage arising form the photothermoelectric can be written as a linear combination of two functions that depend on the doping level in each region, similar to equation 1: Scientific Reports \| 7: 3336 \| DOI:10.1038/s41598-017-03264-0 5 www.nature.com/scientificreports/ tificreports/ Figure 5. A comparison of the product of the Seebeck coefficient and temperature increase obtained experimentally and calculated theoretically as a function of the carrier density. Experimentl (a) The experimental curve has been obtained by Fourier transforming the data in Fig. 3(a) and represents the function fPV2, which corresponds to the region exposed to the top gate. (b) The theoretical curve has been calculated by calculating the Seebeck coefficient using the Mott relation and with a temperature increase of 50 mK. Figure 5. A comparison of the product of the Seebeck coefficient and temperature increase obtained experimentally and calculated theoretically as a function of the carrier density. (a) The experimental curve has been obtained by Fourier transforming the data in Fig. 3(a) and represents the function fPV2, which corresponds to the region exposed to the top gate. (b) The theoretical curve has been calculated by calculating the Seebeck coefficient using the Mott relation and with a temperature increase of 50 mK. Figure 5. A comparison of the product of the Seebeck coefficient and temperature increase obtained h Figure 5. A comparison of the product of the Seebeck coefficient and temperature increase obtained experimentally and calculated theoretically as a function of the carrier density. (a) The experimental curve h been obtained by Fourier transforming the data in Fig. 3(a) and represents the function fPV2, which correspo to the region exposed to the top gate. (b) The theoretical curve has been calculated by calculating the Seebec coefficient using the Mott relation and with a temperature increase of 50 mK. Figure 6. Temperature dependence of the photovoltage. (a) Normalized photovoltage magnitude as a function of temperature at different top gate voltages when nBG = −nTG. The normalization factor depends on VTG. (b) and (c) The photovoltage as a function of the back gate at VTG = 0.3 V. Figure 6. Temperature dependence of the photovoltage. (a) Normalized photovoltage magnitude as a function of temperature at different top gate voltages when nBG = −nTG. The normalization factor depends on VTG. (b) and (c) The photovoltage as a function of the back gate at VTG = 0.3 V. η = − + PV f V f V V ( ) ( ( )) (3) PV BG PV BG TG 1 2 (3) where η = CTG/CBG and we treat the functions fPVi as unknowns and estimate them from the experimental data using two dimensional Fourier transforms (Supplementary Section VII), similar to the approach taken by Gabor et al.29. The function fPV2 has been plotted in Fig. References , Kif yi , , ( ) 2. Koppens, F. H. L. et al. Photodetectors based on graphene, other two-dimensional materials and hybrid systems. Nat Nano 9, 780–793 (2014). 3. Bonaccorso, F., Sun, Z., Hasan, T. & Ferrari, A. C. Graphene photonics and optoelectronics. Nature Photonics 4, 611–622 doi:10.1038/nphoton.2010.186 (2010). p 4. Bao, Q. & Loh, K. P. Graphene photonics, plasmonics, and broadband optoelectronic devices. ACS Nano 6, 3677–94, doi:10.1021/ nn300989g (2012).h p 4. Bao, Q. & Loh, K. P. Graphene photonics, plasmonics, and broadband optoelectronic devices. 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This difference is because the temperature increase of the electronic subsystems obtained through the heat balance equation30 is strongly dependent on the cooling length. We have also neglected any light absorption in the ionic liquid and the effective optical power reaching the device and the corresponding temperature increase could be lower.h p p g p g p Photovoltage measurements at temperatures ranging from 30 K to 150 K have also been performed. The nor- malized photovoltage magnitude at different top gate voltages has been plotted in Fig. 6(a). The photovoltage increases with decreasing temperature. The photovoltage rises rapidly below 90 K and in this range, the product of photovoltage and temperature is constant, indicating that the photovoltage is inversely proportional to temper- ature (Supplementary Section X). The photo-responsivities we have obtained range from 100 mV/W at 273 K to 600 mV/W at 30 K. 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Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and nstitutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per- mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Scientific Reports \| 7: 3336 \| DOI:10.1038/s41598-017-03264-0 8
https://openalex.org/W1813287392	http://journal.ipb.ac.id/index.php/jurnalmpi/article/download/9470/10-6	Indonesian	null	Faktor-Faktor yang Memengaruhi Realisasi Pembiayaan Mikro (Studi Kasus PT Bank Syariah Mandiri KCP Bogor Merdeka)	Manajemen IKM	2,015	cc-by-sa	3,574	ABSTRAK Pembiayaan mikro merupakan produk Bank Syariah Mandiri yang sedang berkembang pesat. Tujuan penelitian ini adalah untuk mengetahui faktor apa yang memengaruhi realisasi pembiayaan mikro serta karakteristik debitur pembiayaan mikro. Dengan analisis deskriptif dapat diketahui bahwa karakteristik debitur pembiayaan mikro didominasi oleh jenis kelamin pria, usia 31-40 tahun, pendidikan SMA, jumlah tanggungan keluarga 2-4 orang, lama usaha di atas 2 tahun, laba bersih per bulan Rp1.000.000-Rp5.000.000, jenis usaha didominasi perdagangan, mayoritas frekuensi pinjaman 1-3 kali, jumlah pembiayaan yang diajukan lebih dari Rp 00.000, nilai agunan nasabah lebih besar dari Rp50.000.000, dan jenis penggunaan adalah produktif. Menggunakan analisis regresi linear berganda dan melakukan uji F serta Uji T pada α = 5% terdapat tiga peubah yang secara nyata memengaruhi realisasi pembiayaan mikro, yaitu jenis usaha (perdagangan), jumlah pembiayaan yang diajukan dan nilai agunan. Kata kunci: karakteristik debitur, pembiayaan mikro, realisasi pembiayaan Faktor-Faktor yang Memengaruhi Realisasi Pembiayaan Mikro (Studi Kasus PT Bank Syariah Mandiri KCP Bogor Merdeka) Samirah Ali * dan Ali Mutasowifin Bank Syariah Mandiri KCP Bogor Merdeka Jl. Merdeka 63, Bogor; e-mail: samierahalie@gmail.com Departemen Manajemen, Fakultas Ekonomi dan Manajemen, Institut Pertanian Bogor Jl. Kamper, Kampus IPB Dramaga Bogor 16680; e-mail: alimu@ipb.ac.id ABSTRACT Microfinance is one popular product of Bank Syariah Mandiri. The purpose of this study is to know factors that influence the realization of microfinance as well as the characteristics of microfinance debitors. Characteristics of microfinance debitors are dominated by male, age 31-40 years old, high school education, 2-4 people dependents family, over 2 years age of business, net income per month from Rp1,000,000 to Rp , dominated by trading companies, 1-3 times of borrowing frequency, amount of financing is more than Rp , value of collateral is more than Rp , and purpose of financing is for productive activities. Using multiple linear regression analysis, F test and T test at α = % there are three variables affect the realization of microfinancing, namely type of business (trade), the amount of financing proposed and value of collateral. Key words: characteristics of debitor, microfinance, realization of financing ) Korespondensi: Bank Syariah Mandiri KCP Bogor Merdeka; Jl. Merdeka 63, Bogor; e-mail: samierahalie@gmail.com Vol. No. http://journal.ipb.ac.id/index.php/jurnalmpi/ Vol. No. http://journal.ipb.ac.id/index.php/jurnalmpi/ Manajemen IKM, Februari - ) ISSN - Vol. No. http://journal.ipb.ac.id/index.php/jurnalmpi/ Key words: characteristics of debitor, microfinance, realization of financing Kata kunci: karakteristik debitur, pembiayaan mikro, realisasi pembiayaan PENDAHULUAN perkembangan jumlah pelaku usaha menurut skala usaha tahun 2010- Usaha Mikro, Kecil dan Menengah (UMKM) memiliki peranan penting dalam perekonomian Indonesia. UMKM mencakup 99,99% dari total pelaku usaha di Indonesia atau sebanyak 52,76 juta unit (BPS, Tabel 1 menunjukkan data Pengembangan UMKM masih menghadapi kendala, seperti rendahnya kemampuan dalam pemasaran, teknologi, dan dalam mengakses pembiayaan dari perbankan. Kendala UMKM dalam memperoleh pembiayaan perbankan bisa Faktor-Faktor yang Memengaruhi Realisasi Pembiayaan Mikro Selain itu, dari 112 nasabah pembiayaan mikro di BSM KCP Bogor Merdeka, 80 nasabah (71%) menerima realisasi pembiayaan lebih rendah dari pengajuan, sedangkan yang sesuai dengan pengajuan hanya 32 nasabah (29%). Dengan kondisi ini, BSM KCP Bogor Merdeka perlu menentukkan langkah yang tepat guna meningkatkan mutu pembiayaan mikro, sehing- ga pertumbuhan pembiayaan dapat meningkat. ditinjau dari sisi permintaan dan penawaran. Dari sisi permintaan, pada umumnya, UMKM tidak memiliki informasi keuangan yang transparan dan terorganisasikan dengan baik, sehingga pemberi pembiayaan menemui kesulitan dalam memperoleh informasi mengenai kondisi keuang- an dan usaha UMKM. Hal tersebut membuat bank kesulitan meminimalisasi risiko default atas pembiayaan yang disalurkan kepada UMKM. Dengan demikian, rumusan masalah dapat disusun sebagai berikut: Dengan demikian, rumusan masalah dapat disusun sebagai berikut: UMKM sangat membutuhkan pembiayaan yang berkarakteristik mudah, cepat, sesuai dan dekat (Nawai dan Shariff, 2011). Namun, aturan main pada skim pembiayaan formal bersifat rigid, sementara UMKM banyak memiliki keterbatasan. Oleh karena itu, sumber-sumber pembiayaan informal lebih akrab dan menjadi alternatif meskipun diketahui bahwa sumber ini memungut bunga pinjaman lebih tinggi (Nurmanaf, 2007). 1. Bagaimanakah karakteristik debitur pembia- yaan Mikro BSM KCP Bogor Merdeka? 2. Faktor-faktor apakah yang memengaruhi realisasi pembiayaan Mikro BSM KCP Bogor Merdeka? Manajemen IKM METODE PENELITIAN Salah satu bank yang terfokus menyalurkan pembiayaan kepada UMKM adalah Bank Syariah Mandiri (BSM), yang memiliki market share 40% di perbankan syariah. BSM memfokuskan perhatian pada pembiayaan mikro dibandingkan dengan pembiayaan lainnya. Number Of Account (NOA) tabungan di BSM sebanyak 3.565.432 nasabah yang tersebar di seluruh Indonesia, sementara yang membutuhkan pembiayaan mikro 778.600.200 nasabah. Faktor-faktor yang memengaruhi realisasi pembiayaan mikro BSM ini didasarkan pada prinsip 5C, yaitu character, capacity, capital, collateral, dan condition of economic (Kasmir, 2013). Berdasarkan pada prinsip 5C ini pemodelan dalam mengestimasi faktor-faktor yang meme- ngaruhi realisasi pembiayaan mikro BSM ditetapkan dengan memodifikasi prinsip 5C. Peubah yang diturunkan dari prinsip 5C meliputi karakteristik individu, usaha, dan pembiayaan. Karakteristik individu meliputi peubah umur, jenis kelamin, jumlah tanggungan dan pendi- dikan. Karakteristik usaha meliputi peubah lama usaha, tingkat laba bersih per bulan, dan jenis usaha. Untuk karakeristik pembiayaan meliputi peubah frekuensi pinjaman, jumlah pembiayaan yang diajukan, nilai agunan dan jenis penggunaan pembiayaan. Faktor-faktor yang memengaruhi realisasi pembiayaan mikro BSM ini didasarkan pada prinsip 5C, yaitu character, capacity, capital, collateral, dan condition of economic (Kasmir, 2013). Berdasarkan pada prinsip 5C ini pemodelan dalam mengestimasi faktor-faktor yang meme- ngaruhi realisasi pembiayaan mikro BSM ditetapkan dengan memodifikasi prinsip 5C. Peubah yang diturunkan dari prinsip 5C meliputi karakteristik individu, usaha, dan pembiayaan. Karakteristik individu meliputi peubah umur, jenis kelamin, jumlah tanggungan dan pendi- dikan. Karakteristik usaha meliputi peubah lama usaha, tingkat laba bersih per bulan, dan jenis usaha. Untuk karakeristik pembiayaan meliputi peubah frekuensi pinjaman, jumlah pembiayaan yang diajukan, nilai agunan dan jenis penggunaan pembiayaan. BSM Kantor Cabang Pembantu (KCP) Merdeka Bogor adalah salah satu cabang yang memberikan pelayanan nasabah mikro di wilayah Bogor yang memiliki aset terbesar di antara KCP lain di wilayah Bogor dengan jumlah nasabah 10.782 orang. Untuk terus memastikan penyalur- an pembiayaan yang hati-hati, BSM KCP Bogor Merdeka terus melakukan perbaikan, salah satu- nya perbaikan pengelolaan risiko pembiayaan, terutama penyeleksian calon debitur agar dapat meningkatkan mutu dan kuantitas pembiayaan. I DAN MUTASOWIFIN Manajemen I Tabel 1. Perkembangan jumlah pelaku usaha menurut skala usaha Tahun 2010– Tabel 1. Perkembangan jumlah pelaku usaha menurut skala usaha Tahun 2010– Faktor-Faktor yang Memengaruhi Realisasi Pembiayaan Mikro Berdasarkan karakteristik individu dapat diketahui pengaruh nyata peubah independen, yaitu peubah umur, jenis kelamin, jumlah tang- gungan dan pendidikan. Karakteristik usaha juga digunakan untuk mengetahui pengaruh peubah lama usaha, tingkat laba bersih per bulan, dan jenis usaha. METODE PENELITIAN Sementara, peubah frekuensi pinjaman, jumlah pembiayaan yang diajukan, nilai agunan dan jenis penggunaan pembiayaan dipilih mewakili karakteristik pembiayaan. Berbeda dengan penelitian yang dilakukan oleh Lubis dan Rachmina (2011) yang meng- gunakan beberapa faktor yang memengaruhi realisasi dan pengembalian kredit usaha rakyat (KUR) dengan menggunakan Analisis Regresi Linear Berganda dan Analisis Logistik Biner. Teknik pengolahan data dilakukan dengan analisis faktor-faktor yang memengaruhi realisasi pembiayaan mikro dengan metode Regresi Linear Berganda, yang merupakan model analisis untuk mengetahui pengaruh peubah-peubah indepen- den berskala metrik (dengan peubah yang belum berskala metrik diubah menjadi peubah boneka) terhadap peubah dependen yang juga berskala metrik (Nazir, . Model ini merupakan model terbaik dalam memprediksi arah, besar koefisien dan sensitivitas perubahan peubah dependen karena perubahan peubah-peubah independen. Gambar Kerangka pemikiran penelitian Estimasi model untuk analisis faktor-faktor yang memengaruhi realisasi pembiayaan mikro berdasarkan pada karakteristik individu, karak- teristik usaha, dan kakteristik pembiayaan yaitu: Ŷ = β + β X + β X + … + β X Keterangan : Ŷ = Peubah dependen, yaitu jumlah realisasi pembiayaan (Rp) Βo = konstanta atau intersept model garis regresi X ,…,X = Peubah independen X = usia (tahun) X = jenis kelamin, peubah boneka (pria=1 dan wanita =0) X = jumlah tanggungan (orang) X = pendidikan (tingkat pendidikan) X = lama usaha (tahun) X = tingkat laba bersih per bulan (Rp) X = jenis usaha (jenis usaha) X = frekuensi pinjaman (kali) X = jumlah pembiayaan yang diajukan (Rp) X = nilai agunan (Rp) X = jenis penggunanan pembiayaan (penggunaan pembiayaan) β ,..β = koefisien peubah independen Ŷ = Peubah dependen, yaitu jumlah realisasi pembiayaan (Rp) p y p Βo = konstanta atau intersept model garis regresi β ,..β = koefisien peubah independen Gambar Kerangka pemikiran penelitian HASIL DAN PEMBAHASAN Dengan mengetahui peubah karakteristik yang memengaruhi realisasi pembiayaan mikro tersebut, diharapkan BSM KCP Bogor Merdeka dapat memformulasikan langkah strategik untuk meningkatkan realisasi pembiayaan mikro. Warung mikro BSM beroperasi mulai tahun 2009. Saat ini oulet warung mikro di seluruh indonesia baik out branch (sales outlet) maupun in branch sebanyak 52 outlet (BSM, 2013). Untuk wilayah Bogor saat ini terdapat 6 outlet in branch dan 1 outlet out branch. Vol. 10 No. 1 Vol. 10 No. 1 Februari 2015 Februari 2015 Faktor-Faktor yang Memengaruhi Realisasi Pembiayaan Mikro Plafon pembiayaan mikro mulai Rp hingga Rp100.000.000 dengan jangka waktu pem- biayaan maksimum 3 tahun (untuk wiraswasta) dan maksimum 5 tahun (untuk karyawan). Struktur organisasi warung mikro BSM terdiri dari 1 Kepala Warung Mikro (KWM), 1 Asisten Analis Mikro (AAM), 1 Mentor Usaha (MU), 3 Pelaksana Marketing Mikro (PMM) dan 1 Admin Pembiayaan Mikro (APM). Menurut Anggraini dan Nasution peranan kredit usaha rakyat atau kredit mikro sangat membantu dan berperan penting dalam pengembangan UMKM. an. Karakteristik individu terbagi menjadi empat peubah, yaitu umur, jenis kelamin, jumlah tang- gungan keluarga, dan pendidikan yang menjadi dasar penilaian karakter nasabah. 2. Karakteristik Usaha Karakteristik usaha sangat berpengaruh terhadap persetujuan pembiayaan. Dalam prinsip C biasa dikenal dengan sebutan capacity, yaitu kemampuan nasabah dalam mengelola usahanya dan menyangkut kemampuan nasabah untuk membayar kewajibannya. Karakteristik usaha dibagi menjadi tiga, yaitu lama usaha, tingkat laba bersih per bulan, dan jenis usaha. 3. Karakteristik Pembiayaan Dalam perealisasian pembiayaan oleh pihak Bank yang mengacu pada prinsip 5C, nasabah dapat dideskripsikan menjadi karakteristik individu, karakteristik usaha dan karakteristik pembiayaan. Data nasabah dalam penelitian ini terdiri dari 112 nasabah produktif (wiraswasta) Warung Mikro BSM KCP Bogor Merdeka yang aktif dari 1 September 2010 sampai dengan 31 Maret 2013. Kemudian dilakukan pengolahan data, dengan beberapa data yang harus dibuang (pencilan), karena menyebabkan data tidak menyebar normal. Data yang dibuang sebanyak 7 responden, sehingga responden yang terlibat dalam penelitian ini berjumlah 105 nasabah. Karakteristik pembiayaan dibagi menjadi empat, yaitu frekuensi pinjaman, jumlah pem- biayaan yang diajukan, nilai agunan (collateral) dan jenis penggunaan pembiayaan. Analisis Realisasi Pembiayaan Mikro Dalam penelitian ini terdapat sebelas faktor yang diduga memengaruhi realisasi pembiayaan mikro di BSM KCP Bogor Merdeka, yaitu Umur (X ), Jenis Kelamin (X ), Jumlah Tanggungan Keluarga (X ), Pendidikan (X ), Lama Usaha (X ), Tingkat Laba Bersih Per Bulan (X ), Jenis Usaha (X ), Frekuensi Pinjaman (X ), Jumlah Pembiayaan yang Diajukan (X ), Nilai Agunan (X ), dan Jenis Penggunaan Pembiayaan (X ). 1. Karakteristik Individu 2. Uji T (parsial) Uji t digunakan untuk menguji nyatanya regresi tiap-tiap peubah independen. Jika nilai probabilitas lebih kecil dari tingkat nyata yang diharapkan (α = 5%), maka terdapat pengaruh nyata peubah dependen dengan independennya. Berdasarkan hasil uji t diketahui peubah- peubah apa yang memengaruhi realisasi pem- biayaan mikro pada BSM KCP Bogor Merdeka. Berdasarkan hasil penelitian (Tabel 2) pada α = 5% terdapat tiga peubah yang memengaruhi realisasi pembiayaan, yaitu jenis usaha (perdagangan), jumlah pembiayaan yang diajukan dan nilai agunan dengan nilai p-value berturut-turut (0,044; Dalam penelitian ini terdapat satu peubah dependen dan sebelas peubah independen. Pada saat dilakukan pengujian dari peubah-peubah tersebut terdapat salah satu peubah yang menghasilkan data yang kurang akurat dengan kondisi pada kenyataan. Dari hasil pengolahan data, peubah yang tidak sesuai adalah DPP (jenis penggunaan pembiayaan) menghasilkan positif untuk penggunaan konsumtif. Artinya jika pengajuan nasabah konsumtif, maka realisasi pembiayaan akan naik, karena bernilai positif, tetapi pada kenyataan bank akan lebih cenderung merealisasikan pembiayaan jika kebutuhan nasabah untuk produktif. Hal tersebut terjadi, karena data yang diperoleh berbanding jauh, yaitu 2:103, sehingga faktor jenis penggunaan pembiayaan dihilangkan dan menghasilkan hasil olahan regresi berganda yang dapat dilihat pada Tabel . 1. Karakteristik Individu Salah satu prinsip dalam 5C adalah character dan sangat penting dalam persetujuan pembiaya- DAN MUTASOWIFIN Manajemen IKM Gambar 2. Hasil uji normalitas dan homogenitas Gambar 2. Hasil uji normalitas dan homogenitas ALI DAN MUTASOWIFIN Manajeme ALI DAN MUTASOWIFIN Manajemen IKM Manajemen IKM Faktor-Faktor yang Memengaruhi Realisasi Pembiayaan Mikro Untuk membuat persamaan regresi linear berganda diperlukan beberapa asumsi mendasar, yaitu normalitas, homogenitas, multikolinearitas dan autokorelasi (Sunyoto, . Pengolahan data dalam penelitian ini menggunakan Minitab 14 (Iriawan dan Astuti, Hasil pengolahan menunjukkan bahwa data memiliki sebaran normal (Gambar 2). Dan untuk asumsi homogenitas terpenuhi dalam gambar residual plots (versus fits) pada Lampiran yang memiliki lebar pitaan sama, sehingga dapat dikatakan bahwa data yang diuji homogen. ada satu peubah atau peubah bebas berpengaruh nyata terhadap peubah tidak bebas (Realisasi Pembiayaan). 1. Uji F (simultan) Uji F pada dasarnya menunjukkan apakah semua peubah bebas yang dimasukkan dalam model mempunyai pengaruh secara bersama- sama (simultan) terhadap peubah terikatnya. 3. Uji R (Koefisien Determinasi) Uji R menunjukkan seberapa besar model mampu menjelaskan variabilitas peubah independen (Gujarati, . R adalah koefisien determinasi yang mengukur besarnya pengaruh peubah independen secara bersama-sama terhadap peubah dependen. Dari hasil penelitian pada Tabel 1 diketahui bahwa R-Sq (adj) = 92,4%, yang artinya kemam- puan seluruh peubah X mampu menjelaskan secara nyata keragaman perealisasian pembiayaan dan sisanya ( ) dijelaskan oleh faktor- faktor lain. Dalam penelitian ini nilai Variance Inflation Factor (VIF) pada tiap-tiap peubah tertinggi, yaitu jumlah pembiayaan yang diajukan 3,540. Karena nilai VIF lebih kecil dari 10, maka tidak terdapat hubungan yang kuat antara peubah bebas atau tiap-tiap peubah bebas tidak saling memengaruhi satu sama lainnya (bebas multikolinearitas). Dalam penelitian ini, peubah terbagi menjadi peubah dependen (yang dipengaruhi) dan varia- bel independen (yang memengaruhi), yaitu: Dalam penelitian ini yang menjadi peubah dependen adalah jumlah realisasi pembiayaan mikro pada PT Bank Syariah Mandiri KCP Bogor Merdeka. Plafon mikro maksimum Rp , sedangkan besaran realisasi pembiayaan mikro berfluktuasi, dengan rataan realisasi pembiayaan mikro menurut penelitian Rp 2. Peubah Independen Jenis usaha (perdagangan) Arti dari model regresi untuk jenis usaha perdagangan adalah jika jenis usaha nasabah adalah perdagangan, maka realisasi pembiayaan akan turun 3. .250. Hal tersebut terjadi karena dikhawatirkan dalam usaha perdagangan debitur memiliki banyak stok dan terjadi penumpukan akibat tidak laku. Selain itu, jenis usaha per- dagangan tidak mengandung sesuatu yang unik, karena banyak yang menjalankan usaha sejenis dan memiliki banyak pesaing. Oleh karena itu, jika usaha debitur adalah perdagangan, maka realisasi pembiayaan akan turun dibandingkan jika jenis usaha debitur adalah manufaktur atau jasa. Keterangan: Keterangan: X = Umur X = Jenis Kelamin. Boneka 1 = perempuan dan 0 = laki-laki X = Jumlah Tanggungan Keluarga X = Pendidikan. DP1 : 1 = SD, 0 = selain SD DP2 : 1 = SMP, 0 = selain SMP DP3 : 1 = SMA, 0 = selain SMA DP4 : 1 = D3, 0 = selain D3 X = Lama Usaha X = Tingkat Laba Bersih per Bulan X = Jenis Usaha. DU1 : 1 = perdagangan, 0 = selain perdagangan DU2 : 1 = manufaktur, 0 = selain manufaktur X = Frekuensi Pinjaman Keterangan: X = Umur X = Jenis Kelamin. Boneka 1 = perempuan dan 0 = laki-laki X = Jumlah Tanggungan Keluarga X = Pendidikan. DP1 : 1 = SD, 0 = selain SD DP2 : 1 = SMP, 0 = selain SMP DP3 : 1 = SMA, 0 = selain SMA DP4 : 1 = D3, 0 = selain D3 X = Lama Usaha X = Tingkat Laba Bersih per Bulan X = Jenis Usaha. DU1 : 1 = perdagangan, 0 = selain perdagangan DU2 : 1 = manufaktur, 0 = selain manufaktur X = Frekuensi Pinjaman X Umur X = Jenis Kelamin. Boneka 1 = perempuan dan 0 = laki-laki X = Jumlah Tanggungan Keluarga DP1 : 1 = SD, 0 = selain SD DP2 : 1 = SMP, 0 = selain SMP DP3 : 1 = SMA, 0 = selain SMA DP4 : 1 = D3, 0 = selain D3 DP1 : 1 = SD, 0 = selain SD DP2 : 1 = SMP, 0 = selain SMP DP3 : 1 = SMA, 0 = selain SMA DP4 : 1 = D3, 0 = selain D3 2. Jumlah pembiayaan yang diajukan 2. Peubah Independen Dari hasil analisis seperti pada Tabel dapat disimpulkan bahwa secara simultan peubah umur, jenis kelamin, jumlah tanggungan keluarga, pendidikan, lama usaha, tingkat laba bersih per bulan, jenis usaha, frekuensi pinjaman, jumlah pembiayaan yang diajukan, serta nilai agunan berpengaruh secara nyata terhadap realisasi pembiayaan, karena nilai p-value (0,000) < alpha (0, , maka tolak Ho sehingga minimal Dalam penelitian ini peubah independen yang memengaruhi realisasi pembiayaan mikro pada α = % terdapat tiga peubah, yaitu jenis usaha (perdagangan), jumlah pembiayaan yang diajukan dan nilai agunan. Berdasarkan hasil analisis regresi dari data panel pada Tabel dapat disusun persamaan regresi sebagai berikut: Vol. 10 No. 1 Februari 2015 Februari 2015 Faktor-Faktor yang Memengaruhi Realisasi Pembiayaan Mikro Faktor-Faktor yang Memengaruhi Realisasi Pembiayaan Mikro Tabel 2. Hasil pengujian model regresi linear berganda Predictor Coef SE Coef T P VIF Constant Umur DJ (jenis kelamin) - - Jumlah Tanggungan Keluarga - - DP1 (pendidikan) DP2 - - DP3 DP4 Lama Usaha (tahun) Tingkat laba bersih per b DU1 (jenis usaha) - - DU2 - - Frekuensi Pinjaman - - Jumlah Pembiayaan yang diajukan * Nilai Agunan S = 4917373 R-Sq = 93,4% R-Sq(adj) = 92,4% Ket : (*) nyata pada taraf 5% Tabel 3. Hasi uji F Analysis of Variance Source DF SS MS F P Regression 3,09469E+16 2,21049E+15 Residual Error 90 2,17625E+15 2,41806E+13 Total 3,31231E+16 Y = 3118844 + 1621 Umur (X ) - 2035874 DJ (jenis kelamin (X )) - 258883 Jumlah Tanggungan Keluarga (X ) + 1945254 DP1 (pendidikan (X )) - 50376 DP2 (X ) + 585002 DP3 (X ) + 75110 DP4 (X ) X = Jumlah Pembiayaan yang Diajukan X = Nilai Agunan Dari persamaan regresi di atas, dap X = Jumlah Pembiayaan yang Diajukan X = Nilai Agunan Y = 3118844 + 1621 Umur (X ) - 2035874 DJ (jenis kelamin (X )) - 258883 Jumlah Tanggungan Keluarga (X ) + 1945254 DP1 (pendidikan (X )) - 50376 DP2 (X ) + 585002 DP3 (X ) + 75110 DP4 (X ) + 196370 Lama Usaha (tahun) (X ) + 0,272 Tingkat laba bersih per bln (X ) - 3270250 DU1(jenis usaha (X )) - 935714 DU2 (X ) - 27226 Frekuensi Pinjaman (X ) + 0,510 Jumlah Pembiayaan yang diajukan (X ) + 0,0822 Nilai Agunan (X ). Dari persamaan regresi di atas, dapat diinter- prestasikan tiap-tiap peubah yang memengaruhi realisasi pembiayaan mikro sebagai berikut: 1. 2. Jumlah pembiayaan yang diajukan Arti dari model regresi untuk jumlah pembiayaan yang diajukan adalah jika jumlah pembiayaan naik satu satuan, maka realisasi pembiayaan naik 0,510. Hal tersebut terjadi X = Frekuensi Pinjaman DAFTAR PUSTAKA karena ketika debitur mengajukan pembiayaan, bank akan menyetujui pembiayaan 85% dari kebutuhan nasabah, sehingga ketika debitur mengajukan tinggi, maka realisasi pembiayaan akan naik. Anggraini, D dan Nasution SH . Peranan Kredit Usaha Rakyat (KUR) Bagi Pengembangan UMKM di Kota Medan (Studi Kasus Bank BRI). Jurnal Ekonomi Pembangunan USU, - .. ALI DAN MUTASOWIFIN Manajemen IKM Manajemen IKM Faktor-Faktor yang Memengaruhi Realisasi Pembiayaan Mikro 3. Nilai agunan Arti dari model regresi untuk nilai agunan adalah jika nilai agunan naik satu satuan, maka realisasi pembiayaan akan naik 0,0822. Hal tersebut terjadi karena semakin besar nilai agunan, akan semakin besar tanggung jawab debitur dalam melakukan pembayaran pinjaman pembiayaan yang diajukan. Selain itu, agunan merupakan second way out yang harus menutupi pinjaman > , sehingga semakin besar nilai agunan, maka semakin besar pula pinjaman yang diberikan. Maka, jika nilai agunan besar, tentunya realisasi pembiayaan naik. [BPS] Badan Pusat Statistik . Data Pelaku Usaha Di Indonesia [Internet]. [diunduh 2013 Juni 11]. Tersedia pada: http://www.bps.go.id. [BSM] Bank Syariah Mandiri . Data Outlet Warung Mikro. Jakarta (ID) : BSM. Gujarati, D. 2007. Dasar-Dasar Ekonometrika. Jakarta (ID): Erlangga. Iriawan, N dan Astuti SP. 2006. Mengolah Data Statistik Dengan Mudah Menggunakan Minitab 14. Yogyakarta (ID): CV Andi Offset. Kasmir. 2013. Bank Dan Lembaga Keuangan Lainnya. Cetakan Keduabelas. Jakarta (ID): Raja Grapindo Persada. KESIMPULAN Karakteristik debitur pembiayaan mikro BSM KCP Bogor Merdeka didominasi oleh pria, dan usia mayoritas nasabah pada usia 31-40 tahun. Tingkat pendidikan mayoritas nasabah adalah SMA. Jumlah tanggungan keluarga rataan 3-4 orang, biasanya terdiri dari suami/istri dan anak. Lama usaha nasabah mikro BSM KCP Bogor Merdeka sudah di atas 2 tahun dan mayoritas < 5 tahun. Untuk laba bersih per bulan mayoritas nasabah Rp1.000.000-Rp5.000.000. Jenis usaha didominasi oleh sektor perdagangan. Mayoritas frekuensi pinjaman nasabah adalah 1-3 kali dan jumlah pembiayaan yang diajukan <= Rp20.000.000. Nilai agunan dari mayoritas nasabah <= Rp , karena pembiayaan yang diajukan tidak terlalu besar dan rataan aset pengusaha mikro memang tidak terlalu besar dan untuk jenis penggunaan pembiayaan banyak digunakan untuk produktif (modal usaha atau investasi) dibandingkan untuk konsumtif. [KEMEN KUKM] Kementrian Negara Koperasi dan Usaha Kecil dan Menengah. 2012. Indikator Makro Usaha Kecil dan Menengah. Jakarta (ID): Kemen KUKM. Lubis, AM dan D. Rachmina. 2011. Faktor-Faktor yang Memengaruhi Realisasi dan Pengem- balian Kredit Usaha Rakyat. Jurnal Forum Agribisnis, - . Nawai, N. dan M. N. M. Shariff. 2011. The Importance of Micro Financing to the Microenterprises Development in Malaysia. Asian Social Science, 7 - Nazir, M . Metodologi Penelitian. Jakarta (ID): Ghalia Indonesia. Nurmanaf, A. R. 2007. Lembaga Informal Pem- biayaan Mikro Lebih Dekat dengan Petani. Analisis Kebijakan Pertanian, 5 - Pato, S. 2013. Analisis Pemberian Kredit Mikro Pada Bank Syariah Mandiri Cabang Manado. Jurnal Riset Ekonomi, Manajemen, Bisnis dan Akutansi, - Dari hasil penelitian terdapat tiga peubah yang memengaruhi realisasi pembiayaan mikro pada BSM KCP Bogor Merdeka, yaitu jenis usaha (perdagangan), jumlah pembiayaan yang diajukan dan nilai agunan. Sunyoto, D. . Analisis Regresi dan Uji Hipotesis. Yogyakarta (ID): CAPS. Vol. 10 No. 1 Februari 2015
https://openalex.org/W2948536937	https://zenodo.org/records/4558728/files/A%20digital%20tool%20to%20design%20structurally%20feasible%20semi-circular%20masonry%20arches%20composed%20of%20interlocking%20blocks.pdf	English	null	A digital tool to design structurally feasible semi-circular masonry arches composed of interlocking blocks	Computers & structures	2,019	cc-by	11,237	A digital tool to design structurally feasible semi-circular masonry arches composed of interlocking blocks C. Casapullaa1, E. Mousaviana, M. Zarghanib C. Casapullaa1, E. Mousaviana, M. Zarghanib a Department of Structures for Engineering and Architecture, University of Naples Federico II, via Forno Vecchio 36, 80134, Naples, Italy b School of Civil Engineering, Iran University of Science and Technology, Tehran, Iran Corresponding author (Claudia Casapulla) Tel./fax: +39 081 2538901 E-mail: casacla@unina.it a Department of Structures for Engineering and Architecture, University of Naples F II, via Forno Vecchio 36, 80134, Naples, Italy School of Civil Engineering, Iran University of Science and Technology, Tehran, Iran Corresponding author (Claudia Casapulla) Tel./fax: +39 081 2538901 E-mail: casacla@unina.it *Corresponding author (Claudia Casapulla) Tel./fax: +39 081 2538901 1C. Casapulla and E. Mousavian are joint first authors. 1C. Casapulla and E. Mousavian are joint first authors. Abstract. This work deals with a digital tool to design stable semi-circular masonry arches composed of interlocking blocks which are kept together by interlocking connectors on their faces. These blocks, comparing to conventional blocks, increase the sliding resistance and reduce the workmanship. However, the digital tools were developed mostly to design arches with conventional blocks. The proposed tool tries to fill this gap by addressing the work in three stages. First, a heuristic method is developed to define the relationships between the geometry of an interlocking face and the sliding resistance. Then, a structural analysis procedure is developed based on the limit analysis and the heuristic method to define the stability condition of the arch. Finally, optimization algorithms are developed to find the thinnest arch by means of two minimization strategies dealing with the relationship between the sliding resistance of the blocks and the geometry of the interlocking faces, differently. The algorithms consider some control points on a given thrust line and automatically adjust them to minimize the thickness, while the stability condition checks the structural feasibility during the geometry adjustment. To evaluate the accuracy of the proposed heuristic method, the results obtained with nonlinear FE analysis are used for comparison. Keywords: Structurally informed architectural design, digitally supported design, limit analysis, interlocking blocks, non-isotropic sliding resistances, thrust line. 1 1. INTRODUCTION This work develops a digital tool to design structurally sound semi-circular masonry arches composed of interlocking blocks. The interlocking blocks are rigid block units which, on their faces, have connectors keeping the blocks together and preventing blocks from sliding. The interlocking blocks, in comparison to the conventional blocks with flat faces, increase the shear resistance to external forces [1] and reduce the construction skills and instruments required [2]. Despite these advantages of interlocking blocks, the digital tools supporting designers for structurally informed architectural design were developed mostly for the conventional blocks. Most of those efforts (reviewed by Rippmann [3]) were focused on designing single-layer masonry vaults. To design masonry assemblages with diverse topologies, Whiting [4] proposed a method in which, given a block assemblage, the block geometry changes automatically to obtain the structurally feasible form. In that work, however, interlocking the blocks during the geometry modification was avoided. There are a few studies [5, 6] that applied simple geometric grammars but no structural constraints to design vaults that are composed of interlocking blocks with limited geometries. In the literature there are different methods of structural analysis to develop the introduced digital tools. Most of them are based on the limit analysis theory aimed at calculating the ultimate load factor satisfying static and kinematic conditions. According to this theory, internal forces are distributed at the interfaces between rigid blocks. These interfaces have infinite compressive, no tensile strength, and infinite [7] or finite sliding resistance including associative [8, 9] or non-associative [10-13] frictional resistance. In this framework, it is worthy to mention a recent numerical limit analysis procedure, named discontinuity layout optimisation (DLO) [14], which has been developed to obtain accurate upper-bound solutions for plane-strain collapse problems. With reference to vaulted single-layered masonry structures, different methods find the stress states at interfaces: line of thrust methods [7, 15, 16]; membrane theory [7, 17, 18]; force network method [19, 20]; convex and concave contact formulations [9, 21], non-smooth contact dynamics [22]. Interesting are also some recent experimental and/or analytical works on the collapse failure of vaulted masonry structures under horizontal loading [23, 24] and after actual displacements of the supports [25-27]. However, all these methods were applied to analyze structures composed of conventional block with isotropic friction. 1. INTRODUCTION Other distinctive methods of masonry analysis, such as discrete and finite element analysis, rarely have been used to develop tools to design structurally feasible masonry assemblages, 2 mostly due to their complex computational techniques. In the former method, masonry is modelled as an assemblage of distinct blocks. For such a block system, the equations of motion are solved using an explicit time integration method [28-30]. Instead, in finite element analysis (FEA), masonry is modelled as connected or distinct elements with different material properties for bricks and mortar joints (detailed and simplified micro models) or as a continuum by smearing the bricks and mortar joints into an isotropic or anisotropic homogeneous continuum (macro models) [31-33]. This work aims at filling the gaps described above about design of structurally sound assemblages of interlocking blocks and proposes a digital framework for the structurally informed design of a semi-circular arch composed of interlocking blocks and subjected to its own weight. To develop this framework, limit state analysis is adopted and extended to interlocking blocks. This structural typology with symmetrical loading and geometry belongs to a special class of non-associative friction problems for which provably unique solutions within limit analysis approach exist [16]. Therefore, the application to this structure allows focusing the attention to the structural behaviour of interlocking blocks rather than to the issue of associated or non-associated flow rules, which is beyond the scope of this work. Three main stages are herein introduced to help the designer to evaluate the structural feasibility of a model and automatically update the model to make it structurally optimal. 1. The first stage of this work is to develop a novel heuristic method, assisting the designers to estimate the interlocking block resistance to sliding. The proposed method allows equating an interlocking block to a conventional block with different sliding resistances along two orthogonal directions (normal and parallel to the connectors). 2. The goal of the second stage is to present a new structural analysis procedure that, applying the heuristic method proposed above, checks if the semi-circular arches composed of interlocking blocks are stable. To achieve this goal, this work extends the limit analysis developed by [16] which applied the line of thrust method to analyze the stability of semi-circular arches composed of blocks with isotropic finite friction. 1. INTRODUCTION Considering the relationship between the block resistance to sliding and the geometry of interlocking faces, the optimization method offers two strategies to minimize the arch thickness: (1) updating the sliding resistance due to the connector geometry change during optimization; (2) changing the number of the block connectors (initially given by the designer) during the optimization process, to keep the sliding resistant constant. Considering the relationship between the block resistance to sliding and the geometry of interlocking faces, the optimization method offers two strategies to minimize the arch thickness: (1) updating the sliding resistance due to the connector geometry change during optimization; (2) changing the number of the block connectors (initially given by the designer) during the optimization process, to keep the sliding resistant constant. The paper is organized as follows. The structural modelling along with the heuristic method introduced above are presented in Section 2, while Section 3 performs the extension of limit state analysis to interlocking blocks. Section 4 develops two optimization strategies to design semi-circular arches with interlocking blocks. Results are presented, discussed and validated in Section 5. Finally, the conclusions are summarized in Section 6. 1. INTRODUCTION The extension deals with semi-circular arches composed of interlocking blocks which, using the heuristic method, are modelled as conventional blocks with orthotropic finite friction (in which the sliding resistances are the minimum and maximum in two orthogonal directions, respectively), in order to analyze their structural stability. 2. The goal of the second stage is to present a new structural analysis procedure that, applying the heuristic method proposed above, checks if the semi-circular arches composed of interlocking blocks are stable. To achieve this goal, this work extends the limit analysis developed by [16] which applied the line of thrust method to analyze the stability of semi-circular arches composed of blocks with isotropic finite friction. The extension deals with semi-circular arches composed of interlocking blocks which, using the heuristic method, are modelled as conventional blocks with orthotropic finite friction (in which the sliding resistances are the minimum and maximum in two orthogonal directions, respectively), in order to analyze their structural stability. 3. Finally, the third stage of this research is to develop an optimization method minimizing the structurally feasible semi-circular arch thickness. The structural feasibility of the model during optimization is checked by the introduced structural analysis procedure. 3. Finally, the third stage of this research is to develop an optimization method minimizing the structurally feasible semi-circular arch thickness. The structural feasibility of the model during optimization is checked by the introduced structural analysis procedure. 3 Considering the relationship between the block resistance to sliding and the geometry of interlocking faces, the optimization method offers two strategies to minimize the arch thickness: (1) updating the sliding resistance due to the connector geometry change during optimization; (2) changing the number of the block connectors (initially given by the designer) during the optimization process, to keep the sliding resistant constant. The paper is organized as follows. The structural modelling along with the heuristic method introduced above are presented in Section 2, while Section 3 performs the extension of limit state analysis to interlocking blocks. Section 4 develops two optimization strategies to design semi-circular arches with interlocking blocks. Results are presented, discussed and validated in Section 5. Finally, the conclusions are summarized in Section 6. 2.1. Structural model of the interlocking block arch The structural model adopted in this work is based on the assumption that masonry structures are composed of assemblages of discrete rigid interlocking blocks in contact interaction, to be constitutively defined, where the displacements of each block should be considered separately. How a semi-circular arch and each of its discrete interlocking blocks are modelled in the developed framework is described as follows. Within the arch modelling, the designer assigns the radius R for the semi-circular arch centreline, the arch width b, and the number of blocks m composing the arch (Figure 1a). The latter number should be odd to model the arch keystone. All arch blocks have same sizes. On the other hand, the block modelling is based on the assumption that each interlocking block has two corrugated and two flat faces. Interlocking faces lock blocks to form the arch and the corrugated faces of the connectors are assumed to have rectangular cross sections (Figure 1b). Different shapes of the connectors could be investigated, but this further analysis will be addressed in future work. To model the interlocking faces, the designer should specify the total number n of projections and depressions of the block. This number should be an odd number in order to guarantee the symmetry of the block shape with respect to the centreline of the arch. Within the introduced discrete approach, the block interfaces are treated as the elements of the problem while the blocks are simply defining the geometry of the problem. Therefore, the analysis is fully related to the behaviour of the interfaces, which can then be regarded as 4 systems of stresses. Considering conventional blocks with rough flat faces in contact with finite friction, these stresses are governed by unilateral contacts and isotropic frictional constraints, due to the rugged projections on their faces, termed asperities. By scaling up the asperities, they can be regarded as connectors of two interlocked blocks. Using corrugated interlocking faces, the block resistance to sliding at the interface is a minimum along the connectors and a maximum along the direction normal to the connectors (Figure 1b). In fact, the friction on the equivalent flat face can be regarded as orthotropic friction, in which the sliding resistances are the minimum and maximum in two orthogonal directions, respectively. 2.1. Structural model of the interlocking block arch The next section presents a method to analyse the sliding resistance of an interlocking block and to relate the sliding resistance of interlocking blocks to the frictional resistance of conventional blocks. Figure 1. Topological models of a) a semi-circular arch and b) a composing interlocking block. Figure 1. Topological models of a) a semi-circular arch and b) a composing interlocking block. 2.2. Heuristic formulations relating the sliding resistance of interlocking blocks to the frictional resistance of conventional blocks The interlocking blocks could have different sliding resistances in different directions, depending on the orientation and the mechanical and geometrical features of the connectors. For example, the interlocking block adopted in this work has the maximum and minimum sliding resistance respectively along the direction normal and parallel to the connectors. This section is aimed at defining a heuristic relationship between the sliding resistance of such interlocking blocks and that of the equivalent conventional blocks with rough flat interfaces. Finding such a relationship, limit state analysis method is then extended to analyse the structural behaviour of a semi-circular arch composed of interlocking blocks (Section 3). The first step for searching out this relation is to study the sliding resistance along the direction orthogonal to the connectors of interlocking blocks. The analysis is carried out with reference to a single interface between a fixed lower block and the upper one subjected to a given normal force N and a lateral load T applied to its centre of gravity (Figure 2a). In order 5 to consider the same sliding resistance for two interlocking blocks, depressions and projections have the same width s, so that a = ns, being n an odd number with n ≥ 3. Figure 2. Geometric parameters of the interlocking blocks and their projections. a) Upper block subjected to lateral force T and its own weights; b) limiting shear force and bending moment of the single projection. Figure 2. Geometric parameters of the interlocking blocks and their projections. a) Upper block subjected to lateral force T and its own weights; b) limiting shear force and bending moment of the single projection. The assumptions for interlocking blocks are: infinite compressive strength, frictional behaviour at the bed joints, finite shear and tensile strengths of the connectors. The sliding failure mode essentially behaves in a rigid perfectly-plastic manner as for conventional blocks when it is governed by the frictional resistances [34, 35], while it is non-ductile when governed by the shear strength. With these assumptions, the lateral resistance of this block at first step is strictly related to the shear and/or bending resistances of the connectors and at second step, after the connector shear or bending failures, it is related to the cracked block frictional resistance. In this paper the moment failure mode of projections is prevented, though. 2.2. Heuristic formulations relating the sliding resistance of interlocking blocks to the frictional resistance of conventional blocks The exclusion of the bending failure can be derived in terms of geometrical relation between the width s and the height h of the projections, with reference to the simplest case of uniformly distributed horizontal forces sketched in Figure 2b. In fact, in order to avoid the bending failure before the shear failure of the single projection, it should be thick enough, i.e.: (1) This constraint is derived by the following assumptions. The limiting shear force and bending moment activating the corresponding failure modes of the single projection can respectively be expressed in function of the connector dimensions as obtained by using the classical Jourawski and Navier formulas [36]: 6 3 2 2 0 0 bs f M sb T tk k =  = (2) (2) where k and ftk are the shear and tensile strengths of the material forming the blocks, respectively. The calibration of parameter k is not generally provided within the mechanical properties of the material and it should be experimentally defined. However, for the sake of simplicity and conservative results, by assuming as a first approximation that k = 0.5 ftk (as in case of pure shear and according to the Tresca yield criterion [37]), Eq. (1) is derived from the condition to prevent moment failure that is T0 < T’0, where T’0 is the shear corresponding to the limiting moment M0 (T’0 = 2M0/h). On the other hand, to analyze how the frictional and shear resistances interact with each other, three different models have been considered: a model including interlocking blocks with similar heights of connectors (Figure 3) and two models including different heights of the lower and upper connectors (Figures 4 and 5). In the first model, due to the simultaneity of the bed contacts between each projection and depression, each connector is subjected to shear and compressive forces. On the other side, in the second and third models in Figures 4 and 5, respectively, the connectors of one block are subjected to shear and compressive forces, while the connectors of the other block are only subjected to shear forces. The shear resistance of a connector which is subjected to shear and compressive forces T”0 is greater than the shear resistance of a connector which is only subjected to pure shear forces T0, because of the effect of the compression. 2.2. Heuristic formulations relating the sliding resistance of interlocking blocks to the frictional resistance of conventional blocks This means that the weaker connectors are the latter ones and as a result, this paper aims at considering T0 as the ultimate shear forces a connector can resist (the more conservative choice). Figure 3. Sliding resistances of interlocking blocks with same height for two-block connectors (Case 1). Figure 3. Sliding resistances of interlocking blocks with same height for two-block connectors (Case 1) The resultant sliding resistance of the interlocking blocks can be derived both with reference to Cases 2 and 3. In both cases, the frictional resistances (Ts and T’s in Figures 4 and 5) may activate only after the shear collapse of the connectors on bed joints represented by the thicker blue lines in the figures. Therefore, considering Case 2 (Figure 4), where the upper projections are higher than the lower ones, the bed joints of the two interlocking blocks are localized at the bottom 7 depressions of the lower block. In this case, the shear resistances of each upper and lower block connector are T”0 and T0, respectively. Whatever the position of projections i) or ii) in Figure 4, the resultant limiting shear force of all connectors and the frictional resistance of all bed joints respectively are:  N T T n T C R = − = ; 2 1 0  N T T n T C R = − = ; 2 1 0  N TC = (3) (3)  N TC = where  is the friction coefficient, N is the fixed normal force (including the own weight of the upper block). Figure 4. Sliding resistances of interlocking blocks with upper connectors higher than lower ones (Case 2). Figure 4. Sliding resistances of interlocking blocks with upper connectors higher than lower ones (Case 2 Figure 5. Sliding resistances of interlocking blocks with lower connectors higher than upper ones (Case 3). Figure 5. Sliding resistances of interlocking blocks with lower connectors higher than upper ones (Case Since the failure mode is first governed by the shear collapse of the projections, the ultimate lateral force strongly depends on the amounts of the two resistances of Eq. (3). In fact, if TR > TC the ultimate lateral force will be T = TR because once the failure is activated the frictional resistance is not enough to prevent sliding. 2.2. Heuristic formulations relating the sliding resistance of interlocking blocks to the frictional resistance of conventional blocks This condition, implying non-ductile behaviour of the interface, is represented by the black dot in Figure 6a, where the greater constant shear resistance is superposed on the choesionless Coulomb yield condition. On the contrary, if TR < TC the lateral force will be governed by TC (T = TC) and a ductile behaviour may occur because at the onset of the shear failure mode of projections the frictional resistances will also be activated and the sliding is prevented until the shear achieves the value TC (Figure 5b). This means that, to avoid sliding of an interlocking block in the direction normal to the connectors, the following inequality should be always met: 8 (4) 𝑇≤max⁡(μ𝑁, 𝑇𝑅) (b) (a) (b) Figure 6. Ultimate lateral force in the a) non-ductile case TR > TC and b) ductile case TR < TC. (a) (b) (a) Figure 6. Ultimate lateral force in the a) non-ductile case TR > TC and b) ductile case TR < TC. Using the first expression of Eq. (2), Ineq. (4) can be rewritten as: Using the first expression of Eq. (2), Ineq. (4) can be rewritten as: 𝑇≤max (μ𝑁, ( 𝑛−1 3𝑛) τ𝑘𝑎𝑏) (5) 𝑇≤max (μ𝑁, ( 𝑛−1 3𝑛) τ𝑘𝑎𝑏) (5) which represents the relationship between the geometries of the projections and interlocking block sliding resistance, limited by being n an integer and odd number. This inequality also shows that the sliding resistance depends on either friction coefficient or shear strength of the block. On the other hand, if Case 3 is considered (Figure 5), the bed joints of the two interlocking blocks are localized at the top projections of the lower block. In this case, the shear resistances of each lower and upper block connector are T”0 and T0, respectively. In fact, it is easy to verify within Figure 5 that the positions of projections i) and ii) for Case 3 are the mirrored positions ii) and i) for Case 2, respectively, about a horizontal line. Therefore, as in Case 2, the lateral resistance is governed by the relationship between the two resistances (Figure 6) and the geometrical parameters satisfying Ineq. (5). Besides, the sliding in the direction parallel to the connectors is only governed by the inequality 𝑇≤𝜇𝑁. This means that the interlocking block proposed by this paper can be considered as a conventional block with orthotropic friction so that the frictional constraints are Ineq. 2.2. Heuristic formulations relating the sliding resistance of interlocking blocks to the frictional resistance of conventional blocks (5) or 𝑇≤𝜇𝑁, depending on the direction. The heuristic formulation given by Ineq. (5) will be applied in the next section to the limit state analysis of a semi-circular arch composed of interlocking blocks and subjected to its own weight. In this case, only the sliding resistance along the direction orthogonal to the 9 connectors of interlocking blocks is activated. Later in Section 4, this heuristic formulation will be implemented to develop the optimization algorithm for such structures. 3. LIMIT ANALYSIS OF A SEMI-CIRCULAR ARCH COMPOSED OF INTERLOCKING BLOCKS As introduced in Section 1, standard limit analysis of rigid block assemblages has been found to be a valuable computational tool for developing two groups of structural optimization problems: 1) to find the maximum load a structure can tolerate (predict the collapse load); and 2) to find the minimum material (i.e., minimum thickness) a structure can have to bear a specific amount of load. Both approaches lead to find the classical rocking failure mechanism of such structures. To develop these optimization problems, either static or kinematic theorems can be applied. The occurrence of Coulomb frictional sliding, which implies non-associative flow rule, is in general excluded from the analysis in order to ensure the validity of the normality condition, one basic hypothesis of classic limit state analysis. In fact, it is well-known that the bounding theorems of plastic limit analysis do not in general provide unique solutions for the collapse load factor if a non-associative flow rule is specified [34] and the classical procedure does not assure that the structure is safe. However, Casapulla and Lauro [16] have identified a special class of non-associative friction problems for which provably unique solutions exist. The class comprises arches with symmetrical loading and geometry. The proposed procedure was applied to arches of this sort to both verify that the numerical and analytical solutions coincide and to investigate the convergence characteristics of the method. Exploiting the uniqueness of the solution due to the symmetry, the present work is mainly aimed at extending this optimization method to design the minimum thickness of a semi- circular arch composed of interlocking blocks and subjected to its own weight. In fact, to minimize the thickness of such an arch, first the desired radius for the arch centreline R is specified and then the optimization problem tries to find the closest thrust line to this centreline which satisfies the equilibrium and yield conditions at contact interfaces. This section explains how to find a thrust line meeting equilibrium and yield conditions. Next section introduces two optimization strategies to find the optimal conditions. The general thrust line is defined by a set of control points variable in function of two points A(0; YA) and B(XB; 0), respectively at the crown and the springing joints (Figure 7a). The X- 10 coordinates of the control points are the same of the application points of each block weight, while the Y-coordinates are found as follows. 3. LIMIT ANALYSIS OF A SEMI-CIRCULAR ARCH COMPOSED OF INTERLOCKING BLOCKS According to Casapulla and Lauro’s method [16], for the case of semi- circular arch subjected to symmetric loads, the ultimate value for Ti is independent of Ni and 11 11 the expression 𝑁𝑖tan(𝜑) can be substituted by two expressions 𝑊𝑖 𝑝𝑟𝑜𝑔 sin(𝛼𝑖+𝜑) sin 𝜑, for inward Ti the expression 𝑁𝑖tan(𝜑) can be substituted by two expressions 𝑊𝑖 𝑝𝑟𝑜𝑔 sin(𝛼𝑖+𝜑) sin 𝜑, for inward Ti and 𝑊𝑖 𝑝𝑟𝑜𝑔 sin(𝛼𝑖−𝜑) sin 𝜑, for outward Ti (Figure 7c); where Wiprog is the sum of weights form blocks and 𝑊𝑖 𝑝𝑟𝑜𝑔 sin(𝛼𝑖−𝜑) sin 𝜑, for outward Ti (Figure 7c); where Wiprog is the sum of weights form blocks 0 to i and αi is the angle between the lower contact joint of the block i and Y-axis and 𝑊𝑖 𝑝 𝑔 sin(𝛼𝑖−𝜑) sin 𝜑, for outward Ti (Figure 7c); where Wiprog is the sum of weights form blocks 0 to i and αi is the angle between the lower contact joint of the block i and Y-axis. and 𝑊𝑖 𝑝 𝑔 sin(𝛼𝑖−𝜑) sin 𝜑, for outward Ti (Figure 7c); where Wiprog is the sum of weights form blocks 0 to i and αi is the angle between the lower contact joint of the block i and Y-axis. After this substitution, Ineq. (10) can be splitted into the two following yield conditions for semi-circular arches composed of interlocking blocks: \|𝑇𝑖\| ≤max⁡(( 𝑊𝑖 𝑝𝑟𝑜𝑔 sin⁡(𝛼𝑖+𝜑) sin 𝜑) , (( 𝑛−1 3𝑛) 𝜏𝑘𝑎𝑏))⁡⁡⁡⁡inward⁡𝑇𝑖 (11) \|𝑇𝑖\| ≤max⁡(( 𝑊𝑖 𝑝𝑟𝑜𝑔 sin⁡(𝛼𝑖−𝜑) sin 𝜑) , (( 𝑛−1 3𝑛) 𝜏𝑘𝑎𝑏))⁡⁡⁡outward⁡𝑇𝑖 (12) (11) (12) Lastly, it should be taken into consideration that since all block weights, the thrust line, and the limiting sliding forces for all blocks lie on the same plane, the problem can be considered as a 2d problem. In this case, the sliding resistance of all interfaces with orthotropic friction is only regarded to be the maximum sliding resistance value on those interfaces (orthogonal to the connectors in Fig. 1b). This means that an arch with interlocking blocks which embeds a thrust line whose control points are obtained by Eqs. (7) and (9) and meets Ineqs. (11) and (12), is a stable arch. 3. LIMIT ANALYSIS OF A SEMI-CIRCULAR ARCH COMPOSED OF INTERLOCKING BLOCKS Given XB and YA, the horizontal reactive thrust Htot can be found as: coordinates of the control points are the same of the application points of each block weight, while the Y-coordinates are found as follows. Given XB and YA, the horizontal reactive thrust Htot can be found as: 𝐻𝑡𝑜𝑡= 𝑊𝑡𝑜𝑡 𝑋𝐵−𝑋𝑤 𝑌𝐴 (7) 𝐻𝑡𝑜𝑡= 𝑊𝑡𝑜𝑡 𝑋𝐵−𝑋𝑤 𝑌𝐴 (7) where Wtot is the weight of the half-arch and Xw is the X-coordinate of the half-arch centre of mass. Being Wi the weight of block i and xit the X-coordinate both of its application point and of the control point i, ∆𝑌𝑖 can be achieved by applying the equilibrium condition: where Wtot is the weight of the half-arch and Xw is the X-coordinate of the half-arch centre of mass. Being Wi the weight of block i and xit the X-coordinate both of its application point and of the control point i, ∆𝑌𝑖 can be achieved by applying the equilibrium condition: ∆𝑌𝑖= ∆𝑋𝑖∑ 𝑊𝑖 𝑖−1 𝑖=1 𝐻𝑡𝑜𝑡 (8) 𝑊𝑖 (8) (8) where ΔXi = xi+1t − xit. Knowing ∆𝑌𝑖, Y-coordinate of control point i, can be achieved by: where ΔXi = xi+1t − xit. Knowing ∆𝑌𝑖, Y-coordinate of control point i, can be achieved by: where ΔXi xi+1 xi . Knowing ∆𝑌𝑖, Y coordinate of control point i, can be achieved by: 𝑦𝑖 𝑡= 𝑦𝑖+1 𝑡 + ∆𝑌𝑖 (9) Figure 7. Thrust line in the half interlocking block arch. (9) Figure 7. Thrust line in the half interlocking block arch. A valid thrust line should also meet the yield conditions at contact interfaces. Applying the heuristic method described in Section 2.2, interlocking blocks composing a semi-circular arch can be considered as conventional blocks whose sliding resistance can be evaluated by Ineq. (5) which for interface i can be rewritten as: 𝑇𝑖≤max (𝑁𝑖tan(𝜑), ( 𝑛−1 3𝑛) τ𝑘𝑎𝑏) (10) 𝑇𝑖≤max (𝑁𝑖tan(𝜑), ( 𝑛−1 3𝑛) τ𝑘𝑎𝑏) (10) where Ti and Ni are the tangential and normal components of Si, respectively, (Figure 7b) and tan(φ) equals µ. 4.1. First strategy In this strategy, the number of projections and depressions n at each interface remains fixed during the optimization. As a result, changing the arch thickness a during optimization, the shear resistance of interfaces changes as well. The thrust line should also meet the yield conditions by satisfying Ineqs. (11) and (12) during the optimization. Hence, the optimization problem for this strategy is as follows: 𝐌𝐢𝐧⁡𝐦𝐚𝐱⁡ max(√(𝑥𝑖 𝑡)2 + (𝑦𝑖 𝑡)2 −𝑅) (13) S.T. (13) \|𝑇𝑖\| ≤max⁡(( 𝑊𝑖 𝑝𝑟𝑜𝑔 sin⁡(𝛼𝑖+𝜑) sin 𝜑) , ( 𝑛−1 3𝑛) 𝜏𝑘𝑏2 max(√(𝑥𝑖 𝑡)2 + (𝑦𝑖 𝑡)2 −𝑅)) inward Ti \|𝑇𝑖\| ≤max⁡(( 𝑊𝑖 𝑝𝑟𝑜𝑔 sin⁡(𝛼𝑖−𝜑) sin 𝜑) , ( 𝑛−1 3𝑛) 𝜏𝑘𝑏2 max(√(𝑥𝑖 𝑡)2 + (𝑦𝑖 𝑡)2 −𝑅)) outward Ti The optimal arch only embeds the obtained thrust line and transforms the statically indeterminate structure into a mechanism with hinges and/or sliding surfaces, as described in Section 5. 4. STRUCTURAL OPTIMIZATION As explained earlier, the optimization problem of this paper is aimed at finding the closest thrust line to the specified arch centreline (minimum thickness) which satisfies the equilibrium and yield conditions at interfaces. Unlike the conventional blocks whose frictional properties remain fixed during optimization, changing the arch thickness during optimization may change the sliding resistance at interfaces. In the following sub-sections, two strategies are proposed to minimize the arch thickness (Figure 8), based on different relationships between the geometry of the interlocking interfaces and the sliding resistance. In the first strategy, the sliding resistance changes during the optimization process; in the second strategy, the geometric properties of interfaces changes in a way that the sliding resistance remains fixed during optimization. For both strategies the objective function is the minimization of the maximum value of the arch half-thickness (amin/2), as described in [16] for the arch with conventional blocks. The variables of the problem are XB and YA of the generic thrust line (Figure 7). 12 12 Figure 8. Two optimization strategies to minimize the thickness of a semi-circular arch with interlocking blocks. Two optimization strategies to minimize the thickness of a semi-circular arch with interlocking blocks. 4.2. Second strategy ax\|√(𝑥𝑖 𝑡)2 + (𝑦𝑖 𝑡)2 −𝑅\| (16) (16) \|𝑇𝑖\| ≤max⁡(( 𝑊𝑖 𝑝𝑟𝑜𝑔 sin⁡(𝛼𝑖+𝜑) sin 𝜑) , 𝑇𝑅 0) inward Ti \|𝑇𝑖\| ≤max⁡(( 𝑊𝑖 𝑝𝑟𝑜𝑔 sin⁡(𝛼𝑖−𝜑) sin 𝜑) , 𝑇𝑅 0) outward Ti 𝑛= ⌊𝑛⌋; 𝑛%2 = 1; 𝑛≥0 ( 𝑖𝜑) \|𝑇𝑖\| ≤max⁡(( 𝑊𝑖 𝑝𝑟𝑜𝑔 sin⁡(𝛼𝑖−𝜑) sin 𝜑) , 𝑇𝑅 0) outward Ti 𝑛= ⌊𝑛⌋; 𝑛%2 = 1; 𝑛≥0 𝑛= ⌊𝑛⌋; 𝑛%2 = 1; 𝑛≥0 This objective function of the optimization problem is the same as the previous strategy and the sliding resistance of the arch is met by applying the two first inequalities. 4.2. Second strategy In this strategy, shear resistance TR remains fixed during optimization via changing the number of the connectors. Given the initial values for XB and YA (XB0 and YA0), the initial Y- coordinates of the control points (yit0 for block i) and then the fixed TR0 can be obtained. In fact, given the initial n (n0) which should be at least three, and considering the arch initial half-thickness (a0/2) to be the maximum distance between the radius and control points on this thrust line, the initial value of shear resistance TR0 can be calculated as follows: 13 𝑇𝑅 0 = ( 𝑛0−1 3𝑛0 ) 𝜏𝑘𝑏2max⁡ \|√(𝑥𝑖 t0) 2 + (𝑦𝑖 t0) 2 −𝑅\| (14) = ( 𝑛0−1 3𝑛0 ) 𝜏𝑘𝑏2max⁡ \|√(𝑥𝑖 t0) 2 + (𝑦𝑖 t0) 2 −𝑅\| (14 (14) where xit0 is the same as xit of the objective function (13). where xit0 is the same as xit of the objective function (13). This value remains fixed during optimization, while both arch thickness a and n change during optimization. Given n0, xit0 and yit0, the relation between a (which equals 2\|√(𝑥𝑖 𝑡)2 + (𝑦𝑖 𝑡)2 −𝑅\|) and n can be defined as follows: This value remains fixed during optimization, while both arch thickness a and n change during optimization. Given n0, xit0 and yit0, the relation between a (which equals 2\|√(𝑥𝑖 𝑡)2 + (𝑦𝑖 𝑡)2 −𝑅\|) and n can be defined as follows: 𝑛= 𝑛0.(2max⁡) 𝑛0.(2max\|√(𝑥𝑖 𝑡) 2+(𝑦𝑖 𝑡) 2−𝑅\|)+(2 max\|√(𝑥𝑖 𝑡) 2+(𝑦𝑖 𝑡) 2−𝑅\|)(1−𝑛0) (15) (15) which should be an (a) integer, (b) odd, and (c) positive number during optimization. As a result, the optimization problem for the second strategy is as follows: 𝐌𝐢𝐧⁡ max\|√(𝑥𝑖 𝑡)2 + (𝑦𝑖 𝑡)2 −𝑅\| (16) which should be an (a) integer, (b) odd, and (c) positive number during optimization. As a result, the optimization problem for the second strategy is as follows: 𝐌𝐢𝐧⁡ max\|√(𝑥𝑖 𝑡)2 + (𝑦𝑖 𝑡)2 −𝑅\| (16) S.T. which should be an (a) integer, (b) odd, and (c) positive number during optimization. As a result, the optimization problem for the second strategy is as follows: 𝐌𝐢𝐧⁡ max\|√(𝑥𝑖 𝑡)2 + (𝑦𝑖 𝑡)2 −𝑅\| (16) S.T. 5. RESULTS AND EVALUATIONS This section is composed of two main parts. First, a case study of a semi-circular arch of 10m centreline radius, containing 27 discrete blocks is analyzed by means of the two introduced optimization strategies. These results are investigated to compare the two proposed optimization strategies to each other and to the Casapulla and Lauro’s optimization method developed for arches with conventional blocks [16]. Then, to evaluate the accuracy of the proposed heuristic method and the limit state analysis method, the interlocking block interface and the case study are analysed by Finite Element method for comparison. 14 14 5.1. Case study In the following, after a brief description on the implemented process, the case study is presented to investigate the relation between the minimum arch thickness, obtained by the two introduced optimization strategies, and the sliding resistance of the block interfaces. Finding these relations, minimum thickness/Radius (t/R) ratio can be defined as a function of the parameters determining the block sliding resistance. 5.1.1. Implementation For modelling the arch with its interlocking blocks and also for part of the structural calculations, Grasshopper’s C# component was applied. Grasshopper is a visual programming language which runs within Rhinoceros 3D. Another part of the structural calculations and optimization are performed by MATLAB used as backend. For the optimization, MATLAB’s fminimax method was used. Multiple hard constraints in the second strategy imposed on variable n could have led to find no solution. To avoid this, soft constraints were used, weighted through trial and error to achieve the most reliable results. 5.1.2. Optimal arches with conventional blocks This sub-section uses the results obtained by Casapulla and Lauro’s optimization method for the sake of comparison with the results obtained by the proposed optimization strategies applied to conventional blocks. Figure 9 shows the relationship between the predicted minimum arch thickness required for stability and the coefficient of friction for the introduced semi-circular arch with conventional blocks. Figure 9. Relationship between the minimum thickness of an arch composed of conventional blocks and the coefficient of friction; red points show formed hinges and green lines represent block sliding interfaces. Figure 9. Relationship between the minimum thickness of an arch composed of conventional blocks and the coefficient of friction; red points show formed hinges and green lines represent block sliding interfaces. 15 15 It results that: 1) for  > 0.395, the Heyman’s theoretical minimum thickness of 10.7% of the centreline radius is approximately reproduced with this discretization (10.74%) and the failure mode is governed by a pattern of hinges at the intrados and extrados; 2) for 0.395 ≥  ≥ 0.332, the failure mechanism is characterized by the sliding interfaces at the horizontal supports along with hinges at the crown and at variable points in between; 3) for  < 0.332 the equilibrium is no longer possible. 5.1.3. Optimal arches with interlocking blocks obtained by the first optimization strategy Figure 10 shows the relationship between the predicted minimum arch thickness required for stability and the friction coefficient for the introduced arch composed of interlocking blocks with fixed n = 5. This relationship varies for different values of parameter 𝑇𝑅 𝑓 𝑇𝑈, where TRf is shear resistance obtained by the first of Eq. (3) for amin and TU is the ultimate frictional resistance of an interface at the onset of the sliding failure. Therefore, when 𝑇𝑅 𝑓 𝑇𝑈≤1, the frictional resistance is greater than the shear resistance and when 𝑇𝑅 𝑓 𝑇𝑈≥1 it is vice versa. Figure 10. Relationship between the minimum thickness of an arch composed of interlocking blocks and the coefficient of friction- 1st optimization strategy; red circles show formed hinges and green lines represent block sliding interfaces. Figure 10. Relationship between the minimum thickness of an arch composed of interlocking blocks and the coefficient of friction- 1st optimization strategy; red circles show formed hinges and green lines represent block sliding interfaces. Figure 10. Relationship between the minimum thickness of an arch composed of interlocking blocks and the coefficient of friction- 1st optimization strategy; red circles show formed hinges and green lines represent block sliding interfaces. Figure 10 represents both the case of 𝑇𝑅 𝑓 𝑇𝑈= 0.98, in which the arch behaves as an arch with conventional blocks and the case of 𝑇𝑅 𝑓 𝑇𝑈= 1.08, whose curve is horizontally moved to the left side. As shown in the figure, the minimum friction coefficient assuring stability has a lower 16 value compared to the minimum one for an arch with conventional blocks, since TR and not TU determines the arch sliding resistance. This result is consistent with the discussion provided in Section 2.2 on the heuristic formulations. value compared to the minimum one for an arch with conventional blocks, since TR and not TU determines the arch sliding resistance. This result is consistent with the discussion provided in Section 2.2 on the heuristic formulations. The results obtained from the first strategy can also be represented from a larger perspective by Figure 11, for  between 0.3 and 0.4 and 𝑇𝑅 𝑓 𝑇𝑈 between 0.98 and 1.20. Figure 11. Relationship between the minimum thickness of an arch composed of interlocking blocks, the coefficient of friction, and 𝑇𝑅 𝑓 𝑇𝑈- 1st optimization strategy. Figure 11. 5.1.3. Optimal arches with interlocking blocks obtained by the first optimization strategy Relationship between the minimum thickness of an arch composed of interlocking blocks, the coefficient of friction, and 𝑇𝑅 𝑓 𝑇𝑈- 1st optimization strategy. Any section of this figure along the direction of -axis, presents the relation between the minimum arch thickness required for stability and 𝑇𝑅 𝑓 𝑇𝑈. For example, Figure 12 shows a section of the graph in Figure 11, corresponding to  = .37. According to this graph when 𝑇𝑅 𝑓 𝑇𝑈 is smaller than one (the frictional resistance is greater than the shear resistance), the minimum arch thickness is equal to the minimum thickness of an arch composed of conventional blocks with  = 0.37. When 𝑇𝑅 𝑓 𝑇𝑈 is larger than 1.07, the sliding resistance is large enough so that hinges form earlier than sliding failure. In this case, the minimum arch thickness is equal to the minimum thickness of an arch composed of conventional blocks with  > 0.395. 17 17 Figure 12. Relationship between the minimum thickness of an arch composed of interlocking blocks and 𝑇𝑅 𝑓 𝑇𝑈- 1st optimization strategy; red circles show formed hinges and green lines represent block sliding failure. Figure 12. Relationship between the minimum thickness of an arch composed of interlocking blocks and 𝑇𝑅 𝑓 𝑇𝑈- 1st optimization strategy; red circles show formed hinges and green lines represent block sliding failure. 5.1.4. Optimal arches with interlocking blocks obtained by the second optimization strategy According to the second strategy proposed above, the initial value of the shear resistance is fixed while the arch thickness and the number of connectors change during optimization. First it is worth highlighting that the final result (a ) is highly dependent on the initial According to the second strategy proposed above, the initial value of the shear resistance is fixed while the arch thickness and the number of connectors change during optimization. First, it is worth highlighting that the final result (amin) is highly dependent on the initial values of the optimization parameters (YA0, XB0), due to the way that the final n is related to YA0 and XB0, i.e.: First, it is worth highlighting that the final result (amin) is highly dependent on the initial values of the optimization parameters (YA0, XB0), due to the way that the final n is related to YA0 and XB0, i.e.: 𝑎𝑚𝑖𝑛⁡𝑛0 𝑛⁡𝑛0+𝑎0(1−𝑛0) (17) 𝑛= 𝑎𝑚𝑖𝑛⁡𝑛0 𝑎𝑚𝑖𝑛⁡𝑛0+𝑎0(1−𝑛0) 𝑛= 𝑎𝑚𝑖𝑛⁡𝑛0 𝑎𝑚𝑖𝑛⁡𝑛0+𝑎0(1−𝑛0) 𝑛= 𝑎𝑚𝑖𝑛⁡𝑛0 𝑎𝑚𝑖𝑛⁡𝑛0+𝑎0(1−𝑛0) (17) In fact, Eq. (17) is obtained when the initial value of shear resistance TR0, which should remain fixed during optimization, is calculated according to Eq. (14). Considering the final n to be a positive value in Eq. (17), this inequality should be always met: In fact, Eq. (17) is obtained when the initial value of shear resistance TR0, which should remain fixed during optimization, is calculated according to Eq. (14). Considering the final n to be a positive value in Eq. (17), this inequality should be always met: 𝑎𝑚𝑖𝑛⁡𝑛0 ≥𝑎0(𝑛0 −1) (18) (18) 𝑎𝑚𝑖𝑛⁡𝑛0 ≥𝑎0(𝑛0 −1) and this means that the final result amin is dependent on the initial thickness a0 which is determined by YA0 and XB0. and this means that the final result amin is dependent on the initial thickness a0 which is determined by YA0 and XB0. Secondly, it should also be underscored that the minimum thickness obtained by the second strategy might differ from that of the first strategy for the same values of friction coefficient and 𝑇𝑅 0 𝑇𝑈 in order to guarantee that TR0 is kept unchanged during the optimization of the second strategy, i.e. that it is: 𝑎𝑚𝑖𝑛⁡ 𝑛−1 𝑛= 𝑎0⁡ 𝑛0−1 𝑛0 (19) 18 18 The right hand side of this equation is a known value while amin and n should be chosen so that this value is achieved. 5.1.4. Optimal arches with interlocking blocks obtained by the second optimization strategy Both amin and n are constrained variables; amin should satisfy the equilibrium condition with sliding constraints and n should be a positive odd value. As a result, amin satisfying Eq. (19) might be different from results of first optimization strategy. It is concluded that the thrust line which is found as the optimal result may not be the unique solution of the arch embedding it. In other words, the rocking failure mechanism of the arches cannot be precisely found. Still, the closest possible solution to that failure mechanism can be found as follows. For example, Figure 13 depicts the tangential force at interface m of the case study optimized by the first strategy when this interface reaches the limiting sliding value (blue curve). This figure also depicts the tangential force at interface m of the case study (XB0 and YA0 equal 10.81 m) optimized by the second strategy (red curve) for the same 𝑇𝑅 0 𝑇𝑈. As shown, these two curves are quite close but not identical. In fact, blue curve represent the closest possible value to the limiting one satisfying Eq. (17) Figure 13. Difference between tangential force at interface m for the optimal arch optimized by first and second optimization strategies Figure 13. Difference between tangential force at interface m for the optimal arch optimized by first and second optimization strategies Build on the two discussed issues, the relationship between the minimum arch thickness required for stability and 𝑇𝑅 0 𝑇𝑈 for the arch explained above, with n0 = 3 and XB0 and YA0 equal to 10.81 m, is as follows (Figure 14- yellow continuous curve). When 𝑇𝑅 0 𝑇𝑈 is smaller than 1.01, the minimum arch thickness equals 1.36 m. It is worth noting that for 1 ≤ 𝑇𝑅 0 𝑇𝑈 𝐵≤1.01 there is no set of amin smaller than 1.36 m and n by which Eq. (19) is satisfied. When 𝑇𝑅 0 𝑇𝑈 is larger than 1.06, the minimum arch thickness equals 1.12 m. According to Ineq. (17), amin should be 19 greater than 1.09. The closest amin to this limiting value which satisfies Eq. (19) is calculated to be 1.12 m. Figure 14. Relationship between the predicted minimum thickness of an arch composed of interlocking blocks and 𝑇𝑅 0 𝑇𝑈- 2nd optimization strategy; red circles show formed hinges. Figure 14. 5.2. Validation and calibration via FEA The heuristic method and the extension of limit analysis of this paper are validated by comparing the results obtained by the proposed methods with the results achieved by Finite Element Analysis (FEA) based on the simplified macro modelling technique proposed by [32]. In this method, blocks are morphed (controlled expansion) to eliminate mortar joints and are considered to be solid or shell elements, while mortar joints are zero-thickness interface elements. This validation includes three parts: in the two first parts the proposed heuristic method is studied by analysing two sets of interlocking blocks with different geometries; the third part studies the proposed limit analysis methods and optimization strategies by analysing the case study of the arch analysed above. The FEA is developed in ABAQUS 6.11-1 by adopting the brittle cracking model. In the first two parts, to purely focus on the shear behaviour at the joint between the main body and the connectors of a block, the following model is designed and analysed: two stacked blocks with a shared interlocking interface are modelled. The connectors and the main body of each of the blocks are modelled with rigid body elements (rigid parts) while the joint between them is modelled with C3D8R elements (flexible part with brittle behaviour) (Figures 16b and 18b). The material properties of elements are shown in Tables 1. The effective density of the expanded blocks is calculated using homogenization process proposed in [32]. Table 1. Material properties of expanded blocks. Compressive strength 4.14 MPa Tensile Strength 0.36 MPa Shear strength 0.5⁡𝑓𝑡𝑘= 0.18 MPa Effective Young’s modulus 3 GPa Poisson’s ratio 0.25 Effective density 𝜌= (150 × 0.5 × 0.24) −(340 × 0.5 × 0.01) 0.5 × 0.25 = 130.4 Kg/m3 Block density 150 Kg/m3 Mortar density 340 Kg/m3 Table 1. Material properties of expanded blocks. The lower face of the lower block is bounded so that no degree of freedom is allowed. The upper face of the upper block is bounded so that one degree of freedom is allowed in the direction of lateral force application. The loads imposed on the model are the weight of the upper block and a lateral force distributed on a vertical face of this block (Figures 16c and 18c). The lateral force by which the principal stress reaches the tensile strength is found. This value is compared to the shear resistance obtained by the proposed heuristic method. 5.1.4. Optimal arches with interlocking blocks obtained by the second optimization strategy Relationship between the predicted minimum thickness of an arch composed of interlocking blocks and 𝑇𝑅 0 𝑇𝑈- 2nd optimization strategy; red circles show formed hinges. ure 14. Relationship between the predicted minimum thickness of an arch composed of interlocking blocks and 𝑇𝑅 0 𝑇𝑈- 2nd optimization strategy; red circles show formed hinges. Satisfying both Ineq. (17) and Eq. (19), yellow continuous curve in Figure 14 is slightly different from that of Figure 12 (results of the first strategy) which is redisplayed by purple dash curve. Still, their overall configuration is similar, as explained above. Figure 15 shows the relationship between the final n for the optimal arch and 𝑇𝑅 0 𝑇𝑈. According to this graph, for 𝑇𝑅 0 𝑇𝑈 greater than 1.0583, the final n remains fixed while all interfaces are resistant enough against sliding so that failure occurs due to the hinge formation. The graph also shows that increasing 𝑇𝑅 0 𝑇𝑈 the rate of change of final n increases. Figure 15. Relationship between the final number of projections and depressions n for the optimal arch composed of interlocking blocks and 𝑇𝑅 0 𝑇𝑈- 2nd optimization strategy; red circles show formed hinges. Figure 15. Relationship between the final number of projections and depressions n for the optimal arch composed of interlocking blocks and 𝑇𝑅 0 𝑇𝑈- 2nd optimization strategy; red circles show formed hinges. 20 5.2. Validation and calibration via FEA The distribution of principal stresses and shear stresses τxy in X direction on the plane whose 21 normal vector is Y is studied as well. The elastic analysis is used to find the stress state of the model. 5.2.1. Analysis of interlocking blocks with n = 3 Figure 16a shows the size of the blocks in meters and the layout of rigid and flexible parts of each block. The maximum lateral force obtained by FEA is 9.42 kN, when the maximum principal stress (0.36 MPa) is observed on the joint between the connector and main body of the lower block. Figure 16. a) Dimensions of the analysed model with n = 3; b) layout of rigid and flexible elements; c) boundary conditions and lateral force distributed on the upper block. Figure 16. a) Dimensions of the analysed model with n = 3; b) layout of rigid and flexible elements; c) boundary conditions and lateral force distributed on the upper block. The maximum τxy is also 0.177 MPa. Figure 17 presents the distribution of principal stresses and τxy on the connector of the lower block which is maximum at two edges of the connector (τxy are 0.177 and 0.134 MPa) and minimum in the middle of connector (τxy is 0.122 Mpa). Figure 17. Principal stress state for a) the whole model with n = 3 and b) for the two flexible layers of the two blocks; c) directions of the principal stresses; d) τxy for the whole model and e) at the two flexible layers of the two blocks. Figure 17. Principal stress state for a) the whole model with n = 3 and b) for the two flexible layers of the two blocks; c) directions of the principal stresses; d) τxy for the whole model and e) at the two flexible layers of the two blocks. 22 Using Eq. (3), the sliding resistance of the proposed heuristic method is calculated: 2/3 × 0.18 × 103 × 0.167 × 0.5 = 10.02 kN. The ratio of sliding resistance obtained by FEA to the proposed heuristic method is 0.94, showing a very good agreement between these results. Using Eq. (3), the sliding resistance of the proposed heuristic method is calculated: 2/3 × 0.18 × 103 × 0.167 × 0.5 = 10.02 kN. The ratio of sliding resistance obtained by FEA to the proposed heuristic method is 0.94, showing a very good agreement between these results. 5.2.2. Analysis of interlocking blocks with n = 7 Figure 18 presents the size of the blocks in meters and the layout of rigid and flexible parts of each block. Figure 18. a) dimensions of the analysed model with n=7; b) layout of rigid and flexible elements; c) boundary condition, the distributed lateral force is imposed on the upper block vertical face in –X direction. Figure 18. a) dimensions of the analysed model with n=7; b) layout of rigid and flexible elements; c) boundary condition, the distributed lateral force is imposed on the upper block vertical face in –X direction. The maximum lateral force obtained by FEA is 26.1 kN. As shown in Figure 19, the maximum principal stress (0.36 MPa) is observed on the joint between the main body of the lower block and the connector that is closer to the lateral face where the horizontal load is applied. The maximum τxy of 0.201 MPa occurs on the same joint. However, the stresses are not uniformly distributed on all the three joints between the three connectors and the main body of the lower block. The more a joint is closer to the applied lateral force, the larger stresses it has. For lateral force of 26.1 kN, the maximum principal stresses on joints between each of the two further connectors and the main body of the lower block are 0.3 and 0.276 MPa. This means that, depending on the interaction between the other connectors after the failure of the first connector, the final value of the force that corresponds to the failure of all the connectors would be larger than 26.1 kN. The sliding resistance according to the heuristic method (Eq. 3) equals 3 × 2/3 × 0.18 × 103 × 0.167 × 0.5 = 30.06 kN. This value is slightly larger than that obtained by FEA because of the failure of all the connectors. Actually, this difference between the maximum force obtained by FEA and the heuristic method could be due to the difference of the lateral force application point. In the heuristic method, in fact, this point is considered to be the centroid of the upper block and, consequently, the stresses are uniformly distributed on all the joints between the connectors and the main body of the block. 23 Figure 19. 5.2.2. Analysis of interlocking blocks with n = 7 Principal stress state for a) the whole model with n = 7 and b) for the two flexible layers of the two blocks; c) directions of the principal stresses; d) τxy for the whole model and e) at the two flexible layers of the two blocks. Figure 19. Principal stress state for a) the whole model with n = 7 and b) for the two flexible layers of the two blocks; c) directions of the principal stresses; d) τxy for the whole model and e) at the two flexible layers of the two blocks. 5.2.3. Analysis of the case study arch composed of interlocking blocks with n = 5 In the case of the arch with assigned radius R = 10m and subjected to its own weight, blocks are modelled with C3D8R elements and the mortar joints between them are modelled as shell interfaces with friction coefficient  = 0.37. The material properties are those reported in Table 1, with the exception of the Young’s modulus which is increased to 6 GPa. The modelling of rigid body elements used in the previous analyses is no longer suitable to the case of the arch due to the presence of non-negligible bending moments at interfaces. This implies some limits of validation for the proposed heuristic method which is based on the pure shear condition. However, the validation of the optimal arch with thickness of 1.07 m, obtained by the first optimization procedure for 𝑇𝑅 𝑓 𝑇𝑈 = 1.08, is herein performed, mainly in terms of failure mode. In fact, using the proposed limit state method, for this optimal model the pure rocking mechanism is observed, that is fully captured by the FE model, as shown in Figure 20. In fact, using the proposed limit state method, for this optimal model the pure rocking mechanism is observed, that is fully captured by the FE model, as shown in Figure 20. In sum, the three cases examined in this section can be considered a good validation of the proposed heuristic method and optimization procedure, with some limitations in the case of In fact, using the proposed limit state method, for this optimal model the pure rocking mechanism is observed, that is fully captured by the FE model, as shown in Figure 20. 5.2.2. Analysis of interlocking blocks with n = 7 In sum, the three cases examined in this section can be considered a good validation of the proposed heuristic method and optimization procedure, with some limitations in the case of rocking/sliding failure modes. Further studies and experimental investigation will be provided by future work. 24 Figure 20. a) boundary condition; b) normal stresses in Y direction (σy); c) shear stresses in X direction on a plane whose normal vector is Y (τxy); d) deformation of the arch 𝑇𝑅 𝑓 𝑇𝑈 = 1.08. Figure 20. a) boundary condition; b) normal stresses in Y direction (σy); c) shear stresses in X direction on a plane whose normal vector is Y (τxy); d) deformation of the arch 𝑇𝑅 𝑓 𝑇𝑈 = 1.08. 6. CONCLUSIONS A Digital tool was developed to design the structurally sound semi-circular arch with interlocking blocks. The main achievements of this research are: A Digital tool was developed to design the structurally sound semi-circular arch with interlocking blocks. The main achievements of this research are: • a heuristic method to find the relationship between the geometric properties of the interlocking connectors and the block sliding resistance; • a limit state analysis approach to analyze the stability of arches composed of interlocking blocks; by using the heuristic method, it extends the limit state approach in order to define a new stability condition for semi-circular arches composed of interlocking blocks when the interface sliding resistance varies in different directions; • two optimization methods to minimize the thickness of the semi-circular arches composed of interlocking blocks via adjusting the control points of constructed the thrust line for the arch; this work extends the optimization algorithm finding the thinnest structurally feasible arch composed of blocks with finite friction. In this extension the newly developed condition of stability (sliding constraint) for arches composed of interlocking blocks are applied. In the first method, the sliding resistance changes during the optimization. The results show that if the shear resistance is less than the frictional resistance, the arch behaves as an arch with conventional blocks. When the shear resistance is larger than the frictional resistance, the • two optimization methods to minimize the thickness of the semi-circular arches composed of interlocking blocks via adjusting the control points of constructed the thrust line for the arch; this work extends the optimization algorithm finding the thinnest structurally feasible arch composed of blocks with finite friction. In this extension the newly developed condition of stability (sliding constraint) for arches composed of interlocking blocks are applied. In the first method, the sliding resistance changes during the optimization. The results show that if the shear resistance is less than the frictional resistance, the arch behaves as an arch with conventional blocks. When the shear resistance is larger than the frictional resistance, the 25 structural behaviour of the arch is governed by the shear strength. For small values of shear strength, the combined rocking/sliding mechanism occurs for the optimal result. For large values of shear strength, pure rocking mechanism can be observed on the optimal result. In the second method, the sliding resistance is kept fixed via changing the number of the connectors. 6. CONCLUSIONS The results are mostly dependent on the constraints keeping the number of the connectors an odd integer value and also on the initial value of sliding resistance defined by the designer. Due to these items, the optimal result for the minimum thickness might be thicker comparing to the result of the first optimization method, given the same shear strength, friction coefficient and geometric inputs. In this case, the mechanism does not occur on the optimal solution. A good agreement of the results obtained by the heuristic method and optimization procedure was found in case of pure shear conditions, while some limits of validation were highlighted in case of mixed rocking/sliding failure modes. Future works will address these issues together with the extension of the approach to the 3d models. Furthermore, the sliding resistance of interlocking blocks will also be experimentally investigated together with the analysis of different shapes of the interfaces. ACKNOWLEDGMENTS This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie Grant Agreement No. 791235. It reflects only the authors’ view and the Agency is not responsible for any use that may be made of the information it contains. References [1] Dyskin, A.V., Pasternak, E. Estrin, Y. (2012). Mortarless structures based on topological interlocking. Frontiers of Structural and Civil Engineering, 6(2): 188-197. 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https://openalex.org/W3209123329	https://www.nature.com/articles/s41467-021-26348-y.pdf	English	null	Consumption in the G20 nations causes particulate air pollution resulting in two million premature deaths annually	Nature communications	2,021	cc-by	12,115	ARTICLE Consumption in the G20 nations causes particulate air pollution resulting in two million premature deaths annually Keisuke Nansai 1,2✉, Susumu Tohno 3, Satoru Chatani 4, Keiichiro Kanemoto 5, Shigemi Kagawa 6, Yasushi Kondo 7, Wataru Takayanagi1 & Manfred Lenzen 2 Keisuke Nansai 1,2✉, Susumu Tohno 3, Satoru Chatani 4, Keiichiro Kanemoto 5, Shigemi Kagawa 6, Yasushi Kondo 7, Wataru Takayanagi1 & Manfred Lenzen 2 Worldwide exposure to ambient PM2.5 causes over 4 million premature deaths annually. As most of these deaths are in developing countries, without internationally coordinated efforts this polarized situation will continue. As yet, however, no studies have quantiﬁed nation-to- nation consumer responsibility for global mortality due to both primary and secondary PM2.5 particles. Here we quantify the global footprint of PM2.5-driven premature deaths for the 19 G20 nations in a position to lead such efforts. G20 consumption in 2010 was responsible for 1.983 [95% Conﬁdence Interval: 1.685–2.285] million premature deaths, at an average age of 67, including 78.6 [71.5–84.8] thousand infant deaths, implying that the G20 lifetime con- sumption of about 28 [24–33] people claims one life. Our results indicate that G20 nations should take responsibility for their footprint rather than focusing solely on transboundary air pollution, as this would expand opportunities for reducing PM2.5-driven premature mortality. Given the infant mortality footprint identiﬁed, it would moreover contribute to ensuring infant lives are not unfairly left behind in countries like South Africa, which have a weak relationship with G20 nations. 1 Material Cycles Division, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba 305-8506, Japan. 2 ISA, School of Physics, Faculty of Science, The University of Sydney, Camperdown, NSW 2006, Australia. 3 Graduate School of Energy Science, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan. 4 Regional Environment Conservation Division, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba 305-8506, Japan. 5 Research Institute for Humanity and Nature, 457-4 Motoyama, Kamigamo, Kita-ku, Kyoto 603-8047, Japan. 6 Faculty of Economics, Kyushu University, 6-19-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan. 7 Faculty of Political Science and Economics, Waseda University, 1-6-1 Nishi-Waseda, Shinjuku-ku, Tokyo 169-8050, Japan. ✉email: nansai.keisuke@nies.go.jp 1 NATURE COMMUNICATIONS \| (2021) 12:6286 \| https://doi.org/10.1038/s41467-021-26348-y \| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-26348-y T o achieve planetary health1, a world that is healthy for both the global biosphere and human civilization, the critical challenge is to mitigate the human health hazards, as well as suppress the environmental impacts created by socioeconomic activities, reining in the latter to within the Earth’s environmental tolerance. Results F i Footprint of PM2.5 mortality induced by G20 nations. In 2010, 1.983 [95% Conﬁdence Interval (CI): 1.685, 2.285] million pre- mature deaths in ﬁve disease categories (Lower Respiratory Infection (LRI), Chronic Obstructive Pulmonary Disease (COPD), Lung Cancer (LC), stroke and Ischemic Heart Disease (IHD)), occurred in 199 countries and regions listed in Supple- mentary Table 1 in the supporting information (SI) around the world as a result of airborne PM2.5 induced by consumption in the 19 nations eligible for the G20 summit presidency (Table 1). The total number of premature deaths due to PM2.5 worldwide is estimated to be 4.019 [CI: 3.413, 4.630] million, a ﬁgure in broad agreement with previous estimates, although the year, method and data employed differ; e.g., 3.440 million in 2007 for four diseases (COPD, LC, stroke, IHD)12 and 3.15 million31 and 3.23 million32 for the same ﬁve diseases in 2010. The greatest number of deaths occur in China: 1.195 [CI: 1.029, 1.362] million, fol- lowed by India: 0.907 [CI: 0.790, 1.022] million, while the other studies estimated them at 1.024 million12 deaths in 2007, 1.367 million33 and 1.300 million (over 30 years only)34 deaths in 2013 for the four diseases in China, and 0.9 million35 deaths for the ﬁve diseases in 2014 in India. By applying consumption-based accounting11, the health impact of PM2.5 induced, by way of the supply chain, by the consumption of one country in another12–17, that of one region in another18–22 and that of one racial-ethnic group in another23 can be calculated; this impact is referred to as a footprint. Multi- national footprint analysis24 helps clarify the rationale and degree of responsibility underlying implementation of joint measures among affecting and affected countries. To date, the multinational footprints of PM2.5 primary particle emissions25 have been quantiﬁed and the multinational footprints of their associated health impacts14–16,26 have been clariﬁed, taking into account the effects of both trade and transboundary pollution. However, only two studies12,17 on multinational footprints have addressed the health impacts of secondary PM2.5 particles formed by chemical reactions in the atmosphere, which in major global cities con- tribute more to atmospheric PM2.5 concentrations than primary black carbon particles27. Consumption in the G20 nations causes particulate air pollution resulting in two million premature deaths annually Among the many environmental problems affecting human health, the greatest threat is that posed by the inhalation of particles with an aerodynamic diameter of 2.5 μm or less, abbreviated to PM2.52. According to the World Health Organi- zation (WHO)3, in 2016 respiratory and cardiovascular diseases and cancer caused by exposure to ambient PM2.5 were responsible for approximately 4.2 million premature deaths, i.e., deaths occurring before the average age of death in the population concerned. The majority of these deaths were in countries with low and middle incomes3, while 91% of the global population lives in areas where air quality is below WHO guidelines4. The loss to the global workforce due to premature deaths attributable to PM2.5 was equivalent to 225 billion US dollars in 20135. While the reduction in air pollution due to the COVID-19 pandemic6 has mitigated human health impacts somewhat, it is neither sufﬁcient nor lasting7–9. address nation-to-nation consumer responsibility for this chal- lenge at the global level. T In order to equitably accommodate consumer responsibility28,29, joint measures will need to be implemented both bilaterally and multilaterally, to facilitate negotiations among related countries, promote decision-making at a high policy level and initiate international agreements. Such a high- level international agenda will increase the likelihood of such initiatives succeeding. The meeting of the Group of Twenty, or G20, is a regular international meeting attended by leaders of developed countries with a high degree of consumer responsi- bility and developing countries incurring consumption-related health damage. G20 members represent around 80% of the world’s economic output, two-thirds of the global population and three-quarters of international trade30. There is a process in which the members of the G20 discuss challenges and measures related to ﬁnancial and socioeconomic issues. These discussions are then published as outcome documents by the leaders in attendance. The G20 is thus, potentially, a productive high-level meeting that could be used to advance joint measures for resol- ving the problems associated with PM2.5-related health issues. g WHO air quality guidelines4 stipulate that the annual mean concentration of PM2.5 in the atmosphere should not exceed 10 μg/m3. Compared with a concentration of 35 μg/m3, as is frequently observed in developing cities, 10 μg/m3 or less can be expected to decrease PM2.5-related deaths by 15%4. In China, with the largest number of premature deaths2, compliance with the WHO guidelines would reduce annual PM2.5-related deaths by 81.5%10. Consumption in the G20 nations causes particulate air pollution resulting in two million premature deaths annually Atmospheric PM2.5 levels can be reduced by cutting emissions of primary PM2.5 particles and the precursors of sec- ondary particles, such as sulphur dioxides, nitrogen oxides and volatile organic compounds (VOCs). Key reduction measures include improvements in combustion technology for power generation, transportation and waste treatment3, as well as ﬂue gas treatment technology for dust collection, denitration, and desulphurization. While our understanding of the impact of PM2.5 on human health, target concentrations and abatement technologies has undeniably grown, though, the reality is that developing countries cannot be expected to implement such countermeasures without ﬁnancial and technical support from the international community. With respect to PM2.5, therefore, two critical challenges remain: to understand how responsibility for the high number of premature deaths is distributed across the nations of the world; and to set up a process for deploying countermeasures, embedded in a high-level international policy framework. To date, however, no studies have quantiﬁed the consumer responsibility of G20 nations for the substantial health impacts caused by atmospheric PM2.5. This lack of scientiﬁc knowledge risks delaying international collaborative efforts to safeguard the victims of the PM2.5 pollution. To this end, we aim to identify the consumer responsibility of G20 nations for the global premature deaths caused by both primary and secondary PM2.5 particles and compare this ﬁgure with the premature deaths caused by the domestic and transboundary pollution due to each nation’s production-based emissions, the primary focus at present. NATURE COMMUNICATIONS \| (2021) 12:6286 \| https://doi.org/10.1038/s41467-021-26348-y \| www.nature.com/naturecommunicatio Results F i PM2.5 premature death footprint 95% CI (conﬁdence interval) Secondary particles contribution to footprint Share of foreign deaths in footprint Premature deaths by production-based PM2.5 emissions 95% CI Share of foreign deaths in deaths by production- based emissions Difference in deaths between footprint and production- based emissions 95% CI [1000 deaths/y] [1000 deaths/y] [%] [%] [1000 deaths/y] [1000 deaths/y] [%] [1000 deaths/y] [1000 deaths/y] 5.66 [4.62, 6.71] 52 41 7.18 [5.80, 8.58] 6.4 −1.5 [−1.9, −1.2] 5.74 [4.61, 6.94] 67 82 1.56 [0.97, 2.22] 1.1 4.2 [3.6, 4.7] 29.8 [23.6, 36.3] 43 24 27.2 [21.1, 33.4] 2.7 2.6 [2.4, 2.8] 13.5 [10.8, 16.4] 69 85 7.96 [5.79, 10.4] 56 5.5 [5.0, 6.0] 905 [777, 1033] 54 7.6 1090 [936, 1242] 5.8 −185 [−209, −158] 27.9 [22.8, 33.3] 73 83 19.7 [15.6, 24.1] 65 8.2 [7.1, 9.2] 44.0 [36.0, 52.3] 76 78 38.9 [31.1, 47.3] 54 5.1 [5.0, 5.1] 493 [431, 555] 48 13 549 [480, 617] 12 −56 [−62, −49] 52.7 [42.1, 63.4] 41 13 59.9 [47.5, 72.4] 3.8 −7.1 [−8.9, −5.4] 22.4 [18.4, 26.5] 77 84 12.6 [10.1, 15.3] 58 9.7 [8.3, 11] 41.8 [34.8, 49.0] 67 74 16.5 [13.2, 19.9] 9.7 25 [22, 29] 14.9 [12.3, 17.6] 64 39 11.8 [9.6, 14.0] 9.5 3.2 [2.7, 3.7] 74.6 [60.4, 89.4] 77 38 76.3 [61.4, 91.7] 25 −1.7 [−2.3, −1.0] 12.4 [10.7, 14.] 84 96 8.72 [7.6, 9.8] 91 3.6 [3.1, 4.3] 20.1 [17.1, 22.9] 66 39 23.3 [19.9, 26.6] 30 −3.2 [−3.6, −2.8] 20.2 [17.0, 23.4] 71 83 13.5 [11.3, 15.8] 60 6.6 [5.7, 7.6] 25.8 [21.6, 30.2] 71 71 22.4 [18.7, 26.2] 61 3.4 [2.9, 4.0] 34.3 [28.1, 40.7] 75 74 20.2 [15.9, 24.6] 38 14 [12, 16] 139 [112, 168] 72 62 64.8 [48, 83] 9.7 75 [64, 85] 323 [263, 386] 72 62 181 [140, 224] 22 143 [123, 162] 1983 [1685, 2285] 57 11 2071 [1759, 2384] 8.4 −88 [−99, −74] 1 Summary of PM2.5 premature death footprint and PM2.5 premature deaths due to the production-based emissions of each G20 nation in 2010. Results F i Furthermore, as those two studies have a lower geographical resolution or scope—calculating footprints for each of 13 aggregated regions of the world12 and for ﬁve afﬂuent countries with respect to 34 Asian countries17—they fail to Since half the 4.019 million deaths are attributable to consumption in the G20 nations, the G20 meeting is a promising high-level policy platform for formulating an international response to mitigate the health impacts of PM2.5 on a global scale. In terms of nations with the largest premature mortality footprint due to PM2.5 (PM2.5 premature death footprint), China has the largest at 905 [CI: 777, 1033] thousand deaths, followed by India at 493 [CI: 431, 555] thousand, the US at 139 [CI: 112, 168] thousand, Russia at 74.6 [CI: 60.4, 89.4] thousand, and Indonesia at 52.7 [CI: 42.1, 63.4] thousand. Given the differences in year and diseases studied, these death footprints can be deemed URE COMMUNICATIONS \| (2021) 12:6286 \| https://doi.org/10.1038/s41467-021-26348-y \| www.nature.com/naturecommunications 2 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-26348-y mary of PM2.5 premature death footprint and PM2.5 premature deaths due to the production-based emissions of each G20 nation in 2010. ARTICLE This is over and against only 58.5 [CI: 43.4, 75.0] thousand domestic US deaths (close to a previous estimate of 54.9 thousand deaths31 in 2010), which at present tends to be the sole concern as long as the focus is exclusively on production- based emissions (Supplementary Fig. 1 in the SI). Compared with the situation in 200712, the PM2.5 impact of US consumption has shifted slightly to India (9.9 thousand in 2007) from China (54.4 thousand in 2007). The footprint of other G20 nations likewise includes impacts on non-G20 nations. Given that it takes time and considerable ﬁnancial resources for a single nation to develop abatement measures jointly with individual non-G20 nations, it would be beneﬁcial if the G20 nations could work together with nations outside the G20 to reduce premature deaths due to PM2.5. broadly similar to the ﬁgures reported earlier by Zhang et al.12: China at 835 thousand, India at 442 thousand, the US at 165 thousand and Russia at 74.0 thousand in 2007. With 75 [CI: 64, 85] thousand deaths, the US stands out in terms of the mortality difference between the footprint and production-based emissions, while China is at the opposite end of the spectrum, with 1.090 [CI: 936, 1.242] million premature deaths due to production emissions, which is 185 [CI: 158, 209] thousand higher than for the footprint. Overall, 13 G20 nations have a PM2.5 premature death footprint exceeding the premature deaths caused by their own production emissions (Table 1). p With the exception of Indonesia and India, about half the deaths in the above nations’ footprints are attributable to secondary particles, conﬁrming that factoring in the impact of secondary particles is essential to understanding the health impact footprint of PM2.5 in the G20 nations. The proportion of foreign deaths in the respective footprint is 7.6% for China, 13% for India, 62% for the US, 38% for Russia, and 13% for Indonesia. Considering the G20 as a whole, there are eleven nations with a percentage of foreign deaths exceeding 50%, while for six of these nations, including Saudi Arabia (96%) and Canada (85%), the ﬁgure is over 80%. This highlights the fact that around half the G20 countries need to address their PM2.5 footprint as an international issue. ARTICLE What the G20 nations have in common is that the human health footprint of the PM2.5 associated with their consumption exceeds the impact on other countries of transboundary PM2.5 pollution from their production activities. nations outside the G20 to reduce premature deaths due to PM2.5. China features prominently in the footprints of Australia, Canada, Japan and South Korea, with China ranking highest among the impacted countries (Fig. 2). In addition to impacting their own countries, France, Germany, Italy, and the United Kingdom (UK) likewise have footprints biased toward China, which perhaps unsurprisingly, is a hotspot for health impacts in the consumption supply chain of developed countries. In the case of production emissions, however, only Korea has a signiﬁcant impact on China, and China’s presence as an affected country is weak for many countries (Supplementary Fig. 1 in the SI). In addition, the G20 nations are less likely to be among the top 10 affected countries in terms of production emissions, as is the case for Australia, the US and Canada. Therefore, in the G20 high- level policy meetings, taking a footprint perspective will make discussions on joint PM2.5 measures more constructive. While appreciation of the number of premature deaths among G20 nations due to transboundary pollution in the form of production emissions creates a motive to implement joint PM2.5 reduction measures among China, Japan, and South Korea, and between the United States and Canada, such motivation is unlikely to arise among countries that are geographically distant, because the mutual direct impact is very small. Quantifying the deaths occurring among nations based on the footprint calcula- tion, on the other hand, enables us to discover closer interactions among them, especially for China and India, which may broadly motivate joint measures to secure PM2.5 reduction (Fig. 1). Infant deaths and average age of death in the footprint. The elderly are more susceptible to death than younger age groups, with the highest number of deaths occurring among those aged 80 and over (Fig. 3). However, there is signiﬁcant mortality among infants aged 0–5; consumption in G20 nations comes at the expense of 78.6 [CI: 71.5, 84.8] thousand infant lives annually, more than in any other age group under 50. ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-26348-y [CI: 39.2, 67.9] thousand deaths). In addition, the US consumer responsibility also extends to non-G20 countries, such as Ban- gladesh (2.1 [CI: 1.8, 2.3] thousand deaths), and the Philippines (1.5 [CI: 1.2, 1.7] thousand deaths), which are among the top 10 countries affected. This is over and against only 58.5 [CI: 43.4, 75.0] thousand domestic US deaths (close to a previous estimate of 54.9 thousand deaths31 in 2010), which at present tends to be the sole concern as long as the focus is exclusively on production- based emissions (Supplementary Fig. 1 in the SI). Compared with the situation in 200712, the PM2.5 impact of US consumption has shifted slightly to India (9.9 thousand in 2007) from China (54.4 thousand in 2007). The footprint of other G20 nations likewise includes impacts on non-G20 nations. Given that it takes time and considerable ﬁnancial resources for a single nation to develop abatement measures jointly with individual non-G20 nations, it would be beneﬁcial if the G20 nations could work together with nations outside the G20 to reduce premature deaths due to PM2.5. China features prominently in the footprints of Australia, Canada, Japan and South Korea, with China ranking highest among the impacted countries (Fig. 2). In addition to impacting their own countries, France, Germany, Italy, and the United Kingdom (UK) likewise have footprints biased toward China, which perhaps unsurprisingly, is a hotspot for health impacts in the consumption supply chain of developed countries. In the case of production emissions, however, only Korea has a signiﬁcant impact on China, and China’s presence as an affected country is weak for many countries (Supplementary Fig. 1 in the SI). In addition, the G20 nations are less likely to be among the top 10 affected countries in terms of production emissions, as is the case for Australia, the US and Canada. Therefore, in the G20 high- level policy meetings, taking a footprint perspective will make discussions on joint PM2.5 measures more constructive. [CI: 39.2, 67.9] thousand deaths). In addition, the US consumer responsibility also extends to non-G20 countries, such as Ban- gladesh (2.1 [CI: 1.8, 2.3] thousand deaths), and the Philippines (1.5 [CI: 1.2, 1.7] thousand deaths), which are among the top 10 countries affected. Results F i ations PM2.5 premature death footprint 95% CI (conﬁdence interval) Secondary particles contribution to footprint Share of foreign deaths in footprint Premature deaths by production-based PM2.5 emissions 95% CI Share of foreign deaths in deaths by production- based emissions Difference in deaths between footprint and production- based emissions 95% CI [1000 deaths/y] [1000 deaths/y] [%] [%] [1000 deaths/y] [1000 deaths/y] [%] [1000 deaths/y] [1000 deaths/y] ina 5.66 [4.62, 6.71] 52 41 7.18 [5.80, 8.58] 6.4 −1.5 [−1.9, −1.2] lia 5.74 [4.61, 6.94] 67 82 1.56 [0.97, 2.22] 1.1 4.2 [3.6, 4.7] 29.8 [23.6, 36.3] 43 24 27.2 [21.1, 33.4] 2.7 2.6 [2.4, 2.8] aa 13.5 [10.8, 16.4] 69 85 7.96 [5.79, 10.4] 56 5.5 [5.0, 6.0] 905 [777, 1033] 54 7.6 1090 [936, 1242] 5.8 −185 [−209, −158] a 27.9 [22.8, 33.3] 73 83 19.7 [15.6, 24.1] 65 8.2 [7.1, 9.2] nya 44.0 [36.0, 52.3] 76 78 38.9 [31.1, 47.3] 54 5.1 [5.0, 5.1] 493 [431, 555] 48 13 549 [480, 617] 12 −56 [−62, −49] sia 52.7 [42.1, 63.4] 41 13 59.9 [47.5, 72.4] 3.8 −7.1 [−8.9, −5.4] 22.4 [18.4, 26.5] 77 84 12.6 [10.1, 15.3] 58 9.7 [8.3, 11] 41.8 [34.8, 49.0] 67 74 16.5 [13.2, 19.9] 9.7 25 [22, 29] o 14.9 [12.3, 17.6] 64 39 11.8 [9.6, 14.0] 9.5 3.2 [2.7, 3.7] 74.6 [60.4, 89.4] 77 38 76.3 [61.4, 91.7] 25 −1.7 [−2.3, −1.0] Arabia 12.4 [10.7, 14.] 84 96 8.72 [7.6, 9.8] 91 3.6 [3.1, 4.3] Africa 20.1 [17.1, 22.9] 66 39 23.3 [19.9, 26.6] 30 −3.2 [−3.6, −2.8] Korea 20.2 [17.0, 23.4] 71 83 13.5 [11.3, 15.8] 60 6.6 [5.7, 7.6] 25.8 [21.6, 30.2] 71 71 22.4 [18.7, 26.2] 61 3.4 [2.9, 4.0] ma 34.3 [28.1, 40.7] 75 74 20.2 [15.9, 24.6] 38 14 [12, 16] 139 [112, 168] 72 62 64.8 [48, 83] 9.7 75 [64, 85] al 323 [263, 386] 72 62 181 [140, 224] 22 143 [123, 162] otal 1983 [1685, 2285] 57 11 2071 [1759, 2384] 8.4 −88 [−99, −74] ons 3 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-26348-y ARTICLE Fig. 2 Breakdown of PM2.5 premature death footprint of G20 nations by impacted country and contribution of primary and secondary particles to footprint. Asterisks above bars indicate G20 nations with presidency rights. Country and region codes within the top 10 are as listed in Supplementary Table 1 in the Supporting Information; others are aggregated as ‘oth’. Error bars indicate 95% conﬁdence intervals. The centre of the bars indicates the mean value. Fig. 2 Breakdown of PM2.5 premature death footprint of G20 nations by impacted country and contribution of primary and secondary particles to footprint. Asterisks above bars indicate G20 nations with presidency rights. Country and region codes within the top 10 are as listed in Supplementary Table 1 in the Supporting Information; others are aggregated as ‘oth’. Error bars indicate 95% conﬁdence intervals. The centre of the bars indicates the mean value. footprints of consumption in South Africa, Saudi Arabia, India and Indonesia include 2.6 [CI: 2.4, 2.8], 1.7 [CI: 1.6, 1.9], 50.7 [CI: 46.2, 54.7], and 2.1 [CI: 1.9, 2.3] thousand infant deaths, respectively, resulting in a high percentage of such deaths. In addition, although the bias towards infant deaths is not as pro- minent as in the above four nations, China (11.0 [CI: 10.0, 11.8] thousand deaths) and the US (3.5 [CI: 3.2, 3.8] thousand deaths) have the next highest numbers of infant deaths following India. pollution from Japan. Focusing solely on production emissions also creates a little opportunity for these developed countries to take responsibility for deaths outside the G20 nations, in particular in Africa and Asia. On the other hand, each footprint of the countries concerned highlights the involvement of infant deaths also in non- G20 countries in Africa and Asia. For instance, the US footprint involves substantial infant deaths in non-G20 countries (1.5 [CI: 1.4, 1.6] thousand), many of them in Africa (0.55 [CI: 0.50, 0.59] thousand), and Asia (0.66 [CI: 0.60, 0.71] thousand) (Fig. 5). By contrast, the production emissions in most of the G7 nations and Australia cause few infant deaths (Supplementary Fig. 2 in the SI), making the average age of death in such nations higher than the age in their footprint. Remarkably, Australia (7 years), Japan (6 years), and Canada (5 years) cause more than 5 years’ difference in the average age of death between production emissions and footprint (Fig. 4). ARTICLE Although the average age of death in the footprint due to the total consumption of the G20 nations is 67, certain countries such as South Africa (57 years), Saudi Arabia (59 years), India (60 years) and Indonesia (62 years) show distinctly younger mortality ages owing to the inclusion of high infant death rates. In concrete ﬁgures, the Countries with the largest share in the footprint. To further clarify the bilateral relationships embodied in the footprint, it was broken down by the countries impacted (Fig. 2). For example, the US consumption has a signiﬁcant impact on China (38.7 [CI: 33.3, 44.1] thousand deaths), India (12.9 [CI: 11.3, 14.5] thousand deaths), Mexico (3.9 [CI: 3.2, 4.6] thousand deaths), and Russia (2.1 [CI: 1.7, 2.5] thousand deaths), as well as on the US itself (52.9 Fig. 1 Comparison of footprint-based and transboundary pollution-based relationships among G20 nations for the number of PM2.5-related premature deaths. Country codes as follows: Argentina (AR), Australia (AU), Brazil (BR), Canada (CA), China (CN), France (FR), Germany (DE), India (IN), Indonesia (ID), Italy (IT), Japan (JP), Mexico (MX), Russia (RU), Saudi Arabia (SA), South Africa (ZA), South Korea (KR), Turkey (TR), United Kingdom (UK), and United States (US). Fig. 1 Comparison of footprint-based and transboundary pollution-based relationships among G20 nations for the number of PM2.5-related premature deaths. Country codes as follows: Argentina (AR), Australia (AU), Brazil (BR), Canada (CA), China (CN), France (FR), Germany (DE), India (IN), Indonesia (ID), Italy (IT), Japan (JP), Mexico (MX), Russia (RU), Saudi Arabia (SA), South Africa (ZA), South Korea (KR), Turkey (TR), United Kingdom (UK), and United States (US) Fig. 1 Comparison of footprint-based and transboundary pollution-based relationships among G20 nations for the number of PM2.5-related premature deaths. Country codes as follows: Argentina (AR), Australia (AU), Brazil (BR), Canada (CA), China (CN), France (FR), Germany (DE), India (IN), Indonesia (ID), Italy (IT), Japan (JP), Mexico (MX), Russia (RU), Saudi Arabia (SA), South Africa (ZA), South Korea (KR), Turkey (TR), United Kingdom (UK), and United States (US). NATURE COMMUNICATIONS \| (2021) 12:6286 \| https://doi.org/10.1038/s41467-021-26348-y \| www.nature.com/naturecommunications 4 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-26348-y These disparities conﬁrm that there is a clear heterogeneity between production and footprint not only in the number of deaths but also in the age distribution of mortality. In other words, a shift in focus to the consumption of these nations, rather than their production, creates the potential for avoiding substantial loss of the length of human life. Unpacking the structure of the footprint-based responsibility brings about a clear rationale for those nations to address this challenge (Fig. 5). In India, for instance, although domestic infant deaths (44.1 [CI: 40.2, 47.7] thousand) induced by the country’s own consumption remain high, the US (1.3 [CI: 1.2, 1.4] thousand), the UK (0.37 [CI: 0.34, 0.40] thousand), Germany (0.35 [CI: 0.32, 0.38] thousand), Japan (0.26 [CI: 0.24, 0.29] thousand), and France (0.21 [CI: 0.19, 0.22] thousand) are responsible for a non-negligible number of infant deaths there. Total premature deaths caused by G20 lifetime consumption. Converting the G20’s aggregate footprint to a per-capita footprint shows that individual consumption in the G20 nations leads on average to 0.46 [CI: 0.39, 0.53] × 10−3 premature deaths per year (Supplementary Table 2 in the SI), with this impact continuing every year until death. Multiplying the per-capita footprint by the present life expectancy of G20 consumers permits a rough esti- mation of the total number of premature deaths due to individual lifetime consumption, which certainly differs across nations (Fig. 6). Taking the average life expectancy of G20 nations as 77.46 years, as in 2018, G20 per-capita lifetime consumption translates to 0.036 [CI: 0.030, 0.041] deaths; taking the reciprocal, the lifetime consumption of every 28 [CI: 24, 33] G20 citizens leads to the premature death of one person. For consumption in the G7, with a smaller per-capita footprint than the G20, the mortality ﬁgure for per-capita lifetime consumption is the same as for the G20: 0.036 [CI: 0.029, 0.043], because of about 4 years’ longer life expectancy. Among the G20, China has the highest value: 0.051 [CI: 0.044, 0.058] deaths/capita, or 20 [CI: 17, 23] people’s lifetime consumption costing one life, followed by Ger- many and the UK (both 0.044 [CI: 0.036, 0.052] deaths/capita), Russia (0.038 [CI: 0.031, 0.045] deaths/capita), and France (0.037 [CI: 0.030, 0.044] deaths/capita). p g g As long as responsibility for infant deaths due to production emissions is the only issue pursued (Supplementary Fig. NATURE COMMUNICATIONS \| (2021) 12:6286 \| https://doi.org/10.1038/s41467-021-26348-y \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-26348-y NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-26348-y When the average life expectancy exceeds 80 years the longevity For other G20 nations with a life expectancy between 70 Fig. 3 Age and gender composition of PM2.5 premature death footprint in each G20 nation. Error bars indicate 95% conﬁdence intervals. The centre of the bars indicates the mean value. ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-26348-y Fig. 3 Age and gender composition of PM2.5 premature death footprint in each G20 nation. Error bars indicate 95% conﬁdence intervals. The centre of the bars indicates the mean value. Fig. 3 Age and gender composition of PM2.5 premature death footprint in each G20 nation. Error bars indicate 9 the bars indicates the mean value. longevity. For other G20 nations, with a life expectancy between 70 and 80 years, lifetime-induced deaths show a degree of spread. Importantly, it is hoped that the long-lived nations of the G20 will lead the way in technological, medical and economic cooperation, so the two aforementioned countries can follow the path to longer life expectancy without increasing their lifetime-induced deaths. When the average life expectancy exceeds 80 years, the difference in the footprint of each country tends to decrease, ranging from 0.021 [CI: 0.017, 0.026] in Australia to 0.044 [CI: 0.036, 0.052] in Germany. Furthermore, focusing on Japan (84.21 years), Italy (82.95 years), and Australia (82.75 years), the three longest-living countries in the world, the lifetime consumption footprints are found to converge at around 0.02–0.03 deaths/ capita, implying that it is hard to decouple longevity and consumption-induced deaths. In addition, it is noteworthy that countries with a longer life expectancy (>80 years) account for the majority of foreign deaths in their footprint. This indicates that countries with longer life expectancies need to be take greater responsibility for the global impacts of their footprints. Uncertainty in the footprints. The present study employed the annual average PM2.5 concentrations in 2010 compiled in GBD2016 (Global Burden of Disease)36,37 to estimate the total premature deaths for each grid square. These deaths were then allocated to G20 nations based on their consumption-based PM2.5 concentrations calculated by the regional chemical transport model CMAQ38. Global PM2.5 concentrations based on ground measurements for each of 1.4 million grid squares in GBD are not available owing to the low density of PM2.5 monitoring. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-26348-y 3 in the SI), we can ﬁnd no rationale for the G7 nations and Australia to confront the mass death of infants in India, China, Indonesia and South Africa except 10 deaths in China due to transboundary 5 5 TURE COMMUNICATIONS \| (2021) 12:6286 \| https://doi.org/10.1038/s41467-021-26348-y \| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-26348-y ARTICLE Fig. 4 G20 comparison of average age of death for PM2.5 premature death footprint and that for premature deaths by production-based PM2.5 emissions. Error bars indicate 95% conﬁdence intervals. The centre of the bars indicates the mean value. 2001–200740, 33 µg/m3 in 201441, and 27–69 µg/m3 in 200742 in Bandung City. This suggests a possible underestimation of Indonesia’s footprint. p The CMAQ-simulated concentration used for the premature death allocation is impacted by the accuracy of the air pollutant emissions inventory. Uncertainty has not been estimated for the emission inventory of EDGAR v4.3.143 used in this study. However, for the updated EDGAR v4.3.2, uncertainty estimates are provided for air pollutant emissions, based on the estimated uncertainty of the activity data and emission factors for each emission sector, pollutant and country44. The two EDGAR databases are consistent for almost all sources and there is no signiﬁcant discrepancy in global emissions between them except for NMVOC36. The uncertainties in the EDGAR v4.3.2 emission inventory can therefore be taken as holding for EDGAR v4.3.1, too. The uncertainties for primary PM2.5 vary by region, with estimates ranging from 49.4 to 96.8% in 2010, as do those for precursors of secondary particles: of these, NH3 (185.0–294.6%) has the highest uncertainty, while SOx (12.5–48.3%) and NOx (17.9–117.9%) are reported to have relatively small estimation errors44. When inputting the upper and lower bound emissions for 95% CI of EDGAR v4.3.1 to CMAQ, the computed PM2.5 concentra- tions in any grid square are approximately proportional to those with average emissions (Supplementary Fig. 5 in the SI). There are inﬁnite combinations of possible emissions, not merely with all grid squares taking upper or lower bounds at the same time. However, in so far as the overall emissions in the inventory data tend to be either underestimated or overestimated compared with the mean emissions, the impact on the premature death footprint allocated by the CMAQ concentrations would be modest, as concentrations vary proportionally to emissions. Within many territories, a 45° line (CMAQ concentration with average emissions) is generally located between the concentrations with the upper and lower emissions. As long as the emissions vary randomly and uniformly between the upper and lower bounds, the average concentration value is expected to approach the concentration with average emissions. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-26348-y Taking the concentration with the average emissions, as has been done in this study, is therefore considered a good approximation of the footprint based on the representative allocation rate. Fig. 4 G20 comparison of average age of death for PM2.5 premature death footprint and that for premature deaths by production-based PM2.5 emissions. Error bars indicate 95% conﬁdence intervals. The centre of the bars indicates the mean value. PM2.5 concentrations for all areas were therefore estimated using the data integration model with ground monitoring data, esti- mates of PM2.5 from remote-sensing satellites and chemical transport models within a Bayesian hierarchical modelling framework37. Hence, the 95% CIs of grid-square concentrations in GBD2016 are not necessarily narrow, as visible in the some- times wide clusters of black dots in Supplementary Fig. 4 in the SI, which is a factor of uncertainty in the footprints. The uncertainty of the footprints with IER (mean) was evaluated in the context of extreme conditions of gridded PM2.5 concentra- tions in GBD2016 (gridded concentrations for each grid square are lower or upper bounds of 95% CI concentrations). p Although the CMAQ has a spatial resolution of 45-km grid squares for more precise country-by-country analysis, at the expense of detail the world was divided into six domains to reduce the computational burden. This division makes it impossible to analyse the impact of consumption-induced concentrations out- side each domain, although, for instance, Zhang et al.12 estimated that 2.2 and 2.0% of premature deaths within the US and Canada, respectively, in 2007 were attributable to long-range transbound- ary pollution from China. We therefore quantiﬁed the contribu- tion of G20 consumption-induced concentrations to the concentrations in the grid squares located on the eastern, western, northern and southern edges of each domain (Supplementary Figs. 6–11 in the SI). For example, at the four edges of the Asian region, the largest contribution of China’s consumption to the PM2.5 concentration was 26%, in a grid square with about 5 μg/ m3. Across all peripheral grid squares, the average concentration contributed by China’s consumption was 0.14 μg/m3, suggesting that while the impact outside the region is non-zero, it is sufﬁciently small compared with that inside the region. pp Taking the lower bound of the 95% CI of GBD2016, the footprints become 0.64 times (South Africa) to 0.81 times (China, India) lower, with an average of 0.72 times for the G20 nations (Fig. 7, left). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-26348-y Gridded p y g p p South Africa has the shortest life expectancy among G20 nations (63.86 years), followed by India (69.72 years), but their lifetime- induced deaths are close to the ﬁgure for Japan, with the highest NATURE COMMUNICATIONS \| (2021) 12:6286 \| https://doi.org/10.1038/s41467-021-26348-y \| www.nature.com/naturecommunications 6 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-26348-y NATURE COMMUNICATIONS \| (2021) 12:6286 \| https://doi.org/10.1038/s41467-021-26348-y \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-26348-y Fig. 5 Relationships between consumer countries and impacted countries (“to”) for infant deaths (zero to less than ﬁve years old) in PM2.5 premature death footprint of G20 nations. umer countries and impacted countries (“to”) for infant deaths (zero to less than ﬁve years old) in PM2.5 premature Fig. 5 Relationships between consumer countries and impacted countries (“to”) for infant deaths (zero to less th death footprint of G20 nations. Fig. 5 Relationships between consumer countries and impacted countries (“to”) for infant deaths (zero to less than ﬁve years old) in PM2.5 premature death footprint of G20 nations. Fig. 6 Relationship between life expectancy, PM2.5-related premature deaths induced by lifetime consumption per capita and percentage of foreign deaths in PM2.5 premature death footprint in each G20 nation. Country codes as follows: Argentina (AR), Australia (AU), Brazil (BR), Canada (CA), China (CN), France (FR), Germany (DE), India (IN), Indonesia (ID), Italy (IT), Japan (JP), Mexico (MX), Russia (RU), Saudi Arabia (SA), South Africa (ZA), South Korea (KR), Turkey (TR), United Kingdom (UK) and United States (US). 4.002 million premature deaths based on the IER to 6.889 million deaths with the GEMM 5-COD. As for premature deaths within China, Liu et al.47 report an increase from 0.986 million deaths with the IER to 1.681 million deaths with the GEMM 5-COD in 2010. In all nations except Australia, premature deaths due to production emissions showed a higher increase than for the footprint, conﬁrming the trend that uncertainty in footprint deaths is less affected by the choice of the relative risk model. Discussion I hi d In this study, we found that consumption in the 19 nations with presidency rights to the G20, which can set the agenda for the G20 summit, induces the mortality of 1.983 [CI: 1.685, 2.285] million people a year through global supply chains as a result of PM2.5. Our analysis suggests that the G20 meeting will be further enriched as a forum for decision-makers to discuss joint inter- national measures to curb PM2.5-related premature deaths by extending the interrelationships among nations to include these consumption-based footprints. Fig. 6 Relationship between life expectancy, PM2.5-related premature deaths induced by lifetime consumption per capita and percentage of foreign deaths in PM2.5 premature death footprint in each G20 nation. Country codes as follows: Argentina (AR), Australia (AU), Brazil (BR), Canada (CA), China (CN), France (FR), Germany (DE), India (IN), Indonesia (ID), Italy (IT), Japan (JP), Mexico (MX), Russia (RU), Saudi Arabia (SA), South Africa (ZA), South Korea (KR), Turkey (TR), United Kingdom (UK) and United States (US). p p In this respect, the G20 offers greater potential than similar high-level policy meetings like the G7 (Canada, France, Germany, Italy, Japan, UK, US), as consumption by the G7 nations induces far fewer premature deaths worldwide: 323 [CI: 263, 386] thou- sand. In addition, 62% of these deaths occur outside the G7, making it difﬁcult for PM2.5 control to become a joint issue for the G7, while in the G20’s footprint, only 11% of deaths are in non-G20 nations. One point the G7 and G20 have in common is that about half the total premature deaths are attributable to secondary particles, conﬁrming the importance of mitigation measures for secondary particle precursors, including VOCs and ammonia, as well as for fuel combustion, although these are generally identical to measures addressing primary PM2.5. themselves proposed the GEMM (Global Exposure Mortality Model)46, which improves the relative risk estimation based on cohort studies of ambient PM2.5 mortality, covering higher outdoor PM2.5 concentrations, which has been a challenge in the IER model. Here, we estimated the premature deaths due to G20 footprints and those of production emissions using the GEMM 5-COD model and in each case compared them with the IER results in terms of the ﬁve diseases studied and exposures above 25 years’ age targeted by the GEMM. Although there are differences among nations, the GEMM-based footprints are all 1.5–2 times larger than the IER footprints (Fig. 7 right). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-26348-y With the upper bound concentrations, they increase between 1.11 times (India) and 1.24 times (South Africa), with a G20 average of 1.17 times. On the other hand, the footprint based on the 2010 average concentration in GBD201339 with the dust concentration data employed in this study gives a G20 mean of 0.90 times. The gridded concentrations directly calculated by the CMAQ give a G20 mean of 0.95 times, showing similar changes in the footprint as in the case of GBD2013. With both the direct CMAQ and GBD2013, the footprints of the G20 countries are generally within the range of the upper and lower bounds of the CI of GBD2016, which would support the reasonability of allocating total premature deaths in a grid based on GBD2016 concentration data by the consumption-based PM2.5 concentra- tion computed by the CMAQ. In the case of Indonesia; however, the footprint with the CMAQ (1.39 times) exceeds the upper bound case. Even at the upper bound of the CI, there is only one grid square with a PM2.5 concentration over 30 µg/m3 among Indonesian grid squares with a population density of 22.5 per- sons/km2 or more (Supplementary Fig. 4 in the SI), despite some studies observing concentrations as high as 40–50 µg/m3 in The 95% conﬁdence intervals for the number of premature deaths reported above reﬂect the uncertainty of the IER (Integrated Exposure-Response) model32,45 that determines the relative risk associated with ambient PM2.5 exposure. The IER function has been used in many previous footprint studies12,17,21,23. Recently, the developers of the IER function 7 7 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-26348-y Especially in Asia and Africa, future population growth is predicted, which implies that delays in abatement measures would permit an unnecessary increase in premature infant deaths in those regions. footprint connect to China and India, which have the largest absolute number of premature deaths, a fact that is overlooked if the focus is solely on production-based emissions. The pursuit of consumer responsibility for PM2.5 in the G20 will thus pave the way to reducing premature deaths in these two countries. There is one G20 nation, however, that does not feature among the top 10 countries with the most premature deaths occurring in the footprint of each of the 19 nations: South Africa. Unlike in the case of China and India, joint action to reduce mortality in South Africa would not appear to be an obvious priority if only general consumer responsibility is pursued. Like India and China, South Africa is a country with a high incidence of infant mortality due to PM2.5. If South Africa were to be omitted from the framework of joint measures, it would only be in this country that premature infant deaths remained unchanged. One approach to including South Africa would be to clarify consumer responsibility by focusing on the infant victims. The visualized network for PM2.5 premature infant deaths (Fig. 5) reveals the opportunities for involvement by the US, the UK, Japan, China, India and Ger- many in the protection of South African infants. Infant mortality hampers achievement not only of Goal 3 (Good health and well- being) of the Sustainable Development Goals (SDGs)48 but also that of Goal 4 (Quality education), by depriving children of access to education. There is no doubt that the achievement of Goal 17 (Partnerships for the goals), backed by footprint-based respon- sibility and rationale, will be key to overcoming this challenge. It h ld b t d th t th ff ti f f i An additional concern is that the number of premature infant deaths due to PM2.5 may potentially be even higher than the ﬁgure reported in this study. Footprints using the GEMM, the latest relative risk model, showed a 1.5- to 2-fold increase in the number of deaths in the 25+ age group compared with the footprints calculated with the IER. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-26348-y Fig. 7 (Left) Change in premature deaths footprint when the other PM2.5 concentration data (LB16, UB16, ME13, CMAQ) are used for grid-square concentrations to estimate total premature deaths in each square, relative to 1 for premature deaths with the IER (Integrated Exposure-Response) model (50% value) and PM2.5 concentration (mean) in GBD2016 (Global Burden of Disease 2016). (Right) Change in number of premature deaths (age 25 years and older) for footprint and production-based emissions when GEMM (Global Exposure Mortality Model) is used for relative risk calculation, relative to 1 for premature deaths with IER model (50% value). Error bars indicate 95% conﬁdence intervals (CI). LB16 and UB16 are the cases with the lower and upper bound concentration in 95% CI of GBD2016, respectively. ME13 is the case with GBD2013 concentration (mean) in 2010 and CMAQ is the case with concentration calculated by the CMAQ (Community Multiscale Air Quality) modelling system. The cases of IER with upper and lower bounds of the 95% CI are shown in Supplementary Fig. 12 in the Supporting Information. Fig. 7 (Left) Change in premature deaths footprint when the other PM2.5 concentration data (LB16, UB16, ME13, CMAQ) are used for grid-square concentrations to estimate total premature deaths in each square, relative to 1 for premature deaths with the IER (Integrated Exposure-Response) model (50% value) and PM2.5 concentration (mean) in GBD2016 (Global Burden of Disease 2016). (Right) Change in number of premature deaths (age 25 years and older) for footprint and production-based emissions when GEMM (Global Exposure Mortality Model) is used for relative risk calculation, relative to 1 for premature deaths with IER model (50% value). Error bars indicate 95% conﬁdence intervals (CI). LB16 and UB16 are the cases with the lower and upper bound concentration in 95% CI of GBD2016, respectively. ME13 is the case with GBD2013 concentration (mean) in 2010 and CMAQ is the case with concentration calculated by the CMAQ (Community Multiscale Air Quality) modelling system. The cases of IER with upper and lower bounds of the 95% CI are shown in Supplementary Fig. 12 in the Supporting Information. Germany (0.02 deaths per 1000), but high in India (1.97 deaths per 1000) and South Africa (1.92 deaths per 1000), representing an almost 200-fold gap49. If this gap is not reduced, infant mortality will remain concentrated in certain countries. Discussion I hi d The extent of this increase is consistent with Burnett et al.46, who conﬁrmed a change in global premature deaths in 2015 from themselves proposed the GEMM (Global Exposure Mortality Model)46, which improves the relative risk estimation based on cohort studies of ambient PM2.5 mortality, covering higher outdoor PM2.5 concentrations, which has been a challenge in the IER model. Here, we estimated the premature deaths due to G20 footprints and those of production emissions using the GEMM 5-COD model and in each case compared them with the IER results in terms of the ﬁve diseases studied and exposures above 25 years’ age targeted by the GEMM. Although there are differences among nations, the GEMM-based footprints are all 1.5–2 times larger than the IER footprints (Fig. 7 right). The extent of this increase is consistent with Burnett et al.46, who conﬁrmed a change in global premature deaths in 2015 from Among the 19 G20 nations, the consumption of eleven nations induces over 50% of premature deaths in other countries. Strik- ingly, many of the bilateral relationships involved in each nation’s NATURE COMMUNICATIONS \| (2021) 12:6286 \| https://doi.org/10.1038/s41467-021-26348-y \| www.nature.com/naturecommunications 8 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-26348-y NATURE COMMUNICATIONS \| (2021) 12:6286 \| https://doi.org/10.1038/s41467-021-26348-y \| www.nature.com/naturecommunications Methods F t To improve the accuracy of the dust concentrations of PM2.5 in each grid square, the dust concentrations in 2010 provided by GBD201339 were extrapolated to the respective corresponding 0.1° × 0.1° grid squares. When the dust concentration of a grid square exceeded the total PM2.5 concentration of the grid in GBD2016, the dust concentration was replaced by the total concentration. Stage 1: consumption-based emissions map. In Stage 1, the global multiregional input-output (MRIO) model Eora54 for the year 2010, which incorporates global supply chains among 187 countries, and the emissions inventory map EDGAR v.4.3.143 were combined using the spatial footprint analysis 55,56, which enabled the mapping of emissions by sectors and by countries determined by an environmental input-output analysis17. By inputting the domestic ﬁnal consumption of each G20 nation to Eora, we extracted only emissions globally induced by the consumption from the EDGAR emissions map. In concreto, a footprint emissions map in 2010, or consumption-based emissions map, of PM2.5 primary particles (black carbon, organic carbon and other primary components) and secondary precursors (NOx, SO2, NH3, CO, and NMVOC) was prepared for each nation. The production-based emissions map, showing the direct emissions generated in each G20 nation, was also compiled from the EDGAR emissions map by extracting the emissions within the territory of each nation. Using the IER model32,45 with the parameters reported in Supplementary Table 2 in the SI, the relative risks (50, 97.5, and 2.5% values) of ﬁve diseases (LRI, COPD, LC, stroke, and IHD) (Supplementary Fig. 20 in the SI) were calculated by age (ﬁve age groups from 0 to over 80 years old) and the number of premature deaths in each 0.1° × 0.1° grid square under base-case concentrations were determined using the same method as Apte et al.32. Then, the number of premature deaths due to consumption-based concentrations was determined using the Direct proportion of burden method12,23, which assumes that the relative risk is proportional to the concentration induced by the country concerned. In concreto, the total number of premature deaths in each 0.1°× 0.1° grid square was allocated to G20 nations based on the share of their consumption-based concentrations in the GBD-based concentrations in that square. The same procedure as above was applied to estimate the premature deaths associated with the PM2.5 concentrations originating from production-based emissions. Stage 2: ambient PM2.5 concentration. Methods F t PM2.5 concentrations, excluding the contribution of each nation’s consumption- based emissions, were then computed using the emissions map, with the nation’s consumption-based emissions being subtracted from the base-case emissions map. This was done in the same way described above, calculating the hourly values and then determining the annual average. Then, by subtracting the concentrations thus obtained from the base-case concentrations, the consumption-based PM2.5 concentrations of the nation were determined (Supplementary Figs. 14–19 in the SI). The PM2.5 concentrations from the production-based emission maps were computed using the same models and calculation conditions. Therefore, the production-based emissions are linked to only production activities, resulting in no inclusion of dust and forest ﬁres emissions. This causes discrepancy from the general particle compositions within each country’s territory. Four stages in an interdisciplinary approach. This study combines the ﬁndings of research conducted using interdisciplinary models and undertaken in four broad stages. Stage 1 focused on the creation of a footprint emissions map and production-based emissions map for each of the 19 nations eligible for the G20 summit presidency. Stage 2 was concerned with calculating the respective ambient PM2.5 concentrations deriving from the two maps. Stage 3 estimated the health impacts due to exposure to the PM2.5 concentrations calculated in Stage 2 in the 199 impacted countries that are ultimately affected by consumption in the G20. The health impacts in the countries affected by the production-based emissions of each G20 nation were also computed. Stage 4 involved estimating the total pre- mature deaths induced by lifetime consumption per capita, considering the average life expectancy of each G20 nation. Stage 3: health impacts due to ambient PM2.5. In Stage 3, premature deaths in each age group were estimated in each grid square. Population distribution data by sex and age71 were prepared for each 0.1° × 0.1° grid square, and to deﬁne the consumption-based concentrations to this level of precision, the CMAQ-based consumption-based concentrations of a nation from Stage 2 were adjusted with the average PM2.5 concentrations in 2010, based on GBD201637, within each 0.1° × 0.1° grid square. Speciﬁcally, the consumption-based concentration of the nation at grid resolution was determined by multiplying the average GBD2016-based PM2.5 concentrations in each 0.1° × 0.1° grid square by the ratio of the nation’s CMAQ- based consumption-based concentration to the base-case concentrations on a 0.5° × 0.5° grid, where the GBD and CMAQ grids overlap. ARTICLE ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-26348-y by policymakers called for here are that the G20 nations agree on a unique choice of footprint within the given range of uncertainty and that the G20 takes joint action as soon as possible to reduce the number of premature deaths due to its consumption. If these nations agreed on their smallest footprint within the uncertainty, it would be 0.78 times the mean case, which excludes 439 thou- sand deaths from the mean G20 footprint. Yet, long-term post- ponement of reduction action means G20 consumption will continue to result in more premature deaths than the number of deaths excluded. We believe the G20’s responsibility for its footprint includes the responsibility to choose and agree on a single ﬁgure without delay, within the limits of its uncertainty. fact, be affected by dust to a certain extent, as has indeed been reported64–68. In developing the GBD2016 data, ground monitoring data, remote-sensing satellite data and chemical transport models were integrated and good correlations with the GBD2016 data therefore do not necessarily mean better model performance for ambient PM2.5 concentrations. Supplementary Fig. 4 in the SI reveals narrower variations in the PM2.5 concentrations from the GBD2016 data than the values simulated by CMAQ. However, such features are not found in comparisons of the observed69 and CMAQ-simulated AOD (Aerosol Optical Depth) (Supplementary Fig. 13 in the SI), suggesting that variations in actual ambient PM2.5 concentrations are greater than in the GBD2016 data and were reproduced well by CMAQ. In particular, large deviations in Indonesia suggest possible underestimations in the GBD2016 data. Exceptional positive biases of AOD in Argentina may be due largely to biomass-burning emissions and transport from the Amazon, where there were intense forest ﬁres in 2010, to northern Argentina70. Correlations in SO42-, NO3-, and NH4+ concentrations indicate good performance of CMAQ on secondary components of PM2.5. Methods F t In Stage 2, consumption-based PM2.5 concentrations for each G20 nation were calculated using the above consumption- based emissions map and atmospheric simulation models. Zhang et al.12 demonstrated that it is more signiﬁcant for PM2.5 consumption-based accounting to understand the effects of induced PM2.5 emissions in other countries through international trade than the health impacts of long-range transboundary pollution. Therefore, aiming to more accurately capture the impact associated with the emitting countries in ﬁner resolution, this study used the regional chemical transport model CMAQ (version 5.2.1)38 as the air quality model and the regional meteorological model WRF (version 3.8.1)57, and set the grid size to 0.5° × 0.5° (about 45 km × 45 km) squares. The vertical airspace was divided into 25 layers extending to 100 hPa. The global chemical transport model MOZART-458 was used to calculate concentrations at the boundaries of the calculated region. As the detailed grid size increases the cost of computation enormously, we divided the world into six regions: Asia, Europe, North America, South America, Africa and Oceania. Stage 4: premature deaths induced by lifetime consumption. In Stage 4, a premature death footprint per capita in 2010 was calculated by dividing the pre- mature deaths induced by consumption in each G20 nation obtained above by the population of each nation (Supplementary Table 3 in the SI). Finally, lifetime- induced deaths per capita were estimated by multiplying the premature death footprint per capita by the average life expectancy in each G20 nation. The models computed hourly PM2.5 concentrations (over 12 months from January 1 to December 31, 2010) in each grid square using the original emissions map of EDGAR (v.4.3.1) and other emissions map data (GFED (Global Fire Emissions Database) v4.1s59 and MEGAN (Model of Emissions of Gases and Aerosols from Nature) v2.160) (below: ‘base-case emissions’). The annual average of hourly concentrations (below: ‘base-case concentrations’) was deﬁned as the PM2.5 concentration for that grid square. The base-case concentrations in the G20 nations were compared with the GBD201636,37 data with 95% CI, while concentrations in countries other than the G20 nations were summarized for each of the six regions as a whole, owing to the limited data available for comparing calculated and measured secondary particle concentrations, and compared with the observation data in IMPROVE61, EMEP62, and EANET63 (Supplementary Fig. 4 in the SI). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-26348-y However, the current GEMM model is unapplicable for evaluating the under-25 age group, including infant mortality, and it is quite possible that the number of infant deaths will increase in the same way as for other age groups. This concern calls for urgent development of a relative risk model to reduce the uncertainties in PM2.5-driven mortality, which will serve to improve the accuracy of estimates of comprehensive PM2.5 health damage and economic impact50–53, as well as approximating the true number of infant victims. At the upper and lower bounds of uncertainty in the PM2.5 concentration data, the G20 country footprints vary by an average of 0.72 and 1.17 times, respectively, which seems less severe than the impact of the choice of relative risk model described above. To signiﬁcantly reduce the uncertainty of the PM2.5 concentration data would require global expansion of ground monitoring points for PM2.5 concentrations using universally standard measurement methods, but this is prohibited by both time and cost constraints. There is therefore little likelihood of any major decrease in the uncertainty of the footprints in the near term. The critical actions It should be noted that the effectiveness of measures focusing on mitigation of premature infant death will be limited unless the basic infant mortality rate is improved in conjunction with decreases in PM2.5 concentrations. For example, the LRI mortality rate among male infants is low in France (0.01 deaths per 1000 people) and 9 9 References 34. Hu, J. et al. Premature mortality attributable to particulate matter in China: source contributions and responses to reductions. Env. Sci. Technol. 51, 9950–9959 (2017). 1. Whitmee, S. et al. 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All relevant data are available from the corresponding author upon reasonable request. All the data on consumption (footprint)- and production-based premature deaths and infant deaths for the 19 G20 nations based on the two cases—with the IER model32,45 and GBD201636,37 and with the GEMM46 and GBD2016—are provided as a Source Data ﬁle provided with this paper. Source data are provided with this paper. The annual PM2.5 concentrations simulated by CMAQ generally fall into the 95% CI of the GBD2016 data. Most of the high GBD2016 PM2.5 concentrations deviating furthest from the simulated values are affected by dust, which CMAQ has difﬁculty simulating accurately. Inﬂuences of dust were corrected in the following Stage 3. While no grid squares are affected by dust in Argentina, Mexico or Turkey (Supplementary Fig. 4 in the SI) according to the GBD2013 criteria (PM2.5 ≥36 μg/m3 and dust fraction ≥50%), in those countries, PM2.5 may still, in Received: 16 October 2020; Accepted: 23 September 2021; 10 NATURE COMMUNICATIONS \| (2021) 12:6286 \| https://doi.org/10.1038/s41467-021-26348-y \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-26348-y Additional information 66. Kutralam-Muniasamy, G., Perez-Guevara, F., Martinez, I. E. & Chari, S. V. Particulate matter concentrations and their association with COVID-19- related mortality in Mexico during June 2020 Saharan dust event. Environ. Sci. Pollut. Res. 28, 49989-50000 (2021). Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41467-021-26348-y. 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Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. 71. Additional information Center for International Earth Science Information Network - CIESIN - Columbia University. 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Chemical Co-ordinating Centre of EMEP. https://projects.nilu.no/ccc/ index.html (2019). 11 11 NATURE COMMUNICATIONS \| (2021) 12:6286 \| https://doi.org/10.1038/s41467-021-26348-y \| www.nature.com/naturecommunications Acknowledgements This research was supported in part by a Grant-in-Aid for Research (No. 16H01797) from the Japanese Ministry of Education, Culture, Sports, Science and Technology, and by a Fund for the Promotion of Joint International Research (Fostering Joint International Research (A)) of the Japan Society for the Promotion of Science (No. 18KK0322). We are also grateful to Nigel Harle of Gronsveld, the Netherlands, for his conscientious improvement of our English. © The Author(s) 2021 NATURE COMMUNICATIONS \| (2021) 12:6286 \| https://doi.org/10.1038/s41467-021-26348-y \| www.nature.com/naturecommunications 12 NATURE COMMUNICATIONS \| (2021) 12:6286 \| https://doi.org/10.1038/s41467-021-26348-y \| www.nature.com/naturecommunicatio
https://openalex.org/W4213374571	https://discovery.ucl.ac.uk/10131460/1/O%27Donovan_rvab038.pdf	English	null	Capabilities for transdisciplinary research	Research evaluation	2,021	cc-by	14,629	Abstract Problems framed as societal challenges have provided fresh impetus for transdisciplinary research. In response, funders have started programmes aimed at increasing transdisciplinary research capacity. However, current programme evaluations do not adequately measure the skills and characteristics of individuals and collectives doing this research. Addressing this gap, we pro- pose a systematic framework for evaluating transdisciplinary research based on the Capability Approach, a set of concepts designed to assess practices, institutions, and people based on public values. The framework is operationalized through a mixed-method procedure which evaluates capabilities as they are valued and experienced by researchers themselves. The procedure is tested on a portfolio of ‘pump-priming’ research projects in the UK. We ﬁnd these projects are sites of capability development in three ways: through convening cognitive capabilities required for academic practice; cultivating informal tacit capabilities; and maintaining often unacknow- ledged backstage capabilities over durations that extend beyond the lifetime of individual projects. Directing greater attention to these different modes of capability development in trans- disciplinary research programmes may be useful formatively in identifying areas for ongoing project support, and also in steering research system capacity towards societal needs. Key words: transdisciplinary research; research evaluation; capability approach; societal challenges This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Cian O’Donovan 1,2,, Aleksandra (Ola) Michalec 3 and Joshua R. Moon 2 Downloaded from https://academic.oup.com/rev/article/31/1/145/6481782 by Universit Downloaded from https://academic.oup.com/rev/article/31/1/145/6481782 by University College London user on 08 June 2022 1Department of Science and Technology Studies, University College London, Gower Street, London WC1E 6BT, UK, 2SPRU—Science Policy Research Unit, University of Sussex, Brighton BN1 9SL, UK and 3Department of Computer Science, Cabot Institute for the Environment, University of Bristol, Bristol BS8 1QU, UK Corresponding author. Email: c.o’donovan@ucl.ac.uk. Research Evaluation, 31(1), 2022, 145–158 https://doi.org/10.1093/reseval/rvab038 Advance Access Publication Date: 23 December 2021 Article 2.1. Locating transdisciplinary research capabilities in practice and theory What can we learn from how evaluators have conceptualized capa- bilities to date? Here we understand capabilities as an individual or group’s ability to do valuable acts or reach valuable states of being in pursuit of TD research. For instance, skills, aptitudes (Lyall and Meagher 2012), competences (Hoffmann et al. 2017; Hansson and Polk 2018), and human capital (Bozeman et al. 2001) are all rele- vant to human capabilities and feature in procedures evaluating the impacts and outputs of research. Our primary interest in outputs is in how they contribute to research capacity and readiness levels and the capabilities that are variously constitutive of these outputs or required in their production. In this way, scientific and technological human capital (Bozeman et al. 2001; Bozeman and Corley 2004) can be understood as outputs and inputs of TD research. These in- clude skills and career prospects of expert researchers measured using bibliometric techniques and content analysis of CVs. Similar techniques have been used to assess capabilities produced in gradu- ate courses and on research teams (de Oliveira et al. 2019). Downloaded from https://academic.oup.com/rev/article/31/1/145/6481782 by University Downloaded from https://academic.oup.com/rev/article/31/1/145/6481782 by University College London user on 08 June 2022 Assessing capacity and underlying capabilities is important be- cause they both contribute to a broader evaluation of the complex and often non-quantifiable criteria of interactions between science and society in TD research (Ernø-Kjølhede and Hansson 2011; van der Ploeg 2011). Such interactions include, but are not limited to, collaboration, integration of knowledge, learning processes, and the performance of cognitive and social functions. Moreover, evaluating TD research is important for funders normatively in measuring processes and outcomes; for demonstrating return on investment and legitimizing funding programmes (Mosse 2004). It is also ana- lytically interesting for evaluators and researchers for what it tells us about what kind of TD research is being done and how society and research systems are reconfigured through collaborative, integrative, and ontological work (Frederiksen, Hansson and Wenneberg 2003; Molas-Gallart et al. 2016). Capabilities also feature in evaluations of research processes. Processes and constituent research practices include the cognitive disciplinary work of research as well as the integrative (Boix- Mansilla 2006) and ontological (Barry, Born and Weszkalnys 2008) work of TD research. They also include social and collaborative practices across project groups and with stakeholders in society. These processes are underpinned in part by what Klein (2008) calls ‘competencies’. 2.1. Locating transdisciplinary research capabilities in practice and theory That is, how well methods for decision-making, knowledge distribution, and networking are managed across proj- ects and sub-projects. These methods in turn require management, coaching, and leadership capabilities. Methods of evaluating research programmes tend to focus on quantitative metrics of research outputs, qualitative network analysis (Oancea, Florez Petour Atkinson 2017), examining ‘productive interactions’ in research between academics and non- academics (Molas-Gallart and Tang 2011; Spaapen and van Drooge 2011), and qualitative evaluations of policy interventions (Smutylo 2001). These efforts however tend not to explicitly focus on capabilities. Nor do these efforts systematically evaluate how policy interventions build capabilities for TD research. The aim of this article is therefore to develop a framework with which to conceptualize and systematically evaluate capabilities for TD research attentive to these gaps. Our entry point is from the perspective of UK research funding agencies, and their institutional obligation to expand capacity on the academic side of transdisciplinary collaborations. As such, our evaluation centres on the capabilities of academic researchers in the first instance. Cutting across evaluations of outputs and processes are issues of quality (Feller 2006; Huutoniemi et al. 2010; de Jong et al. 2011; Belcher et al. 2016). Collaboration for example, can be evaluated as a process, and also be used as an indicator of research quality (de Jong et al. 2011). Collaborative practices in particular draw our at- tention to capabilities that are inherently collective, that is situated across groups. TD quality can be enhanced through the enacting of certain capabilities: mastering multiple disciplines, emphasizing inte- gration, critiquing disciplinarity (Huutoniemi 2010). Effectiveness is one useful criterion of quality with which evaluators can assess social learning (the change in knowledge, attitudes, and skills at the individual, group, and institutional level) and the building of what they call societal capacity (Belcher et al. 2016). We discuss the particulars of these capabilities and how they might arise in Section 2, where we also provide an account of the evaluative framework’s conceptual underpinnings using the Capabilities Approach (Sen 1999; Nussbaum 2001). In order to lo- cate, explore and assess the presence of research capabilities in the field, we propose a novel framework of evaluation. Our research question is: what are the different capabilities valued by researchers performing TD research, and how can these be mapped in specific research projects? 1. Introduction 1 146 evaluating TD research capacity is important in several regards. First so that a state of readiness might be reached and maintained (Klein 2008; Falk-Krzesinski et al. 2011). Readiness to do research, address wicked research problems quickly, or make complex re- search feasible—feasibility and risk can be an inhibiting feature of research systems (Luukkonen 2012). Second, and responding to the need for readiness, building, or enhancing capacity has become a major challenge for funders (Lyall and Meagher 2012). This has been a significant aim of major research initiatives over the past dec- ade, including major UK projects such as RELU, UK SPINE, and UKRI CCF. Finally, capabilities and enhanced capacity are them- selves a product of research, a first-order or intermediate outcome of transdisciplinary activity (Wiek et al. 2014) making TD capacity building an important impact goal of some projects (Hansson and Polk 2018). analysis and implications for evaluation practice, theory, and policy in Section 5. Section 6 offers a short conclusion. analysis and implications for evaluation practice, theory, and policy in Section 5. Section 6 offers a short conclusion. 1. Introduction specific (Nowotny 2000; Klein 2006; Huutoniemi 2010). It facili- tates diverse and mutually accountable interactions between partici- pants and different styles of knowledge. In doing so, TD research potentially fosters transformative scientific and technological pro- gress through social innovations and represented marginalized inter- ests (Lang et al. 2012; Stirling 2015; Smith and Stirling 2018). TD research practices and evaluations are therefore ‘generative proc- esses of harvesting, capitalizing, and leveraging multiple kinds of ex- pertise’ (Klein 2008). While there are numerous discussions on the relations between interdisciplinarity and transdisciplinarity (Klein 2008; Pohl et al. 2011; Lyall Meagher Bruce 2015), they remain out- side the scope of this article. Instead, we focus on the aforemen- tioned features of transdisciplinarity as a mode of research contingent on cultivating relevant capabilities. Research framed to address global, grand, and societal challenges has brought fresh impetus to calls by funding agencies for increased transdisciplinary research capacity (Bammer et al. 2013; Lyall and Fletcher 2013; Belmont Forum 2020; European Commission 2020; Belcher and Hughes 2021). Complex and cross-domain problems such as climate change, public health programmes, and efforts to meet the UN Sustainable Development Goals, provide rationales for research design that go beyond mono- or even interdisciplinary solutions (Werlen 2015). However, transdisciplinary (TD) research cannot be produced on demand. Appropriate TD research capacity is required. We understand TD research as knowledge production activities spanning across disciplinary boundaries and meaningfully involving non-academic partners in research design, operation, analysis, publication, and the practice of attendant methods. TD research is problem-oriented, transcends separate disciplinary sectors, trans- gresses disciplinary and institutional boundaries and is context For expertise to be useful it must be instantiated as a kind of cap- ability. Research capacity then is the aggregate capabilities and resources required for organizations, groups, and individuals to mo- bilize expertise and perform research. Building, maintaining, and 145 V C The Author(s) 2021. Published by Oxford University Press. V C The Author(s) 2021. Published by Oxford University Press. 14 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licen unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Research Evaluation, 2022, Vol. 31, No. 2.1. Locating transdisciplinary research capabilities in practice and theory A common feature of TD research is that assessments of quality are relative to the goals, expectations, norms, and values of research stakeholders and thus vary from one TD research context to another (Huutoniemi 2010). De Jong (2011) for example evaluates perform- ance of a research groups against their own research aims. More broadly, the role of context in TD research has formed an important theme within evaluation literature (Feller 2006; Stokols et al. 2008; Hansson and Polk 2018). Evaluators that assess context draw atten- tion to the role of norms, institutions, rules, and conditions of en- quiry in shaping, enhancing, and diminishing individual and group We describe the procedural tasks of evaluation and propose a novel methodological approach in Section 3. In Section 4, we demonstrate the framework with a case study of a TD research pro- gramme, the ESRC Nexus Networkþ (NNþ)1. We discuss the 147 Research Evaluation, 2022, Vol. 31, No. 1 capabilities (Mormina 2019). Context is also the location for inter- ventions that might enhance capabilities and capacity for integration and collaboration (Klein 2008). 2.3. Our conceptual framework We now introduce a conceptual framework that builds on the evalu- ative features of the capability approach in order to address the issues of TD research discussed in Section 2.1. TD research projects form the evaluative space—what we will refer to as a capability space—for our framework. But what capabilities matter within these projects? This brief discussion indicates some of the features a framework to evaluate capabilities needs to incorporate. It needs to understand capabilities as constitutive of research practice, as well as outputs of research processes. It needs to account for social as well as cognitive capabilities and those performed by individuals as well as groups. It needs to be sensitive to the effect of contextual features on research practice and the complexity of situations in which they arise. It needs to take into account the aims of specific research projects and constituent researchers. And it needs to acknowledge the subjectivity and politics inherent in the values and decisions that determine which knowledge and methods are made to matter through proc- esses of reflection, deliberation, and negotiation (Wiek et al. 2014) or otherwise. To accomplish this, we conceptualize capabilities using the Capability Approach and operationalize the framework using a realist evaluation technique (Section 3). 2.1. Locating transdisciplinary research capabilities in practice and theory Our analysis follows Sen (1999) and Robeyns (2005) in seeing the capabilities valued by and available to researchers as a matter of empirical identification. Moreover, taking the situatedness of TD re- search seriously directs us to paying attention to how researcher might value specific capabilities differently depending on the context of the research. After all, TD research gains in accountability pre- cisely because of the situated nature of the socially robust knowledge it might generate. Downloaded from https://academic.oup.com/rev/article/31/1/145/6481782 by University College London user on 08 June 2022 Nevertheless, we do not start from an entirely blank slate. The literature discussed in Section 2.1 gives us some clues as to what kind of capabilities are likely to be valued by researchers in TD re- search projects generally. We use this literature as a starting point with which to map the capability spaces of the NNþ projects specifically. 2.2. The capability approach—evaluating what researchers are effectively able to do To guide our exploration of these spaces we propose a four-part, conceptually driven heuristic. These parts account for (i) individual capabilities held by researchers; (ii) collective capabilities held with- in the capability set of the research projects; (iii) cognitive capabil- ities required to perform academic practices and again collectively held in the context of transdisciplinary research; (iv) contextual features which shape the capability space of research projects. The idea is to use this heuristic to direct our attention to where and how capabilities are most likely to be situated, whilst—again following Robeyns (2005)—inductively locating them through bibliometric techniques, grounded methods, and careful analysis. What are capabilities exactly, and what capabilities are required to carry out these TD research practices? Moreover, what can an assess- ment of capabilities tell us that current evaluative procedures cannot? Concepts about human capabilities by development scholars such as Amartya Sen (1999) and Martha Nussbaum (2001) are useful here. The Capability Approach (CA) is a framework with which to assess institutions and practices based on one or several public values, conceptualized in capability terms (Robeyns 2016). Emerging from assessments of public values related to quality of life (Sen) and social justice (Nussbaum), the CA has been used for a range of theoretical and conceptual work (Robeyns 2016). These include small-scale pro- ject evaluation (Alkire 2005) and assessing human capabilities in col- laborative workshops (O’Donovan and Smith 2020). Much research has been published on education and capabilities (Saito 2003; Walker 2003) but rather less on the practices of research. Addressing this gap, we develop a conceptualization of capabilities for TD research that are valued by researchers and other interested agents. 2.3.1 Individual capabilities 2.3.1 Individual capabilities Many capabilities required for research are more ‘social or political than simply cognitive’ (Bozeman and Rogers 2001, p. 418). These include ‘the strategies and policies of knowing that are not codified in textbooks but nevertheless inform expert practice’ (Knorr Cetina 1999, p. 2). Examples include skills, tacit knowledge, and experien- tial knowledge acquired by individual researchers (Heckman and Corbin 2016) and performed in the social setting of research proj- ects. Tacit, social, and cognitive factors interact, requiring manage- ment of information and decision-making (Klein 2006). Such tacit individual capabilities are influenced by personal background and include often individualistic ambitions like the capability to have an identity as a researchers (Lau and Pasquini 2008). The core characteristic of the approach is its focus on what peo- ple are effectively able to do and be; that is, on their capabilities (Robeyns 2005 p. 94). For example, the research techniques avail- able for researchers to choose to practice—such as the capability to do ethnographic fieldwork, or to be an environmental economist. In other words, something discrete and measurable, so a research project’s capability set can be thought of like a budget set—the capa- bilities valued and available to that project. The CA is normative in that it supports analysis and decisions based on value judgements about how research groups ought to behave in order to create re- search with and for society. A major strength of the CA is that it allows us account for the normative assumptions of the research subjects, in our case TD researchers. 2.3.2 Collective capabilities Bozeman and Rogers considered the human and technical capital required for research to include the ‘sum total of scientific, technical, and social knowledge and skills and capabilities available to a par- ticular individual’ (Bozeman and Rogers 2001). In TD practice though, individual researchers rarely, if ever, cultivate all of these capabilities required to carry out research. Research project groups facilitate the division of labour, share expertise, and contribute mutual or collective capabilities and are an everyday feature of sci- entific research. To be clear, in our evaluation we focus on the cultivation and availability of capabilities to act, rather than the achievements of the group being evaluated—what capability theorists call achieved func- tionings. Robeyns (2005) provides a comprehensive overview of concepts and procedures used in theorizing and operationalizing capability frameworks, of which the most relevant to the aims of this article are sketched out below. 2 CA scholars have shown how the provision and achievement of capabilities is held, used and influenced by group, social, and Research Evaluation, 2022, Vol. 31, No. 1 148 capabilities within a research project is shaped by these factors. Exactly how is, again, a matter for empirical research. Our frame- work is attentive on the one hand to how researchers might value capabilities differently depending on the context of specific projects. And on the other, to how contextual phenomena influence the avail- ability of capabilities valued by researchers within a capability space. To be clear, the contextual influences that form the final part of our heuristic are not strictly capabilities, but rather the broader social contexts and socio-material configurations of research proj- ects that influence capabilities (Robeyns 2005; Oosterlaken 2011; Zheng and Stahl 2011). Heuristic components are summarised in Table 1. environmental factors (Stewart 2005; Ibrahim 2006). Moreover, a set of capabilities available to an individual or group can be expanded through collective action as well as individual effort (Stewart 2005; Roy 2012). Two points are important here. First, we can expect that a research group’s capability set contains the aggre- gate of individual capabilities. Second, the capability set also con- tains capabilities that are mutually held and not dividable to individual researchers. In other words, there are collective capabil- ities of a group that are not features of individuals on their own. For example, the production of scientific knowledge, legitimacy and individual expertise is influenced socially (Cuevas Garcia 2016) and culturally (Corley et al. 2.3.2 Collective capabilities 2019). Collective capabilities that might be valued by researchers include capabilities for collaboration and coordination (Hohl, Knerr and Thompson 2019), capabilities to build and continuously maintain networks (Spaapen and van Drooge 2011; Hansson and Polk 2018) and to promote interperson- al and intrapersonal learning that can foster enhanced understand- ing and capabilities to respond to complex questions, issues, or problems (Lyall and Meagher 2012). Downloaded from https://academic.oup.com/rev/article/31/1/145/6481782 by University College London user on 08 June 2022 2.3.3 Cognitive capabilities • Stage II: appraise how the capabilities claimed for TD research sites are actually experienced by researchers. Indeed, the expansion of collective cognitive capabilities is an evalu- ative impact of research in itself (Molas-Gallart, Tang and Rafols 2014). These are the capabilities required to perform scientific and technical work include formal educational endowments acquired through graduate and post-graduate training, usually encompassed in concepts of ‘human capital’ (e.g. Becker 1964). These cognitive capabilities variously include the skills and formally taught techni- ques required, for instance, to take soil samples, conduct interviews, or perform econometric modelling and to perform advanced know- ledge tasks such as differentiating, reconciling and synthesizing (Lyall and Meagher 2012). • Stage III: compare the capabilities in Stage I with those mapped in Stage II in order to identify expected and absent capabilities. To achieve this, we follow a typical evaluation pathway (Gilmore et al. 2019): establishing expectations, determining reality, and locating differences—in this case between expected capabilities and those which are experienced by researchers. This is a theory- driven approach that lets us locate discrete sets of capabilities, while also acknowledging the limitations, situatedness, and contextual fea- tures of those capabilities. The provision of these capabilities usually follows disciplinary lines in undergraduate education and often through post-graduate training. These demarcations are also evident in academic journals, which remain, for the most part, closely organized along disciplinary lines (Rafols et al. 2012). Critically for this study, the capability to publish in a disciplinary journal is an important indicator of the capabilities available collectively to researchers in the context of the specific research projects we investigate. 3.1 Realist evaluation and the capabilities approach The three tasks outlined above are typical of a Realist Evaluation (RE) in which the core expectations of an Initial Programme Theory (IPT) are set a priori (Stage I) (Nurjono et al. 2018). Data appropri- ate to examining that IPT is then collected (Stage II). Then IPT expectations are recursively compared with reality (Stage III). Importantly for our purposes, while alternative forms of theory- driven evaluation (for instance programme-theory evaluation or theory-of-change evaluation) ask questions about ‘what works?’, RE is focused on ‘what works for whom and in what contexts?’ (Pawson and Tilley 1997). As such, RE enables us to not only discover what capabilities enabled researchers to conduct TD research, but how the situatedness of those capabilities shaped their usefulness. We detail the methodology for each of these stages in turn. 3. Methods Based on our conceptualization of transdisciplinary research proj- ects, we need a methodology that accomplishes a series of analytic tasks. • Stage I: identify inductively a list of capabilities associated with TD research practices. 2.3.4 Contextual influences on capabilities The social and material contexts in which capabilities are put into practice in the production of knowledge are multiple, patchy and heterogeneous (Pickering 1992, p. 8). These include offices in aca- demic departments, disciplinary norms, methods, campus rules, re- search ethics frameworks, field locations and the myriad of instruments, tools, knowledges, and relations at and between these sites (Feller 2006; Hansson and Polk 2018). In evaluation, flexibility and sensitivity to this diversity and context is critical (Langfeldt 2006). 3.2.1 Stage I—compiling a list of expected capabilities Adapting the CA for analytical purposes requires careful explan- ation and justification of the relevant capabilities identified and the methods used. Robeyns (2003) sets out identification criteria: they should be explicit, discussed and defended. The level of abstraction should be appropriate to the study context and project objectives. Ideal sets of capabilities must become a pragmatic list that can be studied and should include all important elements non-reducible to the other elements, even if there exists some overlap. The normative evaluation of capabilities in context was part of Sen’s motivation in early developments of the CA (Couldry 2019). This milieu of social institutions, norms, environmental factors, guiding policy visions, regulations, and people’s behaviours are what CA analysts call conversion factors that mediate capability inputs, such as knowledge, finance, resources, goods, and services that in turn facilitate the cultivation of capabilities within a capabil- ity set (Robeyns 2005). The degree, distribution, and quality of Guided by these criteria we identified a list of capabilities expected in the test programme, arrived at by searching for signals 149 Research Evaluation, 2022, Vol. 31, No. 1 Table 1. Heuristic used to locate and map capabilities Heuristic component Explanation, indicative examples, and sources of evidence Indicative references Individual tacit capabilities Skills, aptitudes, competences, and capabilities to advance career prospects of researchers. Capabilities might include tacit and experiential knowledge accrued by researchers; the capability to build an identity as a researcher. Capabilities to understand complex social and societal factors. Individual leadership, administrative, and coaching capabilities. Capabilities to critique disciplinarity. Evidenced through interviews with researchers, questionnaire, workshop 1, 2. Bozeman et al. (2001) de Oliveira et al. (2019) Hansson and Polk (2018) Heckman and Corbin (2016) Hoffmann et al. 2.3.4 Contextual influences on capabilities (2017) Huutoniemi (2010) Klein (2006) Lau and Pasquini (2008) Lyall and Meagher (2012) Downloaded from https://academic.oup.com/rev/article/31/1/145/6481782 by University College London user on 08 June 2022 De Jong (2011) Feller (2006) Hansson and Polk (2018) Langfeldt (2006) O’Donovan and Smith (2020) Oosterlaken (2011) Nowotny (2003) categories (Leydesdorff, Carley and Rafols 2013) gave us indicators of different cognitive capabilities and disciplines that constituted each of the NNþ projects. 3 Furthermore, and despite some limita- tions, for each of these project profiles, a co-authorship map gave us inference into potential relations between project researchers. of our conceptual framework in a focused review of the literature on conducting and evaluating TD research. We began the task by searching the literature for signals and expressions of our conceptual framework—cognitive, tacit, collective beings, and doings. We refined this list using project documentation (project proposals, an- nual reports, etc.) from within the NNþ partnership programme. Finally, we integrated observations from two workshops organized by the NNþ, ‘Transdisciplinary Methods for Developing Nexus Capabilities’. Details of these sources of evidence are reported in Section 4.1 (Table 2) and identified capabilities are reported in Section 4.3. Knowledge outputs such as bibliometric indicators, however, only partially reveal characteristics of a knowledge community (Strathern 2004) and are insufficient to capture the nature of trans- disciplinary programmes (Koier and Horlings 2015); for instance reduced efficacy for early career researchers and non-academic par- ticipants (who have fewer publications). These limitations were miti- gated through close analysis of the project proposals and the self- reported biographies of project researchers. Also, findings derived from bibliometric analysis were explicitly triangulated and aug- mented, first at workshop 2 (see Table 2) and over the course of re- searcher interviews. 3.2.2 Stage II—mapping capabilities at research sites Following Step 2 in the realist evaluation procedure and further guided by the heuristic, we next gathered data on actual capabilities valued by researchers within each case. In order to map the collect- ive cognitive capabilities available at each research project, biblio- metric profiles for the respective projects were compiled. These consisted of aggregate records of peer-reviewed journal publications of constituent researchers, collected on 20th August 2018 (no time boundary). Mapping the bibliometric profiles onto Web of Science In parallel, a questionnaire was deployed to all researchers in the five NNþ projects and this article reports 26 respondents. 2.3.4 Contextual influences on capabilities (2017) Huutoniemi (2010) Klein (2006) Lau and Pasquini (2008) Lyall and Meagher (2012) Collective capabilities Collective capabilities to perform collaborative and social practices with stakeholders in society; mutual accountability; distributed ownership and leadership among project participants; collective consensus building and managing tensions; capabilities to build new epistemic communities and cultures of evidence; capabilities for coordination; capabilities to perform ontological work; capabilities to promote interpersonal learning; capabilities to build and maintain networks. Evidenced through interviews with researchers, questionnaire, workshop 2. Documentary analysis of research outputs Barry et al. (2008) Hansson and Polk (2018) Klein (2008) Hohl, Knerr and Thompson (2019) Lang et al. (2012) Michalec et al. (2021) Spaapen and van Drooge (2011) Talwar et al. (2011) Cognitive capabilities The capabilities required to collectively perform scientiﬁc and technical work are central to TD research. For example the capability to publish in a disciplinary journal. Capabilities to differentiate, reconcile and synthesize data and knowledge. Capabilities to perform integrative work. Evidenced through bibliometric indicators, workshop 2. Boix-Mansilla (2006) Bozeman et al. (2001) Bozeman and Corley (2004) Molas-Gallart et al. (2014) Lyall and Meagher (2012) Contextual inﬂuences Research sites are constitutive of a diversity of knowledge, values which in turn inﬂuence capabilities. Framework accounts for multiple issues, multiple actors, multiple settings of TD research. Assessing variance of goals, expectations, norms, and values of research stakeholders relative to TD research context; issues of structure and situatedness to the fore. Capabilities inﬂuenced by personal background of researchers and re- search participants. Assessment accounts for conﬁguration of prevailing social structures and material conditions. Evidenced through interviews with researchers, questionnaire, workshop 2. Documentary analysis of research outputs De Jong (2011) Feller (2006) Hansson and Polk (2018) Langfeldt (2006) O’Donovan and Smith (2020) Oosterlaken (2011) Nowotny (2003) Table 1. Heuristic used to locate and map capabilities Heuristic component Explanation, indicative examples, and sources of evidence Indicative references Individual tacit capabilities Skills, aptitudes, competences, and capabilities to advance career prospects of researchers. Capabilities might include tacit and experiential knowledge accrued by researchers; the capability to build an identity as a researcher. Capabilities to understand complex social and societal factors. Individual leadership, administrative, and coaching capabilities. Capabilities to critique disciplinarity. Evidenced through interviews with researchers, questionnaire, workshop 1, 2. Bozeman et al. (2001) de Oliveira et al. (2019) Hansson and Polk (2018) Heckman and Corbin (2016) Hoffmann et al. 2.3.4 Contextual influences on capabilities The ques- tionnaire asked about the nature and frequency of interpersonal interactions within and across projects in order to map further indi- vidual, collective and group capabilities. It also sought to generate data on how capabilities were valued and experienced by Research Evaluation, 2022, Vol. 31, No. 1 150 Table 2. Characteristics of Nexus Network Partnership Programme, projects, and workshops. These characteristics are augmented by fur- ther bibliometric data and analysis in the Supplementary material Item Background Partnership programme overview In 2016, NNþ launched their Partnership Programme, awarding ﬁve projects between £60,000 and £120,000 each. The aim: building capabilities that supported ‘the development and implementation of transdisciplinary research on nexus-related topics’ (The ESRC Nexus Network 2016). Transdisciplinary approaches were mandated through two assessment criteria. First scope: ‘does the application engage with two or more nexus areas (food, energy, water, and environment)’ and second en- gagement strategy: ‘does the application outline a persuasive strategy for engagement between disciplines and between researchers and other knowledge partners? . . .do the applicants display a depth of understanding about the possible barriers and challenges to inter- and trans-disciplinary work’ (The ESRC Nexus Network 2016). Project 1 This project aimed to engage policy makers with the social sciences’ theories (human geographers, sociologists, and environmen- tal studies scholars) on sustainable kitchen practices. In this project we note diversity of cognitive capabilities is underpinned with evidence of strong cognitive links between researchers. The project has been led by a well-connected and involved senior social scientist. This has been built up over a long timeframe: researchers were located in one of the UK regions and policy col- laborators worked closely with the central government. Project 2 The project combined UK-based economic and soil science analysis to aid farmers’ decision-making in an overseas location. In this project the primary investigator (economist) enacts a strong individual leadership role in aligning people and their capabil- ities. This was reinforced by concurrent activities mobilized by another non-NNþ project run over a similar timeframe. Nevertheless, larger ambitions had to scale down due to delays caused by the complexity of administrating research activities overseas. Project 3 The project aimed to collect a diverse array of natural science data from an overseas region and integrate them into the already existing social science database of the area. The project engaged with the local policy makers and community members during data collection and outreach. 2.3.4 Contextual influences on capabilities The primary investigator (anthropologist) played a convening role and was not involved day to day. The UK-based research group (also a team of anthropologists) had already funded a PhD researcher at an overseas part- ner research organization indicative of, and constitutive of a long-established trans-national research network. Difﬁcult to as- certain where the power lay between partners. Project 4 This project gathered qualitative insights on energy production, consumption and related sustainability dilemmas in an overseas location. The research was conducted by scholars working across Science and Technology Studies (PI and researchers) and Engineering (Co-Is). In this project, cognitive capabilities related to interpretive research techniques existed within the UK aca- demic team but were not uniformly matched in the ﬁeld. The funding itself did not allow for training. Capabilities did not exist to easily connect UK and overseas academic structures creating delays and other barriers to getting work done. Project 5 The project developed a series of deliberations on the future scenarios related to sustainable food systems in one of the UK coun- tries. In this project the primary investigator (sociologist) played a convening role initially and was not involved in day-to-day activities of the project. The main researcher (with background in environmental policy) did not have pre-existing connections with the practitioners participating the in the project. The short-term nature of the NNþ funding did not allow to create a community of practice which would last beyond the timescales of data collection and dissemination. Workshop 1 The Transdisciplinary Methods for Developing Nexus Capabilities workshop brought together researchers from across the broader Nexus Network as well as others with an interest or expertise in TD research for sustainability (Ince 2015). Workshop activities were framed by a discussion paper (Stirling 2015) and yielded observations about capabilities which for participants were important in addressing the needs of TD research. The workshop was organized around two questions: (1) What different kinds and interconnections of method in contrasting con- texts, form the most practical basis for enabling transformative action to address Nexus challenges? (2) How can such encom- passing Nexus methodologies best enable academic, government, business, and civil society actors to develop appropriate skills, training, and research capabilities? Workshop 2 A workshop in May 2018 convened researchers from across the NNþ Partnership Programme. Background Project 1 This project aimed to engage policy makers with the social sciences’ theories (human geographers, sociologists, and environmen- tal studies scholars) on sustainable kitchen practices. In this project we note diversity of cognitive capabilities is underpinned with evidence of strong cognitive links between researchers. The project has been led by a well-connected and involved senior social scientist. This has been built up over a long timeframe: researchers were located in one of the UK regions and policy col- laborators worked closely with the central government. Project 2 The project combined UK-based economic and soil science analysis to aid farmers’ decision-making in an overseas location. In this project the primary investigator (economist) enacts a strong individual leadership role in aligning people and their capabil- ities. This was reinforced by concurrent activities mobilized by another non-NNþ project run over a similar timeframe. Nevertheless, larger ambitions had to scale down due to delays caused by the complexity of administrating research activities overseas. Project 3 The project aimed to collect a diverse array of natural science data from an overseas region and integrate them into the already existing social science database of the area. The project engaged with the local policy makers and community members during data collection and outreach. The primary investigator (anthropologist) played a convening role and was not involved day to day. The UK-based research group (also a team of anthropologists) had already funded a PhD researcher at an overseas part- ner research organization indicative of, and constitutive of a long-established trans-national research network. Difﬁcult to as- certain where the power lay between partners. This project gathered qualitative insights on energy production, consumption and related sustainability dilemmas in an overseas location. The research was conducted by scholars working across Science and Technology Studies (PI and researchers) and Engineering (Co-Is). In this project, cognitive capabilities related to interpretive research techniques existed within the UK aca- demic team but were not uniformly matched in the ﬁeld. The funding itself did not allow for training. Capabilities did not exist to easily connect UK and overseas academic structures creating delays and other barriers to getting work done. The project developed a series of deliberations on the future scenarios related to sustainable food systems in one of the UK coun- tries. In this project the primary investigator (sociologist) played a convening role initially and was not involved in day-to-day activities of the project. 2.3.4 Contextual influences on capabilities Participants were invited to re- ﬂect on capabilities within their projects by building their own maps of stakeholders, project participants, socio-material rela- tions, resources, and institutions (Schiffer 2007). This method was adept at capturing collective capabilities and structural and contextual conditions. Participants were also asked to respond to representations of bibliometric proﬁles constructed of each research team, augmenting bibliometric-based cognitive indicators with qualitative observations and drawing attention to absences in those data (Ayre and O’Donovan 2018). Table 2. Characteristics of Nexus Network Partnership Programme, projects, and workshops. These characteristics are augmented by fur- ther bibliometric data and analysis in the Supplementary material Table 2. Characteristics of Nexus Network Partnership Programme, projects, and workshops. These characteristics are augmented by fur- ther bibliometric data and analysis in the Supplementary material Background The main researcher (with background in environmental policy) did not have pre-existing connections with the practitioners participating the in the project. The short-term nature of the NNþ funding did not allow to create a community of practice which would last beyond the timescales of data collection and dissemination. The Transdisciplinary Methods for Developing Nexus Capabilities workshop brought together researchers from across the broader Nexus Network as well as others with an interest or expertise in TD research for sustainability (Ince 2015). Workshop activities were framed by a discussion paper (Stirling 2015) and yielded observations about capabilities which for participants were important in addressing the needs of TD research. The Transdisciplinary Methods for Developing Nexus Capabilities workshop brought together researchers from across the broader Nexus Network as well as others with an interest or expertise in TD research for sustainability (Ince 2015). Workshop activities were framed by a discussion paper (Stirling 2015) and yielded observations about capabilities which for participants were important in addressing the needs of TD research. The workshop was organized around two questions: (1) What different kinds and interconnections of method in contrasting con- texts, form the most practical basis for enabling transformative action to address Nexus challenges? (2) How can such encom- passing Nexus methodologies best enable academic, government, business, and civil society actors to develop appropriate skills, training, and research capabilities? p 2 A workshop in May 2018 convened researchers from across the NNþ Partnership Programme. Participants were invited to re- ﬂect on capabilities within their projects by building their own maps of stakeholders, project participants, socio-material rela- tions, resources, and institutions (Schiffer 2007). This method was adept at capturing collective capabilities and structural and contextual conditions. Participants were also asked to respond to representations of bibliometric proﬁles constructed of each research team, augmenting bibliometric-based cognitive indicators with qualitative observations and drawing attention to absences in those data (Ayre and O’Donovan 2018). A workshop in May 2018 convened researchers from across the NNþ Partnership Programme. Participants were invited to re- ﬂect on capabilities within their projects by building their own maps of stakeholders, project participants, socio-material rela- tions, resources, and institutions (Schiffer 2007). This method was adept at capturing collective capabilities and structural and contextual conditions. Table 2. Characteristics of Nexus Network Partnership Programme, projects, and workshops. These characteristics are augmented by fur- ther bibliometric data and analysis in the Supplementary material 4. Evaluating transdisciplinary capabilities in the nexus network1 The large clusters on the right-hand side indicate researchers with published outputs in the natural and environmental sciences and engineering indicating a plurality of cognitive capabilities avail- able collectively at the project. Nevertheless, interpretive social sci- entists with a shared interest in the domestic use of water, energy and food were in the majority. For these researchers, collaboration with practitioners was more significant than the integration of knowledge across academic disciplines: 4.1 The nexus network1 partnership programme In 2014, the ESRC Nexus Networkþ was awarded £1.5 m in fund- ing by the UK’s Economic and Social Research Council in order to build a network of researchers capable of examining interactions within and between domains of water, energy, and food, the WEF nexus (Cairns, Wilsdon and O’Donovan 2017; UKRI 2020). The Network þ was the second iteration of a new ‘pump-priming’ model introduced by UK public funding agencies which included the provi- sion of refunding budget for onward distribution over the course of several programmes of activities. Characteristics of the programme and projects are introduced in Table 2, and further bibliometric data are available in the Supplementary material. In the following sections, we report on capabilities valued by researchers across the entire portfolio. We begin by demonstrating the framework in ac- tion by reporting in-detail on Project 1. ‘Insofar as our project was transdisciplinary, it was transdiscipli- nary in terms of involving practitioners as well as academics. Personally, the term I would use would probably be ‘collabora- tive research’ because it was collaborating with practitioners’ (Project 1 researcher). Another explanation for why integration is underplayed is that researchers did not collect novel environmental science and engin- eering data—their analytic focus was elsewhere. Qualitative data also revealed the presence of other collective capabilities in the project. For instance, capabilities to translate, in- tegrate, and operationalize diverse methods. These are collective be- cause they involve researchers from different disciplines collaborating. These also included capabilities to allow researchers collectively reflect on previous collaborations which were required to effectively translate social science perspectives to practitioner partners in water, energy, and food industries. Background Participants were also asked to respond to representations of bibliometric proﬁles constructed of each research team, augmenting bibliometric-based cognitive indicators with qualitative observations and drawing attention to absences in those data (Ayre and O’Donovan 2018). 3.2.3 Stage III—comparing the list of expected capabilities with evidence from research projects 3.2.3 Stage III—comparing the list of expected capabilities with evidence from research projects researchers. This added qualitative data from individuals who were not available for interview, triangulating project narratives. Moreover, this provided an insight into the collective and contextual capabilities in the projects (Ayre and O’Donovan 2018). Data from the questionnaire and bibliometric analyses formed the basis for 15 semi-structured interviews with project researchers from across the five NNþ projects. PIs, Co-Is, and research associates as well as and practitioner partners were interviewed. These interviews provided further feedback loops within the evaluation framework. They also allowed identification of research practices and capability experien- ces that were important to researchers actually doing the research. The final step of realist evaluation is in practice multiple iterations of the same step—to iteratively reflect upon our initial list of capa- bilities as our data collection and analysis evolves (Gilmore et al. 2019). Stage III occurred in parallel with Stages I and II. This inter- play between expectations and reported reality is where realist evaluation highlights those aspects which are structural, unexpected, or absent entirely. As more data was gathered, project stories be- came thicker, more detailed, and increased in specificity. This allowed us to infer more accurately the capabilities valued by 151 Research Evaluation, 2022, Vol. 31, No. 1 Figure 1. Collective cognitive capabilities available in Project 1, mapped onto a Map of Science overlay map (Leydesdorff et al. 2013). Each node represents a Web of Science category (cognate discipline), in sum indicating the collective cognitive capabilities available at the project. Clusters of cognate disciplines are based on citation ﬂows in the overall WoS corpus and are represented by nodes sharing the same colour—available in online edition. The two dominant clus- ters in the ﬁgure indicate the research team is constitutive of capabilities from geography and social sciences (left cluster) and environmental sciences and civil engineering (right). Downloaded from https://academic.oup.com/rev/article/31/1/145/6481782 by University College London user on 08 June 2022 Figure 1. Collective cognitive capabilities available in Project 1, mapped onto a Map of Science overlay map (Leydesdorff et al. 2013). Background Each node represents a Web of Science category (cognate discipline), in sum indicating the collective cognitive capabilities available at the project. Clusters of cognate disciplines are based on citation ﬂows in the overall WoS corpus and are represented by nodes sharing the same colour—available in online edition. The two dominant clus- ters in the ﬁgure indicate the research team is constitutive of capabilities from geography and social sciences (left cluster) and environmental sciences and civil engineering (right). researchers within each project and therefore refine our Initial Programme Theory (the capabilities list). society’ (UKRI 2020). This already indicates the possibility of an in- cursion of social science methods and capabilities into the WEF nexus idiom, traditionally composed of engineering and environ- mental science expertise, for instance capabilities for optimizing, modelling, and quantifying. 4.2 Mapping the capabilities within NN1 partnership programme Project 1 The aim of Project 1 was to facilitate the transfer of research find- ings from environmental social sciences into the UK policy context (Table 2). Bibliometric analysis indicates disciplinary characteristics of the set of collective cognitive capabilities such as capabilities in geography and other social sciences with environmental sciences and civil engineering (Figure 1). Augmenting the project’s co-authorship network with practitioner relations found in the data, we found that the project’s capability set was influenced by three distinct networks (shown in Figure 2). First, the network illustrates what one interviewee—referring to the region in which their colleagues worked—called a ‘disciplinary North-West Figure 1 reveals the presence of social science capabilities on the left-hand side in line with the ESRC’s goal for the NNþ to ‘engage the social science community with these complex ‘Nexus challenges’ and link them to research users from business, government, and civil 152 Research Evaluation, 2022, Vol. 31, No. 1 Figure 2. An emerging co-authorship network for Project 1 which illustrates nexus-domain expertise in water, energy, and food. Nodes represent authors and those labelled nodes represent researchers in the network. Links represent a co-authoring relationship. Clusters of authors mapped using VOSViewer (co- authorship links are best observed in a full-colour version of article online). Downloaded from https://academic.oup.com/rev/article/31/1/145/6481782 by Universit Figure 2. An emerging co-authorship network for Project 1 which illustrates nexus-domain expertise in water, energy, and food. Nodes represent authors and those labelled nodes represent researchers in the network. Links represent a co-authoring relationship. Clusters of authors mapped using VOSViewer (co- authorship links are best observed in a full-colour version of article online). proposal in a tight timeframe. So, you know, if you’ve got a proto-team already in place you can mobilize it quickly in re- sponse to things’ (Project 1 researcher). hub’ of predominantly social theorists, whose expertise is located in various domains of the water-energy-food nexus. Second, a social sci- ence—engineering network is evidenced, revealing working relations established over a decade. One co-investigator (the relatively isolated ‘water cluster Co-I’ at the top of the graph) is distant from the net- work’s core because their publications are mostly collaborations with engineers. Third, integrated across these two networks is a set of academic-practitioner relationships (labelled clusters, Figure 2). hub’ of predominantly social theorists, whose expertise is located in various domains of the water-energy-food nexus. 4.2 Mapping the capabilities within NN1 partnership programme Project 1 Second, a social sci- ence—engineering network is evidenced, revealing working relations established over a decade. One co-investigator (the relatively isolated ‘water cluster Co-I’ at the top of the graph) is distant from the net- work’s core because their publications are mostly collaborations with engineers. Third, integrated across these two networks is a set of academic-practitioner relationships (labelled clusters, Figure 2). These networks indicate not only existing cross-disciplinary capabilities (established connections), but also the potential for new collaborations, based on long-established networks, but made more likely by being funded together. Indeed, this was an aim of the Partnership Grant, to convene and realize transdisciplinary poten- tial. One North-west hub researcher told us that the project was exciting exactly because it was an opportunity to work with co-is that they had long-known, but not published with previously. In the context of the project, this co-authorship network indicates a set of emergent collective capabilities. Collectively, relations under- pinned trustworthy collaborations and effective communication of complexity surrounding environmental science and policy. Furthermore, already-established relations allowed researchers build their project team, including non-academic partners, at short notice and effectively manage the group through the NNþ funding process: Turning to research practice, researchers reported capabilities of pluralism, in the sense of valuing different kinds of knowledge: ‘It’s very easy to have a critical or a meta orientation towards sci- ence if you were versed in rudimentary sociology. I don’t think that does you much good. (. . .) economists or scientists may have models of human action that we find objectionable as social sci- entists. Rather than going, ‘Okay, well they’re stupid, let’s dis- miss it,’ they’re not stupid. Have the courtesy to understand what it is that other people do.’ (Project 1 researcher) ‘Longstanding links between our two universities is the bottom line. We know each other personally; we tend to work in the same sort of field. ‘[. . .] It’s a completely pre-existing academic and stakeholder net- work, which is handy to have when you’ve got to turn around a 153 Research Evaluation, 2022, Vol. 31, No. 1 Furthermore, researchers praised the egalitarian ethos in the team (9), demonstrated through collaborative approaches to writing: researcher who strives to identify, engage with or alter existing re- search priorities power structures. 4.2 Mapping the capabilities within NN1 partnership programme Project 1 Leadership was mentioned by several researchers in terms of being able to build and manage a research team and also in terms of instilling an attitude of openness, balance. In the context of these re- search projects we find it more useful to think about leadership in terms of separate capabilities: managing research, being egalitarian in relation to project participants and being able to sustain values of pluralism in the project. ‘I don’t think there was a particular hierarchy between us. I would come up with the same copy, and depending on col- leagues’ availability, would do the rounds between them. It would come back to me with the edges smoothed and some sug- gestions for further work to do. . .. it was a nice mix.’ (Project 1 research associate) Summarizing, researchers cultivated group capabilities to be hum- ble, plural, egalitarian and reflexive. In particular, our analysis revealed the value of professional networks within academia and with practitioners. While interviewees did not emphasize cross- disciplinary cognitive capabilities, capabilities that allowed them to transgress institutional boundaries were valued. Also valued were capabilities that allowed them to manage the project team, commu- nicate complexity across nexus domains and sustain their own liveli- hoods through each round of competitive funding. Researchers value being able to break down the hierarchies trad- itionally constituted by academic disciplines, social status, seniority and uneven geographies—even if they are not always able to fully realize this. A researcher in Project 4 said: Downloaded from https://academic.oup.com/rev/article/31/1/145/6481782 by University College London user on 08 June 2022 ‘a lot of the funding is directed through the [global north] univer- sities, so although we were able to give our partners money through subcontracts, we were still controlling it. So, in a sense there is some kind of implicit hierarchy that goes with it. (. . .) Are there ways in which we could support research partners in the global south more directly?’. 4.3.3 Collective capabilities: valuing networks Project 1 shows us how capabilities are influenced by the configur- ation of people, knowledge and resources within research sites. Throughout the portfolio, capabilities like egalitarianism, team management, and trust in collaborations were experienced through practices such as ‘encouraging to ask silly questions’ (researcher P2); ‘not being precious during collaborative writing’ (researcher P1); and ‘managing tensions with respect and positivity’ (practitioner P5). It seems that not only are these capabilities individually held, they are also cultivated through shared concerns, interests, and re- search cultures. Second, within nexus research settings we found radically different kinds of organizations, procedures, stakeholders, power relations, purposes, and wider political–cultural contexts. These included inter- national assessments, government enquiries, regulatory committees, participatory processes, NGO studies, or social movement activities and accessing data in firms. Researchers valued a cognitive capability to apply known tools and frameworks in situations such as these, in which they were sometimes working for the first time. Significantly, networks provide positive feedback for researchers, strengthening capabilities to conduct TD research: ‘I guess these things have a life of their own. Once you start engaging people and you find people you like and can work with, then you want to do more’ (researcher P2). Third, we found researchers in Projects 1 and 4 valued a cognitive capability for a sustained appreciation of the particular— assessing social and material practices in specific contexts. This capability urges caution over the implications of generalization. Methodologically this is a capability when investigating intercon- nections between WEF nexus and represents a novel contribution to traditional quantitative and aggregative nexus methods. Networks can be established in numerous ways, by primary investigators opening access to established academic and policy net- works (P1 and P3), through networking grants (P1 and P2), or con- ferences (P4 and P5). Project networks demonstrate the critical importance of nurturing connections over time. Participants reflected that without pre-established personal networks it is chal- lenging to sustain collaborations once the project finishes. This was the experience of the investigators in P4 and P5, where the connec- tions between the researchers and practitioners were established only shortly before the project start and through professional events rather than personal networks: 4.3. Mapping and comparing capabilities across the NN1 partnership programme What researchers are acknowledging here is a capability to cede power, not merely respecting global south interjections, but actively opening and creating spaces for them. They note that capabilities that might better empower overseas colleagues were absent, or structural impediments were too strong to overcome. 4.3.1 Cognitive capabilities We now report on the capabilities in projects across the portfolio using the heuristic (Section 2, Table 1). The analysis indicates cogni- tive capabilities are valued in at least three ways. First, the cognitive capability to do research beyond the boundary of researchers’ home disciplines. This capability is about having available the cognitive ability to use or, critically, to learn new ways of doing and knowing research. However, while individual researchers value the possibil- ities of publishing in new disciplines and journals, this possibility is only available to them because they are part of a research team. The capability to publish across disciplinary lines is collectively held. 4.3.3 Collective capabilities: valuing networks 5. Discussion: implications for evaluators and funders Researchers perceived TD research as a risk to the academics and stakeholders, who, without the institutional support to build long-term partnerships and secure careers, are left to the project- based and precarious working conditions. Similarly, under the current research evaluation framework, transdisciplinarity was seen as a risky endeavour for the universities, which often retreat to mono-disciplinary research and commercial partnerships as they are easier to prove impactful and lucrative according to a researcher from P4: From the capabilities reported in Table 3, we draw several implica- tions for funders and evaluators of TD research. At a programme level, the Nexus Networkþ appears to have achieved its goal. The network convened a host of cognitive capabilities, anchored in social science disciplines, in a problem domain that is dominated by nat- ural and systems sciences. At first glance, this represents a significant achievement for the NNþ, and their funders, the ESRC, whose goal was to prime social science capacity and capabilities in WEF Nexus domains. By convening a portfolio of projects that were constitutive of cognitive capabilities, the NNþ extended ‘nexus thinking’ putting social science firmly on the knowledge map (see Figure 1). ‘The [Research Excellence Framework] is still siloed. When you put together projects and publications, PI’s and Co-I’s and are becoming increasingly pickier as to what publications they are put- ting together, what sorts of projects. Publications can tick boxes. Sometimes they can be put in a tick box for engineering, sometimes for geography. I think REF conflates the whole TD agenda because it’s still very much siloed.’. Using a capability approach however, our evaluation has revealed a more ambiguous picture. The value of the framework is that it emphasizes the skills and characteristics of individual researchers and collectives that are not included in typical pro- gramme evaluations. For example, individual and collective capabil- ities that underpin processes of learning are valued by researchers (as expected in Table 1) but often reported absent. As noted in Project 4 (Table 2), the funding did not allow for formal training. Frustrations about the lack of support for capabilities to sustain a livelihood from TD research was put even more succinctly by this researcher from Project 1: Table 3. 4.3.2 Individual tacit capabilities: engaging with power and structure Individual capabilities valued by researchers included capabilities of humility, valued in colleagues who ‘[don’t] think somewhere in the back of their minds that they are superior’ (researcher P4), the implication here being that superiority arose when uncertainty was located in the work of others, but not their own; capabilities of plur- alism in colleagues who are ‘not particularly holding to any Western way of understanding the data’ (researcher P3); and capabilities to be egalitarian, in colleagues who can ‘(. . .) learn to listen what the stakeholders need and understand how you can respond to their needs, and not just the project’s’ (researcher P1). In other words, a ‘It’s very hard to keep networks running after a project has ended. With all the other drawings on our time, other things tend to take priority, especially then if they’re to do with the lifeblood of running an institute i.e., income streams’ (researcher P5). Research Evaluation, 2022, Vol. 31, No. 1 154 ‘if these grand challenges are so bloody important, then how do we invest in people’s careers that are also transdisciplinary with- out increasing precarity and casualization?’. 4.3.4 Contextual influences: a need for transdisciplinary research policy 4.3.4 Contextual influences: a need for transdisciplinary research policy Participants pointed out the need to enhance capability building at the institutional level through training and learning for TD practi- ces, more flexible procedures for bidding for research funding, pro- vision of career progression mitigating risk for those engaged in TD research, and investment in cross-institutional networks were seen as necessary enablers for TD research. They facilitate the cultivation of cognitive capabilities as well as those that enhance livelihood, pluralism, and capabilities to challenge-power, a capability that requires mitigation of risk. When asked about existing capabilities for TD research at university and research council level, all partici- pants emphasized the lack of appropriate structures and policies. In summary, mapping capabilities through each of the heuristic levels we found a plurality of capabilities that are valued and experi- enced (summarized in Table 3) and some that appear in Table 1 that are absent entirely. In the following section we discuss some points particularly salient for the provision of policy in these areas. 5. Discussion: implications for evaluators and funders By way of ex- ample, the principal investigator in Project 4 was attentive to power imbalances between the UK team and the overseas partners, imbal- ances they could not successfully address. The implications are two- fold, first, given the noted absence, it appears collective capabilities requires greater structural support at the institutional level. The current funding system in the UK treats research capacity as held in disembodied projects that may be turned on and off as de- mand dictates (Vaesen and Katzav 2017)—bringing us back to the issue of projectification. This is simply not the case. Our framework facilitates a more nuanced understanding of what is going on and the extent to which capabilities are being convened, cultivated, or maintained under a buzzword like nexus, or indeed sustainability. Second, with regard to evaluation literature, these findings fit with ideas about the transformative potential of research such as ag- onistic–antagonistic logics (Barry, Born and Weszkalnys 2008), transformative values (Huutoniemi and Rafols 2017) and trans- formative understandings (Marres and de Rijcke 2020). This sug- gests an ambition for TD research that ‘springs from a self- conscious dialogue with, criticism of or opposition to the intellec- tual, ethical or political limits of established disciplines or the status of academic research in general’ (Barry, Born and Weszkalnys 2008, p. 29)4. This has implications and applications in particular for how re- search is organized to address emerging hot topics. It also draws at- tention to the importance of capabilities for network building, noted in Table 1 (Spaapen and van Drooge 2011; Hansson and Polk 2018). But this idea goes beyond highlighting the value of network building. It draws attention to how network relations make avail- able a store of social and material resources—conversion factors in the language of the Capability Approach—that contribute to capabilities. It is a limitation of our framework that we are not permitted to evaluate the actual transformative impact of the research. Rather, we observe a set of collective capabilities that might contribute to such (political) goals of TD researchers. Of course, this in itself poses a challenge to evaluators given such normative and often con- tested ambitions. However, the usefulness of our capability frame- work is in fact to reveal the values that underpin such positions, making visible the normativity of these value-decisions in pursuit of transformation. 5. Discussion: implications for evaluators and funders While we find compelling evidence that transgres- sive capabilities are valued in NNþ projects, we find less evidence for their actual presence, as reported in Table 3. This suggests the need for a long-term TD research interventions at the institutional level. This has implications for this kind of pump-priming model as a means of organizing and controlling research and ultimately ready- ing capacity—at least as it is instantiated here which we argue resembles a form of research projectification (Fowler, Lindahl and Sko¨ld 2015; Torka 2018). In this case, we find that model of re- search control that emphasizes discreet projects over short durations is not sufficient to meet broader ambitions for TD research as valued by researchers themselves. Returning to the case of the PI in project 4, they found it difficult to support capability development of overseas partners from the UK side. By comparison, researchers in Project 3 had spent over a dec- ade building up these kinds of capabilities through the establishment of new networks of local people. Although highly contextual, this comparison illustrates an issue of capability building across the port- folio. Where transdisciplinary capabilities have been well-cultivated, in P1 and P3 and to a lesser extent P2, these capabilities have been supported by long-established networks of actors, knowledge, rela- tions and other capabilities. Capability development has taken place through other means, that is, outside of the project boundaries established through the NNþ Partnership Programme. In these cases, we find evidence of capability building over a multi-project timeframe initiated prior to the NNþ Programme. In the case of Project 3 for example, this backstage capability space, was estab- lished by researchers over the course of a decade by building up net- works and maintaining relations and the provision of new resources such as funding and researchers. Downloaded from https://academic.oup.com/rev/article/31/1/145/6481782 by University College London user on 08 June 2022 Furthermore, calls for societal legitimacy in knowledge produc- tion and more latterly societal impact have been a feature of TD re- search programmes. These are motivating capability drivers for researchers in our study, expressed through the collective capabil- ities they value such as being able to critically interact with power and being able to contribute to societal capacity for democratic life. For instance, through the maintenance of accountability relation- ships between science and society (Strathern 2004). 5. Discussion: implications for evaluators and funders Capabilities experienced and absent in the ﬁve NNþ research projects Capabilities for TD research Heuristic Reported presence or absence in NNþ portfolio projects Inductively and iteratively generated from literature, workshops and programme call and empirical data Being able to perform research across disciplinary boundaries Cognitive Present across the portfolio Being able to apply tools and frameworks in new situations Cognitive Present across the portfolio Being able to sustain appreciation for the importance of the particular Cognitive Present in P1, P4, ambiguous or difﬁcult to assess elsewhere Being able to sustain values of pluralism in relation to different kinds of knowledge and methods Individual Present across the portfolio A capability to acknowledge and communicate complexity Individual Present across the portfolio Being able to trust in collaboration Individual Present across the portfolio Being humble in relation to recognizing uncertainty in knowledge Individual Present across the portfolio Being able to sustain a livelihood Individual Valued but mostly absent across the portfolio Being able to reﬂexively consider one’s own work, situation and impact. Individual; collective Present across the portfolio Being able to critically interact with and challenge power in know- ledge, in organizations and in institutions Individual; collective Valued but mostly absent across the portfolio Being able to build and manage a research team and project Individual; collective Present across the portfolio Being egalitarian in relation to all project participants and stakeholders Collective Valued across the portfolio, present in P1, P3 Being able to contribute to societal capacity for democratic life by maintaining relationships between science and society. For ex- ample, accountability relations. Collective Valued but not sustained in projects Being able to build and sustain networks beyond project timescales Collective Valued by all, present in P1, P2, P3 Table 3. Capabilities experienced and absent in the ﬁve NNþ research projects 155 Research Evaluation, 2022, Vol. 31, No. 1 Learning itself can be considered a capability (as evidenced in the heuristic, Table 1). However, we found that in projects learning was more usefully understood as a process of capability building that is directed by policy and programme rules and is enhanced through available resources such as knowledge, finance, and time. These resources were not sufficiently provisioned under the NNþ Partnership Programme. disciplinary knowledge and challenge institutional or structural con- straints. To be clear, these are goals in themselves, but the collective capabilities that are valued for their contribution achieving trans- formative goals. 5. Discussion: implications for evaluators and funders with this, analyses such as bibliometric studies that rely on indica- tors of research outputs would benefit from being conducted at greater time intervals after the research to better reflect publishing norms in academic practice. Or, put another way, the capability mapping framework proposed in this article might usefully comple- ment established evaluation procedures (see Section 1 and 2) that al- ready focus on issues of impact, quality or transformation. Acknowledgements This article has proposed a novel framework for evaluating how researchers value capabilities for TD research. It reveals capabilities of researchers typically not included in programme evaluation. Our main empirical finding is that under a ‘nexus research’ framing, the NNþ Partnership Programme convened a diversity of cognitive capabilities. However, these capabilities on their own were insuffi- cient to support TD research. We noted two other features of cap- ability development. The value placed on transgressive capabilities by researchers and the maintenance of a backstage capability space required for TD research. The authors thank the participants at the workshops mentioned in the article. We sincerely thank researchers from the Nexus Network Partnership Grant programme who agreed to be interviewed. We are grateful to Becky Ayre, Marion Clarke, Gordon Mackerron and James Wilsdon, and to Fre´de´rique Bone and Andy Stirling. Thanks also to the journal’s editors and two anonym- ous reviewers for their substantive engagement with this work. Notes 1. See the NNþ final report for details of the network’s activities (Cairns et al. 2017) and UKRI funding details (UKRI 2020). 2. The distinction between achieved functionings and capabilities is between what is actually realized—the beings and doings— and what is effectively possible—the options valued by researchers. Whether a person can convert a set of means into a functioning depends on certain contextual drivers, such as per- sonal, political and environmental conditions, what capability theorists call conversion factors. Our focus on capabilities ra- ther than functionings is driven by the aim of the study to as- sess what real possibilities for research are cultivated in the course of NNþ funding. Nevertheless, a capability approach demonstrates both the im- portance and absence of systematic capability evaluation in research evaluation frameworks to date. By expanding the scope of our in- quiry beyond outputs, quality, and processes, we show how an evaluation of capabilities can contribute to evaluations of epistemic and social contexts of knowledge production. In this, we draw on examples from other recent evaluation contributions (Belcher et al. 2016; Bone et al. 2020). A point of similarity in these approaches is opening the black box of research projects to explore knowledge production downstream of traditional indicators. Future work might explore complementarities between these approaches. 3. It is suitable to use WOS categories based on already done work for capabilities because capabilities are at their core pro- spective. We find it reasonable and plausible to use these dis- ciplinary categories as indicators of capability, that is, as indicators of epistemic work researchers may choose to do to- gether in the future. We expect this framework to be useful for funding agencies and public research organizations, especially in midterm or ongoing evaluations. Our framework looks at capabilities as expressed in practice, and thus concerns itself with how projects enact transdisci- plinary capabilities. As such, a capabilities approach to evaluation is best used formatively in an effort to identify areas for support to achieve project success. Also, we believe these findings to have par- ticular salience in pump-priming instruments such as sandpits and UKRI Networkþ model where the urgency of developing capabil- ities to research societal issues are stressed, yet uncertainties regard- ing the most appropriate action remain. Examples of issues in US, European, and UK research policy systems include climate change, artificial intelligence, and economic productivity. 4. 6. Conclusions—towards a framework for evaluating capabilities Downloaded from https://academic.oup.com/rev/article/31/1/145/6481782 by University College London user on 08 June 2022 5. Discussion: implications for evaluators and funders Indeed, mono- or multi-disciplinary approaches are no less normative simply because they align with more deeply situ- ated epistemic incumbencies (Stirling 2019). Finally, a strength of the capability framework is in how it emphasizes contingency and situatedness of capabilities. This brings with it at least two limitations. First is the generalizability of capa- bilities from one capability space to another. Following Robeyns (2005), we are resistant to the idea of a master list of capabilities for TD research. However, we suggest the evaluation literature would benefit from further analysis of what factors cultivate or close down commonly valued capabilities. For that, more comparative research is required. And in the case of collective capabilities, forms of obser- vational analysis such ethnography or situated analysis might be usefully employed in this task. Epistemic, institutional, or broader societal transformation is not always achieved in TD research. Nevertheless, we propose that there is use in drawing attention to capabilities valued by researchers in pursuit of transformative goals and following agonistic logics. We cannot be confident these capabilities on their own will lead to transformative outcomes (for instance, being able to challenge power in knowledge; being able to contribute to societal capacity for democratic life), and so we characterize them as transgressive capabilities. Further research might also address a second limitation, which is a lack of epistemic and explanatory power over why certain capabil- ities arise in some situations and not others. Empirically derived, these capabilities may in some cases be conceptually ambiguous. Work is needed to define and explain such capabilities with social theory and methods appropriate to the evaluation setting. In line We understand transgressive capabilities as the collective cap- ability of experts to come together to transgress the boundaries of 156 Research Evaluation, 2022, Vol. 31, No. 1 The evaluation approach might also be extended to other forms of participatory knowledge production such as co-creation and co- design. The cultivation and maintenance of transgressive capabilities in particular is a prudent way in which funders might plan for future urgencies, the details of which are often difficult to anticipate. Critically, such capabilities will augment research system capacity only if appropriately resourced and configured. We believe this evaluation framework can contribute in formatively identifying, cul- tivating, and maintaining such capabilities. Notes A point we thank one of our anonymous reviewers for highlighting. Funding This work was supported by the Economic and Social Research Council, ESRC [ES/L01632X/1]. Conflict of interest statement. None declared. Supplementary data Supplementary data are available at Research Evaluation Journal online. One important observation from this research is to show that developing expertise for real word problem solving (be they framed as grand challenges, societal missions, or agendas more modestly named) is not about assembling the best experts from a rage of nar- row academic domains. Rather, it is about fostering a set of capabil- ities that will contribute to knowledge production that gains legitimacy and accountability from the plurality of contributing actors, the wider societal contexts in which it is produced, and inter- connected relations between these actor and contexts. We have con- tributed a set of methods that can evaluate these practices and procedures. However, our focus on researcher capabilities neglects a systematic study on the capabilities of other participants of TD re- search project. Beyond the scope of this article, such studies are ur- gently required. References Alkire, S. (2005) ‘Why the Capability Approach?’, Journal of Human Development, 6, 115–35. Alkire, S. 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https://openalex.org/W4393044032	https://link.springer.com/content/pdf/10.1007/s10489-024-05396-z.pdf	English	null	An integrated decision framework for evaluating and recommending health care services	Applied intelligence	2,024	cc-by	13,980	Abstract Quality management techniques such as the quality function deployment model can help hospitals assess and improve the quality of their services by integrating the voice of customers. The different quality parameters of this model are usually determined and assessed by experts; nonetheless, obtaining such experts is not always easy or inexpensive. Moreover, in this method, patient opinions are not usually considered directly, although they are the real users of the services and those who can best assess those services. Nevertheless, these opinions are easily accessible today, owing to the development of medical social networks where patients directly convey their opinions about the different services and features of a hospital. Therefore, it is feasible to replace expert knowledge with the information provided by these opinions. Based on this idea, this study proposes a novel fuzzy recommendation model based on the quality function deployment method to rank hospitals depending on patient opinions and preferences regarding hospital services. This model integrates a topic modeling strategy for determining hospital requirements, customer needs, and the relationship between them as well as a sentiment analysis algorithm for assessing customer satisfaction regarding hospital services. To demonstrate the usefulness of the proposed method, several experiments were conducted using patient reviews from real hospitals, and the method was compared against other recommendation models. The results prove that this approach represents a step toward more personalized and effective health care system selection considering patient preferences and opinions. Keywords Sentiment analysis · Hospital ranking · Recommender systems · Quality function deployment · House of quality Keywords Sentiment analysis · Hospital ranking · Recommender systems · Quality function deploym An integrated decision framework for evaluating and recommending health care services Bashar Alshouha1 · Jesus Serrano-Guerrero1 · Francisco Chiclana2 · Francisco P. Romero1 · Jose A. Olivas1 Bashar Alshouha1 · Jesus Serrano-Guerrero1 · Francisco Chiclana2 · Francisco P. Romer Accepted: 12 March 2024 / Published online: 21 March 2024 © The Author(s) 2024 Accepted: 12 March 2024 / Published online: 21 March 2024 © The Author(s) 2024 Applied Intelligence (2024) 54:4153–4171 https://doi.org/10.1007/s10489-024-05396-z Applied Intelligence (2024) 54:4153–4171 https://doi.org/10.1007/s10489-024-05396-z B Jesus Serrano-Guerrero jesus.serrano@uclm.es Extended author information available on the last page of the article Bashar Alshouha, Jesus Serrano-Guerrero, Francisco Chiclana, Francisco P. Romero and Jose A. Olivas contributed equally to this work. 1 Introduction Given that these values are assumed to come from experts, i.e., they are not usually expressed in numbers but in natural language expressions such as good and bad, they are represented by fuzzy sets. In addition to the fact that it is not easy to ﬁnd many experts, on many occasions their limited involvement further complicates QFD implementation. Experts in the ﬁeld of health care services are often consulted to assess the quality of the services; nevertheless, the perspective of the patients is not considered on many occasions [14], although previ- ous studies have highlighted the signiﬁcance of incorporating user feedback in improving health care service quality [15]. Experts are usually expensive and difﬁcult to ﬁnd, and on many occasions, they convey their own opinions which are different from patient opinions [11, 16–18]. In summary, the main contributions of this study are as fol- lows: • A fuzzy recommendation mechanism for ranking real hospitals utilizing a fuzzy-QFD method based on patient opinions and user preferences. • A topic modeling algorithm to identify the quality param- eters of the QFD model from patient opinions on the hospitals. Furthermore, the static nature of traditional QFD methods poses a challenge in today’s dynamic health care envi- ronment. Customer preferences and health care service conditions can change rapidly; nonetheless, QFD may not adapt quickly enough to these changes, potentially leading to services that no longer meet customer needs. For these rea- sons, we propose the use of a patient feedback web platform to capture information directly from users instead of resort- ing to experts, which is often more difﬁcult and expensive. • An opinion mining algorithm for calculating the polarity of the DRs. • A fuzzy mechanism to represent the polarity of patient opinions and the relationship between the CRs and the DRs of a hospital. • A set of experiments using real patient reviews from hospitals to customize the recommendations of these hospitals considering user preferences under different scenarios. Another typical challenge lies in dealing with the uncer- tainty associated with QFD evaluations. Vagueness, ambi- guity, and incomplete information are common in expert judgments and relationships between customer requirements (CRs) and design requirements (DRs). 1 Introduction To assess the quality of many services, it is necessary to use formal quality models, and the quality function deploy- ment (QFD) method [3, 4] is one of the most well-know. This method has been successfully applied to many domains in the past, such as supply chains [5], supplier selection [6], strategic selection [7], manufacturing strategies [8], market segment evaluation [9], health care [10] and health care ser- vices [11]. Nevertheless, it still has many limitations. As the health care industry continuously growing and chang- ing, health care providers are constantly seeking new and innovative methods to maintain and improve the quality of hospital services. Currently, patients not only seek medical treatments but also demand safe and comfortable health care experiences [1]. Moreover, hospitals, as patient-oriented ser- vice providers, are expected not only to provide the best medical treatments but also, to improve patient satisfac- tion [2]. Wolnia [12] and Huang et al. [13] studied the main proper- ties as well as the main limitation of the QFD method. Among these limitations, some issues were highlighted related to the dependence on experts in making any decision. Traditionally, small teams of experts conduct QFD analyses, which may not sufﬁciently capture the various perspectives and knowledge needed for complex products and services, especially in dis- tributed contexts. This lack of general input can limit the effectiveness of QFD in real-world scenarios. One of these scenarios is the health care systems; when several hospitals are involved, it is necessary to rely on several dozen experts, Bashar Alshouha, Jesus Serrano-Guerrero, Francisco Chiclana, Francisco P. Romero and Jose A. Olivas contributed equally to this work. B Jesus Serrano-Guerrero jesus.serrano@uclm.es Extended author information available on the last page of the article Bashar Alshouha, Jesus Serrano-Guerrero, Francisco Chiclana, Francisco P. Romero and Jose A. Olivas contributed equally to this work. B Jesus Serrano-Guerrero jesus.serrano@uclm.es Extended author information available on the last page of the article 12 3 3 4154 B. Alshouha et al. which is very difﬁcult to achieve. For that reason, it is nec- essary to use other strategies such as those presented here. determining the health care dimensions and quality param- eters using topic modeling techniques and assessing them using sentiment analysis techniques applied to past patient reviews. 1 Introduction Additionally, existing QFD approaches often ignore the complex relationships and dependenciesbetweenCRs.Forthisreason,afuzzyrepresen- tation model is proposed to represent the information from the system, instead of a classical crisp representation, which is less ﬂexible and less similar to how a user expresses their assessments. The remainder of the paper is organized as follows: Section 2 provides a detailed overview of the state-of-the-art methods of hospital service assessment. Section 3 presents the proposal, outlining the methodology and dataset utilized in the hospital ranking. In Section 4, the results of the case studies testing the methodology are discussed. Finally, in Section 5, conclusions are drawn based on the ﬁndings of this study. Despite all these limitations, there are opportunities to address them. The development of medical social platforms can provide valuable information on the health care services given by appropriate patients or health care users. Further- more, techniques from areas such as opinion mining and topic modeling can help clarify the most relevant dimensions for the users and their opinions about them in the medical domain. Therefore, it is possible to exploit this informa- tion instead of the information provided by experts, to try to understand the quality of the provided services. This can aid hospitals in enhancing the quality of their services, opti- mizing them, and increasing their competitiveness within the health care industry. Consequently, the goal of this study is to propose an application that is able to recommend the most suitable hospitals for a speciﬁc user depending on the quality of the provided services according to patient opinions. To do so, the novelty of the proposal lies in replacing the knowledge usually obtained from experts to model the QFD method by 2 Literature review One of the key steps of this approach is the HOQ, which reﬂects the relationships between CRs and ser- vice characteristics. This matrix helps clarify the needs and desires of the target customer and how they can be met through the design and delivery of the service. There are two main styles of HOQ: American and Japanese. The American style typically includes six basic steps and has an additional section, known as Area E (see Fig. 1) while the Japanese style is considered simpler and easier to use [24]. This study focuses on the Japanese style of HOQ to measure the quality of services provided by hos- pitals considering patient feedback. This approach allows us to identify areas needing improvement and the necessary changes to provide better patient services: Araujo et al. integrated fuzzy set theory, QFD, and SERVQUAL to assess medical clinic service quality, empha- sizing the importance of prioritizing key requirements [29]. A similar approach was presented by Junior et al. to enhance decision support for surgical centers in Brazil [30]. Kanan et al. applied QFD to analyze private health care services in Saudi Arabia and identiﬁed management issues and informa- tion ﬂow as key factors [31]. Amran et al. introduced fuzzy multicriteria decision-making (MCDM) for prioritizing crit- ical medical devices in biomedical engineering maintenance services, ensuring the reliability of the hospitals of the Min- istry of Health [32]. The HOQ information obtained from surveys completed by experts is exploited in this method. • Area A represents the CRs, which are the needs and desires of the target customer that the service or prod- uct should meet. In this paper, the CRs were determined through a comprehensive academic literature review pro- cess. • Area B represents the relative importance of CRs, which can be determined through various methods, includ- ing direct methods where the user assigns scores to the requirements and indirect methods utilizing more complex mathematical and statistical models such as analytical hierarchy process (AHP) and decision-making trial and evaluation laboratory (DEMATEL). Alejo-Vilchis et al. applied QFD to identify improve- ment areas in clinical analysis laboratories based on patient satisfaction data obtained through SERVQUAL question- naires [33]. Khan et al. focused on building a resilience-based model for the health care sector to ﬁght COVID-19 in Pak- istan. This model combines multicriteria decision-making with QFD to prioritize resilient attributes and strategies [7]. Tortorella et al. 2 Literature review In the health care industry, different models such as SERVQUAL [19] and Kano [20] have been used to mea- sure the quality of different services. The SERVQUAL model evaluates the gap between expected and perceived service quality through different dimensions, such as assur- ance, responsiveness, empathy, tangibility and reliability. Kano, on the other hand, categorizes service attributes into three categories—must-haves, performance, and excitement factors—to assess customer satisfaction. Both models have been applied in health care to identify areas for improving and enhancing patient satisfaction [21, 22]. The QFD methodology is a systematic approach used to optimize the design and development of products and/or ser- vices [3, 4]. It is a customer-focused method that uses a series 12 123 123 An integrated decision framework for evaluating and recommending health care services 4155 Fig. 1 HOQ diagram requirements and serve as a benchmark for evaluating the design effectiveness. The weight of each DR is also a signiﬁcant consideration because it determines the relative priority of each requirement within the design process. These weights are determined using the HOQ methodology and serve as a guiding principle for the design team to ensure that all DRs are met in an appro- priate manner. QFD has been successfully applied in various ﬁelds. In health care, QFD has proven to be a valuable tool for ser- vice planning and customer needs analysis. By translating consumer points of view into conclusions for regular ser- vice planning, hospital managers can effectively identify and prioritize consumer needs through the use of QFD [25]. It can also help optimize emergency department operations, decrease patient waiting times [26], and improve medical device design and patient safety in hospital settings [27]. In addition, Said et al. applied QFD to develop quality inpatient services based on patient needs and to achieve high levels of medical tourist satisfaction in India [11]. Gavahi et al. developed a hybrid approach using QFD and SERVQUAL to assess and improve radiology center services [28]. This study used the voice of customers (VOC) through surveys to evaluate service quality for radiology patients; its ﬁndings can be easily applied by practitioners. Fig. 1 HOQ diagram of matrices to translate customer needs and requirements into speciﬁc design and development goals. Cohen [23] introduced a four-phase approach for service development utilizing QFD. 2 Literature review proposed a problem-oriented methodology using QFD for prioritizing the integration of Industry 4.0 technologies in hospitals, demonstrating its applicability in ranking digital applications [9]. Nie et al. introduced a cloud- supported QFD model to implement a customer-centric approach for improving health care service quality [34]. • Area C describes the DRs or service characteristics, which are the solutions to meet customer requirements. • Area D is the relationship matrix, which is the core element of the HOQ and shows the strength of the rela- tionships between the CRs and DRs. • Area E is the correlation matrix, which illustrates the impact that one DR may have on another. This area helps identify potential trade-offs between requirements. • Area F represents the target value for each DR. These val- uesarederivedfromathoroughanalysisofthecustomer’s 12 123 4156 B. Alshouha et al. Fig. 2 Example of Careopinion story Neira-Rodado et al. addressed challenges in health care technology product design by combining fuzzy Kano, AHP, DEMATEL, and QFD, providing a novel approach for translating customer needs into product characteristics [35]. Salah et al. proposed a QFD-LFA framework to improve patient safety in hospital design [36]. Fauziah et al. analyzed the effect of pharmacy service quality and identiﬁed the largest gap between the speed and accuracy of pharmacy staff [37]. These studies offer valuable insights into enhanc- ing the quality of health care services by considering CRs, identifying critical improvement actions, and prioritizing improvement efforts. Furthermore, Raziei et al. presented a hybrid approach that uses group decision-making, service quality measurement, and QFD to prioritize patient needs as quality factors and cope with uncertainties [38]. QFD-based approach considers the past experiences of patients as well as their satisfaction degree with respect to the services offered by the hospital. Unlike traditional QFD mechanisms applied to hospital management, this approach does not rely on experts to deter- mine and assess the primary dimensions of hospitals. In this case, the information provided by the patients’ opinions posted on a feedback web platform called Careopinion1 is sufﬁcient to model and assess all these dimensions modeled by the HOQ. These dimensions in the health care indus- try are well-known, and it is therefore easy to ﬁnd these opinions. Furthermore, the opinions can reveal positive or negative feelings toward these dimensions. 2 Literature review Hence, it is not only possible to identify the dimensions of the HOQ of the QFD model but also to determine the values of the matrix, i.e., the assessment of the dimensions. These studies collectively demonstrate the potential of QFD techniques to improve hospital service quality by prior- itizing patient needs, identifying quality characteristics, and enhancing health care processes. Most of these methods have been used for studying speciﬁc quality features of a hospital but not for ranking hospitals according to the most critical quality dimensions, as is proposed here. The Careopinion website is a platform for patients, where they can share information about their own experiences in a hospital. Each opinion is called a “story” because it includes personal emotions and anecdotes when visiting a hospital. The structure of every story consists of a “Story Summary” section with three subsections covering the patient feelings, positive aspects of the hospital, and areas that could be improved. The “how did you feel” section provides a glimpse into the emotional state of the patient during their hospital visit and can help identify areas of improvement for hospital managers. The “what’s good” section highlights the positive aspects of the patient experience and can serve as an indicator of areas where the hospital is performing correctly. Finally, the “what could be improved” section allows patients to pro- vide feedback on areas needing attention such as waiting times or communication with staff. This last section can be especially useful for detecting DRs to pay special attention to. For instance, if several patients mention that the wait- ing area is uncomfortable, it is possible to infer that a better waiting area could help increase patient satisfaction. Although QFD has been widely used, it has limitations mainly due to its manual approach, which can be time- consuming to implement and relies on subjective decision- making. Moreover, customer preferences and/or opinions are often expressed using subjective, vague, or imprecise terms, both numerically and linguistically. Consequently, in QFD, it is challengingtointerpret fuzzyhumanlanguagewiththeaim of translating it into technical requirements. For this reason, many QFD-based solutions use fuzzy logic-based repre- sentations in diverse industries such as supply chains [5], supplier selection [39], strategic selection [40], manufactur- ingstrategies[41],marketsegmentevaluation[42]andhealth care [43]. As shown in Fig. 2, the information about the dimensions and their assessments are available in these opinions. 1 https://www.careopinion.org.uk 2 Literature review It is necessary to detect the opinions and their associated feelings to model the HOQ. Regardless of the dimension an opinion 3.1.1 Step 1. Identifying the customer requirements (“WHATs”) To accurately reﬂect the expectations and needs of patients, identifying the k key CRs (C R = {C R1, C R2, C R3, · · · , C Rk}) for hospitals is paramount. By conducting a thorough analysis of the most recent and relevant literature [2, 38, 45], the crucial dimensions for assessing the quality of health care services were determined according to the majority of related studies. Moreover, these dimensions play a vital role in shaping DRs for hospitals. Particularly, six dimensions were found, therefore, in this study k = 6. These six essential quality dimensions identiﬁed for the CRs were: As one of the goals of the system is to replace human interaction and most of the system information is provided by users, this information will be represented by linguistic terms as usual. For instance, the most common mechanism for collecting the assessment of the CRs to build the HOQ and gather its inputs is to conduct surveys, interviews, or focus groups with the customers, who may be experts, end- users, or practitioners. Since the values of these variables are mostly linguistic (the dimension may be ‘highly important’ or performance may be ‘very good’), they tend to be uncer- tain, imprecise, subjective, and vague; therefore, the use of fuzzy logic techniques seems appropriate under these cir- cumstances. Therefore, the values of the HOQ are modeled by fuzzy linguistic term sets. • Tangible (C R1): This refers to the physical appearance and equipment of the hospital and the staff, including for instance, their uniforms. The goal is to create a profes- sional atmosphere that instills trust in patients. Finally, taking advantage of the information represented in the fuzzy HOQs of the assessed hospitals and consider- ing the user preferences for different CRs, an aggregation method based on the fuzzy suitability index is proposed to determine the ﬁnal score of all hospitals. The rank of the hospitals is established according to this score. The entire process is depicted in Fig. 3, and the subsequent steps are described in detail below. • Reliability (C R2): This dimension focuses on the con- sistency and accuracy of the hospital services as well as the ability of the staff to deliver services as needed. Patients can only rely on their health care providers if they deliver high-quality services. 3.1 Algorithm concerns, it is detected by a speciﬁcally designed LDA-based model [44]. As to whether the opinion toward a dimension is positive or negative, an opinion mining-based algorithm is proposed to process the sentences talking about each dimen- sion and calculate the corresponding polarity. 3 Proposal In this section, an application for recommending real hospi- tals according to user preferences is described. This fuzzy 123 An integrated decision framework for evaluating and recommending health care services 4157 3.1.1 Step 1. Identifying the customer requirements (“WHATs”) • Responsiveness (C R3): Responsiveness refers to the promptness and willingness of the hospital staff when providing services to patients. This can be demonstrated through the hospital commitment to providing services in a timely manner and sharing diagnostic information promptly with patients. This process is repeated for all hospitals that are to be ranked to calculate their different fuzzy suitability indices. Theﬁnalrankingorderisestablishedbycomparingthevalues of the indices. Fig. 3 Steps for modeling the HOQ Fig. 3 Steps for modeling the HOQ Fig. 3 Steps for modeling the HOQ 12 3 4158 B. Alshouha et al. In the second scenario, more weight is assigned to the “assur- ance” dimension; i.e., the user prioritizes this dimension over the others: In the second scenario, more weight is assigned to the “assur- ance” dimension; i.e., the user prioritizes this dimension over the others: • Assurance (C R4): This dimension is focused on the level of trust, conﬁdence, and security that patients feel about the provided services. It includes staff competence, knowledge, and skills, as well as staff willingness to help and communicate effectively with the patients. WC R2 = {0.025, 0.025, 0.025, 0.875, 0.025, 0.025} • Empathy (C R5): This dimension is centered around the caring and understanding attitude of the staff toward the patients. The goal is to create a supportive environment for patients. In the third scenario, the “reliability” dimension receives the highest weight, i.e., this is the most crucial aspect for the user: In the third scenario, the “reliability” dimension receives the highest weight, i.e., this is the most crucial aspect for the user: • Professionalism (C R6): This dimension is related to the level of skills and expertise of the hospital staff, and their ability to provide services in a manner aligned with patient expectations. Patients expect to receive services from knowledgeable and well-trained staff who adhere to high ethical standards. C R3 = {0.025, 0.875, 0.025, 0.025, 0.025, 0.025} WC R3 = {0.025, 0.875, 0.025, 0.025, 0.025, 0.025} By evaluating various weight combinations, it is possible to gain a comprehensive understanding of customer preferences and opinions, providing valuable insights for service quality improvement. 3.1.3 Step 3. Identifying the relevant aspects for the design requirements (“HOWs”) This approach aims at prioritizing the customer’s voice in the evaluation process. Thus, to determine the importance of the CRs, it is necessary to obtain the information form the user. The easiest manner is to directly ask users about their preferences regarding CRs. The users can assign weights to different dimensions based on their needs, ensuring a trans- parent and user-centric process. Nonetheless, if the system where this methodology is implemented has information about the user priorities over the CRs, for instance, in the form of textual opinions, it is also possible to develop indirect mechanisms able to analyze these opinions and determine the user preferences in a ‘less subjective’ manner than directly asking the user about them. To determine the m DRs (DR = {DR1, DR2, · · · , DRm}) of the HOQ, a manual comprehensive analysis of the patient reviews available on Careopinion was carried out. This analysis included identifying the key areas of concern and opportunities for improvement highlighted by patient opin- ions. In addition, the subsections “how did you feel”, “what’s good”, and “what could be improved” provided very rel- evant information. In particular, “what could be improved” was especially relevant highlighting speciﬁc areas to improve according to the patients. These ﬁndings were used to iden- tify the DRs (“HOWs”) necessary to enhance the quality of hospital services. In total, eight DRs were identiﬁed, and con- sequently, m = 8. The requirements found primarily match the ones used in the literature [2, 46, 47]: Mathematically, these weights are represented by a k- dimensional vector WC R = {w1, · · · , wk}, where k symbol- izes the number of CRs. This vector representation provides a comprehensive understanding of customer preferences regarding the six CRs mentioned above in the same order (see Section 3.1.1). The sum of the weights must always be 1. • Staff (DR1): The competency, knowledge, and attitudes of health care providers, including doctors and nurses, play crucial roles in delivering high-quality services to patients. To enhance this aspect, hospitals can focus on ongoing training and development programs, hiring experienced and knowledgeable personnel, and fostering a supportive and empathetic workplace culture. WC R1 ={0.16666,0.16666,0.16666,0.16666,0.16666,0.16666} 3.1.3 Step 3. Identifying the relevant aspects for the design requirements (“HOWs”) For the evaluation section, three scenarios are considered for simulating different types of users: (i) a user consider- ing that all dimensions are important to them, (ii) another considering the “assurance” dimension to be more relevant and (iii) another considering “reliability” to be more relevant. In this manner, it is possible to simulate different users and recognize that customer preferences can change over time. • Care (DR2): The level of compassion, empathy, and attention given to patients is critical to providing high- quality care. Hospitals can improve this aspect by promoting patient-centered care, encouraging open and effective communication between staff and patients, and regularly gathering feedback from patients to address areas of improvement. Hence, the ﬁrst scenario involves all aspects being equal regarding the CRs; i.e., the user does not consider one aspect more important than the others: • Facilities (DR3): The physical environment of the hos- pital, including the design and maintenance of buildings, WC R1 ={0.16666,0.16666,0.16666,0.16666,0.16666,0.16666} 3 12 An integrated decision framework for evaluating and recommending health care services 4159 value of the matrix ri j represents the relationship degree of the i-th CR with respect to the j-th DR: equipment, and services, is important for developing a welcoming and safe environment for patients. Hospitals can enhance this aspect by investing in modern and well- maintained facilities, ensuring cleanliness and hygiene, and providing a comfortable and accessible environment. matrixe = {ri j \| i = 1, . . . , k and j = 1, . . . , m} where k = number of C Rs (“WHATs”), and m = number of DRs (“HOWs”). where k = number of C Rs (“WHATs”), and m = number of DRs (“HOWs”). • Conﬁdence (DR4): Conﬁdence refers to the trust, belief, and sense of security that patients have regarding a hos- pital and its services. It is built upon the reliability, preventative measures, and experience of the hospital in providing safe and high-quality care. Hospitals can pro- mote patient conﬁdence by demonstrating expertise in their ﬁeld, ensuring the safety and security of patients through robust protocols and procedures, proactively addressing concerns and disappointments, and providing reliable and preventative care measures. In the literature, the expert opinions are usually expressed by linguistic terms such as ‘very low’ or ‘very high’. Therefore, it is necessary to mathematically represent these opinions. The most natural mechanism to do this is fuzzy logic. relationship_matrix = matrix1 ⊕matrix2 ⊕. . . ⊕matrixx • Timeliness (DR6): The promptness and efﬁciency of healthcare services are critical for ensuring that patients receive care in a timely manner. Hospitals can improve this aspect by streamlining processes, investing in tech- nology, and providing adequate stafﬁng to ensure that patients receive prompt and effective care. To do so, the individual fuzzy values ri je of all the matrices, which represent the opinions of decision-maker e regarding the i-th CR and the j-th DR, must be aggregated into a unique value ri j: • Efﬁciency (DR7): The effective use of resources and reduction of waste are important in promoting cost- effectiveness and sustainability in health care services. Hospitals can enhance this by promoting resource opti- mization, reducing waste, and improving operational processes. ri j = ri j1 ⊕ri j2 ⊕. . . ⊕ri jx where x = number of decision-makers. Thus, the ﬁnal matrix values, denoted as “ri j”, indicate the aggregation of the fuzzy values from each expert, representing the relationship between the i −th “WHAT” aspect and the j −th “HOW” aspect. These matrix items are also represented by fuzzy sets as a result of the aggregation of fuzzy sets. • Transparency (DR8): This dimension refers to open, honest, and clear communication, disclosure, and clarity of information and processes in a hospital. It encom- passes openness and honesty in sharing information, reviewing procedures, and addressing questions or con- cerns raised by patients. Transparency involves providing accurate and accessible information, promoting openness in practices and procedures, and actively seeking feed- back from patients. This approach, not only improves the interpretability of the relationship matrix but also, addresses the differences and ambiguity associated with the opinions of experts, pro- viding an accurate and clear representation of the relationship between CRs and DRs. This process is used repeatedly in the literature [37, 47, 48]. From the literature, it is seen that experts’ opinions are repeated in many studies. Therefore, it is possible to extract the most commonly repeated opinions for the DRs and CRs detected in Sections 3.1.1 and 3.1.3, representing three different experts. In the experimental section, these repeated opinions are used to represent three different experts to calculate the overall relationship matrix. Therefore, in the experimental section, the number of CRs is k = 6 3.1.3 Step 3. Identifying the relevant aspects for the design requirements (“HOWs”) Thus, these linguistic labels can be associated with balanced triangular fuzzy numbers (TFNs). Therefore, the system represents the opinions of several experts with several fuzzy matrices that must be merged into a unique fuzzy relationship matrix that accurately represents the overall relationship between each pair of a CR and DR. To determine the overall relationship between CRs and DRs, it is necessary to aggregate all opinion matrices from all experts: • Services (DR5): The quality and responsiveness of the services provided to patients, such as diagnosis, treat- ment, and recovery support, are important factors for promoting patient satisfaction. Hospitals can enhance this aspect by investing in advanced technology, pro- moting timely and effective treatment, and providing a patient-centered approach to care. 3.1.6 Step 6. Computing the rating of a hospital with respect to the design requirements (“HOWs”) After the weights of each DR are calculated, the next step consists of evaluating each hospital with respect to each DR. Most studies applying QFD rely on assessments from experts or decision-makers through questionnaires or surveys; nev- ertheless, in this research, the evaluation of the quality of hospital services is based on patient reviews, exploiting, the information available on the internet from real customers, i.e., patients, and avoiding the search for experts for this type of task. Patient reviews provide a comprehensive and nuanced understanding of the quality of the services deliv- ered by hospitals, highlighting areas of improvement that may have gone unnoticed. This methodology leads to a more accurate and well-rounded evaluation of hospital services, providing valuable insights for both health care providers and patients. The proposed methodology for evaluating the quality of hospitals consists of two phases: Fig. 4 The linguistic term set used in this study (see Section 3.1.1), the number of DRs is m = 8 (see Section 3.1.3), and the number of experts x = 3. Furthermore, the opinions of these experts in the litera- ture are usually expressed using the following labels: ‘very very low (VVL)’, ‘very low (VL)’, ‘low (L)’, ‘medium-low (ML)’, ‘medium-high (MH)’, ‘high (H)’, ‘very high (VH)’, and ‘very very high (VVH)’. Hence, these labels can be mod- eled mathematically by a scheme of fuzzy sets, as shown in Fig. 4. This scheme will be followed for the case studies shown in the experimental section. For instance, if the expert considers the relationship between the CR “empathy” and DR “staff” to be very very high (VVH), then it would be mathematically represented by the TFN = (0.875, 1, 1). Phase A. Design requirement detection To detect the DRs available in the patient opinions, the algorithm used in [49] was applied. First, the reviews from the dataset were pre- processed, and an LDA-based method was used to identify the most relevant topics. Sixteen topics were computed and manually analyzed to determine which one belonged to each DR criterion according to the most descriptive terms. The procedure is depicted in Fig. 5, and an excerpt of the most representative terms of each topic, which were assigned to each DR criterion manually, is presented in Table 1. Through this approach, any sentence belonging to one of the 16 topics is automatically grouped into the corresponding DR related to that topic. 3.1.4 Step 4. Determining the CR-DR (“HOW”-“WHAT”) relationship scores To calculate the relationship degrees between the CRs and the DRs in the HOQ, the most common solution is to consult x relevant experts in the health care ﬁeld. The opinion of each expert e can be represented by a k × m matrix where each 12 123 4160 B. Alshouha et al. Fig. 4 The linguistic term set used in this study Steps 5 and 6 can be executed in parallel, as shown in Fig. 3. Fig. 5 DR detection algorithm 2 https://pypi.org/project/vaderSentiment/ 3.1.5 Step 5. Calculating the weights of the design requirements (“HOWs”) To ﬁnalize the HOQ, it is necessary to calculate the weights of the DRs as WDR = {w j \| j = 1, . . . , m}. This is done by averaging the aggregated weighted relationship scores between the C Rs and DRs through the values of the relationship_matrix = {ri j \| i = 1, . . . , k and j = 1, . . . , m} (see Section 3.1.5) and the vector WC R (see Section 3.1.2): Although this method provides helpful information semi- automatically, i.e., the clusters are generated automatically, a user must still classify the clusters manually into the cor- responding DRs; nevertheless, it is possible to ﬁnd other alternatives that also involve user interaction. For example, Wu et al. provided a BERT-based model that automatically detects the features or design requirements from opinions; the major drawback of this approach is that it is necessary to w j = 1 m ⊗[(r1 j ⊗wC R1) ⊕. . . ⊕(rmj ⊗wC Rm)] As a result, the vector WDR represents the importance of each hospital design requirement. The values of the vector are again triangular fuzzy numbers. Fig. 5 DR detection algorithm 1 3 m Fig. 5 DR detection algorithm 12 3 An integrated decision framework for evaluating and recommending health care services 4161 Table 1 Example characteristic terms for each DR DR Criteria Terms Staff Staff, doctor, nurse, physician, receptionist, team, midwife, counselor, consultant, dentists, worker, ... Care Care, treatment, patient, pain, empathy, compassion, recovery, chemotherapy, family, needles, home, ... Efﬁciency Effective, efﬁcient, accurate, optimized, streamlined, performance, calibration, track, operation, scan, tech- nology, check, able, ... Time Time, wait, response, appointment, replay, answer, hours, timely, timescales, timeliness, annual, delays, punctuality, ... Facility Room, parking, building, outdoor, ward, suite, clinic, toilet, bedrooms, equipment, lifts, clinic, hospital, ... Transparency communication, disclosure, clarity, openness, honesty, information, review, procedures, question, contact, informed, referred, ... Conﬁdence Conﬁdent, trust, belief, secure, concern, disappointed, incredulous, reliability, preventative, knowledge, skills, experience, safe, ... Service call, service, read, admission, appointment, cancel, brilliant service, welcomed, food, water,... 4161 An integrated decision framework for evaluating and recommending health care services Staff, doctor, nurse, physician, receptionist, team, midwife, counselor, consultant, dentists, worker, ... call, service, read, admission, appointment, cancel, brilliant service, welcomed, food, water,... 3.1.7 Step 7. Fuzzy suitability index calculation generate a large set of instances that are manually labeled to be able to tune the BERT model to detect them [50]. Finally, the fuzzy suitability index (FSI) for each hospital is calculated to determine the extent to which each hospital sat- isﬁes the requirements [51]. This method is an aggregation mechanism suitable for dealing with TFNs. In particular, the FSI index of a hospital is represented as a TFN, which is determined by multiplying the previously calculated polarity score Phospital = {PDR j \| j = 1, . . . , m} by the correspond- ing weights WDR = {w1, . . . , wm} (see Section 3.1.5) for each DR criterion: Phase B. Design requirement polarity detection Having successfully identiﬁed the eight DRs, it is crucial to calculate the sentiment orientation conveyed by the reviews regarding each DR. Understanding the polarity for each individual cri- terion will help us understand the overall opinion about a hospital. To compute these polarities, the input of the algorithm is the set of sentences grouped according to each DR, enabling the calculation of the sentiment degree of the opinions toward that particular criterion. In this case, the Valence Aware Dictionary and sEntiment Reasoner (VADER2) is the tool selected to compute the polarity of every sentence. The polarity PDR j regarding a speciﬁc aspect DR j of hospital is calculated as the average of the polarity of all sentences S according to VADER yielding the compound sentiment value for each opinion. Mathematically, the overall polarity for each DR j of a hospital is computed as follows: FSI = 1 m ⊗(PDR1 ⊗w1) ⊕. . . ⊕(PDRm ⊗wm) This ﬁnal value represents how well a hospital l meets the expectations of a user, represented by a fuzzy set. As the goal Fig. 6 The mechanism for calculating the polarity for each DR Phospital = {PDR j \| j = 1, . . . , m} PDR j = 1 \| SDR j \| si∈SDR j V ADE R(si) With the goal of modeling the polarity value of each aspect i, a fuzzy label set can be used. This fuzzy label set includes labels represented by a TFN with appropriate membership degrees (see Fig. 4). Figure 6 illustrates the mechanism for computing the polarity for each aspect DR represented by a TFN. Fig. 6 The mechanism for calculating the polarity for each DR Fig. 3.2 Summary of mathematical notations and operations of the proposal Evaluation of a hospital regarding the DRs Following the algorithm described in [49], the evaluation/polarity of a hos- pital regarding each DR is calculated as a vector Phospital = {PDR j \| j = 1, . . . , m}. The previous subsections describe the entire process to rank the hospitals. Next, to synthesize all steps, the most important mathematical deﬁnitions and operations to be considered are summarized. First, from the opinions of the hospital available in the dataset, all sentences si talking about the different DRs are detected by an LDA-based topic modeling algorithm, and then, the polarity is calculated as the aggregation of the individual polarities of each sentence PDR j = 1 \|SDR j \| si∈SDR j V ADE R(si). To calculate the individual polarities, the well-known tool VADER has been used. Customerrequirements Thek CRs(C R ={C R1, C R2, · · · , C Rk}) are determined by experts. Overall, in most of the available studies, six customer requirements are used when assessing the quality of a hospital. These ones (tangible, reliability, responsiveness assurance, empathy and profes- sionalism) are used here as explained in Section 3.1.1, and consequently, k = 6. As a real user would usually rate a hospital using a lin- guistic labels and not crip values, then a fuzziﬁcation method must be selected to codify the polarity value according to a previously selected linguistic term set. Thus, PDR j is ﬁnally represented as a set of triangular fuzzy sets. Relative importance of the CRs The user must determine what CRs are the most important for her/him. To do so, the user manually assigns values in the range [0, 1] to each CR in the vector WC R = {w1, · · · , wk}. The sum of all weights must be 1. Final score a hospital The ﬁnal score given to a hospital considers the opinion over each DR (Phospital = {PDR j \| Final score a hospital The ﬁnal score given to a hospital considers the opinion over each DR (Phospital = {PDR j \| j = 1, . . . , m}) and the corresponding importance of each DR (WDR = {w1, . . . , wm}). Considering both vectors [51], the FSI computes the ﬁnal score of the hospital represented by a TFN (< F Nα, F Nβ, F Nγ >): FSI = 1 m ⊗(PDR1 ⊗ w1) ⊕. F Nα + 2F Nβ + F Nγ 4 Importance of the DRs As explained in Section 3.1.5, the importance of the DRs (WDR = {w1, w2, · · · , wm}) can be computed averaging the relationship_matrix = {ri j \| i = 1, . . . , k and j = 1, . . . , m} and the vector WC R following the formula: w j = 1 m ⊗[(r1 j ⊗wC R1)⊕· · ·⊕(rmj ⊗wC Rm)]. These discrete values can then be ranked to set the ﬁnal order of the hospitals according to the user’s preferences. 3.2 Summary of mathematical notations and operations of the proposal . . ⊕(PDRm ⊗wm) Design requirements They are represented by a m-dimensi- onal vector DR = {DR1, DR2, DR3, · · · , DRm}. Man- ually analyzing the used dataset, the found requirements are the same eight that can be usually found in the litera- ture. These ones (staff, care, facilities, conﬁdence, services, timeliness, efﬁciency and transparency) are described in Section 3.1.2, and consequently, m = 8. Final ranking Once the FSI of each hospital (H = {H1, H2, ....}) to be assessed is calculated, these values can be defuzzied by the formula: F Nα+2F Nβ+F Nγ 4 . These values allow ranking the hospitals. The highest value indicates the most recommendable hospital and lowest the least recom- mendable one. Experts It is usual to resort to a set of experts E = {e1, e2, · · · , ex} to assess the relationship degrees between the CRs and the DRs. In most of the studies, the number of experts is usually three (x = 3). Linguistic term set The experts express the relationship degrees by linguistic labels such as ‘very good’ or ‘quite bad’; therefore, a linguistic term set like the one depicted in Fig. 4 is necessary to be deﬁned to model the expert’s opinions. 3.1.7 Step 7. Fuzzy suitability index calculation 6 The mechanism for calculating the polarity for each DR 12 3 4162 B. Alshouha et al. DR in a k × m matrix using the previous selected linguistic term set: matrixe = {ri j \| i = 1, . . . , k and j = 1, . . . , m}. DR in a k × m matrix using the previous selected linguistic term set: matrixe = {ri j \| i = 1, . . . , k and j = 1, . . . , m}. DR in a k × m matrix using the previous selected linguistic term set: matrixe = {ri j \| i = 1, . . . , k and j = 1, . . . , m}. of the application is to recommend the most suitable hospital in a set of hospitals H = {H1, H2, ....}, the order of this set depends on the FSI of each hospital. As the fuzzy values are represented by triangular values < F Nα, F Nβ, F Nγ >, to compare the FSIs of all hospitals, these values are discretized by the following formula: Relationship matrix The ﬁnal opinion over the relation- ship scores between the CRs and the DRs depends on all the experts’ opinions. Hence, all the score matrices are aggregated into a ﬁnal matrix: relationship_matrix = matrix1 ⊕matrix2 ⊕. . . ⊕matrixx. 4.2 Case study 1 The ﬁrst case study uses opinions provided by previous patients on Careopinion from the 7 hospitals, as shown in Table 2. As mentioned previously, three scenarios are assessed. For simplicity, the case of a balanced user, i.e., one whose prefer- ences arerepresentedbythevector WC R1 ={0.16666,0.16666, 0.16666, 0.16666, 0.16666, 0.16666} (see Section 3.1.2), it explained next step by step, and the results of the remaining of cases are simply given. for every case study, three types of users are represented depending on their preferences toward the CRs. These prefer- ences are modeled by the vectors explained in Section 3.1.2. To assess the quality of the results of the proposed model, it is necessary to compare the generated ranking against the gold standard or ideal ranking. To compute the ideal rank- ing, the assessments of the hospitals from Careopinion were used, as explained in Section 5.1 of [49]. These assessments are not obtained from the textual opinions but from the key- words provided by the users in the subsections of the opinions (“how did you feel”, “what’s good”, and “what could be improved”), which are interpreted as direct user evaluations of the different hospital aspects. Apart from this ranking, the model for ranking hospitals proposed in [49], which is based on the textual information opinions, was also implemented for comparison. Using the dataset shown in Table 2, ﬁrst, the CRs or “WHATs” were identiﬁed through the analysis of the rel- evant available literature (see Section 3.1.1). Second, the relative importance of each CR was determined using three scenarios (see Section 3.1.2). Third, the DRs or “HOWs” were identiﬁed by conducting a comprehensive analysis of the relevant literature, a manual analysis of patient reviews on the CAREOPINION website and an evaluation of user experiences (see Section 3.1.3). Fourth, the CR-DR relation- ship scores were determined by gathering multiple expert perspectives as explained in Section 3.1.4. A graphical rep- resentation of the HOQ model showing the results after the ﬁrst four steps is presented in Fig. 7. The column “relative importance for the CR” represents the weights for scenario 1 (see Section 3.1.2) and the remaining cells represent the assessments of the three experts conveyed in linguistic terms. For instance, the cell “tangible staff” was assessed as high, high, and medium by the three experts. CR-DR relationship score matrix Each expert e expresses the relationship score between the i-th CR with respect to the j-th To test the performance of the model, two case studies are pro- posed for assessing two different sets of hospitals. Furthermore, CR-DR relationship score matrix Each expert e expresses the relationship score between the i-th CR with respect to the j-th 3 An integrated decision framework for evaluating and recommending health care services 4163 Table 2 List of hospitals and numbers of retrieved opinions for case study 1 Hn Hospital name # Opinions H1 East Surrey Hospital 1965 H2 Leicester Royal Inﬁrmary Hospital 1857 H3 Lincoln County Hospital 2152 H4 Lister Hospital 1797 H5 Medway Maritime Hospital 1855 H6 Royal Cornwall Hospital 1809 H7 University Hospital Wishaw 1734 where X = {x1, x2, · · · , xn} and Y = {y1, y2, · · · , yn} are the two compared rankings. This value can be normalized to the range [0, 1] by dividing by the maximum value among the items of each ranking. A score of 0 indicates that the two lists are identical, whereas a score of 1 indicates that the rankings are completely different. Fig. 7 The HOQ model results after the ﬁrst four steps 4.2 Case study 1 Mathematically, these To measure how close every proposal is to the ideal base- line, a ranking comparison metric, Spearman’s footrule, is used. This measure is based on the differences between the orders of the n items of the rankings being compared. Spearman(X, Y) = n i=1 \| rank(xi) −rank(yi) \| (1) (1) Fig. 7 The HOQ model results after the ﬁrst four steps 1 3 Fig. 7 The HOQ model results after the ﬁrst four steps . 7 The HOQ model results er the ﬁrst four steps 123 12 3 B. Alshouha et al. 4164 Fig. 8 The fuzzy-HOQ Fig. 8 The fuzzy-HOQ Fig. 8 The fuzzy-HOQ labels are processed as the associated fuzzy sets, shown in Fig. 4. Depending on the experts’ assessments, other fuzzy sets could be used. opinion; for that reason, these values have been fuzziﬁed as the closest label associated with a scheme of fuzzy sets such as the one seen Fig. 4 for the variable polarity. Each label is able to capture the variations in sentiments and ensure a com- prehensive and interpretable representation. The linguistic values obtained by computing the polarities of all aspects for each DR criterion of each hospital are displayed in Table 4. Finally, the HOQ was completed by calculating the DR weights by averaging the weighted relationship values between the CRs and DRs (see Section 3.1.5). This pro- cess utilized both the relationship scores and the weights assigned to the CRs. Additionally, to represent the relation- ship between the CRs and the DRs, the fuzzy sets depicted in Fig. 4 were employed. The result of this process is the fuzzy- HOQ model shown in Fig. 8, which shows the outcomes after the ﬁve steps of the methodology. In this case, the cells rep- resent the ﬁnal values of the calculated triangular fuzzy sets, with every triangle having the form < F Nα, F Nβ, F Nγ >. For instance, the value for the cell “staff professionalism” is the fuzzy set “VVH” from Fig. 4. The last step (see Section 3.1.7) consists of calculating the FSI of each hospital using the outputs of steps 5 and 6. These values are useful to determine the ﬁnal hospital rec- ommendations for the user. Table 5 shows the FSIs of each hospital and the rankings. The highest FSI value indicates the most recommendable hospital. 4.2 Case study 1 In addition to the results for scenario 1, the table shows the ﬁnal rankings for the other 2 scenarios when “reliability” and “assurance” are priorities for the user. The most relevant hospital is highlighted in bold for each case. The next phase (see Section 3.1.6) involved computing the ratings of the hospitals with respect to the DRs or “HOWs”. The topic modeling technique LDA [44] was used to detect the DR aspects from the opinions, and VADER was used to calculate the polarity for each hospital. Table 3 presents the polarity results which include the average compound value per DR criterion and hospital. The polarity values are expressed in the range [0, 1], where 0 is the most negative value, 0.5 is a neutral value and 1 is the most positive value. By analyzing the results from Table 5 and Fig. 9, it is evident that consumers’ preferences play a vital role in deter- mining the overall ranking. In the ﬁrst scenario, where all aspects were given the same weight, H4 was ranked ﬁrst, followed by H6 and H3. However, when the “Assurance” dimension was given the highest weight in the second sce- nario, H6 was ranked ﬁrst, followed by H4 and H3. In the third scenario, “Reliability” was the most important aspect and H4 was the most highly ranked hospital, followed by H6 and H5, whereas H3 was ranked fourth. 4.2 Case study 1 Overall, analyzing As these polarity values are numeric, they do not represent the real linguistic expression a user would use to convey their Table 3 Polarity results for the DR criteria Hospitals Staff Care Facility Conﬁdence Service Efﬁciency Transparency Timeliness H1 0.770026 0.651134 0.581142 0.570555 0.776956 0.651494 0.531169 0.550101 H2 0.61202 0.779356 0.409658 0.762268 0.582002 0.534863 0.495821 0.521625 H3 0.833429 0.757223 0.512768 0.622173 0.657088 0.631936 0.679682 0.505246 H4 0.749685 0.768344 0.591939 0.633991 0.827404 0.643253 0.670783 0.613963 H5 0.604432 0.605469 0.429091 0.584503 0.83484 0.588043 0.504514 0.593932 H6 0.760179 0.788246 0.574391 0.648023 0.797549 0.654177 0.691511 0.580942 H7 0.595662 0.6312679 0.455611 0.592289 0.751089 0.550567 0.480041 0.603064 12 An integrated decision framework for evaluating and recommending health care services An integrated decision framework for evaluating and recommending health care services 4165 An integrated decision framework for evaluating and recommending health care services 4165 Table 4 Polarity values for the DRs of each hospital DRs Staff Care Facility Conﬁdence Service Efﬁciency Transparency Timeliness H1 H MH MH MH H MH M M H2 MH H ML H MH M M M H3 VH H M MH MH MH MH M H4 H H MH MH VH MH MH MH H5 MH MH ML MH VH MH M MH H6 H H MH MH H MH H MH H7 MH MH M MH H M M MH Table 5 Hospitals ranking Hospitals H1 H2 H3 H4 H5 H6 H7 FSI First scenario Final score 0.06356535 0.05927336 0.06492133 0.06992476 0.06240125 0.06950774 0.06062301 Ranking H4, H6, H3, H1, H5, H7, H2 FSI Second scenario Final score 0.05885712 0.05736664 0.06085994 0.06439214 0.05722710 0.06451405 0.05604174 Ranking H6, H4, H3, H1, H2, H5, H7 FSI Third scenario Final score 0.06107896 0.05769406 0.06185880 0.06818467 0.06226475 0.06759087 0.05967103 Ranking H4, H6, H5, H3, H1, H7, H2 Table 5 Hospitals ranking 12 123 B. Alshouha et al. 4166 Table 7 Ranking of hospitals in case study 2 Scenarios Ranking First scenario H4, H7, H1, H6, H2, H3, H5 Second scenario H7, H4, H6, H1, H3, H2, H5 Third Scenario H4, H7, H1, H6, H3, H2, H5 Table 6 List of hospitals and number of retrieved opinions for case study 2 Hn Hospital name # Opinions H1 Kings Mill Hospital 1726 H2 Worcestershire Acute Hospital 1701 H3 Tameside General Hospital 1443 H4 Crosshouse Hospital 1635 H5 Queens Hospital (Romford) 1788 H6 Victoria Hospital 1087 H7 Queen Margaret Hospital 1000 hospitals. 4.2 Case study 1 In the ﬁrst scenario, where equal weights were assigned to all aspects, H4 had the top position, closely fol- lowed by H7 and H1. In the second scenario, where the “assurance” dimension was given the highest weight, H7 achieved the top position, followed by H4 and H6. In the third scenario, where “reliability” was prioritized over the other aspects, H4 obtained the top rank, followed by H7 and H1. the opinions, hospitals H4 and H6 are the best rated; con- sequently, they always appear in the ﬁrst positions because the best polarity values are obtained for each DR in general (see Table 3). Hospitals H2, H7 and H5 obtained the lowest polarityscoresfortheDRs;forthatreason,theirpositionscan change slightly depending on user preferences; nonetheless, they are still the worst-scored hospitals. Notably, hospitals H4 and H7 were consistently ranked as top-rated establishments. In contrast, hospitals H2, H3, and H5, assigned low ranks, showed positional variations due to user preferences, consistently maintaining their status as the lowest-ranked hospitals. Fig. 10 FSI values of the hospitals in case study 2 4.4 Comparative analysis In the second scenario, there are a few more differences between the rank- ings; nonetheless, the proposed method is closer to the ideal one. This fact can be corroborated by calculating Spearman’s footrule metric. In scenario 1 case study 1 our method is able to rank all hospitals as expected by the ideal ranking except hospital 1 and 3 which exchanged their positions in the ﬁnal rank. The same situation happens in [49]; nonetheless, the positions of hospitals 7 and 5 are also exchanged with respect to the ideal ranking. In the rest of scenarios similar situations take place, some hospital positions are exchanged by only one position with respect to the ideal rank. Nevertheless, there are two hospitals that [49] struggles to rank properly, H3 and H5, whereas our proposal places them closer to their ideal positions. Looking at the relative weights of HOQ in scenario 2, care/empathy and conﬁdence are the most relevant areas to be considered, whereas staff and service the least relevant. Regarding scenario 3, timeliness and efﬁciency are the most important aspects to be considered in the HOQ, whereas staff and facilities, the least relevant ones. As shown in Table 9, in both case studies, the proposed method consistently demonstrated superior performance, achieving lower average Spearman’s footrule values than the method in [49]. For instance, in the ﬁrst case study, the pro- posed method attained an average footrule of 0.17, while the method in [49] had a higher average of 0.28. Similarly, in case study 2, the average Spearman’s footrule value of the proposed method was 0.14, surpassing the average of the other method (0.25). These results highlight that the pro- posed model performed very similarly to the ideal ranking in both case studies, proving its effectiveness in this manner. Todeeper analyzetheresults, it is necessarytofocus onthe proportion of positive and negatives sentences per aspect and their importance. Looking at the extremes of the rankings, in A similar situation occurs in scenario 2, overall, the best hospitals are H7 and H4 and the worst ones H5 and H2 in all scenarios, because the ratio of positive over negative sen- tences is 2.4 and 2.26 for H7 and H4, respectively, and 1,68, 1.7 and 1,71, for H5, H3 and H2, respectively. 4.4 Comparative analysis 10 FSI values of the hospitals in case study 2 12 123 An integrated decision framework for evaluating and recommending health care services 4167 An integrated decision framework for evaluating and recommending health care services An integrated decision framework for evaluating and recommending health care services 4167 Table 8 Results for the different scenarios Scenario Ideal ranking Proposed model Other model [49] Case study 1 #1 H4, H6, H1, H3, H5, H7, H2 H4, H6, H3, H1, H5, H7, H2 H4, H6, H3, H1, H7, H5, H2 #2 H6, H3, H4, H2, H1, H5, H7 H6, H4, H3, H1, H2, H5, H7 H6, H4, H1, H3, H5, H2, H7 #3 H4, H5, H6, H1, H3, H2, H7 H4, H6, H5, H3, H1, H7, H2 H4, H6, H3, H1, H5, H7, H2 Case study 2 #1 H4, H1, H7, H6, H2, H5, H3 H4, H7, H1, H6, H2, H3, H5 H4, H7, H1, H6, H3, H2, H5 #2 H4, H7, H6, H1, H3, H2, H5 H7, H4, H6, H1, H3, H2, H5 H7, H4, H1, H3, H6, H5, H2 #3 H4, H7, H1, H6, H2, H3, H5 H4, H7, H1, H6, H3, H2, H5 H4, H7, H6, H1, H3, H2, H5 study case 1 the best hospitals are H4 and H6 according to the ideal ranking for most scenarios, and the worst for all scenar- ios arehospital H2andH7. Similar results areobtainedbyour approach and the one in [49] because H4 and H6 have by far the major ratio of positive opinions over negatives, 2.34 and 2.43, respectively, whereas H2 and H7 have the minor ratio, 1.49 and 1.47, respectively. Nonetheless, the ratio for the other hospital is more similar and then, the weights assigned by the house of quality play an important role to correctly place the hospital in the ﬁnal ranking. accuracy of the ranking process. While [49] does not delve into these aspects, our approach systematically incorporates them, contributing to a more robust and insightful evaluation. Therefore, the proposed method was used in the same context as described in [49], yielding the rankings shown in Table 8 for case studies 1 and 2. In the ﬁrst case study, looking at the different scenar- ios, the results are practically the same, varying by only one position at most between the ideal rankings of the proposed method and the other comparison method. 4.4 Comparative analysis In the second case study, this fuzzy QFD-based approach was extended to seven additional hospitals to validate its sta- bility and adaptability for different health care settings. The patient opinions from Careopinion for the hospitals presented in Table 6 again served as the basis for implementing the pro- posed approach. As mentioned previously, to test the effectiveness of the proposal, a gold standard and other approaches for rank- ing hospitals were implemented [49]. The proposed method demonstrated its effectiveness and superiority over other approaches when evaluating the service quality of hospitals. The most notable difference with respect to [49] lies in the consideration of CRs in the ranking process. Unlike [49], the proposed approach integrates CRs, considering their impact on the overall ranking of hospitals. This additional dimension provides a more patient-centric evaluation and corresponds with user perspectives. Moreover, the relationship between CRs and DRs is a crucial factor in our method, enhancing the This implementation not only tested the stability of the methodology but also conﬁrmed its effectiveness in different hospitals. Table 7 shows the ﬁnal ranking of the hospitals according to this methodology under the different scenarios. A comprehensive analysis of the results presented in Table 7 and Fig. 10 yielded signiﬁcant ﬁndings regarding the effect of customer preferences on the overall ranking of Fig. 10 FSI values of the hospitals in case study 2 Fig. 4.5 Discussion The proposed methodology distinctively involves patients or users directly in the assessment process, eliminating the reliance on expert evaluations, in contrast to the methods of other studies [30, 52–57] that primarily rely on experts to assess service quality and recommend products or suppliers. Instead, this approach strategically avoids these challenges by automating the evaluation process so that it is more adaptable and reﬂective of real-world users’ perspectives. Considering this, if it is necessary to evaluate 14 hospitals using traditional expert-based methods, and each hospital needs 3 experts for the evaluation, a large team of 42 spe- cialized experts in the medical ﬁeld would be needed, each providing nonrandom evaluations. This poses challenges in terms of reliability, consistency, and substantial resource requirements. The second case study serves as a proof that the proposed method is stable, adaptable, and reliable. The consistent rank- ings across different scenarios and additional hospitals show that the proposed method is stable and that the results are reliable. The method’s adaptability is shown by its ability to perform well in different health care settings. Second, user opinions have been found to be powerful tools for recommending speciﬁc hospitals to others based on their DRs. The presented fuzzy approach mitigates uncer- tainty in these opinions, yields more accurate information, and enables better-informed decisions regarding the most suitable hospitals for individual patients or users. It also allows hospital managers to gain deeper insights into the needs and expectations of their patients or users, facilitating targeted improvements in their services and facilities. Moreover, in contrast to the methods of other studies [58– 60], the method proposed here uses an alternative means of attribute extraction. While classic methods rely on the team knowledge and experience to extract the criteria for the HOQ, the proposed method incorporates a classiﬁcation technique that automatically identiﬁes the criteria of HOQ within the QFD, effectively capturing the criteria speciﬁc to the hospital context. This automation not only improves the efﬁciency of the process but also reduces the costs associated with human interaction. Overall, this approach represents an alternative to traditional expert-based evaluations, providing a practical and effective method that is easily aligned with the contem- porary shift toward patient-centered health care assessments that take advantage of internet technologies. In conclusion, the integration of the HOQ matrix with user opinions provides a more accurate and reliable analysis of hospital DRs. 4.4 Comparative analysis In this second case study, our approach exchanges just one position between several hospitals regarding the ideal ranking in all scenarios; nevertheless, H3 and H6 are incorrectly placed several posi- tions by the other algorithm [49]. For example, in scenario 3, the selection of the appropriate weights for H3 is vital. Our approach assigns more importance to weak aspects such as timeliness whose ratio is 1.1 for H3, i.e, there is barely the same number of positive sentences and negatives. This fact Table 9 Comparison in terms of Spearman’s footrule metric Scenario #1 #2 #3 AVG Case study 1 Propposed model 0.083333 0.166667 0.25 0.166667 Other model [49] 0.166667 0.333333 0.333333 0.277778 Case study 2 Propposed model 0.166667 0.166667 0.083333 0.138889 Other model [49] 0.25 0.333333 0.166667 0.25 123 12 3 3 4168 B. Alshouha et al. is key for this hospital, because the second one lowest ratio is 1.49 for H5. Therefore, the calculation of the correct weight for the HOQ highly inﬂuences and determine the results of the approach here presented. on the ranking of hospitals. Hence, health care providers should consider their customers’ preferences when develop- ing strategies to improve their service quality. Furthermore, the results of this study can help health care providers identify areas for improvement to allocate resources more effectively to meet their customers’ expectations. In doing so, health care providers can enhance their competitiveness, increase customer satisfaction and loyalty, and improve the quality of health care services provided. 4.5 Discussion considered instead of just considering feelings only in com- puting the polarity of opinions [67–69]. programming interface (API) provided by Careopinion; therefore, privacy does not seem to be a major issue for patients. The proposed methodology does not use any per- sonal data, and the information used can be considered perfectly anonymous because the data are aggregated, rather than managed individually. Consequently, it is impossible to trace patient opinions and ﬁnd personal details about the patients. Author Contributions Bashar Alshouha: Investigation, Conceptual- ization, Software, Writing - original draft. Jesus Serrano-Guerrero: Conceptualization, Supervision, Investigation, Data Analysis, Writing - original draft. Francisco Chiclana: Conceptualization, Supervision, Data Analysis, Writing - review & editing. Francisco P. Romero: Inves- tigation, Methodology, Writing - review & editing, Funding acquisition. Jose A. Olivas: Supervision, Conceptualization, Funding acquisition Funding Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This work has been supported by FEDER and the State Research Agency (AEI) of the Spanish Ministry of Economy and Competition under grant SAFER: PID2019-104735RB- C42 (AEI/FEDER, UE), the General Subdirection for Gambling Regulation of the Spanish Consumption Ministry under the grant Detec- EMO: SUBV23/00010 and the project TED2021-130682B-100 funded by MCIN/AEI and by the European Union NextGenerationEU/PRTR. Declarations Ethicalandinformedconsentfordataused All collected data are freely and publicly available on Careopinion website. Neither personal nor private information is involved in this study. Competing Interests The authors declare that there is no conﬂict of interest. The primary contribution lies in the use of user opinions, integrated within the HOQ model, to rank hospitals and guide health care providers to understand what speciﬁc services or requirements need improvement. Unlike the traditional QFD model, which relies on decision-makers to rate hospitals or health care providers, this approach provides a more direct and patient-centered perspective on hospital performance. The ﬁndings suggest that incorporating patient or user opin- ions in the ranking process is useful for understanding patient satisfaction and creating a more patient-centered health care system. Thus, this study contributes to the growing literature on the use of HOQ in improving health care quality improve- ment and highlights the importance of incorporating patient or user opinions in the ranking process. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adap- tation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indi- cate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitteduse,youwillneedtoobtainpermissiondirectlyfromthecopy- right holder. To view a copy of this licence, visit http://creativecomm ons.org/licenses/by/4.0/. 5 Conclusions and future work This study describes a fuzzy QFD approach that integrates patient opinions to rank hospitals; this approach offers sig- niﬁcant advantages over traditional QFD methods that rely solely on decision-makers. The paper initially presents the basic concepts of the QFD model and proposes the HOQ method to address the “what” and “how” questions. In situations where information is incomplete, imprecise, or subjective, a fuzzy decision-making environment can be an interesting tool. For that reason, a fuzzy QFD approach is proposed to represent the polarity of user opinions and to calculate the relationship between CRs and DRs. Then, a systematic procedure using the fuzzy-QFD method to rank the hospitals is proposed. Data Availability Data will be made available on reasonable request 4.5 Discussion Implementing this approach in the QFD pro- cess has signiﬁcant implications for enhancing health care service quality and meeting the demands of patients and users. Therefore, this approach can help improve hospital facilities and services and ultimately, enhance patient satis- faction. Among the implementation challenges, it is necessary to highlight that this proposal is costly in terms of development the ﬁrst time it is implemented because the ﬁrst steps of the methodologydonotneedtobeimplementedagainifthesetof hospitals to be assessed is different. Therefore, the bottleneck of the system is step 6 which depends on the number of opinions available per hospital. Furthermore, several practical contributions useful for patients, health care providers, and managers can be derived from this study. First, the results of this study demonstrate the effectiveness of the integrated HOQ matrix and patients’ opinions in detecting the most relevant DRs for hospitals. By analyzing the opinions of patients or users, it is possible to identify the hospital aspects that are most relevant to them. Regarding the implementation of the proposal in a real environment, the major challenge is to obtain a feedback plat- form such as Careopinion, where the patients truly want to collaborate to provide reliable information. Furthermore, the proposal is modeled considering the information provided by Careopinion; nevertheless, the primary necessary infor- mation is patient feedback, which is assumed to be available on every similar platform. The other information provided by sections such as “how did you feel” or “what’s good” is used only for evaluation purposes and to manually corrobo- rate some of the data obtained automatically in some steps of the methodology. Overall, the results of this analysis can provide valuable insights for health care providers and policy-makers, helping them focus on speciﬁc aspects of service quality that are the most important to their customers. It is also essential to rec- ognize that customer opinions and preferences can change over time. Therefore, health care providers should regularly assess their customers’ changing opinions and preferences and adjust their service quality accordingly. Moreover, these scenarios demonstrate that the weights and scores given to each aspect of service quality have a crucial impact In terms of privacy, Careopinion users accept the terms of sharing their data with other users through the application 123 An integrated decision framework for evaluating and recommending health care services 4169 considered instead of just considering feelings only in com- puting the polarity of opinions [67–69]. References There are several potential avenues for further research building upon the ﬁndings of this study. First, incorporating public and personal preferences based on patient opinions, rather than relying only on personal preferences, could pro- vide a more patient-centered approach to ranking hospitals. This could involve gathering and integrating feedback from a larger sample of patients to capture a broader range of perspectives and opinions. 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Olivas joseangel.olivas@uclm.es 1 Department of Information Technologies and Systems, University of Castilla-La Mancha, Paseo de la Universidad 4, Ciudad Real 13071, Spain 2 Institute of Artiﬁcial Intelligence (IAI), School of Computer Science and Informatics, De Montfort University, Leicester, The Gateway, Leicester LE1 9BH, UK 12 123
https://openalex.org/W1992194520	https://mechanika.ktu.lt/index.php/Mech/article/download/3622/2322	English	null	OPTIMIZED FUZZY LOGIC MODEL FOR PREDICTING SELF-COMPACTING CONCRETE SHRINKAGE	Mechanika	2,013	cc-by	5,028	1. Introduction concrete shrinkage strain in a faster and less expensive way when compared to experimental measurements. Prediction models are based either on analytical or empirical ap- proaches, although the later is used most frequently be- cause of its simplicity. According to McDonald and Roper [5], complex prediction models do not necessarily lead to better prediction than simple ones. Concrete shrinkage is defined as the decrease in concrete volume with time. This volume decrease does not depend on external stress and it is not completely reversi- ble. The shrinkage is associated with a series of factors, such as chemical reactions, gradient in temperature, and loss of water. Each one of these factors leads to different types of shrinkage, such as autogenous, plastic, drying, and thermal shrinkage [1, 2]. One of several existing shrinkage prediction mod- els that is frequently used is the EN1992 model, considered by the Eurocode. This model consists of a combination of CEB FIP 1990 and CEB MC90-99 prediction models and its equations are described in [6]. The EN1992 model is a standard and its prediction formulas are routinely used by the industry’s experts for any concrete, including SCC which is not explicitly excluded in the Eurocode. Indeed, the European guidelines for SCC [7], state that the values and formulas given in the Eurocode for normal concrete are still valid in the case of SCC. The occurrence of shrinkage leads to the devel- opment of internal tension stresses that can result in con- crete cracking if the developed internal tension stresses surpass concrete tensile strength. Cracks in concrete create pathways for the easier access of aggressive agents that can contribute to the reduction in concrete’s structure durabil- ity and service life. Moreover, the strain resulting from excessive shrinkage may reduce bounding tension and in- crease deflection in asymmetrically reinforced concrete structures [2]. Although prediction models are regularly used, the shrinkage curve obtained from the models does not necessarily match experimental measurements. To verify that, experimental data taken from different authors [8-10], were compared with those predicted by the EN1992 model. For these reasons, a reliable prediction of concrete shrinkage strain is an important factor in the entire design- ing process. Predicting shrinkage, especially during the period of construction, allows early countermeasures to be taken, e.g., premature loading or prestressing to compen- sate for the negative shrinkage effect. Optimized fuzzy logic model for predicting self-compacting concrete shrinkage W. R. L. da Silva, P. Štemberk* Czech Technical University in Prague, Thákurova 7, 166 29, Prague, Czech Republic, E-mail: wilsonecv@gmail.com Czech Technical University in Prague, Thákurova 7, 166 29, Prague, Czech Republic, E-mail: stemberk@fsv.cvut.cz htt //d d i /10 5755/j01 h 19 1 3622 W. R. L. da Silva, P. Štemberk** Czech Technical University in Prague, Thákurova 7, 166 29, Prague, Czech Republic, E-mail: wilsonecv@gmail.com Czech Technical University in Prague, Thákurova 7, 166 29, Prague, Czech Republic, E-mail: stemberk@fsv.cvut.cz http://dx.doi.org/10.5755/j01.mech.19.1.3622 http://dx.doi.org/10.5755/j01.mech.19.1.3622 67 67 ISSN 1392 - 1207. MECHANIKA. 2013 Volume 19(1): 67-72 ISSN 1392 - 1207. MECHANIKA. 2013 Volume 19(1): 67-72 1. Introduction The scatter plot of the experimental- ly measured and predicted strain values for the analyzed data is illustrated in Fig. 1. determination of the number and shape of the fuzzy sets. Commonly, there are mk fuzzy rules, where m and k are the number of fuzzy sets and input variables, respectively. In the classical fuzzy logic approach, the number of fuzzy rules can be reduced by the user’s experience, and to sim- plify calculations the shape of the fuzzy sets is usually lin- ear. However, when this approach is implemented to mod- el the behavior of non-linear materials, the final result is a rather rough shaped piecewise curve. Using the classical approach is also feasible for material modeling; however, a larger number of linear fuzzy sets is required to obtain smoothed curves. This leads to a longer data collection time and high computational cost. Thus, to improve the modeling process a modified approach that includes an evolutionary computing method is proposed. From Fig. 1 a considerable scatter can be seen, therefore showing the relative error as well as an underes- timation trend of EN1992 in predicting shrinkage strains. Hence, it can be stated that the reliability of EN1992 model is open to discussion and improvements appear to be re- quired. Fig. 1 Comparison of measured and predicted shrinkage strain for SCC using the EN1992 model 0 100 200 300 400 500 600 700 800 0 100 200 300 400 500 600 700 800 EN1992 sh-measured , microstrain sh-predicted , microstrain 0 100 200 300 400 500 600 700 800 0 100 200 300 400 500 600 700 800 EN1992 sh-measured , microstrain sh-predicted , microstrain Evolutionary computing involves robust optimi- zation methods that can be generally applied without re- course to domain-specific heuristics. These methods oper- ate on a population of potential solutions and apply the principle of survival of the fittest to produce successively better approximations for a solution [13]. Among several evolutionary computing methods, genetic algorithms (GA) have been successfully applied in numerical optimization in civil engineering, e.g. [14]. They consist of adaptive heuristic search algorithms based on the principles of Darwin’s theory of natural selection. They represent an intelligent exploitation of a random search that uses historical information to guide the search into the region of better performance, within a defined search space. 1. Introduction As a result, accurate shrinkage prediction helps reduce maintenance costs and ensures that the designed structure will meet service life and durability requirements. An example that highlights the importance of modeling early age concrete time- dependent behavior is presented by Štemberk and Kalafu- tová [3]. The error was computed through mean squared error (MSE). The obtained results are presented in Table 1. The MSE equations are: 2 1 1 1 n j j j f d n    ; (1) 2 1 1 n all j j f f N    , (2) 2 1 1 1 n j j j f d n    ; (1) 2 1 1 n all j j f f N    , (2) (1) (2) Apart from external factors such as ambient tem- perature and humidity, concrete shrinkage is caused by hydration reaction in the cement paste. When considering self-compacting concrete (SCC) mixtures i.e. a high- performance concrete that can flow under its own weight and self-consolidate without any mechanical vibration [4], a high volume of cement paste is necessary in the composi- tion to achieve excellent deformability. However, SCC is prone to higher shrinkage strains when compared to con- ventional concrete. Hence, measuring the shrinkage strain is especially important when working with SCC. where N and n relate to the number of data sets and data points considered in the analysis, respectively; dj is the percent difference between predicted and measured strain for the data point i; fj is the MSE for the data set j; and fall is the overall MSE. Table 1 MSE for EN-1992 prediction model for SCC Input data: Author, [Ref.] N fmodel, % fall, % Guneyisi, [8] 8 50.1 62.5 Bouzoubaa, [9] 8 72.4 Lemann, [10] 3 63.1 MSE for EN-1992 prediction model for SCC Nonetheless, the experimental measurement of shrinkage strain is laborious, time consuming, and expen- sive. These characteristics, added to short deadlines, tight budgets, and the industry’s trend of accelerating the con- struction processes, sometimes make experimental meas- urements unfeasible. As a result, construction designers tend to use shrinkage prediction models. The overall MSE, fall = 62.5% (Table 1), indicates that the EN1992 model did not lead to satisfactory results Shrinkage prediction models aim to determine 68 for the analyzed data. 1. Introduction The basic form of a GA involves three operators to achieve evolution: selection (or reproduction), crossover, and mutation [13]. Fig. 1 Comparison of measured and predicted shrinkage strain for SCC using the EN1992 model In light of this, this work aims to propose an ex- perimental-based prediction model for SCC shrinkage. The model is developed based on an independent methodology that combines fuzzy logic and genetic algorithm. The hy- bridization of these techniques is advantageous once they complement each other as further discussed in section 2. The advantage of combining fuzzy logic and ge- netic algorithms is that they complement each other. Fuzzy logic is not capable of adaption or parallel computation, which are features found in GA. On the other hand, GA lacks knowledge representation and human interaction, which are the kernel of fuzzy systems [15]. The proposed model aims to predict the behavior of concrete for a period of up to 90 days. This corresponds to a reasonable construction time during which counter- measures can be taken. Moreover, the model focuses on general applications of SCC when no experimental data is available. The final results obtained from the proposed model were compared not only to other published data, but also to EN1992 as it is the regularly used standard. 3. Proposed methodology for optimization of fuzzy decision-making The proposed approach combines fuzzy logics and genetic algorithms to optimize fuzzy decision-making, which is achieved by optimizing the shape of the member- ship functions. The proposed methodology is described as follows and summarized in Fig. 2. 2. Fuzzy logic and evolutionary computing In case none of individuals from the new popula- tion shows better fitness than the stored solution, the indi- vidual with the worst solution from the new population is replaced by the best overall solution. The selection operator chooses the chromosomes for reproduction. In this case, the tournament selection scheme was applied. It selects the best fitness from indi- viduals chosen at random from the population. Fig. 2 Flowchart of the proposed methodology for optimiza- tion of fuzzy sets Initial random population Selection Crossover Mutation Is f (x’)< f (x)? Define the population size and encoding of individuals i = i +1 Update best solution (elitism method) Stop criteria? (convergence) Evaluate fitness function, f (x’), for the new population i = 1 Evaluate fitness function, f (x), see Eq. (2) Define the number of representative intervals Start The worst individual of the current population is substituted by the best solution (elitism method) Optimized group of fuzzy sets Generating new population Yes No Yes No Initial random population Selection Crossover Mutation Define the population size and encoding of individuals i = i +1 Evaluate fitness function, f (x’), for the new population i = 1 Evaluate fitness function, f (x), see Eq. (2) Define the number of representative intervals Start Generating new population The automatic process of generating a new popu- lation and evaluating the best fit is repeated until conver- gence occurs. In this case, convergence is considered as achieved when more than 200 consecutive runs do not lead to any improvements in the fitness function result. The final result consists of a group of optimized fuzzy sets that will compose the fuzzy decision-making. Moreover, the decision-making is also composed by the rule base, Rn, defined in Eq. (3). Finally, the predicted shrinkage strain, εsh,output, is computed by means of Eq. (4). 2. Fuzzy logic and evolutionary computing Fuzzy theory, first introduced by Zadeh [11] cor- responds to a natural way of thinking where verbally ex- pressed rules are applied to deal with vagueness. The abil- ity to deal with uncertainties makes fuzzy logic reasoning a robust and flexible tool that can be used in material model- ing. For example, Štemberk and Rainová [12] used fuzzy logic for simulating hydration heat liberation of concrete. First, the user determines the number of repre- sentative intervals, Nint, of shrinkage strain, εsh, and the concrete age, t, of the experimental shrinkage strain curves of concrete mixtures with different volumes of cement paste, Vcp. The more complex the shape of the curve the higher the number of intervals needed to achieve optimal results. Fuzzy logic systems comprise three basic steps: fuzzification, decision-making and defuzzification. The fuzzification consists of converting the crisp input values into degrees of membership through membership func- tions. This step is followed by the decision-making, which involves assigning a degree of membership to the output depending on the rule base of the system. Finally, the de- fuzzification is performed to convert the output fuzzy set into a single value. After that, the user specifies the size of the popu- lation, Spop, to be used in the genetic part of the algorithm. From this point on the optimization process is automatic. Based on the value set for Nint, the encoding of each indi- vidual, or chromosome, from the population is defined. It comprises a string of nenc = 2 × Nint real numbers, which correspond to the exponent values, EL and ER, related to the membership function to be optimized (Fig. 3). The key factors to achieve an acceptable perfor- mance in a fuzzy logic system are connected to the proper Next, an initial random population is generated and the fitness function, f (x), is evaluated. The fitness 69 create a mutated version that will be incorporated in the population. function corresponds to the MSE shown in Eq. (2). Then, three genetic operators (selection, crossover, and mutation) are applied to generate a new population. After a new population has been generated, the fitness function re-evaluates all individuals from the new population. The obtained results, i.e., f (x’), are then com- pared with those from previous populations, f (x). Subse- quently, elitism is applied, i.e., the best overall solution is stored. 2. Fuzzy logic and evolutionary computing Selection Crossover Mutation i = i +1 Evaluate fitness function, f (x’), for the new population i = 1 Generating new population , , : IF is THEN is n cp cp n sh sh n R V V   ; (3) , , 1 1 n n sh output n sh n n n n        , (4) (3) (4) where n is the number of rules; Vcp is the cement paste vol- ume input value, in l/m3; Vcp,n and sh,n are the optimized group of fuzzy sets for cement paste volume and shrinkage strain, respectively; μn is the degree of membership as- signed to each sh,n group from the rule Rn. Fig. 3 General equation and shape of the membership func- tions to be optimized x1 0.00 0.25 0.50 0.75 1.00 x3 x2  x  x - x 3 x - x 3 2  x  x - x 1 x - x 2 1 L - left slope R - right slope E R E L Limit values, x Degree of membership, μ Update best solution (elitism method) Stop criteria? (convergence) Fig. 3 General equation and shape of the membership func- tions to be optimized Fig. 2 Flowchart of the proposed methodology for optimiza- tion of fuzzy sets The methodology illustrated in Fig. 2 was applied for the experimental data presented by Leemann et al., [10], and the results from Loser et al., [16], were used to verify the optimized model. The selected chromosomes, or parents, are then crossed over by one-point crossover scheme, with a proba- bility, Cprob, set as 90.0%, to create a new individual to be included in the population. This scheme sets an independ- ent randomized crossover point for couples of parents, whose data is swapped to create a new population. verified. EL and ER values of the optimized fuzzy sets The number of representative intervals was defined as Nint = 3. The population size was set as Spop = 10. The proposed methodology, see Fig. 3, was then performed and convergence was achieved after around 500 runs. Fuzzy set MF EL ER Vcp Vcp1 - 1.000 Vcp2 1.000 - εsh1 sh11 - 1.573 sh12 0.842 1.436 sh13 0.498 0.726 sh14 1.144 - εsh2 sh21 - 1.621 sh22 0.393 1.519 sh23 0.636 1.512 sh24 0.762 - Fig. 4 Shrinkage strain curves of SCC, [11] 100 200 300 400 500 600 0 10 20 30 40 50 60 70 80 90 Vcp=230 l/m3 Vcp=378 l/m3 0 Concrete age, t, days Shrinkage strain, εsh, microstrain The fuzzy logic prediction model for SCC is then composed of the optimized fuzzy sets shown in Table 3, the rule base and the final output equation, presented in Eq. (3) and (4). The obtained prediction model, named the FL mod- el, is suitable for predicting shrinkage strain up to 90 days of SCC with Vcp ranging from 230 to 378 l/m3 and testing con- ditions designed by Leemann et al., [10]. Concrete age, t, days Fig. 4 Shrinkage strain curves of SCC, [11] Fig. 4 Shrinkage strain curves of SCC, [11] To verify the quality of the FL model in predicting shrinkage strain, the experimental data published by Loser et al. [16] was used for comparison. This data comprises shrinkage curves of five SCC mixtures that were tested in conditions compatible with the limits of the developed mod- el. The Vcp of each SCC mixture is listed in Table 4. The experimental and predicted shrinkage strains were compared and the MSE values, computed by Eq. (2), are presented in Table 5. The representative intervals of the experimental curves and the exponent values, EL and ER, of the group of optimized fuzzy sets are listed in Table 2 and 3, respectively. Since only two curves were available for the optimization process, the fuzzy sets connected to Vcp were set as linear. verified. Table 2 Representative intervals of the experimental curves Vcp, l/m3 t1, days εsh1, microstrain t2, days εsh2, microstrain 230 0 0 15 175 15 175 70 305 70 305 90 325 328 0 0 20 315 20 315 70 500 70 500 90 530 Table 2 Representative intervals of the experimental curves As a complementary analysis, the shrinkage strain curve of each SCC mixture from [16] was also compared to the strain curves predicted by the EN1992 model. For that, the input data listed in Table 4 was taken into account. The obtained results are presented in Table 5 together with those from FL model to ease the comparison of the models. Table 4 Input data used to predict shrinkage strain - EN1992 and FL model, [16] Input Parameters SCC 1 2 3 4 5 Cement paste volume, Vcp, l/m3 329.0 349.0 316.0 342.0 332.0 Curing time, tc , days 1 Relative Humidity, RH, % 70.0% Cement type CEM I 42.5 Design compressive strength, fc’, MPa 53.3 63.1 51.0 49.4 66.0 Compressive strength at 28 days, fcm28 , MPa 61.3 71.1 59.0 57.4 74.0 Specimen size, mm 120 × 120 × 360 Member shape Infinite prism * Vcp was only considered by FL model; Table 5 MSE values for different shrinkage prediction models fmodel, % SCC fall, % 1 2 3 4 5 EN1992 56.8 51.1 57.1 55.9 49.7 54.2 FL model 6.9 19.1 4.4 16.2 14.1 13.4 From Table 5, it can be seen that the FL model pre- sented lower MSE in all cases, indicating that the FL model is more reliable in predicting SCC shrinkage than EN1992 for the evaluated data. The comparison of experimental shrinkage curve for SCC 1 and the correspondent predicted shrinkage strain curves obtained from EN1992 and FL models is illustrated in Fig. 5. Although the MSE presented by FL model was lower than EN1992 in all cases, this value is still considered high, around 15.0%. 4. Results and discussion In the present analysis the volume of cement paste, Vcp, was chosen as input parameter and two experimental curves, illustrated in Fig. 4, were considered as training data. These curves were taken from the experimental database presented by Leemann et al. [10],where further details con- cerning the materials properties and curing conditions can be Further, a mutation operator is applied to maintain genetic diversity. The mutation is performed by disturb- ance with a probability, Mprob, set as of 10.0%. This opera- tor randomly flips some of the values in a chromosome to 70 Table 3 Table 6 Table 6 Exponents values, EL and ER, of the optimized group of fuzzy sets for FL-2 model Fuzzy set MF EL ER Vcp Vcp1 - 0.251 Vcp2 2.099 - εsh1 sh11 - 1.573 sh12 0.842 1.436 sh13 0.498 0.726 sh14 1.144 - εsh2 sh21 - 1.621 sh22 0.393 1.519 sh23 0.636 1.512 sh24 0.762 - Exponents values, EL and ER, of the optimized group of fuzzy sets for FL-2 model Table 7 two experimental curves were used as training data, which led to linear membership functions for the fuzzy sets con- nected to Vcp. If an intermediary curve was included as train- ing data, the linear shape of the Vcp fuzzy set, see Table 3, would be optimized. Consequently, the lower MSE of FL model would be reached. Individual and overall MSE values for EN1992, FL, and FL-2 shrinkage prediction models Individual and overall MSE values for EN1992, FL, and FL-2 shrinkage prediction models fmodel , % SCC fall , % 1 2 3 4 5 EN1992 56.8 51.1 57.1 55.9 49.7 54.2 FL model 6.9 19.1 4.4 16.2 14.1 13.4 FL-2 model 8.9 3.8 11.2 – 3.9 7.6 used as training data to develop the FL-2 model. Fig. 5 Experimental and predicted curves from FL and EN1992 for SCC 1 0 100 200 300 400 500 0 10 20 30 40 50 60 70 80 90 FL Model EN1992 Measured (SCC 1) Concrete age t, (days) Shrinkage strain, εsh, microstrain 0 100 200 300 400 500 0 10 20 30 40 50 60 70 80 90 FL Model EN1992 Measured (SCC 1) Concrete age t, (days) Shrinkage strain, εsh, microstrain Moreover, it also indicates that the proposed meth- odology is able to adjust according to the training data. For instance, this allows for including long-term shrinkage ex- perimental measurements, e.g., up to 365 days, to build a model for different applications than the one presented in this work. Finally, the lower MSE values from FL and FL-2 models against EN1992 confirm their quality in simulating the materials behaviour, and also the success in combining fuzzy logics and GA to develop optimized materials models. Concrete age t, (days) Acknowledgements This work was supported by the Czech Science Foundation, project number P105/10/2098, and by the Czech Technical University in Prague, project number SGS11/107/OHK1/2T/11, which is gratefully acknowl- edged. Also, the authors wish to express their appreciation to Erasmus Mundus External Cooperation Window EMECW – EUBrazil Startup program. verified. The reason for this is probably that only Table 4 Input data used to predict shrinkage strain - EN1992 and FL model, [16] Input Parameters SCC 1 2 3 4 5 Cement paste volume, Vcp, l/m3* 329.0 349.0 316.0 342.0 332.0 Curing time, tc , days 1 Relative Humidity, RH, % 70.0% Cement type CEM I 42.5 Design compressive strength, fc’, MPa 53.3 63.1 51.0 49.4 66.0 Compressive strength at 28 days, fcm28 , MPa 61.3 71.1 59.0 57.4 74.0 Specimen size, mm 120 × 120 × 360 Member shape Infinite prism * Vcp was only considered by FL model; Input data used to predict shrinkage strain - EN1992 and FL model, [16] is more reliable in predicting SCC shrinkage than EN1992 for the evaluated data. Table 5 Table 5 MSE values for different shrinkage prediction models fmodel, % SCC fall, % 1 2 3 4 5 EN1992 56.8 51.1 57.1 55.9 49.7 54.2 FL model 6.9 19.1 4.4 16.2 14.1 13.4 MSE values for different shrinkage prediction models MSE values for different shrinkage prediction models The comparison of experimental shrinkage curve for SCC 1 and the correspondent predicted shrinkage strain curves obtained from EN1992 and FL models is illustrated in Fig. 5. g Although the MSE presented by FL model was lower than EN1992 in all cases, this value is still considered high, around 15.0%. The reason for this is probably that only From Table 5, it can be seen that the FL model pre- sented lower MSE in all cases, indicating that the FL model 71 5. Conclusions Fig. 5 Experimental and predicted curves from FL and EN1992 for SCC 1 By developing a shrinkage strain prediction model for SCC the objective of this paper has been achieved. The proposed methodology for optimization of fuzzy decision- making has shown satisfactory results. To verify the assumption that additional training data would lead to a more reliable model, one of the experi- mental curves from Loser et al. [16] particularly SCC 4, was included as additional training data. The optimization pro- cess was performed again and the exponent values, ER and EL, obtained for the optimized fuzzy sets are indicated in Table 6.The experimental data from SCC 4 was only used as training data to optimize the shape of the Vcp fuzzy sets, therefore the optimized fuzzy sets εsh1 and εsh2, see Table 3, remained unchanged. The obtained model was named the FL-2 model. In addition, the optimized fuzzy sets led to a proper prediction of the shrinkage with a reduced number of rules, making the modeling process more effective. The statistical analysis pointed to an overall MSE around 50.0% for EN1992, against ~15.0% for FL model, indicating that the FL model better represents the materials behaviour and can be used to predict SCC shrinkage within the limits of the model. The further inclusion of additional training data in the optimization methodology contributed to reduce the overall error of the FL model from ~15.0% to ~7.0%, demonstrating the flexibility of the model in self-adjusting according to the training data. Such flexibility is a great ad- vantage of the FL model when compared to models based on defined equations and its constants. References The predicted shrinkage strain from FL-2 model was compared to the data from Loser et al. [16] and the MSE was computed. The obtained results are listed in Ta- ble 7 together with those from EN1992 and FL model. 1. Brook, J. 2003. Elasticity, Shrinkage, Creep and Thermal Movement. In: Newman, J.; Choo, B.S. Ad- vanced Concrete Technology – Testing and Quality. Elsevier, London, Vol. 1, 320 p. http://dx.doi.org/10.1016/B978-075065686-3/50254-8. 1. Brook, J. 2003. Elasticity, Shrinkage, Creep and Thermal Movement. In: Newman, J.; Choo, B.S. Ad- vanced Concrete Technology – Testing and Quality. Elsevier, London, Vol. 1, 320 p. http://dx.doi.org/10.1016/B978-075065686-3/50254-8. From Table 7, it can be seen that the overall MSE for FL-2 model were considerably reduced when compared with the first version of the model. This confirms the as- sumption that the inclusion of additional training data would lead to a prediction model with lower overall error. 2. Mehta, P.K.; Monteiro, P.J.M. 2006. Concrete: Mi- crostructure, Properties and Materials, 3rd Ed. McGrawHill, New York, 660p. 2. Mehta, P.K.; Monteiro, P.J.M. 2006. Concrete: Mi- crostructure, Properties and Materials, 3rd Ed. McGrawHill, New York, 660p. 72 W. R. L. da Silva, P. Štemberk W. R. L. da Silva, P. Štemberk 3. Štemberk, P.; Kalafutová, P. 2008. Modeling very early early age concrete under uniaxial short-time and sustained loading, Mechanika, 2(70): 16-21. R e z i u m ė http://dx.doi.org/10.1617/2912143624.002. Straipsnio tikslas – sukurti eksperimentinį modelį vienodai pasiskirsčiusio betono susitraukimui įvertinti. Modelis skirtas nustatyti betono elgsenai 90 dienų periodu, atitinkančiu statybos trukmę, per kurią galimi atsakomieji veiksmai. Modelis sukurtas taikant nepriklausomą metodo- logiją, jungiančią neraiškiąją (fuzzy) logiką ir genetinį al- goritmą. Ši metodologija pritaikyta eksperimento duome- nims, o naudojantis šiuo modeliu gauti rezultatai palyginti su kitais publikuotais rezultatais. Statistinė analizė patvirti- no pasiūlytojo modelio, vertinamo Eurokodu, patikimumą. 5. Mcdonald, D.B.; Roper, H. 1993. Accuracy of predic- tion models for shrinkage of concrete, ACI Materials Journal 90(3): 265-271. 6. EN1992-1-1:2004. 2004 EuroCode 2: Design of con- crete structures - Part 1-1: General Rules and Rules for Buildings, European Committee for Standardization, Brussels. 7. The European Project Group. 2005. The European guidelines for Self-compacting concrete. 63p. 8. Guneyisi, E.; Gesoglu, M.; Ozbay, E. 2010. Strength and drying shrinkage properties of self-compacting concretes incorporating multi-system blended mineral admixtures, Construction and Building Materials 24(10): 1878-1887. http://dx.doi.org/10.1016/j.conbuildmat.2010.04.015. W. R. L. da Silva, P. Štemberk OPTIMIZED FUZZY LOGIC MODEL FOR PREDICTING SELF-COMPACTING CONCRETE SHRINKAGE 9. Bouzoubaa, N.; Lachemi, M. 2001. Self-compacting concrete incorporating high volumes of class F fly ash: Preliminary results, Cement and Concrete Research 31(3): 413-420. http://dx.doi.org/10.1016/S0008-8846(00)00504-4. OPTIMIZUOTAS NERAIŠKIOSIOS LOGIKOS MODELIS VIENODAI PASISKIRSČIUSIO BETONO SUSITRAUKIMUI NUSTATYTI 4. Skarendahl, A. 2005. Changing Concrete Construction Through Use of Self-Compacting Concrete. In: PRO 42 1st International Symposium on Design, Performance and Use of Self-Consolidating Concrete – SCC’2005, RILEM Publications, 17-24. http://dx.doi.org/10.1617/2912143624.002. S u m m a r y This paper aims to develop a shrinkage prediction model for self-compacting concrete based on experimental data. The model focuses on predicting the behavior of con- crete up to a period of 90 days, which corresponds to a construction time during which countermeasures can be taken. The model was designed based on an independent methodology that combines fuzzy logic and genetic algo- rithm. This methodology was applied for an experimental data set, and the obtained model was compared to other published data and the prediction model considered by the Eurocode. The results were verified by statistical analysis that confirmed the reliability of the proposed model. 10. Leemann, A.; Lura, P.; Loser, R. 2011. Shrinkage and creep of SCC – The influence of paste volume and binder composition, Construction and Building Materi- als 25(5): 2283-2289. http://dx.doi.org/10.1016/j.conbuildmat.2010.11.019 11. Zadeh, L.A. 1965. Fuzzy sets, Information and Control 8: 38-53. http://dx.doi.org/10.1016/S0019-9958(65)90241-X. Š http://dx.doi.org/10.1016/S0019-9958(65)90241-X Š 12. Štemberk, P.; Rainová, A. 2011. Simulation of hydra- tion and cracking propagation with temperature effect based on fuzzy logic theory, Mechanika, 17(4): 358- 362. Keywords: Self-compacting concrete, shrinkage, fuzzy logic, genetic algorithm, material modeling. 13. Coley, D.A. 1999. An Introduction to Genetic Algo- rithms for Scientists and Engineers, World Scientific, Singapore, 244 p. Received January 27, 2012 Accepted January 16, 2013 14. Rokonuzzaman, Md.; Sakai, T. 2010. Calibration of the parameters for a hardening-softening constitutive model using genetic algorithms, Computers and Ge- otechnics 37(4): 573-579. http://dx.doi.org/10.1016/j.compgeo.2010.02.007. 15. Cordon, O.; Herrera, F.; Hoffman, F.; Magdalena, L.; 2002. Genetic Fuzzy Systems: Evolutionary Tuning and Learning of Fuzzy Knowledge Bases, In: Zadeh, L.A. Advances in fuzzy systems: applications and theo- ry. World Scientific, London, Vol.19, 448 p. 16. Loser, R.; Leemann, A. 2009. Shrinkage and re- strained shrinkage cracking of self-compacting con- crete compared to conventionally vibrated concrete, Materials and Structures 42: 71-82. http://dx.doi.org/10.1617/s11527-008-9367-9.
https://openalex.org/W2922262556	https://gfzpublic.gfz-potsdam.de/pubman/item/item_4006892_4/component/file_4006894/4006892.pdf	English	null	Sol-Gel Processes in Micro-Environments of Black Shale: Learning from the Industrial Production of Nanometer-Sized TiO2 Polymorphs	ChemEngineering	2,019	cc-by	8,564	hulz, H.-M. (2019): Sol-Gel Processes in Micro-Environments of Black Shale: Learning from the dustrial Production of Nanometer-Sized TiO2 Polymorphs. - ChemEngineering, 3. Received: 15 February 2019; Accepted: 4 March 2019; Published: 8 March 2019 Abstract: Micro-environments in black shale are reactors for geochemical reactions that differ from the bulk scale. They occur in small isolated pores of several 10 s to 100 s of nanometers without or with limited ionic exchange by diffusion to the surrounding matrix. The example of the formation of titania polymorphs brookite (and anatase) in black shale demonstrates that pH < 4 of the pore waters or lower must prevail to enable dissolution of Ti-bearing precursors followed by the precipitation of these metastable solids. Comparably low pH is applied during the industrial production of nanometer-sized brookite or anatase by sol-gel methods. The process parameters during industrial production such as low pH, negative Eh, or low ionic strength (to promote agglomeration) allow a comparison with parameters during geochemical processes leading to titania formation in black shale. Sol-gel processes are suggested herein as key geochemical processes in micro-environments of black shale in order to understand the formation of single brookite crystals or agglomerates on a nanometer scale. Keywords: black shale; brookite; micro-environment; sol-gel process Schulz, H.-M. (2019): Sol-Gel Processes in Micro-Environments of Black Shale: Learning from the Industrial Production of Nanometer-Sized TiO2 Polymorphs. - ChemEngineering, 3. DOI: http://doi.org/10.3390/chemengineering3010028 DOI: http://doi.org/10.3390/chemengineering3010028 DOI: http://doi.org/10.3390/chemengineering3010028 Micro-Environments of Black m the Industrial Production of O2 Polymorphs n Research Centre for Geosciences, Section 3.2 Organic Geochemistry, fz-potsdam.de; Tel.: +49-331-288-1789 March 2019; Published: 8 March 2019 k shale are reactors for geochemical reactions that differ from olated pores of several 10 s to 100 s of nanometers without or on to the surrounding matrix. The example of the formation of ase) in black shale demonstrates that pH < 4 of the pore waters lution of Ti-bearing precursors followed by the precipitation ably low pH is applied during the industrial production of by sol-gel methods. The process parameters during industrial Eh, or low ionic strength (to promote agglomeration) allow g geochemical processes leading to titania formation in black d herein as key geochemical processes in micro-environments the formation of single brookite crystals or agglomerates on a ro-environment; sol-gel process c sediment with high water content. Porosity-depth trends water content) from initially up to 80% to less than 20% at a burial history and composition [1]. Porosity in shale comprises he organic material as well as in or between minerals [2,3]. res (diameter > 50 nm), to mesopores (2–50 nm), to micropores great detail in shale research as an unconventional resource [5,6]. ered spots for geochemical reactions that are different from the o-environments for the localized dissolution and precipitation be ﬁlled with formation water (besides liquid and gaseous a matrix, reactant, and transport medium during rock–ﬂuid stically exemplary micro-environment for the formation of in black shale is introduced—its signiﬁcance for localized om the bulk scale. By explaining the formation of nano-sized ence is made to today´s technical sol-gel processes for the ow temperatures under controlled physicochemical conditions. chemengineering chemengineering ChemEngineering 2019, 3, 28; doi:10.3390/chemengineering3010028 Article Hans-Martin Schulz Helmholtz Centre Potsdam GFZ—German Research Centre for Geosciences, Section 3.2 Organic Geoch D-14473 Potsdam, Germany; schulzhm@gfz-potsdam.de; Tel.: +49-331-288-1789 Hans-Martin Schulz Hans-Martin Schulz Helmholtz Centre Potsdam GFZ—German Research Centre for Geosciences, Section 3.2 Organic Geochemistry, D-14473 Potsdam, Germany; schulzhm@gfz-potsdam.de; Tel.: +49-331-288-1789 www.mdpi.com/journal/chemengineering 2. Geochemical Micro-Environments in General and in Black Shale—A Brief Review Chemical microenvironments are well-known phenomena in various geological matrixes and are reported from shale [11–13], hardgrounds [14], soils [15,16], and ﬂuvial systems [17], but also occur in sediment macrofauna [18], living planktic foraminifera [19], or in fecal pellets [20]. One often reported feature is the formation of iron-bearing solid solutions in various geological matrixes such as marine sediments [21], glaciers [22], or even on Mars [23], but many other minerals also dissolve or precipitate in such microenvironments which suggest geochemical processes different from those of the bulk matrix. A geochemical micro-environment in a rock can be deﬁned as a small entity within a bulk rock matrix. It is ﬁlled by a ﬂuid with a composition that is different from the bulk composition. Such a ﬂuid can be physically isolated from the bulk solution or not. A deﬁnition by Reference [24] also includes scale considerations from a large scale (e.g., clay lenses in sandy sediments) to nanometer scale. The authors stated that micro-environments can be found at mineral interfaces or contacts [25], within mineral grains [26], or even at the microbial cell surface on the sub-micron to micron scale. At the nanometer scale, a microenvironment may involve different reacting molecular sites represented by the reactivities of different mineral surface faces or at their intersections. Recently it was found that micro-environments also play a signiﬁcant role in preserving organic fossils due to interactions between clay minerals and organic tissues. Conversion or decay of organic matter may be inhibited by blocked enzymatic reactions [27]. The processes leading to the development of microenvironments in shale are dependent on chemical gradients which develop in the aqueous phase due to fast reaction rates (either microbially mediated or mineral dissolution/precipitation rates) and compensate the export or import of solutes [24]. Due to this deﬁnition, preferential sites for the development of micro-environments are physically heterogeneous systems with different ﬂow rates and molecular diffusion as the main transport processes. Accordingly, micro-environments can be retraced by the occurrence of features such as concretions in shale of various scales. Characteristic hydrogeochemical conditions develop due to organic–inorganic interactions in chemical micro-environments of black shales. The driving force for the creation of localized hydrogeochemical conditions in such micro-environments (or microreactors) is the conversion of labile organic matter (OM). 1. Introduction Shale is a ﬁne-grained, siliciclastic sediment with high water content. Porosity-depth trends indicate a loss in porosity (and thus in water content) from initially up to 80% to less than 20% at a depth of around 3000 m, dependent on burial history and composition [1]. Porosity in shale comprises of a variety of pores which occur in the organic material as well as in or between minerals [2,3]. These pores range in size from macropores (diameter > 50 nm), to mesopores (2–50 nm), to micropores (<2 nm; [4]) and have been described in great detail in shale research as an unconventional resource [5,6]. Pores of small size especially are considered spots for geochemical reactions that are different from the bulk scale and may act as isolated micro-environments for the localized dissolution and precipitation of solids. Much of the porosity can be ﬁlled with formation water (besides liquid and gaseous hydrocarbons). This aqueous phase is a matrix, reactant, and transport medium during rock–ﬂuid interactions at various scales [7–9]. In this contribution, a characteristically exemplary micro-environment for the formation of nanometer-sized titania polymorphs in black shale is introduced—its signiﬁcance for localized organic–inorganic interactions apart from the bulk scale. By explaining the formation of nano-sized titanium dioxide in black shale, reference is made to today´s technical sol-gel processes for the production of titania nanomaterials at low temperatures under controlled physicochemical conditions. Several experimental parameters are compared with those under geological subsurface conditions to ﬁnd similar controls on the formation, fate, and behavior of brookite (and anatase) in organic-rich sediments and comparable processes in other organic-rich and aqueous environments, such as oil–water contacts in oil ﬁelds. ChemEngineering 2019, 3, 28; doi:10.3390/chemengineering3010028 www.mdpi.com/journal/chemengineering www.mdpi.com/journal/chemengineering 2 of 11 ChemEngineering 2019, 3, 28 The selected example relates to the metastable titania (TiO2) polymorphs brookite and anatase, and how they form in black shale environments [10]. This issue is reconsidered in this short communication, but with reference to the prenucleation concept and the so-called sol-gel process. Selected titania polymorphs (only brookite in this research) in black shales will be presented in the form of an image collection highlighting micro-environments on a micrometer to nanometer scale, and their formation will be discussed as controls of organic matter conversion in micro-environments as analogues of industrial nano-titania production. 3. Formation of Inorganic Materials from Solutions 3. Formation of Inorganic Materials from Solutions Stawski and Benning [29] reviewed how amorp Stawski and Benning [29] reviewed how amorphous and crystalline inorganic materials may form from solutions. In summary, precipitation and growth of any solid material from a solution are dynamic processes during which different species of various shapes, sizes, and internal structures form and further develop into a ﬁnal product. In this paper, two hypotheses are discussed for the explanation of nucleation and crystallization phenomena taking place in solutions—the classical nucleation theory (CNT) and the prenucleation concept (see references in [29]). Although different in their individual process chains, the conceptual pathways described for both approaches consider the formation of nanostructures from a solution to a stable end-product. A well-balanced interplay of the physical and chemical parameters controls the morphology and growth kinetics of these nanostructures in evolving systems. It is important to note that the prenucleation concept also refers to intermediate stages of amorphous particles (Figure 1A). In a sol-gel process, a sol (colloidal suspension) is formed from the hydrolysis and polymerization reactions of the precursors, which are inorganic metal salts or metal organic compounds such as metal alkoxides (Figure 1B). The transition from the liquid sol into a solid gel phase is caused by complete polymerization and loss of the solvent [30]. Stawski and Benning [29] reviewed how amorphous and crystalline inorganic materials may form from solutions. In summary, precipitation and growth of any solid material from a solution are dynamic processes during which different species of various shapes, sizes, and internal structures form and further develop into a final product. In this paper, two hypotheses are discussed for the explanation of nucleation and crystallization phenomena taking place in solutions—the classical nucleation theory (CNT) and the prenucleation concept (see references in [29]). Although different in their individual process chains, the conceptual pathways described for both approaches consider the formation of nanostructures from a solution to a stable end-product. A well-balanced interplay of the physical and chemical parameters controls the morphology and growth kinetics of these nanostructures in evolving systems. It is important to note that the prenucleation concept also refers to intermediate stages of amorphous particles (Figure 1A). In a sol-gel process, a sol (colloidal suspension) is formed from the hydrolysis and polymerization reactions of the precursors, which are inorganic metal salts or metal organic compounds such as metal alkoxides (Figure 1B). 2. Geochemical Micro-Environments in General and in Black Shale—A Brief Review The OM consumption already takes place in the overlying water column where microreactors may form in sinking particles, such as in anoxic bottom water (e.g., pyrite framboid formation; [2]). Later, they develop during deposition and continue from early diagenesis to the oil window and beyond, all dependent on the sensitivity of the preserved but still reactive to organic matter. The processes in micro-environments are thus not affected by the buffer potential of the bulk rock. However, during subsidence in a sedimentary basin, a variety of soluble organic compounds are released from the organic material which control different hydrogeochemical processes—as these compounds may change in pH. For example, during early diagenesis to the oil window, acidic and corrosive hydrogeochemical conditions develop due to the release of low molecular weight organic acids (e.g., acetic acid) or carbon dioxide and lead to dissolution of unstable minerals, which is compensated by the precipitation of solid solutions at equilibrium [28]. 3 of 11 3 of 11 ChemEngineering 2019, 3, 28 ChemEngineering 2019, 3, x FO 3. Formation of Inorganic Materials from Solutions 3. Formation of Inorganic Materials from Solutions Stawski and Benning [29] reviewed how amorp The transition from the liquid sol into a solid gel phase is caused by complete polymerization and loss of the solvent [30]. Figure 1. (A) Nonclassical pathway for the growth of crystalline materials from solutions. (B) Scheme representing the evolution of aqueous silica system from monomers to developed sols and gels, in relation to reaction conditions. (A) is redrawn after Figure 5.2 in Reference [29]; see details for references therein. (B) Various steps in the sol-gel process to control the final morphology of the product (redrawn after Figure 2.1. in Reference [30]). Figure 1. (A) Nonclassical pathway for the growth of crystalline materials from solutions. (B) Scheme representing the evolution of aqueous silica system from monomers to developed sols and gels, in relation to reaction conditions. (A) is redrawn after Figure 5.2 in Reference [29]; see details for references therein. (B) Various steps in the sol-gel process to control the ﬁnal morphology of the product (redrawn after Figure 2.1. in Reference [30]). 4. Methodologies to Characterize and Analyze Micro-Environments The analysis of micro-environments requires chemical or imaging techniques that are capable of characterizing small volumes or small sizes. However, their applicability can be limited by the close association with the bulk phases. Moreover, the geochemical modelling of micro-environments in shale by PHREEQC—a program based on chemical thermodynamics—may be problematic due to the low pH in micro-environments and due to migration of acidic capillary pore water [31], but general approaches have existed since the 1970s [32]. The analytical approach used here was based on a ﬁrst overview characterization of the black shale by conventional light microscopy. In a second step, scanning electron microscopy (SEM) with a coupled energy dispersive X-ray spectroscopy (EDS) enabled the analysis of different shale entities. Contents of organic carbon (total organic carbon, TOC) were determined and Rock Eval analyses of whole rock samples were carried out according to standard procedures to characterize the amount, character, and maturity of the organic material. The ﬁnal and main technique applied for visualization and characterization of micro-environments was transmission electron microscopy (TEM). Selected results from TEM investigations are presented, and brookite identiﬁcation is based on the method described in Reference [10]. Samples for TEM investigations were prepared applying the focused ion beam (FIB) technique (detailed in Reference [33]). Figure 1. (A) Nonclassical pathway for the growth of crystalline materials from solutions. (B) Scheme representing the evolution of aqueous silica system from monomers to developed sols and gels, in relation to reaction conditions. (A) is redrawn after Figure 5.2 in Reference [29]; see details for references therein. (B) Various steps in the sol-gel process to control the final morphology of the product (redrawn after Figure 2.1. in Reference [30]). Figure 1. (A) Nonclassical pathway for the growth of crystalline materials from solutions. (B) Scheme representing the evolution of aqueous silica system from monomers to developed sols and gels, in relation to reaction conditions. (A) is redrawn after Figure 5.2 in Reference [29]; see details for references therein. (B) Various steps in the sol-gel process to control the ﬁnal morphology of the product (redrawn after Figure 2.1. in Reference [30]). 4 of 11 ChemEngineering 2019, 3, 28 5. Example Brookite (and Anatase): Formation of Unstable Titania Polymorphs in Micro-Environments of Black Shale The titania polymorph brookite (Figures 2 and 3) mainly occur as a single crystal—less as agglomerated nano-crystals—in organic matter-rich sediments of differing age and thermal maturity [10], and single brookite crystals increase in diameter with increasing thermal maturity. In contrast, anatase prevails as both single crystals or as agglomerates at oil–water contacts in oilﬁelds, and along fractures with ﬂuid ﬂow enriched in dissolved organic carbon. In general, titania nano-crystal precipitation, growth (and agglomeration) takes place in the pore water of micro-environments at low to high temperatures, and where low pH is coupled to the occurrence of dissolved organic components. Low sedimentation rates preserving a critical geochemical environment or higher temperatures seem to be major controls for the precipitation of anatase (cf. Figure 4) and its tendency not to agglomerate. 5 of 11 5 of 11 ChemEngineering 2019, 3, 28 Figure 2. Titania (TiO2) polymorphs (brookite in all selected samples) in selected black shales: Bakk Shale (A,B), Mediterranean sapropel S6 (C,D), and Schöneck Formation (E,F). (A,C,D) are hig magnification scanning transmission electron microscopy (STEM) images (high-angle annular da field (HAADF) mode), whereas (B,D,F) are TEM Bright Field images. The Pleistocene Mediterrane Sapropel S6 from the Eastern Mediterranean Sea was recovered from a sediment depth of 4.12 (water depth, 2788 m), has a total organic carbon (TOC) content of 4‒6%, and is composed of mari organic matter (OM). The sample from the Oligocene Schöneck Fm. (Upper Austrian Molasse bas originates from a well core from a depth of 1384 m, has a TOC content of 3.6%, and an immatu lacustrine OM (hydrogen index, HI: 550 mg HC/g TOC). The Bakken Shale sample is of Upp Devonian to Lower Mississippian age, was drilled in the Williston Basin (North Dakota, U.S.A.), a comes from a depth of 2332 m. The TOC content is 8.3%, the marine OM has a HI of 416 mg HC TOC and an oxygen index (OI) of 27 mg CO2/g TOC Figure 2. Titania (TiO2) polymorphs (brookite in all selected samples) in selected black sh Bakken Shale (A,B), Mediterranean sapropel S6 (C,D), and Schöneck Formation (E,F). (A,C,D high-magniﬁcation scanning transmission electron microscopy (STEM) images (high-angle ann dark-ﬁeld (HAADF) mode), whereas (B,D,F) are TEM Bright Field images. 5. Example Brookite (and Anatase): Formation of Unstable Titania Polymorphs in Micro-Environments of Black Shale The Pleisto Mediterranean Sapropel S6 from the Eastern Mediterranean Sea was recovered from a sedi depth of 4.12 m (water depth, 2788 m), has a total organic carbon (TOC) content of 4–6%, a composed of marine organic matter (OM). The sample from the Oligocene Schöneck Fm. (U Austrian Molasse basin) originates from a well core from a depth of 1384 m, has a TOC content of and an immature lacustrine OM (hydrogen index, HI: 550 mg HC/g TOC). The Bakken Shale sa is of Upper Devonian to Lower Mississippian age, was drilled in the Williston Basin (North Da U.S.A.), and comes from a depth of 2332 m. The TOC content is 8.3%, the marine OM has a HI o Figure 2. Titania (TiO2) polymorphs (brookite in all selected samples) in selected black shales: Bakken Shale (A,B), Mediterranean sapropel S6 (C,D), and Schöneck Formation (E,F). (A,C,D) are high- magnification scanning transmission electron microscopy (STEM) images (high-angle annular dark- field (HAADF) mode), whereas (B,D,F) are TEM Bright Field images. The Pleistocene Mediterranean Sapropel S6 from the Eastern Mediterranean Sea was recovered from a sediment depth of 4.12 m (water depth, 2788 m), has a total organic carbon (TOC) content of 4‒6%, and is composed of marine organic matter (OM). The sample from the Oligocene Schöneck Fm. (Upper Austrian Molasse basin) originates from a well core from a depth of 1384 m, has a TOC content of 3.6%, and an immature lacustrine OM (hydrogen index, HI: 550 mg HC/g TOC). The Bakken Shale sample is of Upper Devonian to Lower Mississippian age, was drilled in the Williston Basin (North Dakota, U.S.A.), and comes from a depth of 2332 m. The TOC content is 8.3%, the marine OM has a HI of 416 mg HC/g TOC and an oxygen index (OI) of 27 mg CO2/g TOC. Figure 2. Titania (TiO2) polymorphs (brookite in all selected samples) in selected black shales: Bakken Shale (A,B), Mediterranean sapropel S6 (C,D), and Schöneck Formation (E,F). (A,C,D) are high-magniﬁcation scanning transmission electron microscopy (STEM) images (high-angle annular dark-ﬁeld (HAADF) mode), whereas (B,D,F) are TEM Bright Field images. The Pleistocene Mediterranean Sapropel S6 from the Eastern Mediterranean Sea was recovered from a sediment depth of 4.12 m (water depth, 2788 m), has a total organic carbon (TOC) content of 4–6%, and is composed of marine organic matter (OM). The sample from the Oligocene Schöneck Fm. 5. Example Brookite (and Anatase): Formation of Unstable Titania Polymorphs in Micro-Environments of Black Shale (Upper Austrian Molasse basin) originates from a well core from a depth of 1384 m, has a TOC content of 3.6%, and an immature lacustrine OM (hydrogen index, HI: 550 mg HC/g TOC). The Bakken Shale sample is of Upper Devonian to Lower Mississippian age, was drilled in the Williston Basin (North Dakota, U.S.A.), and comes from a depth of 2332 m. The TOC content is 8.3%, the marine OM has a HI of 416 mg HC/g TOC and an oxygen index (OI) of 27 mg CO2/g TOC. Figure 2. Titania (TiO2) polymorphs (brookite in all selected samples) in selected black shales: Bakken Shale (A,B), Mediterranean sapropel S6 (C,D), and Schöneck Formation (E,F). (A,C,D) are high- magnification scanning transmission electron microscopy (STEM) images (high-angle annular dark- field (HAADF) mode), whereas (B,D,F) are TEM Bright Field images. The Pleistocene Mediterranean Sapropel S6 from the Eastern Mediterranean Sea was recovered from a sediment depth of 4.12 m (water depth, 2788 m), has a total organic carbon (TOC) content of 4‒6%, and is composed of marine organic matter (OM). The sample from the Oligocene Schöneck Fm. (Upper Austrian Molasse basin) originates from a well core from a depth of 1384 m, has a TOC content of 3.6%, and an immature lacustrine OM (hydrogen index, HI: 550 mg HC/g TOC). The Bakken Shale sample is of Upper Devonian to Lower Mississippian age, was drilled in the Williston Basin (North Dakota, U.S.A.), and comes from a depth of 2332 m. The TOC content is 8.3%, the marine OM has a HI of 416 mg HC/g TOC and an oxygen index (OI) of 27 mg CO2/g TOC. Figure 2. Titania (TiO2) polymorphs (brookite in all selected samples) in selected black shales: Bakken Shale (A,B), Mediterranean sapropel S6 (C,D), and Schöneck Formation (E,F). (A,C,D) are high-magniﬁcation scanning transmission electron microscopy (STEM) images (high-angle annular dark-ﬁeld (HAADF) mode), whereas (B,D,F) are TEM Bright Field images. The Pleistocene Mediterranean Sapropel S6 from the Eastern Mediterranean Sea was recovered from a sediment depth of 4.12 m (water depth, 2788 m), has a total organic carbon (TOC) content of 4–6%, and is composed of marine organic matter (OM). The sample from the Oligocene Schöneck Fm. (Upper Austrian Molasse basin) originates from a well core from a depth of 1384 m, has a TOC content of 3.6%, and an immature lacustrine OM (hydrogen index, HI: 550 mg HC/g TOC). 5. Example Brookite (and Anatase): Formation of Unstable Titania Polymorphs in Micro-Environments of Black Shale Titania (TiO2) polymorphs (brookite in all selected samples) in the Dictyonema Shale (Tremadocian, Ordovician). (A,D) are TEM Bright Field images whereas (B,C) are high-magnification scanning transmission electron microscopy (STEM) images (high-angle annular dark-field (HAADF) mode). (A) The sample originates from a well core from a depth of 52.05 m, has a TOC content of 12.20%, and is early mature (hydrogen index, HI: 191 mg HC/g TOC; Tmax: 414 °C). (B,C,D) The sample originates from a well core from a depth of 52.40 m, has a TOC content of 6.15%, and is early mature (hydrogen index, HI: 95 mg HC/g TOC; Tmax: 414 °C). Figure 3. Titania (TiO2) polymorphs (brookite in all selected samples) in the Dictyonema Shale (Tremadocian, Ordovician). (A,D) are TEM Bright Field images whereas (B,C) are high-magniﬁcation scanning transmission electron microscopy (STEM) images (high-angle annular dark-ﬁeld (HAADF) mode). (A) The sample originates from a well core from a depth of 52.05 m, has a TOC content of 12.20%, and is early mature (hydrogen index, HI: 191 mg HC/g TOC; Tmax: 414 ◦C). (B,C,D) The sample originates from a well core from a depth of 52.40 m, has a TOC content of 6.15%, and is early mature (hydrogen index, HI: 95 mg HC/g TOC; Tmax: 414 ◦C). Figure 3. Titania (TiO2) polymorphs (brookite in all selected samples) in the Dictyonema Shale (Tremadocian, Ordovician). (A,D) are TEM Bright Field images whereas (B,C) are high-magnification scanning transmission electron microscopy (STEM) images (high-angle annular dark-field (HAADF) mode). (A) The sample originates from a well core from a depth of 52.05 m, has a TOC content of 12.20%, and is early mature (hydrogen index, HI: 191 mg HC/g TOC; Tmax: 414 °C). (B,C,D) The sample originates from a well core from a depth of 52.40 m, has a TOC content of 6.15%, and is early mature (hydrogen index, HI: 95 mg HC/g TOC; Tmax: 414 °C). Figure 4. Enthalpy of titania polymorphs as a function of surface area (already presented in Reference [10], redrawn after Reference [34], following Reference [35]). Although the crystalline polymorph rutile is thermodynamically the most stable one, anatase and brookite are often slightly metastable by only a few kilojoules per mole. Brookite has a surface enthalpy of approximately 1.0 J/m2, which is higher than for anatase (0.4 J/m2). 5. Example Brookite (and Anatase): Formation of Unstable Titania Polymorphs in Micro-Environments of Black Shale The Bakken Shale sample is of Upper Devonian to Lower Mississippian age, was drilled in the Williston Basin (North Dakota, U.S.A.), and comes from a depth of 2332 m. The TOC content is 8.3%, the marine OM has a HI of 416 mg HC/g TOC and an oxygen index (OI) of 27 mg CO2/g TOC. 6 of 11 ChemEngineering 2019, 3, 28 Figure 3. Titania (TiO2) polymorphs (brookite in all selected samples) in the Dictyonema Shale (Tremadocian, Ordovician). (A,D) are TEM Bright Field images whereas (B,C) are high-magnification scanning transmission electron microscopy (STEM) images (high-angle annular dark-field (HAADF) mode). (A) The sample originates from a well core from a depth of 52.05 m, has a TOC content of 12.20%, and is early mature (hydrogen index, HI: 191 mg HC/g TOC; Tmax: 414 °C). (B,C,D) The sample originates from a well core from a depth of 52.40 m, has a TOC content of 6.15%, and is early mature (hydrogen index, HI: 95 mg HC/g TOC; Tmax: 414 °C). Figure 3. Titania (TiO2) polymorphs (brookite in all selected samples) in the Dictyonema Shale (Tremadocian, Ordovician). (A,D) are TEM Bright Field images whereas (B,C) are high-magniﬁcation scanning transmission electron microscopy (STEM) images (high-angle annular dark-ﬁeld (HAADF) mode). (A) The sample originates from a well core from a depth of 52.05 m, has a TOC content of 12.20%, and is early mature (hydrogen index, HI: 191 mg HC/g TOC; Tmax: 414 ◦C). (B,C,D) The sample originates from a well core from a depth of 52.40 m, has a TOC content of 6.15%, and is early mature (hydrogen index, HI: 95 mg HC/g TOC; Tmax: 414 ◦C). ChemEngineering 2019, 3, x FOR PEER REVIEW 6 of 11 Figure 3. Titania (TiO2) polymorphs (brookite in all selected samples) in the Dictyonema Shale (Tremadocian, Ordovician). (A,D) are TEM Bright Field images whereas (B,C) are high-magnification scanning transmission electron microscopy (STEM) images (high-angle annular dark-field (HAADF) mode). (A) The sample originates from a well core from a depth of 52.05 m, has a TOC content of 12.20%, and is early mature (hydrogen index, HI: 191 mg HC/g TOC; Tmax: 414 °C). (B,C,D) The sample originates from a well core from a depth of 52.40 m, has a TOC content of 6.15%, and is early mature (hydrogen index, HI: 95 mg HC/g TOC; Tmax: 414 °C). gineering 2019, 3, x FOR PEER REVIEW Figure 3. 5. Example Brookite (and Anatase): Formation of Unstable Titania Polymorphs in Micro-Environments of Black Shale Enthalpy of titania polymorphs as a function of surface area (already presented in Reference [10], redrawn after Reference [34], following Reference [35]). Although the crystalline polymorph rutile is thermodynamically the most stable one, anatase and brookite are often slightly metastable by only a few kilojoules per mole. Brookite has a surface enthalpy of approximately 1.0 J/m2, which is higher than for anatase (0.4 J/m2). Moreover, for titania particles with a surface area higher than around 4000 m2/mol (thus for smallest particles), anatase has the lowest enthalpy and can directly transform into brookite [35]. At a crossover size of about 30 nm, anatase nanoparticles may directly transform into rutile [34,36]. 5. Example Brookite (and Anatase): Formation of Unstable Titania Polymorphs in Micro-Environments of Black Shale Brookite has a surface enthalpy of approximately 1.0 J/m2, which is higher than for anatase (0.4 J/m2). Moreover, for titania particles with a surface area higher than around 4000 m2/mol (thus for smallest particles), anatase has the lowest enthalpy and can directly transform into brookite [35]. At a crossover size of about 30 nm, anatase nanoparticles may directly transform into rutile [34,36]. Figure 4. Enthalpy of titania polymorphs as a function of surface area (already presented in Reference [10], redrawn after Reference [34], following Reference [35]). Although the crystalline polymorph rutile is thermodynamically the most stable one, anatase and brookite are often slightly metastable by only a few kilojoules per mole. Brookite has a surface enthalpy of approximately 1.0 J/m2, which is higher than for anatase (0.4 J/m2). Moreover, for titania particles with a surface area higher than around 4000 m2/mol (thus for smallest particles), anatase has the lowest enthalpy and can directly transform into brookite [35]. At a crossover size of about 30 nm, anatase nanoparticles may directly transform into rutile [34,36]. Figure 4. Enthalpy of titania polymorphs as a function of surface area (already presented in Reference [10], redrawn after Reference [34], following Reference [35]). Although the crystalline polymorph rutile is thermodynamically the most stable one, anatase and brookite are often slightly metastable by only a few kilojoules per mole. Brookite has a surface enthalpy of approximately 1.0 J/m2, which is higher than for anatase (0.4 J/m2). Moreover, for titania particles with a surface area higher than d 4000 2/ l (th f ll t ti l ) t h th l t th l d di tl Figure 4. Enthalpy of titania polymorphs as a function of surface area (already presented in Reference [10], redrawn after Reference [34], following Reference [35]). Although the crystalline polymorph rutile is thermodynamically the most stable one, anatase and brookite are often slightly metastable by only a few kilojoules per mole. Brookite has a surface enthalpy of approximately 1.0 J/m2, which is higher than for anatase (0.4 J/m2). Moreover, for titania particles with a surface area higher than around 4000 m2/mol (thus for smallest particles), anatase has the lowest enthalpy and can directly transform into brookite [35]. At a crossover size of about 30 nm, anatase nanoparticles may directly transform into rutile [34,36]. Figure 4. 5. Example Brookite (and Anatase): Formation of Unstable Titania Polymorphs in Micro-Environments of Black Shale Moreover, for titania particles with a surface area higher than around 4000 m2/mol (thus for smallest particles), anatase has the lowest enthalpy and can directly Figure 4. Enthalpy of titania polymorphs as a function of surface area (already presented in Reference [10], redrawn after Reference [34], following Reference [35]). Although the crystalline polymorph rutile is thermodynamically the most stable one, anatase and brookite are often slightly metastable by only a few kilojoules per mole. Brookite has a surface enthalpy of approximately 1.0 J/m2, which is higher than for anatase (0.4 J/m2). Moreover, for titania particles with a surface area higher than around 4000 m2/mol (thus for smallest particles), anatase has the lowest enthalpy and can directly transform into brookite [35]. At a crossover size of about 30 nm, anatase nanoparticles may directly transform into rutile [34,36]. Figure 4. Enthalpy of titania polymorphs as a function of surface area (already presented in Reference [10], redrawn after Reference [34], following Reference [35]). Although the crystalline polymorph rutile is thermodynamically the most stable one, anatase and brookite are often slightly metastable by only a few kilojoules per mole. Brookite has a surface enthalpy of approximately 1.0 J/m2, which is higher than for anatase (0.4 J/m2). Moreover, for titania particles with a surface area higher than around 4000 m2/mol (thus for smallest particles), anatase has the lowest enthalpy and can directly transform into brookite [35]. At a crossover size of about 30 nm, anatase nanoparticles may directly transform into rutile [34,36]. Figure 4. Enthalpy of titania polymorphs as a function of surface area (already presented in Reference [10], redrawn after Reference [34], following Reference [35]). Although the crystalline polymorph rutile is thermodynamically the most stable one, anatase and brookite are often slightly metastable by only a few kilojoules per mole. Brookite has a surface enthalpy of approximately 1.0 J/m2, which is higher than for anatase (0.4 J/m2). Moreover, for titania particles with a surface area higher than d 4000 2/ l ( h f ll i l ) h h l h l d di l Figure 4. Enthalpy of titania polymorphs as a function of surface area (already presented in Reference [10], redrawn after Reference [34], following Reference [35]). Although the crystalline polymorph rutile is thermodynamically the most stable one, anatase and brookite are often slightly metastable by only a few kilojoules per mole. around 4000 m /mol (thus for smallest particles), transform into brookite [35]. At a crossover size o 6. Industrial Production of Titania Polymorphs transform into rutile [34,36]. Low temperature industrial processes may serve as process analogues to extract relevant key variables which might also control hydrogeochemical processes during diagenesis of black shales. 7 of 11 ChemEngineering 2019, 3, 28 However, sedimentary geosystems are complex and variable in time and space. The geochemical composition of the interacting organic–inorganic rock–ﬂuid–gas inventory in sedimentary geosystems thus changes with geological times. Moreover, the material properties of industrial TiO2 nanoparticles strongly depend on the detailed production methodology and lead to different nanoparticle sizes, crystal structures, and morphologies. Thus, the variety of experimental conditions used for the artiﬁcial synthesis of the different TiO2 phases complicate a direct comparison with natural mechanisms during nanoparticle formation in a black shale. As a logical consequence, a direct transfer of factors, phenomena, and their controls in laboratory experiments under controlled conditions to the geosphere must be critically scrutinized. Nevertheless, there are basic physicochemical factors that resemble in black shales during laboratory experiments and during sol-gel processes for the industrial production of nanotitania. Today, the most widely used industrial process for the production of metal oxides is the ceramic method, which is based on high temperature treatment (several 100s of ◦C) of powder mixtures and results in thermodynamically stable phases rather than brookite [30]. However, comparably low temperatures down to ~80 ◦C prevail in TOC-rich ﬁne-grained sediments until a depth of around 2 km (considering a general geothermal gradient with a mean surface temperature of 20 ◦C). According to this, the sol-gel process is chosen as the industrial analogue for comparison (details in overview articles such as References [37,38]). In general, aqueous and non-aqueous sol-gel chemical methods must be differentiated Reference [39]. Non-aqueous sol-gel processes are performed in organic solvents. In contrast, the aqueous alternative is described as the polymerization reaction of a dissolved metal chloride, nitrate, or sulfate (also of an organic compound, [30]). Hence, for the purpose of this short contribution, reference is made to the aqueous method as a comparative analogue for hydrogeochemical processes in sedimentary basins. There are several physical and chemical parameters in aqueous sol-gel experiments that are key variables for the formation of brookite (and anatase). Such parameters may thus also control their formation and aggregation in black shales and resemble in (i) pH, (ii) ionic strength, and (iii) addition of acetic acid (and others, e.g. oxalic acid, fulvic/humic acid, etc). around 4000 m /mol (thus for smallest particles), transform into brookite [35]. At a crossover size o 6. Industrial Production of Titania Polymorphs The pH of the aqueous solution is a major control for brookite formation in sol-gel processes. Pottier et al. [40] showed that brookite exclusively forms from a strongly acidic aqueous 0.05 mol/dm3 TiCl4 solution at 100 ◦C, and that higher TiCl4 concentrations favor rutile precipitation. Brookite is thus the major phase experimentally obtained in the range 17 ≤Cl:Ti ≥35 with 0.15 mol dm3 Ti. The Cl:Ti molar ratio thus controls the kind of generated crystalline phases and their relative proportions, but also the particle size. Moreover, a suitable ratio also avoids recrystallisation of brookite into rutile during ageing in suspension. As a result of these investigations, the complex Ti(OH)2(Cl)2(OH2)2 is suggested to be the precursor of the brookite phase and dissolved chloride ions stabilize brookite in suspension. Li et al. [41] also demonstrated a phase-selective synthesis of titania nanocrystals at 180 ◦C (3 h) at pH lower than 2 and observed that nano-brookite was formed by a controlled coupling of pH and TiCl3 concentrations. In general, anatase formed at high pH. Bhave and Lee [42] synthesized nanocrystalline brookite particles at pH 2 using dissolved TiCl4 with isopropanol as the co-solvent in hydrochloric acid. The resultant gel mass was peptized and crystallized under reﬂuxing condition for 15 h at 83 ◦C to a pure brookite phase. However, brookite also formed at 70 ◦C and 100 ◦C, but often together with rutile and/or anatase. The addition of hydroxypropyl cellulose yielded a more uniform particle size along with decreasing agglomeration tendencies. This addition of organic compounds plays a further key role in the precipitation of TiO2 nanocrystals via aqueous sol-gel syntheses. For example, adsorption of humic acids and zeta potential are known to be dependent on pH and thus control aggregation. At low pH the amount of adsorbed humic or fulvic acids is high and thus lowers aggregation [43,44]. Moreover, synthesis by the sol-gel route using ethylene glycol as a gelling agent results in 19 nm-sized anatase [45]. Other organic gelation agents are ammonia [46] or citric acid and acetyl acetone as complex agents [47]. However, there is a variety of organic compounds used as solvents, surfactants, or additives in sol-gel methods to synthesize titania particles [48]. ChemEngineering 2019, 3, 28 8 of 11 In a series of papers about sol-gel synthesis of titania nanoparticles by Isley and Penn [49,50] and Isley et al. around 4000 m /mol (thus for smallest particles), transform into brookite [35]. At a crossover size o 6. Industrial Production of Titania Polymorphs [51,52], the effects of pH and ionic strength on the growth of brookite and anatase, as well as their contents and particle sizes were studied. In general, anatase is the primary product of such sol-gel syntheses at pH lower than 0. Brookite content and particle sizes can be controlled by varying the pH of the sol-gel synthesis. Sol-gel methodological experiments under controlled aqueous conditions at room temperature showed that titanium dioxide primary particles of 4–5 nm in size readily form stable aggregates with an average diameter of 50–60 nm at pH ∼4.5 in a NaCl suspension adjusted to an ionic strength of 0.0045 M [53]. A rapid formation of micron-sized aggregates occurs by solely increasing ionic strength to 0.0165 M, but also at pH values up to 8.2. Similar results may be achieved by applying CaCl2 suspensions and indicate that divalent cations may enhance aggregation of nano-TiO2 in soils and surface waters. In summary, the ﬁndings of industrial processes, mainly over the sol-gel route, provide general insights into controls of nano-brookite formation under controlled conditions in the laboratory. Natural processes are more complex and change in time and space, but general ﬁndings may serve to better understand brookite formation in black shale. Funding: This research received no external funding. Funding: This research received no external funding. Funding: This research received no external funding. Acknowledgments: The author is grateful to Richard Wirth and Anja Schreiber for their guidance and efforts put into FIB foil preparation and TEM analyses over many years and especially during our joint cooperation on titania polymorphs. Conﬂicts of Interest: The authors declare no conﬂict of interest. 7. Conclusions and Outlook Hydrogeochemical processes in micro-environments of black shale can completely differ from processes of the bulk rock. The controlling factors and reasons for this difference is locally lacking permeability. This situation leads to geochemical processes in semi-closed systems as spots with localized and differing hydrogeochemical conditions. The formation of nano-sized titania polymorphs serves as one example for processes in micro-environments and suggests that sol-gel processes take place in black shale. However, similar processes may occur whenever comparable conditions are caused by the co-occurrence of water, dissolved carbon dioxide or acetic acid, and solid phases with low stability against pH changes of the pore water. The dissolution of carbon dioxide and acetic acid in pore water leads to lower pH and, in the following, to the dissolution of matrix solids. However, a mandatory prerequisite is an isolated space, small or large, to keep pH low. There is a broad spectrum of sedimentary environments in which similar factors may control sol-gel processes in isolated micro-environments, e.g. oil–water contacts in oil ﬁelds [10], or during barite precipitation at the sulphate–methane transition zone (SMT; [54]). However, the controlling factors may also prevail in non-sedimentary environments, e.g. in volcanic or seismically active areas [55], where aqueous ﬂuids are in contact with organics. References 1. Mondol, N.H.; Bjørlykke, K.; Jahren, J.; Høeg, K. 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https://openalex.org/W4250238710	https://www.qeios.com/read/QQHH0A/pdf	English	null	MicroRNA 509-5p	Definitions	2,020	cc-by	71	Qeios · Definition, February 2, 2020 Open Peer Review on Qeios Open Peer Review on Qeios MicroRNA 509-5p National Cancer Institute National Cancer Institute Qeios ID: QQHH0A · https://doi.org/10.32388/QQHH0A Source National Cancer Institute. MicroRNA 509-5p. NCI Thesaurus. Code C142769. A 21 ribonucleotide sequence that is a final product of the processing of either MIR509-1 pre-miRNA or MIR509-2 pre-miRNA. This oligonucleotide may be involved in the negative regulation of gene expression. 1/1
https://openalex.org/W2912701107	https://europepmc.org/articles/pmc6358104?pdf=render	English	null	Identification of the X-linked germ cell specific miRNAs (XmiRs) and their functions	PloS one	2,019	cc-by	13,752	RESEARCH ARTICLE Hiromitsu Ota1,2, Yumi Ito-Matsuoka1, Yasuhisa MatsuiID1,2,3* 1 Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Miyagi, Japan, 2 The Japan Agency for Medical Research and Development-Core Research for Evolutional Science and Technology (AMED-CREST), Chuo-ku, Tokyo, Japan, 3 Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi, Japan * yasuhisa.matsui.d3@tohoku.ac.jp a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Abstract MicroRNAs (miRNAs) play a critical role in multiple aspects of biology. Dicer, an RNase III endonuclease, is essential for the biogenesis of miRNAs, and the germ cell-specific Dicer1 knockout mouse shows severe defects in gametogenesis. How miRNAs regulate germ cell development is still not fully understood. In this study, we identified germ cell-specific miR- NAs (miR-741-3p, miR-871-3p, miR-880-3p) by analyzing published RNA-seq data of mouse. These miRNA genes are contiguously located on the X chromosome near other miRNA genes. We named them X chromosome-linked miRNAs (XmiRs). To elucidate the functions of XmiRs, we generated knockout mice of these miRNA genes using the CRISPR/ Cas9-mediated genome editing system. Although no histological abnormalities were observed in testes of F0 mice in which each miRNA gene was disrupted, a deletion covering miR-871 and miR-880 or covering all XmiRs (ΔXmiRs) resulted in arrested spermatogenesis in meiosis in a few seminiferous tubules, indicating their redundant functions in spermatogen- esis. Among candidate targets of XmiRs, we found increased expression of a gene encoding a WNT receptor, FZD4, in ΔXmiRs testis compared with that in wildtype testis. miR-871-3p and miR-880-3p repressed the expression of Fzd4 via the 30-untranslated region of its mRNA. In addition, downstream genes of the WNT/β-catenin pathway were upregulated in ΔXmiRs testis. We also found that miR-871, miR-880, and Fzd4 were expressed in sper- matogonia, spermatocytes and spermatids, and overexpression of miR-871 and miR-880 in germ stem cells in culture repressed their increase in number and Fzd4 expression. Previous studies indicated that the WNT/β-catenin pathway enhances and represses proliferation and differentiation of spermatogonia, respectively, and our results consistently showed that stable β-catenin enhanced GSC number. In addition, stable β-catenin partially rescued reduced GSC number by overexpression of miR-871 and miR-880. The results together suggest that miR-871 and miR-880 cooperatively regulate the WNT/β-catenin pathway during testicular germ cell development. OPEN ACCESS OPEN ACCESS Citation: Ota H, Ito-Matsuoka Y, Matsui Y (2019) Identification of the X-linked germ cell specific miRNAs (XmiRs) and their functions. PLoS ONE 14(2): e0211739. https://doi.org/10.1371/journal. pone.0211739 Editor: Jean-Pierre Rouault, Centre de Recherche en Cancerologie de Lyon, FRANCE Received: October 4, 2018 Accepted: January 18, 2019 Published: February 1, 2019 Copyright: © 2019 Ota et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. OPEN ACCESS Citation: Ota H, Ito-Matsuoka Y, Matsui Y (2019) Identification of the X-linked germ cell specific miRNAs (XmiRs) and their functions. PLoS ONE 14(2): e0211739. https://doi.org/10.1371/journal. pone.0211739 Editor: Jean-Pierre Rouault, Centre de Recherche en Cancerologie de Lyon, FRANCE Received: October 4, 2018 Accepted: January 18, 2019 Published: February 1, 2019 Copyright: © 2019 Ota et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Citation: Ota H, Ito-Matsuoka Y, Matsui Y (2019) Identification of the X-linked germ cell specific miRNAs (XmiRs) and their functions. PLoS ONE 14(2): e0211739. https://doi.org/10.1371/journal. pone.0211739 Editor: Jean-Pierre Rouault, Centre de Recherche en Cancerologie de Lyon, FRANCE Editor: Jean-Pierre Rouault, Centre de Recherche en Cancerologie de Lyon, FRANCE Copyright: © 2019 Ota et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Identification of the X-linked germ cell specific miRNAs (XmiRs) and their functions Hiromitsu Ota1,2, Yumi Ito-Matsuoka1, Yasuhisa MatsuiID1,2,3* * yasuhisa.matsui.d3@tohoku.ac.jp Introduction the Japan Agency for Medical Research and Development8https://www.amed.go.jp/en/index. html). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Germ cells first arise as primordial germ cells (PGCs) in early embryos [1], and they proliferate and migrate into the genital ridges during embryonic development [2]. After arriving at the genital ridges, male germ cells enter G0 mitotic arrest and differentiate into prospermatogonia, which resume proliferation after birth [3, 4]. Subsequently, they further differentiate into sper- matogonial stem cells (SSCs), and a subpopulation of SSCs starts the first wave of spermato- genesis [5, 6]. Spermatogenesis is a highly complex differentiation process, during which gene expression is highly orchestrated and strictly regulated [7]. Competing interests: The authors have declared that no competing interests exist. Competing interests: The authors have declared that no competing interests exist. In addition to transcriptional regulation, post-transcriptional regulation also plays an important role during spermatogenesis. MicroRNAs (miRNAs) are small non-coding RNAs 18–23 nucleotides in length that play a critical role in the regulation of development and differ- entiation in many organisms and different tissues [8, 9]. For example, miR-125a modulates self-renewal of hematopoietic stem cells and protects them from apoptosis [10]. miR-1 and miR-206 inhibit cell proliferation and promote myoblast differentiation [11]. miR-29 has mul- tiple activities at different stages of osteoblast differentiation [12]. miRNAs repress gene expression by binding the 30-untranslated region (UTR) of their target mRNAs, thereby decreasing mRNA stability and translational efficiency [13]. Dicer1 encodes an RNase III endonuclease, which is a key enzyme for the biogenesis of miRNAs. Germ cell-specific Dicer1 knockout affects PGC proliferation, spermatogenesis, and fertility. For example, specific removal of Dicer1 in male germ cells with Ddx4-Cre results in deficient transition from the leptotene to zygotene stage in meiosis, increased apoptosis in the pachytene stage, and morphological defects in spermatozoa [14, 15]. Germ cell-specific knock- out mice of Drosha, which encodes an RNase III endonuclease that processes primary miRNAs to pre-miRNAs, with germ cell-specific Stra8-Cre also leads to progressive loss of pachytene spermatocytes and spermatids [16]. In addition, previous studies reported that various miR- NAs were expressed in germ cells and are involved in their development [17]. For instance, survival and/or proliferation [18–22], and differentiation [23–25] of SSCs are controlled by several miRNAs via inhibiting their target gene expression. miRNAs function in the spermatogenesis Introduction However, miRNAs-mediated regu- lation of complex spermatogenic processes have not been fully understood. WNT/β-catenin signaling is a highly conserved pathway that is essential for embryonic development and cellular differentiation. For example, WNT5A stimulates the proliferation and self-renewal of hematopoietic stem cells in vitro [26], and WNT10B plays an important role in bone development [27]. In the WNT/β-catenin signaling pathway, WNTs bind to members of the Frizzled (FZD) family of receptors and form a stable ligand-receptor complex [28, 29]. This complex phosphorylates the intra-cellular Dishevelled protein and inhibits glyco- gen synthase kinase-3β, which induces ubiquitination and subsequent degradation of β-cate- nin via phosphorylation; thereby, WNTs reduce degradation of β-catenin [30]. Hence, cytoplasmic levels of β-catenin rise, and β-catenin translocates to the nucleus where it associ- ates with T-cell factor/lymphoid enhancer binding factor transcription factors to activate the expression of downstream genes [31, 32]. WNT/β-catenin signaling stimulates proliferation of SSCs but represses their differentiation potential [33–35], and is also involved in later stages of spermatogenesis [36–38]. WNT/β-catenin signaling is a highly conserved pathway that is essential for embryonic development and cellular differentiation. For example, WNT5A stimulates the proliferation and self-renewal of hematopoietic stem cells in vitro [26], and WNT10B plays an important role in bone development [27]. In the WNT/β-catenin signaling pathway, WNTs bind to members of the Frizzled (FZD) family of receptors and form a stable ligand-receptor complex [28, 29]. This complex phosphorylates the intra-cellular Dishevelled protein and inhibits glyco- gen synthase kinase-3β, which induces ubiquitination and subsequent degradation of β-cate- nin via phosphorylation; thereby, WNTs reduce degradation of β-catenin [30]. Hence, cytoplasmic levels of β-catenin rise, and β-catenin translocates to the nucleus where it associ- ates with T-cell factor/lymphoid enhancer binding factor transcription factors to activate the expression of downstream genes [31, 32]. WNT/β-catenin signaling stimulates proliferation of SSCs but represses their differentiation potential [33–35], and is also involved in later stages of spermatogenesis [36–38]. In this study, we identified germ cell-specific, X chromosome-linked miRNAs (XmiRs; miR- 741-3p, miR-871-3p, miR-880-3p). We generated knockout mice of these miRNA genes. The phenotype of the mice suggested that miR-871-3p and miR-880-3p were functionally redundant and that their deficiency caused spermatogenic failure by abnormally activating the WNT/β-cate- nin signaling pathway. In vitro studies by using germ stem cells (GSCs) revealed that WNT/β- catenin signaling was under the control of miR-871-3p and miR-880-3p to regulate GSC number. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. Funding: H.O. was supported by a Grant-in-Aid (KAKENHI) for Young Scientists (B) (grant #JP16K20908) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (http://www.mext.go.jp/en/index.htm). Y.M. was supported by KAKENHI in the Innovative Areas, “Mechanisms regulating gamete formation in animals” (grant #16H06530) from MEXT, and by AMED-CREST (grant #JP17gm0510017h) from 1 / 26 PLOS ONE \| https://doi.org/10.1371/journal.pone.0211739 February 1, 2019 PLOS ONE \| https://doi.org/10.1371/journal.pone.0211739 February 1, 2019 Construction of gRNA expression vectors and preparation of gRNAs and Cas9 mRNA Target sequences of gRNAs were selected by using a web program CRISPRdirect (https:// crispr.dbcls.jp/) [43]. DNA oligonucleotides, which have targeting sequences with BsaI cutting sites at 50end were synthesized by Fasmac Co., Ltd. (Atsugi, Japan) and were annealed. The annealed oligonucleotides were cloned into BsaI site of a gRNA expression vector pDR274 [44]. The oligonucleotide sequences for gRNA constructs were shown in S9 Table. gRNAs were transcribed from the DraI-digested gRNA expression vectors as templates by using MEGAshortscript kit (Invitrogen AM1354). The Cas9 mRNA was transcribed from SalI- digested Cas9 expression vector, pSP64-hCas9, by using mMESSAGE mMACHINE SP6 Tran- scription Kit (Invitrogen AM1340). Following completion of transcription, the samples were treated by DNase I according to the manufacturer’s instructions. Both the gRNA and the Cas9-encoding mRNA were purified by MEGA clear Transcription Clean-Up Kit (Invitrogen AM1908). Purified RNAs were concentrated by ethanol precipitation and re-dissolved in Opti-MEM I Reduced Serum Medium (Gibco 31985062). miRNA sequence analysis Small RNA sequence data using in this study are as follows [40–42]: GSE40499; Brain (SRR553582), Cerebellum (SRR553583), Heart (SRR553584), Kidney (SRR553585), Testes (SRR553586), GSE52950; ES cells (SRR1042095, SRR1042096, SRR1042097), MEF cells (SRR1042098, SRR1042099), GSE59254; PGC (SRR15097510), Spermatogonia (SRR1509750), Spermatozoa (SRR1509748). Cutadapt (Ver 1.8.3) was used to clip adapter sequences from raw small RNAs sequencing data. Prinseq (Ver. 0.20.4) was then used to filter low quality reads out from clipped reads. The processed reads were aligned to mouse-genomic reference (mm10) or miRBase 21 reference by using aligner program Bowtie (Ver 1.1.2). Heatmaps were built using the ‘regHeatmap’ function of the ‘Heatplus’ package of R. Animals MCH and B6D2F1 (C57BL/6 x DBA2 F1) were purchased from CLEA Japan Inc. and Japan SLC., respectively. Oct4-deltaPE-GFP [39] transgenic mice were maintained in a C57BL/6J genetic background. All animal care and experiments were carried out in according to the guidelines for experimental animals defined by the facility, the Animal Unit of the Institute of Development, Aging and Cancer (Tohoku University). Animal protocols were reviewed and approved by the Tohoku University Animal Studies Committee. Introduction PLOS ONE \| https://doi.org/10.1371/journal.pone.0211739 February 1, 2019 2 / 26 miRNAs function in the spermatogenesis Generating XmiR-deficient mice by genome editing Fertilized eggs were collected from B6D2F1 females mated with Oct4-deltaPE-GFP transgenic male mice in mWM medium (ARK Resource), and were washed with Opti-MEM I Reduced Serum Medium (Gibco 31985062) three times. Eggs were then subjected to electroporation according to a described method [45]. Eggs were aligned in a line in an electrode chamber filled with Opti-MEM I Reduced Serum Medium containing 400 ng of Cas9 mRNA and 200 ng of gRNA (total 5 μl). A condition of electroporation was at 30 V (3 msec ON + 97 msec OFF) × 7 times. After electroporation, the eggs were immediately collected from the electrode and washed with mWM medium three times, and then cultured overnight in mWM medium at 37˚C and 5% CO2 incubator. Two-cell embryo were collected and transplanted to pseudo- pregnant MCH mice. 3 / 26 PLOS ONE \| https://doi.org/10.1371/journal.pone.0211739 February 1, 2019 miRNAs function in the spermatogenesis Histological examination Testes were fixed in Bouin at 4˚C overnight. The fixed testes were dehydrated in a graded series of ethanol (70% to 100%), cleared in xylene, and embedded in paraffin wax. Embedded tissue samples were sectioned at a thickness of 5 μm and then mounted on slides. These sec- tions were deparaffinized with xylene two times, rehydrated with a graded series of ethanol (100% to 70%) and distilled water, and then stained with hematoxylin for 10 min. Sections were rinsed with water for 20 min, stained with 1% eosin for 5min, and then rinsed with water briefly. These sections were dehydrated with a graded series of ethanol (70% to100%) followed by xylene two times and mounted with Permount (Falma). Diameter of seminiferous tubules that were round or nearly round were measured by using Image-J software. Prediction of target genes of XmiRs Candidate target mRNAs of each XmiR and of neighboring miRNA were predicted by the three web programs (miRDB (http://www.mirdb.org), TargetScan (http://www.targetscan. org), and microT-CDS (http://diana.imis.athena-innovation.gr/DianaTools/index.php?r= microT_CDS/)) based on complementarity of sequences between miRNAs and 3’-UTR of mRNAs[46–48]. Candidate target mRNAs selected by at least one program were applied for further analysis of hierarchical clustering based on similarity of their target sequences by hclust in R program. To predict common target mRNAs of miR-871-3p and miR-880-3p, their target mRNAs predicted by at least the two programs were selected, and common mRNAs between target mRNAs of miR-871-3p and miR-880-3p were identified. PLOS ONE \| https://doi.org/10.1371/journal.pone.0211739 February 1, 2019 Immunostaining Testes of WT and ΔXmiRs mice were fixed with 2% paraformaldehyde for overnight and embedded with Optimum Cutting Temperature (O.C.T.) compound (Sakura Finetek 4583). The embedded samples were sectioned using Cryostat CM1900 (Leica) with a section thick- ness of 10 μm. The sectioned samples were permeabilized and blocked in 5% bovine serum albumin (BSA) and 1% Triton X-100 in PBS for 1 h at room temperature. The sections were then incubated with the primary antibodies diluted by 1% BSA and 0.1% Triton X-100 in PBS overnight at 4˚C, and were incubated with the secondary antibodies in the same buffer with 1 μg/ml DAPI for 2 h at 4˚C. Samples were washed for 5 min × 3 by 0.1% Triton X-100 in PBS after the primary and the secondary antibody treatments. Samples were mounted with VEC- TASHIELD (VECTOR H-1000) and observed with confocal laser scan microscope TCS SP8 (Leica). The primary antibodies were: SCP3 (abcam ab15093, 1:100), PLZF (Santa Cruz Bio- technology sc-28319, 1:50), γH2AX (Upstate 05–636, 1:100), Fzd4 (abcam ab83042, 1:100) and β-catenin (Cell signaling technology #8480, 1:100). The secondary antibodies were Goat anti- mouse secondary antibody, Alexa Fluor 647 (Invitrogen A-21235 1:500), Goat anti-mouse sec- ondary antibody, Alexa Fluor 568 (Invitrogen A-11031 1:500), Goat anti-rabbit secondary antibody, Alexa Fluor 568 (Invitrogen A-11011 1:500) and Goat anti-rabbit secondary anti- body, Alexa Fluor 488 (Invitrogen A-11034 1:500). Images were acquired under a Leica TCS SP8 confocal microscope. For quantification of β-catenin expression, the Leica Application Suite X program (Leica microsystems, Buffalo Grove, IL, USA) was used for analyzing pixel intensity for β-catenin in a region of interest (ROI) after background subtraction. Mean of pixel intensities in nucleus or cytoplasm of a germ cell was normalized by that of one to three Leydig cells of the same fields. The definition of sub-stages of spermatocytes in prophase I was based on localized patterns of SCP3 [49]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0211739 February 1, 2019 4 / 26 miRNAs function in the spermatogenesis Luciferase reporter assay Full length 3’UTR of Fzd4 gene was amplified from mouse genomic DNA by PCR, cloned into an XhoI-NotI site downstream of a luciferase reporter gene in psiCHECK2 vector (Promega C8021). 1–927 or 927–2263 region of Fzd4 3’UTR were amplified from a cloned WT Fzd4 3’UTR. For Fzd4 3’UTR delta, a set of over-lapping oligo-DNA primers were designed around a target site of the miRNA, in which the target sequence itself is deleted, in Fzd4 3’UTR. Fragments of Fzd4 3’UTR up-stream and down-stream to the target site were amplified from a cloned WT Fzd4 3’UTR by using the target site primers and outer primers. PCR products were then annealed in the overlapping primer regions and amplified full length Fzd4 3’UTR in which the target sites were deleted, by using the outer primers. miR-871 and miR-880 genes were amplified from mouse genomic DNA by PCR, cloned into CSII-EF-MCS vector [50] by using In-Fusion cloning kit (Clontech 639648). A luciferase reporter vector and a miRNA expression vector were co-trans- fected in HEK293T human embryonic kidney cells. HEK293T cells were maintained in DMEM (Gibco 11965092) supplemented with 10% FBS. Luciferase activity was measured by using Dual- Luciferase Reporter Assay System (Promega E1910) 48 hour after transfection. The synthetic Renilla luciferase activity was normalized to synthetic firefly luciferase activity for each sample. Total RNA isolation, semi-quantitative RT-PCR of miRNAs and Quantitative RT-PCR Total RNA containing small RNAs was purifies by using the miRNeasy kit (Qiagen 217004), according to the manufacturer’s instructions. For semi-quantitative RT-PCR of miRNAs, total RNA was polyadenylated for 15 min at 37˚C in a 5 μl reaction mixture containing 2 U poly(A) polymerase (New England BioLabs M0276). 5 μl volume of polyadenylated RNA was then incubated at 65˚C for 5 minutes with 3 pmol oligo(dT)-RACE primer and 1μl 10 mM dNTPs (Roche 11969064001) in a reaction volume of 13 μl. Following addition of 200 U SuperScript III (Invitrogen 18080051), 1 μl RNasin Plus RNase Inhibitor (Promega N2611), 1 μl 0.1 M DTT and 4 μl 5X RT buffer, the reaction was incubated at 50˚C for 1 hour followed by 70˚C for 15 minutes. Semi-quantitative RT-PCR reaction was performed with Power SYB Green PCR Master Mix (Applied Biosystems 4367659) in a 20 μl reaction containing 5 pmol forward and reverse primer, 0.1 μl cDNA template and 10 μl Power SYBR Green PCR Master Mix and the cycling conditions; 50˚C for 2 min, 95˚C for 10 min; 40 cycles (95˚C, 15 sec; 60˚C, 1 min). The amplified products were separated on a 3% agarose gel and visualized by ethidium bro- mide staining. U6 snRNA was used as an internal control. For quantitative RT-PCR, 5 μl vol- ume of total RNA was incubated at 65˚C for 5 minutes with 0.3 μl of Random primers (Promega C118A) and 1μl 10 mM dNTPs (Roche 11969064001) in a reaction volume of 13 μl. Following addition of 200 U SuperScript III (Invitrogen 18080051), 1 μl RNasin Plus RNase Inhibitor (Promega N2611), 1 μl 0.1 M DTT and 4 μl 5X RT buffer, the reaction was incubated at 50˚C for 1 hour followed by 70˚C for 15 minutes. PCR reaction were performed with Power SYB Green PCR Master Mix (Applied Biosystems 4367659) in a 20 μl reaction containing 5 pmol forward and reverse primer, 0.1 μl cDNA template and 10 μl of Power SYBR Green PCR Master Mix. PCR signals were detected using CFX Connect (Bio-Rad) and the cycling condi- tions; 50˚C for 2 min, 95˚C for 10 min; 40 cycles (95˚C, 15 sec; 60˚C, 1 min). Arbp was used as an internal control. All primers used in this study were listed in S9 Table. Purification of stage-specific spermatogenic cells by FACS sorting Dissociation of testicular cells and subsequent staining was carried out based on a described method [51]. Testes were dissected from B6 mice at 10 to 12 weeks of age. After removing PLOS ONE \| https://doi.org/10.1371/journal.pone.0211739 February 1, 2019 5 / 26 miRNAs function in the spermatogenesis albuginea, testes were incubated for 25 min in 6 ml of Gey’s Balanced Salt Solution (GBSS; Sigma-Aldrich G9779) containing 1.2 mg/ml of Collagenase Type I (Sigma-Aldrich C0130) at 32˚C, and seminiferous tubules were dissociated. Interstitial cells were removed by filtration with a 40 μm Cell strainer (Falcon 352340). Seminiferous tubes retained on the filter were col- lected and incubated at 32˚C for 25 min in the same collagenase-containing buffer as that used for the first step. Cell aggregates were sheared gently by 10 times of pipetting with wide orifice plastic transfer pipet and filtered through 40 μm Cell strainer to remove cell clumps. Cells were washed with GBSS and then resuspended in GBSS containing 1% FBS. Two million cells were diluted in 2 ml of GBSS containing 1% FBS and stained with 5 μg/ml of Hoechst 33342 (Invi- trogen H3570) for 1 h at 32˚C. Cells were kept on ice and protected from light until sorting. Before sorting, 10 μl of propidium iodide (Becton-Dickinson 51-66211E) was added to the stained cells and filtered through 40 μm Cell strainer. Sorting was performed on a Becton- Dickinson FACSAria II cell sorter. Total RNA was purified from 1 x106 cells of round sperma- tids and 1 x 105 cells of other population in testes by using the miRNeasy kit (Qiagen 217004), according to the manufacturer’s instructions. PLOS ONE \| https://doi.org/10.1371/journal.pone.0211739 February 1, 2019 Identification of germ cell-specific miRNAs To identify germ cell-specific miRNAs, we compared the expression of miRNAs among somatic tissues, cell lines, and germ cells by using published small RNA-seq data of mouse [40–42]. The proportion of miRNAs to the total reads in germ cells was less than that in somatic tissues (S1 Table). In addition to miRNAs, other small RNAs, such as Piwi-interacting (pi) RNAs that maintain genomic quality were contained in the small RNA-seq data of germ cells [56]. Cluster analysis showed that the expression profiles of these miRNAs were classified into three groups, i.e., those highly expressed in somatic tissues or mouse embryonic fibro- blasts (MEFs), in spermatogenic cells, and in PGCs and embryonic stem (ES) cells (Fig 1A). Of the 20 most abundantly expressed miRNAs in PGCs, many miRNAs were also highly expressed in ES cells (Fig 1B and S2 Table). miR-741-3p, miR-871-3p, and miR-880-3p were highly expressed in PGCs and spermatogonia, but were rarely expressed in ES cells or somatic tissues. The results suggest that these miRNAs have germ cell-specific functions. Interestingly, these miRNA genes were located on the X chromosome in contiguity with other miRNA genes (Fig 1C). Their high expression in testes was confirmed with RT-qPCR (Fig 1D). We named these three miRNAs X chromosome-linked miRNAs (XmiRs). Overexpression of miR-871, miR-880 and stable β-catenin in GSCs Virus particles were collected by centrifuging the cultured medium at 2,330 × g for 30 minutes at 4˚C after incubating with PEG6000 solution [final 2.5% PEG6000 (Wako), 100 mM NaCl, 10 mM HEPES (pH 7.4)] overnight at 4˚C, and they were re-suspended in GS medium [54] (StemPro-34 SFM (Gibco 10640–019) supplemented with StemPro-34 Nutrient Supplement (Gibco 10641–025), 25 μg/ml insulin, 100 μg/ml transferrin, 60 μM putrescine (Sigma P5780), 30 nM sodium selenite (Sigma S9133), 6 mg/ml D-(+)-glucose (Gibco A24940), 30 μg/ml pyruvic acid (Gibco 11360070)), 1 μl/ml DL-lactic acid (Sigma 69785), 5 mg/ml bovine albumin (Sigma A3311), 2 mM L-glutamine, 50 μM 2-mercaptoethanol (Sigma M3148)), 1 x minimal essential medium (MEM) vitamin solution (Gibco 11120052), 1 x MEM nonessential amino acid solution (Gibco 11140050), 100μM ascorbic acid (Sigma A4544), 10 μg/ml d-biotin (Sigma B4639), 30 ng/ml β-estradiol, 60 ng/ml progesterone (Sigma P6149), 20 ng/ml mouse epidermal growth factor, 10 ng/ml human basic fibroblast growth PLOS ONE \| https://doi.org/10.1371/journal.pone.0211739 February 1, 2019 6 / 26 miRNAs function in the spermatogenesis factor (Sigma F0291), 10 ng/ml recombinant rat glial cell line-derived neurotrophic factor (GDNF) and 1% fetal calf serum). GSCs (DBAGS) [54] were cultured in GS medium on MEF feeder cells. Infection of the lentivirus vectors to GSCs were carried out as described previously with some modifications. Lentivirus was infected to GS cells at 1 x 104 lentiviral particle/cell [55] and incu- bated at 37˚C with 5% CO2 overnight. The culture medium containing the transfection mixture was discarded and replaced with GS culture medium containing 500 ng/ml puromycin 16–24 h after infection. GSCs were collected by trypsin treatment at day 4, 6, 8 and 10 after the virus infec- tion and cell number were counted. GSCs were collected by trypsin treatment at day 8 and total RNA was purified by using the miRNeasy kit (Qiagen 217004), according to the manufacturer’s instructions. All primers used in this study were listed in S9 Table. Overexpression of miR-871, miR-880 and stable β-catenin in GSCs miR-871 and miR-880 genes were amplified from mouse genomic DNA by PCR, cloned into an AgeI-EcoRI site of pLKO1 vector [52]. For construction of stable β–catenin, amino acid substitu- tions of S33A, S37A, T41A, and S45A to prevent phosphorylation by GSK3 [53] were introduced. A set of over-lapping oligo-DNA primers were designed around the mutation site. Fragments of stable β–catenin up-stream and down-stream to the mutation site were amplified from mouse cDNA by using the target site primers (up-stream: Ctnnb1-AAAA-Rev, down-stream: Ctnnb1-AAAA-Fw) and outer primers (up-stream: Kozak-Ctnnb1-Fw-for-CS2-EF-MCS, down- stream: Ctnnb1-Rev-for-CS2-EF-MCS). An up-stream fragment of stable β–catenin was annealed to Ctnnb1 AAAA AS oligo, which is partially overlapped with Ctnnb1-AAAA-Rev and Ctnnb1-AAAA-Fw. It was then amplified with Kozak-Ctnnb1-Fw-for-CS2-EF-MCS primer, which results in an elongated up-stream fragment covering all mutation sites. The elongated up- stream fragment was then annealed with the downstream fragment in the overlapping regions, and full length stable β–catenin was amplified by using the outer primers (Kozak-Ctnnb1-Fw-for- CS2-EF-MCS and Ctnnb1-Rev-for-CS2-EF-MCS). PCR product was cloned into CSII-EF-MCS vector [50] by using In-Fusion cloning kit (Clontech 639648). Lentivirus particles were produced as described previously. Briefly, pLKO1 (miR-971, miR-880)- or CSII-EF-MCS (stable-β–catenin)- lentivirus vector, pCMV-VSV-G-RSV-Rev and pCAG-HIVgp [50] were co-transfected into HEK293T cells by the calcium phosphate method. After 48 hours, cell supernatant containing lenti- viral particles was collected, and virus concentration was determined by using Lenti-X qRT-PCR Titration Kit (Clontech 631235) according to the manufacturer’s instructions. PLOS ONE \| https://doi.org/10.1371/journal.pone.0211739 February 1, 2019 miRNAs function in the spermatogenesis Fig 1. The expression profile of miRNAs in various tissues and cell lines. (A) A heat map of hierarchical clustering of miRNAs detected in small RNA-seq data used in this study. (B) A heat map of 20 miRNAs highly expressed in PGCs. Relative miRNA expression is described according to the color scale. Red and green indicate high and low expression, respectively. Mouse embryonic fibroblasts (MEFs), embryonic stem (ES) cells, primordial germ cells (PGCs), spermatogonia (SPG), spermatozoa (SPZ). (C) The locus of XmiR genes on the X chromosome. (D) The expression of XmiRs in testes, ES cells, and MEFs determined by quantitative RT-PCR. Each expression level was normalized to the expression of U6 snRNA. The expression in ES cells was set as 1.0. Error bars show standard errors of three biological replicates. P < 0.01. Fig 1. The expression profile of miRNAs in various tissues and cell lines. (A) A heat map of hierarchical clustering of miRNAs detected in small RNA-seq data used in this study. (B) A heat map of 20 miRNAs highly expressed in PGCs. Relative miRNA expression is described according to the color scale. Red and green indicate high and low expression, respectively. Mouse embryonic fibroblasts (MEFs), embryonic stem (ES) cells, primordial germ cells (PGCs), spermatogonia (SPG), spermatozoa (SPZ). (C) The locus of XmiR genes on the X chromosome. (D) The expression of XmiRs in testes, ES cells, and MEFs determined by quantitative RT-PCR. Each expression level was normalized to the expression of U6 snRNA. The expression in ES cells was set as 1.0. Error bars show standard errors of three biological replicates. P < 0.01. https://doi.org/10.1371/journal.pone.0211739.g001 few seminiferous tubules at 8 and 24 weeks of age (Fig 3E and 3F), suggesting that XmiRs act in a functionally redundant manner. To test this possibility, we examined target mRNAs of each XmiR and other miRNAs nearby shown in Fig 1C by using three web programs (miRDB (http://www.mirdb.org), Tar- getScan (http://www.targetscan.org), and microT-CDS (http://diana.imis.athena-innovation. gr/DianaTools/index.php?r=microT_CDS/)) that predict targets of miRNAs by evaluating each potential miRNA-target mRNA pair based on complementarity of a seed sequence and target sequences [46–48]. Hierarchical clustering analysis showed that the predicted target mRNAs of XmiRs converged on the same clusters (Fig 4A), and a portion of their target mRNAs actually overlapped (Fig 4B and S3 Table). To further investigate the functional redundancy of XmiRs, we generated mutant mice with a large deletion covering all three XmiR genes (ΔXmiRs) (Fig 4C and 4D). We confirmed that ΔXmiRs region did not contain any protein coding genes. We obtained three independent mutant mouse lines (OT84, OT87, and OT100), which showed identical testicular abnormali- ties. Expression of the XmiRs was not detected in the testes of a ΔXmiRs mouse with RT-PCR (Fig 4E). ΔXmiRs mice grew normally, and their fertility was indistinguishable from that of WT or heterozygous littermates (S4 Table). As in mice with deficiency of a single XmiR, testis weight was not affected but seminiferous tubules were thinner in ΔXmiRs compared with those in WT (S1C and S1D Fig). However, in the testes of ΔXmiRs mice, a few abnormal semi- niferous tubules were observed at 8 weeks of age, and the number of defective seminiferous tubules increased with age (Fig 4F and S5 Table). Similar to miR-871/miR-880 doubly mutated mice, fewer germ cells were present in the abnormal seminiferous tubules. In the severely affected seminiferous tubules, germ cells were rarely observed, while a few germ cells were found in the mildly affected tubules (Fig 4F, 30 weeks). To determine the spermatogenic stages affected in ΔXmiRs mice, we performed immunos- taining of testes of WT and ΔXmiRs mice using an antibody against Synaptonemal complex protein 3 (SCP3), a component of the synaptonemal complex [63], and an antibody against Promyelocytic leukemia zinc finger protein (PLZF), which is required for self-renewal of undifferentiated SSCs [64, 65]. Number of SSCs, leptotene, zygotene and pachytene spermato- cytes was not significantly changes in the mildly affected seminiferous tubules of ΔXmiR mice, but diplotene spermatocytes were decreased, and spermatids which are observed as cells with condensed nuclei following DAPI staining as well as spermatozoa were rarely found (Fig 5A and 5B). We also examined the expression of γH2AX (phosphorylated histone H2AX), and found dot-like signals likely representing localization on XY body in pachytene spermatocytes in ΔXmiRs as well as WT testes (S2 Fig). This result suggests that spermatogenesis was arrested in meiosis in the abnormal seminiferous tubules of ΔXmiR mice. Defective spermatogenesis in XmiR-deficient mice To examine the functions of XmiRs in germ cells, we generated knockout mice for each XmiR gene with the CRISPR/Cas9-mediated genome editing system [57]. gRNAs were designed in the Dicer processing site [58], seed sequence [59], and Drosha processing site [60] for miR- 741, miR-871, and miR-880, respectively. In the ΔmiR-741 mouse, a deletion occurred in the Dicer processing site that shortened the predicted terminal loop and stem. Drosha has a strong preference for pri-miRNA hairpin structures with a large (>10 nucleotides) terminal loop, suggesting that this deletion causes a severe defect in miR-741-3p production (Fig 2A) [61]. In the ΔmiR-871 mouse, the seed sequence was completely deleted, suggesting that miR-871-3p cannot recognize its target mRNA (Fig 2B) [59]. In the ΔmiR-880 mouse, the Drosha process- ing site was deleted, and the lower stem that binds to DGCR8 was shortened, suggesting that the ΔmiR-880 mouse cannot generate functional pre-miRNA (Fig 2C) [62]. Testis weight (S1A Fig) and spermatogenesis evaluated by histological analysis (Fig 3A–3D) revealed no differ- ences between wildtype (WT) and mutant testes in which a single XmiR gene was deficient, though seminiferous tubules were thinner in testes of ΔmiR-741 and ΔmiR-871 (S1B Fig). Among these XmiR gene-deficient mice, we obtained two mice (OT15 and OT124) in which miR-871 and miR-880 were doubly mutated by chance due to loss of the seed sequence in miR-871-3p and shortening of the hairpin structure of miR-880-3p due to deletion of the Drosha target site (Fig 2D and 2E). These two mice showed abnormal spermatogenesis in a PLOS ONE \| https://doi.org/10.1371/journal.pone.0211739 February 1, 2019 7 / 26 miRNAs function in the spermatogenesis PLOS ONE \| https://doi.org/10.1371/journal.pone.0211739 February 1, 2019 8 / 26 https://doi.org/10.1371/journal.pone.0211739.g001 Identification of target genes of XmiRs Because ΔXmiRs mice showed similar abnormalities as those in miR-871/miR-880 double mutant mice (Figs 3E and 3F and 4F), miR-871/miR-880 but not miR-741 are likely important for spermatogenesis. Therefore, we attempted to identify common target genes of miR-871-3p and miR-880-3p. We first selected putative target mRNAs of miR-871-3p and miR-880-3p by PLOS ONE \| https://doi.org/10.1371/journal.pone.0211739 February 1, 2019 9 / 26 miRNAs function in the spermatogenesis miRNAs function in the spermatogenesis Fig 2. Predicted altered secondary structures of XmiR precursors by genome editing. Secondary structures of predicted miRNA precursors of (A) miR-741 WT (left) and ΔmiR-741 (OT16) (right), (B) miR-871 WT (left) and ΔmiR-871 (OT17) (right), (C) miR-880 WT (left) and ΔmiR-880 (OT49) (right), and (D, E) miR-871 (top) and miR-880 (bottom) in ΔmiR-871/ΔmiR-880 in two different mice, OT15 (D) and OT124 (E). Fig 2. Predicted altered secondary structures of XmiR precursors by genome editing. Secondary structures of predicted miRNA precursors of (A) miR-741 WT (left) and ΔmiR-741 (OT16) (right), (B) miR-871 WT (left) and ΔmiR-871 (OT17) (right), (C) miR-880 WT (left) and ΔmiR-880 (OT49) (right), and (D, E) miR-871 (top) and miR-880 (bottom) in ΔmiR-871/ΔmiR-880 in two different mice, OT15 (D) and OT124 (E). Fig 2. Predicted altered secondary structures of XmiR precursors by genome editing. Secondary structures of predicted miRNA precursors of (A) miR-741 WT (left) and ΔmiR-741 (OT16) (right), (B) miR-871 WT (left) and ΔmiR-871 (OT17) (right), (C) miR-880 WT (left) and ΔmiR-880 (OT49) (right), and (D, E) miR-871 (top) and miR-880 (bottom) in ΔmiR-871/ΔmiR-880 in two different mice, OT15 (D) and OT124 (E). https://doi.org/10.1371/journal.pone.0211739.g002 https://doi.org/10.1371/journal.pone.0211739.g002 using the same web programs used for the analysis in Fig 4A, and chose mRNAs commonly selected by at least two of the three web programs as promising candidates. We consequently identified 46 common target mRNAs of miR-871-3p and miR-880-3p by comparing their tar- gets (S3 Fig and S6 Table). We next used RT-qPCR to examine the expression of those target genes in the testes of WT and ΔXmiR mice. Among the candidates, the expression of Fzd4, Mllt3, and Stox2 was signifi- cantly increased in the testes of ΔXmiR mice compared with that in WT testes, and Chdh and Magix also tended to be upregulated (Figs 6A and S4). Several studies have reported the contri- bution of the WNT/β-catenin signaling pathway to spermatogenesis [33–38]. Fzd4 encodes a WNT receptor. Because no previous reports have shown roles for Mllt3, Stox2, or Magix in spermatogenesis, and the only known function of Chdh is involvement in sperm motility [66], we focused on Fzd4 for further analysis. Fig 3. Spermatogenesis in mice with mutations in miR-741, miR-871, and miR-880. PLOS ONE \| https://doi.org/10.1371/journal.pone.0211739 February 1, 2019 PLOS ONE \| https://doi.org/10.1371/journal.pone.0211739 February 1, 2019 10 / 26 PLOS ONE \| https://doi.org/10.1371/journal.pone.0211739 February 1, 2019 Hematoxylin-eosin (HE)- stained sections of seminiferous tubules in the testes of (A) WT, (B) ΔmiR-741 (OT16), (C) ΔmiR-871 (OT17), (D) ΔmiR-880 (OT49), and ΔmiR-871/ΔmiR-880 (E) (OT15) mice at 8 weeks of age, and (F) ΔmiR-871/ΔmiR-880 (OT124) mouse at 24 weeks of age. The arrowheads show abnormal seminiferous tubules. Scale bars = 100 μm. https://doi.org/10.1371/journal.pone.0211739.g003 Fig 3. Spermatogenesis in mice with mutations in miR-741, miR-871, and miR-880. Hematoxylin-eosin (HE)- stained sections of seminiferous tubules in the testes of (A) WT, (B) ΔmiR-741 (OT16), (C) ΔmiR-871 (OT17), (D) ΔmiR-880 (OT49), and ΔmiR-871/ΔmiR-880 (E) (OT15) mice at 8 weeks of age, and (F) ΔmiR-871/ΔmiR-880 (OT124) mouse at 24 weeks of age. The arrowheads show abnormal seminiferous tubules. Scale bars = 100 μm. https://doi.org/10.1371/journal.pone.0211739.g003 11 / 26 PLOS ONE \| https://doi.org/10.1371/journal.pone.0211739 February 1, 2019 Inhibition of luciferase (luc)-Fzd4-30-UTR reporter gene expression by XmiRs We performed a luc assay to investigate whether miR-871 and miR-880 directly repressed Fzd4 expression. To predict the miRNA target site, we used DIANA tools in the microT-CDS pro- gram (http://diana.imis.athena-innovation.gr/DianaTools/index.php?r=microT_CDS/) [48], and found one and two candidate target sites for miR-871-3p and miR-880-3p, respectively (Fig 6C). Expression vectors for miR-871 or miR-880 were co-transfected with a luc reporter containing the Fzd4-30-UTR into HEK293T cells. miR-871 and miR-880 repressed luc activity (Fig 6D and 6E). We then used a luc reporter containing the Fzd4-30-UTR in which a miR- 871-3p target site was deleted (Fzd4-30-UTR ΔmiR-871). In this case, luc activity was not repressed by miR-871 (Fig 6D), indicating that miR-871-3p inhibited the expression of the reporter via its target site. Because the Fzd4-30-UTR has two candidate target sites for miR- 880-3p, we co-transfected miR-880 with luc reporters containing a part of the Fzd4-30-UTR that has each candidate target site. miR-880 significantly repressed luc reporters with each frag- ment of the Fzd4-30-UTR. However, miR-880 also repressed luc activity from reporter vectors with Fzd4-30-UTR fragments in which putative miR-880-3p target sites were deleted (Fzd4-30- UTR 1–927 ΔmiR-880; Fzd4-30-UTR 927–2263 ΔmiR-880) (Fig 6E). The results suggest that miR-880 targets other sequences in the Fzd4-30-UTR that were not predicted by the program. miRNAs function in the spermatogenesis PLOS ONE \| https://doi.org/10.1371/journal.pone.0211739 February 1, 2019 12 / 26 miRNAs function in the spermatogenesis Fig 4. Relationship between target mRNAs of XmiRs and generation of ΔXmiRs mice. (A) A dendrogram of hierarchical clustering analysis of target mRNAs of XmiRs and their neighboring miRNAs. (B) Venn diagram showing the relationship among putative target mRNAs of miR-741-3p, miR-871-3p, and miR-880-3p. Corresponding gene lists are shown in S3 Table. (C) A schematic presentation of the WT and ΔXmiRs locus. gR-741 and gR-871 represent positions of guide RNAs used for genome editing. (D) Representative PCR for genotyping of WT and ΔXmiRs (OT84) mice. Arrows in the right panel represent primers used for PCR. (E) Expression of XmiRs in WT and a ΔXmiRs testes (F2 of OT84) determined with semi-quantitative RT-PCR analysis. U6 snRNA was used as an internal control. (F) HE-stained sections of seminiferous tubules in WT (left) and ΔXmiR (F2 of OT100) (right) testes at 8, 12, 16, and 30 weeks of age. The second and fourth panels for 30 weeks show higher magnification views corresponding to the rectangular area in the first and third panels. Lower two panels show mildly affected seminiferous tubules. Arrowheads show abnormal seminiferous tubules. Scale bar = 50 μm (8, 12, 16 weeks), 200 μm (30 weeks, lower magnification), 100 μm (30 weeks, higher magnification). https://doi.org/10.1371/journal.pone.0211739.g004 Because the expression of Fzd4 was increased in ΔXmiRs mouse testes, the expression of downstream genes of WNT/β-catenin signaling is also likely upregulated in ΔXmiRs mouse testes. To investigate this possibility, we used RT-qPCR to examine the expression of previ- ously reported possible downstream genes of WNT/β-catenin signaling in the testes of WT and ΔXmiR mice. Four of the five tested downstream genes were significantly increased, and c- Myc also tended to be upregulated in ΔXmiRs testes (Fig 6B). We also found that the expres- sion of β-catenin protein was increased both in cytoplasm and nucleus in some SSCs and sper- matocytes in ΔXmiRs testes compared with that in WT testes (S5 Fig). It suggests that β- catenin protein is stabilized to transmit signal in ΔXmiR testis. These results suggest that XmiRs repress WNT/β-catenin signaling via repression of Fzd4 expression. The expression of XmiRs and Fzd4 in spermatogenic cells To determine the stages of spermatogenic cells in which miR-871, miR-880, and Fzd4 are expressed, we purified testicular germ cells at different spermatogenic stages with fluorescence- activated cell sorting (FACS) (Fig 7A). We first confirmed the purity of the sorted germ cells by examining the expression of stage-specific germ cell marker genes (Fig 7B). Spermatogonia-spe- cific Gfra1 [67], spermatocyte-specific Scp3 [63] and Rad21l [68], and spermatid-specific Acrv1 [69] showed the expected expression levels in each cell fraction. The expression of Fzd4 mRNA increased from spermatogonia to the preleptotene-zygotene stage and then decreased from the pachytene stage onward (Fig 7C). Meanwhile, FZD4 protein was apparently upregulated in PLOS ONE \| https://doi.org/10.1371/journal.pone.0211739 February 1, 2019 13 / 26 miRNAs function in the spermatogenesis 0 1371/j l 0211739 F b 1 2019 14 / 26 PLOS ONE \| https://doi.org/10.1371/journal.pone.0211739 February 1, 2019 14 / 26 miRNAs function in the spermatogenesis Fig 5. Spermatogenesis was arrested at meiotic prophase in abnormal seminiferous tubules in ΔXmiR testes. (A) Testis sections were co-stained with anti-SCP3 (green) and anti-PLZF (cyan) antibodies in WT and ΔXmiRs (F2 generation of the OT100 line) mice at 12 weeks of age. The second and fourth column show higher magnification views corresponding to the rectangular area in the pictures in the first and third columns. Mildly affected seminiferous tubules in ΔXmiR testis shown in Fig 4F are presented. Spermatogonia (yellow arrowheads), leptotene spermatocytes (white arrowheads), pachytene spermatocytes (white arrows), and round spermatids (yellow arrows) are indicated. Scale bars = 50 μm (the first and third columns) and 25 μm (the second, and fourth columns). (B) Number of cells at different spermatogenic stages determined by staining for Scp3 and Plzf in WT and mildly affected seminiferous tubules in ΔXmiR testis. Cells in two sections in a single mouse of WT and ΔXmiR testes were counted. Vertical lines in the graphs indicate means. P < 0.05 and P < 0.01. https://doi.org/10.1371/journal.pone.0211739.g005 pachytene spermatocytes (S6 Fig), suggesting its translational regulation by XmiRs. On the other hand, miR-871-3p was expressed from spermatogonia to leptotene-zygotene spermato- cytes, but its expression decreased after the pachytene stage onward (Fig 7D). miR-880-3p was expressed in spermatogonia, and its expression decreased at the onset of meiosis (Fig 7E). miR- 871-3p and miR-880-3p in spermatogenic cells likely play a role in adjusting Fzd4 expression levels and subsequent WNT/β-catenin signaling levels that are suitable for proper development of spermatogonia and spermatocytes. Overexpression of XmiRs represses growth and/or survival of germ stem cells (GSCs) To examine functions of XmiRs in SSCs, we overexpressed XmiRs in a GSC line in culture [54]. We confirmed that the expression of miR-871 and miR-880 was upregulated at 8 days after infection of lenti-virus vectors (Fig 8A). The expression of Fzd4 was repressed by overex- pression of miR-880 and/or miR-871, and those miRNAs additively repressed Fzd4 expression (Fig 8B). The number of GSCs was unchanged when expression vectors of miR-871-3p and/or miR-880-3p were infected, whereas GSCs increased in number with an empty vector (Fig 8C and 8D). The results suggest that excess miR-871-3p and miR-880-3p repress proliferation and/or survival of GSCs. We then tested whether WNT/β-catenin signaling was under the control of XmiRs in GSCs. We co-transfected an expression vector of stable β-catenin [53] with an expression vec- tor of miR-871 or miR-880. Stable β-catenin alone enhanced GSC number, indicating that WNT/ β-catenin signaling stimulates proliferation and/or survival of GSCs (Fig 8E and 8F). In addition, GSCs also increased in number by stable β-catenin with the expression of miR-871 or miR-880. Although decreased GSCs by miR-871 or miR-880 were not completely recovered by stable β-catenin, ratios of increased GSCs by stable β-catenin with miR-871 or miR-880 was higher compared with those with a control XmiR vector (S7 Table). It suggests that stable β- catenin partially rescues the influence of the XmiRs in GSCs, implying that WNT/β-catenin signaling functions under the control of XmiRs. Discussion In this study, we identified miR-741-3p, miR-871-3p, and miR-880-3p as being highly and preferentially expressed in germ cells. The genes for these three miRNAs are clustered on the X chromosome. In mice, 28.1% of miRNA genes form clusters on various chromosomes [70]. miRNA gene clusters may arise by de novo formation of miRNA-like hairpin structures in existing primary miRNA transcript units or by tandem duplication of a single miRNA gene [71]. XmiRs and additional miRNA gene clusters nearby may also be generated by similar molecular mechanisms. A single mRNA is likely targeted by multiple miRNAs due to the short length of seed sequences. Several studies proposed that miRNAs in the same gene cluster are often PLOS ONE \| https://doi.org/10.1371/journal.pone.0211739 February 1, 2019 15 / 26 miRNAs function in the spermatogenesis Fig 6. Upregulation of WNT/β-catenin signaling genes in testes of ΔXmiRs mice, and repression of Fzd4 by XmiRs. (A, B) Relative expression of the putative common target genes of miR-871-3p and miR-880-3p (A) and of the downstream genes of WNT/β-catenin signaling (B) in the testes of WT and ΔXmiRs (F2 of the OT84 line) mice at 12 weeks of age was determined by quantitative RT-PCR analysis. The expression in WT testis was set as 1. (C) Potential target sites of miR-871-3p and miR-880-3p in Fzd4-30-UTR. (D, E) Relative luciferase activity from the reporter vectors with Fzd4-30-UTR with or Fig 6. Upregulation of WNT/β-catenin signaling genes in testes of ΔXmiRs mice, and repression of Fzd4 by XmiRs. (A, B) Relative expression of the putative common target genes of miR-871-3p and miR-880-3p (A) and of the downstream genes of WNT/β-catenin signaling (B) in the testes of WT and ΔXmiRs (F2 of the OT84 line) mice at 12 weeks of age was determined by quantitative RT-PCR analysis. The expression in WT testis was set as 1. (C) Potential target sites of miR-871-3p and miR-880-3p in Fzd4-30-UTR. (D, E) Relative luciferase activity from the reporter vectors with Fzd4-30-UTR with or Fig 6. Upregulation of WNT/β-catenin signaling genes in testes of ΔXmiRs mice, and repression of Fzd4 by XmiRs. (A, B) Relative expression of the putative common target genes of miR-871-3p and miR-880-3p (A) and of the downstream genes of WNT/β-catenin signaling (B) in the testes of WT and ΔXmiRs (F2 of the OT84 line) mice at 12 weeks of age was determined by quantitative RT-PCR analysis. miRNAs function in the spermatogenesis without expression vectors for miR-871 (D) or miR-880 (E). Fzd4-30-UTR ΔmiR-871; Fzd4-30-UTR with deleted miR-871-3p target site (D). Fzd4-30-UTR 1–927 or 927–2263 with or without ΔmiR-880; 1–927 or 927–2263 bp fragment of Fzd4-30-UTR with or without deleted miR-880-3p target sites (E). Luciferase activity was measured 48 h after transfection. Luciferase activity with an empty expression vector was set as 1. Error bars represent standard errors of three biological replicates. P < 0.05 and P < 0.01. without expression vectors for miR-871 (D) or miR-880 (E). Fzd4-30-UTR ΔmiR-871; Fzd4-30-UTR with deleted miR-871-3p target site (D). Fzd4-30-UTR 1–927 or 927–2263 with or without ΔmiR-880; 1–927 or 927–2263 bp fragment of Fzd4-30-UTR with or without deleted miR-880-3p target sites (E). Luciferase activity was measured 48 h after transfection. Luciferase activity with an empty expression vector was set as 1. Error bars represent standard errors of three biological replicates. P < 0.05 and P < 0.01. https://doi.org/10.1371/journal.pone.0211739.g006 functionally correlated, and those miRNAs exert cooperative and/or redundant repressive effects on the same target genes. For example, members of the miR-17-92 gene cluster are highly conserved among vertebrates, and cooperatively regulate transforming growth factor (TGF)-β signaling and cell cycle regulation; miR-17 and miR-20a directly target TGF-β recep- tor II mRNA, whereas miR-18a targets the mRNA of Smad2 and Smad4, two members of the TGF-β signaling pathway [72]. In addition, miR-17 and miR-20a cooperatively modulate the expression of E2F1 [73], a member of the E2F family of transcription factors that play a central Fig 7. Expression of XmiRs and Fzd4 in spermatogenic cells. (A) Scatter plot of FACS for testicular cells stained with Hoechst 33342. Spermatogonia (SPG), pre-leptotene (PreL), leptotene-zygotene (L/Z), pachytene-diplotene (P/D), round spermatids (RS). (B-E) Relative expression of stage-specific germ cell marker genes (B), Fzd4 (C), miR-871-3p (D), and miR-880-3p (E) in FACS-purified spermatogenic cells. Gfra1 for spermatogonia, Scp3 and Rad21l for spermatocytes, Acrv1 for spermatids. Gene expression was determined by RT-qPCR. Error bars represent standard errors of three biological replicates. P < 0.01. h //d i /10 13 1/j l 0211 39 00 Fig 7. Expression of XmiRs and Fzd4 in spermatogenic cells. (A) Scatter plot of FACS for testicular cells stained with Hoechst 33342. Spermatogonia (SPG), pre-leptotene (PreL), leptotene-zygotene (L/Z), pachytene-diplotene (P/D), round spermatids (RS). (B-E) Relative expression of stage-specific germ cell marker genes (B), Fzd4 (C), miR-871-3p (D), and miR-880-3p (E) in FACS-purified spermatogenic cells. without expression vectors for miR-871 (D) or miR-880 (E). Fzd4-30-UTR ΔmiR-871; Fzd4-30-UTR with deleted miR-871-3p target site (D). Fzd4-30-UTR 1–927 or 927–2263 with or without ΔmiR-880; 1–927 or 927–2263 bp fragment of Fzd4-30-UTR with or without deleted miR-880-3p target sites (E). Luciferase activity was measured 48 h after transfection. Luciferase activity with an empty expression vector was set as 1. Error bars represent standard errors of three biological replicates. P < 0.05 and P < 0.01. Discussion The expression in WT testis was set as 1. (C) Potential target sites of miR-871-3p and miR-880-3p in Fzd4-30-UTR. (D, E) Relative luciferase activity from the reporter vectors with Fzd4-30-UTR with or PLOS ONE \| https://doi.org/10.1371/journal.pone.0211739 February 1, 2019 16 / 26 Gfra1 for spermatogonia, Scp3 and Rad21l for spermatocytes, Acrv1 for spermatids. Gene expression was determined by RT-qPCR. Error bars represent standard errors of three biological replicates. P < 0.01. https://doi.org/10.1371/journal.pone.0211739.g007 PLOS ONE \| https://doi.org/10.1371/journal.pone.0211739 February 1, 2019 17 / 26 miRNAs function in the spermatogenesis Fig 8. Roles of miR-871, miR-880 and stable β-catenin in GSCs. (A, B) Relative expression of miR-871 and miR-880 (A) and Fzd4 (B) in GSCs overexpressing XmiRs at 8 days after infection with the lenti-virus vectors. Gene expression was determined with RT-qPCR. (C, D) The effect of XmiRs overexpression in GSCs in culture. The pLKO1 empty vector was used as the control. Cell number at day 4 to day 10 after infection with the lenti-virus vector (C), and ratios of GSC number at day10 compared with that at day4 (D) are shown. (E, F) The effect of co-overexpression of stable β-catenin and miR-871 or miR-880. The pLKO1 empty vector and CSII-EF-MCS vector were used as the control. Cell number at day4 Fig 8. Roles of miR-871, miR-880 and stable β-catenin in GSCs. (A, B) Relative expression of miR-871 and miR-880 (A) and Fzd4 (B) in GSCs overexpressing XmiRs at 8 days after infection with the lenti-virus vectors. Gene expression was determined with RT-qPCR. (C, D) The effect of XmiRs overexpression in GSCs in culture. The pLKO1 empty vector was used as the control. Cell number at day 4 to day 10 after infection with the lenti-virus vector (C), and ratios of GSC number at day10 compared with that at day4 (D) are shown. (E, F) The effect of co-overexpression of stable β-catenin and miR-871 or miR-880. The pLKO1 empty vector and CSII-EF-MCS vector were used as the control. Cell number at day4 PLOS ONE \| https://doi.org/10.1371/journal.pone.0211739 February 1, 2019 18 / 26 miRNAs function in the spermatogenesis to day10 after infection with the indicated lenti-virus vectors (E), and ratios of GSC number at day10 compared with that at day4 (F) are shown. Error bars represent standard errors of three biological replicates. P < 0.05 and P < 0.01. to day10 after infection with the indicated lenti-virus vectors (E), and ratios of GSC number at day10 compared with that at day4 (F) are shown. Error bars represent standard errors of three biological replicates. P < 0.05 and P < 0.01. https://doi.org/10.1371/journal.pone.0211739.g008 role in the regulation of G1 to S phase progression [74]. miR-20a also targets both E2F2 and E2F3 [75]. In the case of XmiRs, we found that miR-871 and miR-880 had redundant functions in spermatogenesis via repression of the expression of a WNT signaling molecule, FZD4. ΔXmiR mice showed subtle abnormalities in spermatogenesis. Although the expression was upregulated in ΔXmiRs testes, the increase of Fzd4 expression was marginal (Fig 6A). The results together suggest that additional miRNAs redundantly target Fzd4. Consistent with this idea, we found other miRNAs in addition to XmiRs, which are predicted to target the Fzd4 30- UTR, among miRNAs expressed in testes (S7 Fig and S8 Table). WNT/β-catenin signaling is an important pathway involved in proliferation and differenti- ation of stem cells in many different tissue types including testicular germ cells. Regarding the WNT receptor gene in testis, Fzd2, Fzd3, Fzd7, and Fzd8 are expressed in spermatogonia [37], whereas we found that Fzd4 was expressed in spermatogonia, spermatocytes, and spermatids (Fig 7C), suggesting functions for FZD4 in those cells. Previous in vitro studies have shown that WNT3A and WNT5A promote proliferation of SSCs [33, 34]. Consistent with this obser- vation, conditional knockout of the β-catenin gene in testicular germ cells with Axin2-Cre sup- presses proliferation of undifferentiated spermatogonia [35], and we also consistently found that stable β-catenin enhanced GSC number in culture (Fig 8E and 8F). However, WNT/β- catenin signaling-activated SSCs show reduced SSC activity after transplantation into seminif- erous tubules [34]. Constitutive activation of β-catenin expression in testicular germ cells in transgenic mice causes progressive loss of spermatocytes and spermatids and reduction of mei- otic germ cells from the leptotene to pachytene stages [38]. Taken together, the results indicate that WNT/β-catenin signaling enhances proliferation of SSCs, but represses their differentiation. We showed that XmiRs targeted Fzd4 mRNA and downstream genes of WNT/β-catenin signaling were upregulated in ΔXmiR testes (Fig 6A and 6B) in which a few spermatocytes but no spermatids were observed in some seminiferous tubules (Fig 5). The results together sug- gest that deficiency in XmiRs results in abnormal enhancement of WNT/β-catenin signaling in spermatogenic cells, which may cause impaired meiosis. Whether or not X-linked miRNAs escape meiotic sex chromosome inactivation (MSCI) is controversial [76–78], but we found the expression of miR-871-3p was downregulated in pachytene/diplotene spermatocytes (Fig 7D), suggesting that it suffers silencing at least in some extent. Meanwhile, sex bodies associat- ing with MSCI was observed in remaining pachytene spermatocytes in ΔXmiR testes (S2 Fig), suggesting MSCI occurs in ΔXmiR spermatocytes. Although the testicular abnormalities in ΔXmiR mice were subtle, the mice showed progres- sively more severe testicular abnormalities with age (Fig 4F and S5 Table). PLOS ONE \| https://doi.org/10.1371/journal.pone.0211739 February 1, 2019 In addition, it is also likely that XmiRs repress additional WNT/β-catenin signaling related genes other than Fzd4. According to this prediction, we found that Dixdc1 encoding Disheveled-2-associated protein [80] and Tbl1xr1 encoding β-catenin-associated protein [81] are potential targets of XmiRs (S6 Table). Previous studies showed functions of miRNAs in SSCs. For instance, proliferation of SSCs is stimulated by miR-20 and miR-106 via repressing the expression of Ccnd1 (Cyclin D1) and Stat3 (signal transducer and activator of transcription 3) [18], and by miR-224 via repressing Dmrt1 (Doublesex and mab-3 related transcription factor 1) [19], while survival of SSCs is sup- ported by miR-146 via repressing Med1 (Mediator of RNA polymerase II transcription subunit 1) encoding a retinoic acid receptor associating protein [20]. In addition, miR-202 [24] and miR-221/222 [25] maintain SSCs in undifferentiated status via repressing Rbfox2 (RNA bind- ing fox-1 homolog 2) and unknown direct targets, respectively. We found that XmiRs repressed GSCs to increase in number in culture, and repression of a WNT receptor, FZD4 and of possible additional targets by XmiR may be involved. The results together suggest that SSCs are maintained by the functions of miRNAs via various molecular pathways. Previous studies showed functions of miRNAs in SSCs. For instance, proliferation of SSCs is stimulated by miR-20 and miR-106 via repressing the expression of Ccnd1 (Cyclin D1) and Stat3 (signal transducer and activator of transcription 3) [18], and by miR-224 via repressing Dmrt1 (Doublesex and mab-3 related transcription factor 1) [19], while survival of SSCs is sup- ported by miR-146 via repressing Med1 (Mediator of RNA polymerase II transcription subunit 1) encoding a retinoic acid receptor associating protein [20]. In addition, miR-202 [24] and miR-221/222 [25] maintain SSCs in undifferentiated status via repressing Rbfox2 (RNA bind- ing fox-1 homolog 2) and unknown direct targets, respectively. We found that XmiRs repressed GSCs to increase in number in culture, and repression of a WNT receptor, FZD4 and of possible additional targets by XmiR may be involved. The results together suggest that SSCs are maintained by the functions of miRNAs via various molecular pathways. In the normal context, appropriate levels of WNT/β-catenin signaling molecules are crucial for spermatogonia and spermatocytes, and miR871-3p and miR-880-3p in those cells may con- tribute to fine tuning of the levels of WNT/β-catenin signaling activity via regulation of Fzd4 expression. The expression of Fzd4 mRNA was decreased at zygotene-pachytene transition in meiosis (Fig 7C), while FZD4 protein expression was upregulated in pachytene/diplotene sper- matocytes and round spermatids (S6 Fig). With regard to the expression of XmiRs, miR-880- 3p and miR-871-3p was downregulated in preleptotene and pachytene/diplotene spermato- cytes, respectively (Fig 7D and 7E). The results suggest that miR-880-3p is involved in de-stabi- lization of Fzd4 mRNA, while miR-871-3p may repress translation of Fzd4 mRNA. It is likely that Fzd4 mRNA starts to translate immediately after downregulation of miR-871-3p at zygo- tene-pachytene transition, and FZD4 protein may stably persist in pachytene spermatocytes onwards. Identification of downstream molecules of WNT/β-catenin signaling that directly function in spermatogenesis is an important subject for future studies. Constitutive activa- tion of β-catenin in testicular germ cells in transgenic mice results in progressive loss of spermatogenic cells, in which fewer than 5% of seminiferous tubules are defective in spermato- genesis at 13 weeks of age. The ratio of defective tubules increases to more than 40% of total tubules at 75 weeks of age [38]. These data suggest that the influences of abnormal activation of WNT/β-catenin signaling by XmiR deficiency may gradually accumulate in some germ cells with aging. Those germ cells may undergo abnormal meiosis when the influences from acti- vated WNT/β-catenin signaling exceed a threshold. Functions of other miRNAs in meiosis in vivo have not been described well. miR-17-92 knockout mouse showed abnormal spermato- genesis with reduced number of sperm, though detailed mechanisms including its target mRNAs were not reported [79]. 19 / 26 PLOS ONE \| https://doi.org/10.1371/journal.pone.0211739 February 1, 2019 miRNAs function in the spermatogenesis We also found that overexpression of miR-871 and/or miR-880 in GSCs repressed the increase in number, and Fzd4 expression (Fig 8A–8D). In addition, stable β-catenin rescued decreased GSC number by miR-871 or miR-880 (Fig 8E and 8F and S7 Table). These results suggest that control of the expression levels of Fzd4 and subsequent WNT/β-catenin signaling activity by miR-871-3p and miR-880-3p are critical for proliferation and/or survival of GSCs. Meanwhile, repression of GSCs by XmiRs was not completely rescued by stable β-catenin (Fig 8E and 8F), suggesting that XmiRs repress the expression of additional targets other than WNT/β-catenin signaling related genes to regulate GSCs. In addition, it is also likely that XmiRs repress additional WNT/β-catenin signaling related genes other than Fzd4. According to this prediction, we found that Dixdc1 encoding Disheveled-2-associated protein [80] and Tbl1xr1 encoding β-catenin-associated protein [81] are potential targets of XmiRs (S6 Table). We also found that overexpression of miR-871 and/or miR-880 in GSCs repressed the increase in number, and Fzd4 expression (Fig 8A–8D). In addition, stable β-catenin rescued decreased GSC number by miR-871 or miR-880 (Fig 8E and 8F and S7 Table). These results suggest that control of the expression levels of Fzd4 and subsequent WNT/β-catenin signaling activity by miR-871-3p and miR-880-3p are critical for proliferation and/or survival of GSCs. Meanwhile, repression of GSCs by XmiRs was not completely rescued by stable β-catenin (Fig 8E and 8F), suggesting that XmiRs repress the expression of additional targets other than WNT/β-catenin signaling related genes to regulate GSCs. Supporting information S1 Fig. Testis weight and diameter of seminiferous tubules of XmiR-deficient mouse. (A, C) Testis weight / body weight of ΔmiR-741 (OT16; n = 2), ΔmiR-871 (OT17; n = 2) and ΔmiR-880 (OT49; n = 2) (A), of ΔXmiRs (OT84; n = 6) (B), and of their wildtype littermates (n = 6 for A and n = 6 for C) at 8 weeks of age for A and 12 weeks of age for C. (B, D) Diameter of seminiferous tubules of ΔmiR-741 (OT16; n = 1), ΔmiR-871 (OT17; n = 1) and ΔmiR-880 (OT49; n = 1) (B), of ΔXmiRs (OT84; n = 3) (D), and of their wildtype littermates (n = 1 for B and n = 3 for D) at 8 weeks of age for B and 12 weeks of age for D. Fifteen seminiferous tubules in each section and three sections of each mouse were measured. P < 0.05 and P < 0.01. (TIF) S2 Fig. Localization of γH2AX in spermatocytes in ΔXmiR testes. Testis sections were co- stained by anti-SCP3 (red) and anti-γH2AX (cyan) antibodies in WT and ΔXmiRs (F2 generation 20 / 26 PLOS ONE \| https://doi.org/10.1371/journal.pone.0211739 February 1, 2019 miRNAs function in the spermatogenesis of OT100) mice. Arrowheads show spermatocytes of the indicated stages. The fourth column shows higher magnification views corresponding to the rectangular area in the pictures in the third columns. Scale bars = 50 μm (the third columns), 25 μm (the first, second and fourth col- umns). (TIF) S3 Fig. Venn diagram showing the relationship of putative target mRNAs of miR-871-3p and miR-880-3p. Corresponding gene lists are shown in S6 Table. (TIF) S3 Fig. Venn diagram showing the relationship of putative target mRNAs of miR-871-3p and miR-880-3p. Corresponding gene lists are shown in S6 Table. (TIF) S4 Fig. The expression of the putative common target genes of miR-871-3p and miR-880- 3p in the testes of WT and ΔXmiRs mice. Relative expression of the putative common target genes of miR-871-3p and miR-880-3p in the testes of WT and ΔXmiRs (F2 of the OT84 line) mice at 12 weeks of age was determined by quantitative RT-PCR analysis. The expression in WT testis was set as 1. Error bars represent standard errors of three biological replicates. (TIF) S5 Fig. The expression of β-catenin in ΔXmiR testes. (A) Sections of WT and ΔXmiR (OT84) testes at 12 weeks of age were co-stained by anti- β-catenin (red) and anti-Plzf (cyan) antibod- ies. The second and fourth column show higher magnification views corresponding to the rectangular area in the pictures in the first and third columns. Arrows and arrowheads show Plzf-positive SSCs with intense and faint fluorescence, respectively, for β-catenin. Scale bars = 50 μm (the first, the third columns), 25 μm (second and fourth columns). (B) Quantita- tive estimation of the expression of β-catenin protein in Plzf-positive SSCs in WT and ΔXmiR testes. Relative signal intensity in nucleus and cytoplasm of SSCs compared with that in Leydig cells is shown. Four and eleven Plzf-positive cells in a single ΔXmiR and WT mouse, respec- tively, were measured. P < 0.01. (TIF) S6 Fig. The expression of FZD4 in WT testes. Testis sections were co-stained by anti-SCP3 (red) and anti-FZD4 (green) antibodies in WT. The second and fourth column show higher magnification views corresponding to the rectangular area in the pictures in the first and third columns. White arrowheads: leptotene spermatocytes, yellow arrowheads: zygotene spermato- cytes, white arrows: pachytene spermatocytes, yellow arrows: diplotene spermatocytes. Scale bars = 50 μm (the first, the third columns), 25 μm (second and fourth columns). (TIF) S6 Fig. The expression of FZD4 in WT testes. Testis sections were co-stained by anti-SCP3 (red) and anti-FZD4 (green) antibodies in WT. The second and fourth column show higher magnification views corresponding to the rectangular area in the pictures in the first and third columns. White arrowheads: leptotene spermatocytes, yellow arrowheads: zygotene spermato- cytes, white arrows: pachytene spermatocytes, yellow arrows: diplotene spermatocytes. Scale bars = 50 μm (the first, the third columns), 25 μm (second and fourth columns). Acknowledgments We would like to thank Dr. Takashi Shinohara for sharing GS cells, Asuka Takehara for main- tenance of mouse colonies, all the members of the Cell Resource Center for Biomedical Research for helpful discussions, the Center of Research Instruments in the Institute of Devel- opment, Aging, and Cancer and Biomedical Research Unit of Tohoku University Hospital for use of instruments and technical support. S6 Table. Lists of putative target mRNAs of miR-871-3p and miR-880-3p (corresponding to S3 Fig). (XLSX) S7 Table. Relative number of GSCs with the expression vector of stable β-catenin com- pared with those with control expression vector. Values for GSCs with miR-871, miR-880 and control miR expression vectors based on the data in Fig 8F are shown. (DOCX) S8 Table. miRNAs that are predicted to target the Fzd4 3’-UTR expressed in germ cells (corresponding to S7 Fig). Read counts of each miRNA normalized to reads per million (RPM) were shown. ES: embryonic stem cell, mouse embryonic fibroblasts (MEFs), PGCs: pri- mordial germ cells, SPG: spermatogonia, SPZ: spermatozoa. (DOCX) S9 Table. List of primers used in this study. (DOCX) S9 Table. List of primers used in this study. (DOCX) S3 Fig. Venn diagram showing the relationship of putative target mRNAs of miR-871-3p and miR-880-3p. Corresponding gene lists are shown in S6 Table. (TIF) (TIF) S7 Fig. A heat map of miRNAs highly expressed in testis or spermatogonia. Relative miRNA expression is described according to the color scale. Red and green indicate high and low expression, respectively. Mouse embryonic fibroblasts (MEFs), embryonic stem (ES) cells, primordial germ cells (PGCs), spermatogonia (SPG), spermatozoa (SPZ). (TIF) S1 Table. Small RNA-seq data used for this study. ES: embryonic stem cells, MEFs: mouse embryonic fibroblasts, PGCs: primordial germ cells. (DOCX) S2 Table. Top 20 miRNAs highly expressed in PGCs (corresponding to Fig 1B). Read counts of each miRNA normalized to reads per million (RPM) were shown. miR-741-3p, miR- 871-3p, and miR-880-3p were highlighted by yellow. ES: embryonic stem cell, mouse embry- onic fibroblasts (MEFs), PGCs: primordial germ cells, SPG: spermatogonia, SPZ: spermatozoa. (DOCX) 21 / 26 PLOS ONE \| https://doi.org/10.1371/journal.pone.0211739 February 1, 2019 miRNAs function in the spermatogenesis S3 Table. Lists of predicted target genes of miR-741-3p, miR-871-3p, and miR-880-3p (cor- responding to Fig 4B). (XLSX) S3 Table. Lists of predicted target genes of miR-741-3p, miR-871-3p, and miR-880-3p (cor- responding to Fig 4B). (XLSX) S4 Table. Fertility of ΔXmiRs mice. Three hemizygous ΔXmiRs F2 males of the OT100 line (#2, 4, 5) and their WT littermates (#1, 3, 6) were mated twice each with MCH females. Three homozygous ΔXmiRs F2 females of the OT84 line (#2, 3, 6) and their heterozygous littermates (#4, 10, 11) were mated once with Oct4-ΔPE-GFP transgenic males. The number of pups is shown. (DOCX) S4 Table. Fertility of ΔXmiRs mice. Three hemizygous ΔXmiRs F2 males of the OT100 line (#2, 4, 5) and their WT littermates (#1, 3, 6) were mated twice each with MCH females. Three homozygous ΔXmiRs F2 females of the OT84 line (#2, 3, 6) and their heterozygous littermates (#4, 10, 11) were mated once with Oct4-ΔPE-GFP transgenic males. The number of pups is shown. (DOCX) S5 Table. Ratios of abnormal seminiferous tubules. Ratios of abnormal seminiferous tubules (% of abnormal seminiferous tubules in total seminiferous tubules) in ΔXmiRs mice at 8, 12, 16, and 30 weeks of age. Abnormal seminiferous tubules were counted in three sections from each mouse. Testis sections were prepared from three WT mice and one mouse of each ΔXmiRs line (OT84, OT97, and OT100). ND: not determined. (DOCX) S6 Table. Lists of putative target mRNAs of miR-871-3p and miR-880-3p (corresponding to S3 Fig). (XLSX) References 1. Ginsburg M, Snow MH, McLaren A. Primordial germ cells in the mouse embryo during gastrulation. Development. 1990; 110(2):521–8. PMID: 2133553 2. Richardson BE, Lehmann R. Mechanisms guiding primordial germ cell migration: strategies from different organisms. Nat Rev Mol Cell Biol. 2010; 11(1):37–49. https://doi.org/10.1038/nrm2815 PMID: 20027186 3. McLaren A. Meiosis and differentiation of mouse germ cells. Symp Soc Exp Biol. 1984; 38:7–23. PMID: 6400220 4. 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https://openalex.org/W2018057949	https://europepmc.org/articles/pmc3505545?pdf=render	English	null	Laparoscopic Sleeve Gastrectomy: Symptoms of Gastroesophageal Reflux can be Reduced by Changes in Surgical Technique	Obesity surgery	2,012	cc-by	4,515	OBES SURG (2012) 22:1874–1879 DOI 10.1007/s11695-012-0746-5 CLINICAL RESEARCH Abstract Background Bariatric surgery is the most effective treat- ment for gastro-esophageal reflux disease (GERD) in obese patients, with the Roux-en-Y gastric bypass being the tech- nique preferred by many surgeons. Published data reporting the results of laparoscopic sleeve gastrectomy (LSG) in patients with GERD are contradictory. In a previous obser- vational study, we found that relative narrowing of the distal sleeve, hiatal hernia (HH), and dilation of the fundus pre- dispose to GERD after LSG. In this study, we evaluated the effects of standardization of our LSG technique on the incidence of postoperative symptoms of GERD. Conclusions Our results confirm that careful attention to surgical technique can result in significantly reduced occur- rence of symptoms of GERD up to 12 months postopera- tively, compared with previous reports of LSG in the literature. Keywords Sleeve . Gastroesophageal reflux . Laparoscopic . Technique . Hiatal hernia Methods This was a concurrent cohort study. Patients who underwent bariatric surgery at our center were followed pro- spectively. LSG was performed in all patients in this series. Results A total of 234 patients underwent surgery. There were no cases of death, fistula, or conversion to open surgery. All 134 patients who completed 6–12 months of postoperative follow-up were evaluated. Excess weight loss at 1 year was 73.5 %. In the study group, 66 patients (49.2 %) were diag- nosed with GERD preoperatively, and HH was detected in 34 patients (25.3 %) intraoperatively. HH was treated by reduc- tion in three patients, anterior repair in 28, and posterior repair Laparoscopic Sleeve Gastrectomy: Symptoms of Gastroesophageal Reflux can be Reduced by Changes in Surgical Technique Jorge Daes & Manuel E. Jimenez & Nadin Said & Juan C. Daza & Rodolfo Dennis Published online: 23 August 2012 # The Author(s) 2012. This article is published with open access at Springerlink.com Jorge Daes & Manuel E. Jimenez & Nadin Said & Juan C. Daza & Rodolfo Dennis Published online: 23 August 2012 # The Author(s) 2012. This article is published with open access at Springerlink.com Published online: 23 August 2012 # The Author(s) 2012. This article is published with open access at Springerlink.com in three. Only two patients (1.5 %) had symptoms of GERD at 6–12 months postoperatively. Surgical Procedure We use five ports when performing LSG. A 12-mm port is placed at the umbilicus for insertion of the laparoscope, sta- pling of the stomach, and eventual removal of the stomach. A second 12-mm port is placed at the left flank for devasculari- zation of the greater curvature of the stomach, as a secondary position for insertion of the laparoscope during stapling of the stomach, and for suturing. Three additional 5 mm ports are placed: one at the epigastrium for liver retraction, one at the left upper quadrant for the surgeon's left hand working trocar, and one at the left lateral subcostal area for the assistant. We have performed more than 1,300 sleeve gastrectomies since 2006. In a previous observational study, we identified three technical problems which explained most cases of GERD and some cases of weight regain after LSG. These problems were: a relative narrowing at the junction of the vertical and the horizontal portions of the sleeve, a dilated fundus, and a persistent HH. Some of these problems have previously been discussed in the literature [14]. When we started removing more of the fundus of the stomach (leaving only enough for oversewing of the staple line), routinely correcting the HH, and avoiding narrowing or torsion of the sleeve, we observed a substantial decrease in the need for postoperative endoscopy to investigate food intolerance or symptoms of GERD. After emptying the stomach, the greater curvature is devas- cularized using an ultrasonic device, starting 3 cm proximal to the pylorus and continuing until the fundus is dissected free from the left crus of the diaphragm. We ensure that there is at least 3 cm of intrabdominal esophagus, which sometimes requires dissection of the peri-esophageal fat pad. When a HH is present, we completely free the esophageal-gastric union from the left and right crura, divide the phrenoesopha- geal membrane and peri-esophageal connective tissues, and continue the dissection well into the mediastinum to ensure a sufficient length of intra-abdominal esophagus. The HH de- fect is then closed with non-absorbable monofilament sutures, anterior or posterior to the esophagus depending on the size of the defect and the resulting position of the esophagus. We do not use a calibrating bougie for hiatal closure and do not use mesh even for large hernias. Surgical Procedure This study prospectively evaluated the effects of stan- dardization of the LSG technique as described above on the incidence of postoperative symptoms of GERD. The other objectives were to establish the preoperative prevalence of GERD, esophagitis, and HH; the morbidity associated with the LSG procedure; the percentage of excess weight loss at 1 year; and the correlation between preoperative endoscopic diagnosis of HH and intraoperative findings. A 32 Fr bougie is introduced to reach the distal antrum. Division of the stomach starts 3 cm proximal to the pylorus, making sure that the bougie stays adjacent to the lesser curvature of the stomach. We use a 60–4.8 mm cartridge for the initial firing of the stapler, followed by three to five 60–3.5 mm cartridges. We take extra care to avoid relative narrowing at the junction between the vertical and horizon- tal parts of the stomach, which usually occurs during the first and occasionally the second firing of the stapler if it is inappropriately pressed against the bougie in an attempt to leave a small antrum. Narrowing can be avoided by using an articulating stapler, slightly angled to the greater curvature, to create a wide angle at the junction between the horizontal antrum and the vertical body of the stomach. Introduction There is a strong association between morbid obesity and gastroesophageal reflux disease (GERD), with GERD occurring in approximately 50 % of morbidly obese patients. The main cause of GERD in obese patients is transient relaxation of the lower esophageal sphincter com- bined with increased intra-abdominal pressure [1]. Symp- tomatic hiatal hernia (HH) occurs in 15 % of patients with a body mass index (BMI) of >35 kg/m2 [2]. Bariatric surgery is the most effective treatment for GERD in obese patients, with the Roux-en-Y gastric bypass being the preferred tech- nique by most surgeons [3–5]. Published data regarding the results of sleeve gastrecto- my in patients with GERD are contradictory, and compari- son between studies is difficult. Many authors do not perform laparoscopic sleeve gastrectomy (LSG) in patients with symptomatic GERD [6]. The criteria for diagnosis of GERD are not always clear, and the use of preoperative endoscopy varies among studies. Indications for LSG and the techniques used vary widely among surgeons. Symp- tomatic GERD has been reported to occur in 7.8–20 % of patients at 12–24 months after LSG in selected series of more than 100 patients [2, 7–9]. At the second and the third international consensus summits for sleeve gastrectomy, J. Daes (): M. E. Jimenez: N. Said: J. C. Daza Minimally Invasive Surgery Department, Clinica Bautista, Carrera 38 calle 71 esquina, Barranquilla, Colombia e-mail: jorgedaez@gmail.com J. Daes (): M. E. Jimenez: N. Said: J. C. Daza Minimally Invasive Surgery Department, Clinica Bautista, Carrera 38 calle 71 esquina, Barranquilla, Colombia e-mail: jorgedaez@gmail.com R. Dennis Research Department, Fundacion Cardioinfantil, Bogotá, Colombia R. Dennis Clinical Epidemiology Department, School of Medicine, Universidad Javeriana, Bogotá, Colombia OBES SURG (2012) 22:1874–1879 1875 disease were recorded together with any additional pathological findings. All operations were performed by our group. reflux disease was reported to occur in 6.5 % and 17 % of patients, respectively, after sleeve gastrectomy [10, 11]. reflux disease was reported to occur in 6.5 % and 17 % of patients, respectively, after sleeve gastrectomy [10, 11]. A recent review of the literature found that four studies reported an increase in reflux symptoms after sleeve gas- trectomy, and seven studies reported a reduction in symp- toms [12]. Most studies reported an increase in reflux symptoms during the first year following sleeve gastrecto- my, followed by a gradual decrease in symptoms up to the third postoperative year [13]. Materials and Methods The presence and degree of esophagitis and Barrett's OBES SURG (2012) 22:1874–1879 1876 Table 1 Characteristics of patients who underwent LSG: total cohort and study cohort Characteristic Total cohort (n0234) Study cohort (n0134) Age in years (SD) 36.4 37.8 Female (%) 139 (59) 87 (65) BMI, kg/m2 (range) 39 (30–64) 38 (30–64) GERD (%) 112 (48) 66 (49) Hiatal hernia (%) 117 (50) 65 (49) Table 1 Characteristics of patients who underwent LSG: total cohort and study cohort may result in food regurgitation, vomiting, or GERD. Division of the stomach, including most of the fundus, is then complet- ed, leaving only a small portion for oversewing. We routinely bury the stapler line with continuous sero- serosal stitches using non-absorbable monofilament sutures, starting at the top of the sleeve. We then retract the hollow bougie proximally and test the sleeve with methylene blue to confirm complete and uniform filling. Adequate distention occurs with 25–35 ml. We then perform a small omental patch with a single sero-serosal stitch over the top of the sleeve. We carefully extract the removed stomach through the umbilical port after incising the aponeurosis without the use of bags. There were no cases of death, fistula, or conversion to open surgery. One patient developed postoperative bleeding requiring revision surgery, but the cause of the bleeding was not identified (Table 2). Four patients complained of diffi- culty tolerating solid food in the early postoperative period. This difficulty resolved completely in three of the four patients after endoscopy alone, and the other patient had slight torsion of the distal sleeve which improved after endoscopic balloon dilatation. We routinely instill diluted bupivicaine into the left upper quadrant of the abdomen. We do not use drains. The trocars are removed under direct vision to ensure hemostasis. The incised aponeurosis at the umbilical port is carefully sutured. The technical aspects described can be viewed at http://www.sages.org/video/details.php?id=103375. The patient is discharged the following day after tolerat- ing a liquid diet and receiving a complete set of instructions which includes aspects of nutrition, medication, activity, appointments, and mental health. The nurse coordinator delivers and explains the instructions to the patient. Interestingly, only a fraction of HHs diagnosed preoper- atively were confirmed intraoperatively, and of the 69 patients without HH diagnosed on preoperative endoscopy, six had HH detected intraoperatively. Statistical Analysis A Microsoft Access database (Office 2000) was used for data collection. All statistical analyses were performed using Epi Info 7.0 software (Centers for Disease Control and Prevention,Atlanta,GA).A two-sided p value of <0.05 was considered significant. Bivariate analyses used the χ2 test or Fisher's exact test for categorical data and the independent- sample t test for continuous data. At the 6–12 months postoperative follow-up, only two patients (1.5 %) had symptoms of GERD. Both were eval- uated endoscopically and were found to have a small HH without esophagitis. These two patients are currently treated with proton pump inhibitors. Both these patients had a large HH on preoperative endoscopy. Noteworthy is the fact that Materials and Methods HH was detected intraoperatively in 34 patients (25 %) in the study cohort, of which 29 (85.3 %) had preoperative symptoms of GERD. Twelve patients had a large HH, of which all had preoper- ative symptoms of GERD. The HH was reduced in three patients, repaired anteriorly in 28 patients, and repaired posteriorly in three patients. The patient is evaluated on the eighth postoperative day and then at 1, 3, 6, and 12 months postoperatively and twice a year thereafter. We record symptoms of GERD using the same survey as in the preoperative evaluation. Early endoscopy is indicated if the patient has difficulty tolerating solid food and after the sixth postoperative month in patients with symptoms of GERD. Table 3 shows the preoperative characteristics of patients with and without GERD. There were significant differences in BMI and in the frequency of diagnosis of HH and large HH between these two groups (p<0.01). HH was six times more frequent in patients with GERD than those without, and large HH only occurred in patients with GERD. Materials and Methods We prospectively followed all patients who underwent bari- atric surgery from April 2011 to April 2012. All these patients underwent LSG. Patients with HH and reflux dis- ease were included in the study, including two patients who had a band removed and one patient with ultra-short- segment Barrett's disease. All patients completed a multi- disciplinary preoperative evaluation and met the criteria for LSG of the Colombian Surgical Society. Preoperative GERD was diagnosed if patients experi- enced symptoms of heartburn or acid reflux more than twice a week, if they required antacid treatment for more than 2 weeks, or if preoperative endoscopy showed esophagitis. All patients underwent preoperative endoscopy by an endoscopist/surgeon with more than 25 years of experience. A small HH was diagnosed when there was a patulous cardia or a hernia measuring less than 2 cm. A large HH was diagnosed when there was a hernia measuring more than 2 cm. The presence and degree of esophagitis and Barrett's Preoperative GERD was diagnosed if patients experi- enced symptoms of heartburn or acid reflux more than twice a week, if they required antacid treatment for more than 2 weeks, or if preoperative endoscopy showed esophagitis. In some cases with the pylorus located in the upper right quadrant of the abdomen and a very curved stomach, the only way to form a small antrum and avoid relative narrow- ing is to place an additional trocar through a port at the right flank to help aim the first stapler in the correct direction. We also emphasize that when dividing the stomach, the same distance should be maintained between the lesser curvature and the entire staple line to avoid twisting of the sleeve, which All patients underwent preoperative endoscopy by an endoscopist/surgeon with more than 25 years of experience. A small HH was diagnosed when there was a patulous cardia or a hernia measuring less than 2 cm. A large HH was diagnosed when there was a hernia measuring more than 2 cm. Table 5 Advantages of laparoscopic sleeve gastrectomy 3. Excellent resolution of co-morbidities including type 2 diabetes mellitus. 4. No marginal ulceration, internal hernia, or nutritional deficiencies. 5. Enables full endoscopic examination of the stomach postoperatively. 6. Easily revised or converted. preferred bariatric surgery procedure has changed from the adjustable gastric band and the Roux-en-Y gastric bypass in the period from 1999 to 2005 to the LSG. The reasons for this change are explained in Table 5. The great majority of our patients prefer the LSG. no patient with small or no HH was found to have postop- erative symptoms (p00.021) (Table 4). no patient with small or no HH was found to have postop- erative symptoms (p00.021) (Table 4). no patient with small or no HH was found to have postop- erative symptoms (p00.021) (Table 4). Published data regarding the effects of sleeve gastrecto- my in patients with GERD are contradictory, and compari- son between studies is difficult. The criteria for diagnosis of GERD are not always clear, and the use of preoperative endoscopy varies among studies. Many authors routinely exclude patients with symptomatic GERD as candidates for LSG. Indications for LSG and the surgical techniques used vary widely among authors. Discussion The results of this study show a very low incidence of GERD (1.5 %) at 6–12 months after LSG which was per- formed with careful attention to the described technical details. These results are even more significant because this was an unselected cohort of patients, with no exclusions. The prevalence of GERD in this series is similar to that reported in the literature, but the mean BMI is slightly lower. There were no cases of death, fistula, or conversion to open surgery. The morbidity was very low and the excess weight loss at 1 year was 74 %. In selected series involving more than 100 LSG patients, symptomatic GERD was reported to occur in 7.8–20 % of patients at 12– 24 months postoperatively. Cottan et al. [7] reported a series of 126 patients who underwent LSG and found a 20 % incidence of GERD at 12 months postopera- tively. Hamoui et al. [8] reported 131 LSG patients with a 12.7 % incidence of GERD at 13 months, Nocca et al. [9] reported 163 LSG patients with an 11.8 % incidence of GERD at 24 months, and Sorelly et al. [2] reported 264 LSG patients with a 7.8 % incidence of GERD at 24 months. There is a strong association between morbid obesity and GERD, with GERD occurring in approximately 50 % of morbidly obese patients. There is a stronger association between GERD and waist circumference (a marker of cen- tral adiposity) than between GERD and BMI [15]. The main cause of GERD in obese patients is transient relaxation of the lower esophageal sphincter combined with increased intra-abdominal pressure [1]. Some postoperative factors tend to reduce reflux after sleeve gastrectomy, such as reduced intra-abdominal pres- sure due to decreased body weight, reduced acid production, and accelerated gastric emptying (which has been well dem- onstrated in scintigraphy studies) [16, 17]. However, many postoperative factors predispose to reflux, such as lack of gastric compliance (which is the objective of the procedure), increased intraluminal pressure with an intact pylorus, low esophageal sphincter pressure (which has been described in a recent manometric study and is attributed to resection of the sling fibers of the distal part of the lower sphincter) [16], and the final shape of the sleeve. The shape of the superior pouch and the tubular configuration contribute to reflux but not the configuration of the inferior pouch [13]. Results Table 2 Surgical findings and outcomes: total cohort and study cohort Outcome Total cohort (n0234) Study cohort (n0134) Perioperative death 0 0 Fistula 0 0 Bleeding 1 1 Hiatal hernia, confirmed (%) 58 (25) 34 (25) Hiatal hernia, large (%) 21 (9) 12 (9) Table 2 Surgical findings and outcomes: total cohort and study cohort A total of 234 patients underwent surgery during the 12- month study period. This report focuses on the 134 patients who have completed 6–12 months of follow-up, because reflux symptoms in the first postoperative months may repre- sent an adaptation to the restricted stomach size. Table 1 shows the characteristics of patients in the total cohort (n0234) and the cohort analyzed in this study (n0134). Most patients were severely obese young females, and about half had both HH and GERD. 1877 OBES SURG (2012) 22:1874–1879 Table 3 Baseline characteristics of patients with and without GERD: study cohort (n0134) Characteristic With GERD (n066) Without GERD (n068) Age in years* 37.6 37.9 Female (%)* 43 (65) 44 (65) BMI, kg/m2 39.3 36.5 Hiatal hernia (%) 29 (44) 5 (7) Hiatal hernia, large (%)** 12 (18) 0 Differences not statistically significant, *p<0.01 Table 5 Advantages of laparoscopic sleeve gastrectomy 1. Low morbidity and mortality rates. 2. Effective in the medium term. 3. Excellent resolution of co-morbidities including type 2 diabetes mellitus. 4. No marginal ulceration, internal hernia, or nutritional deficiencies. 5. Enables full endoscopic examination of the stomach postoperatively. 6. Easily revised or converted. 7. Short operation and hospitalization times. Table 3 Baseline characteristics of patients with and without GERD: study cohort (n0134) Table 5 Advantages of laparoscopic sleeve gastrectomy References When we started to remove the fundus of the stomach more completely (leaving only enough to allow oversew- ing), routinely corrected the HH, and avoided relative nar- rowing or torsion of the sleeve, we observed a sharp decrease in the need for postoperative endoscopy to inves- tigate food intolerance or symptoms of GERD. 1. Schneider JH, Küper M, Königsrainer A, et al. Lower esophageal sphincter relaxation in morbid obesity. Obes Surg. 2009;19 (5):595–600. 2. Soricelli E, Casella G, Rizzello M, et al. Initial experience with laparoscopic crural closure in the management of hiatal hernia in obese patients undergoing sleeve gastrectomy. Obes Surg. 2010;20 (8):1149–53. 2. Soricelli E, Casella G, Rizzello M, et al. Initial experience with laparoscopic crural closure in the management of hiatal hernia in obese patients undergoing sleeve gastrectomy. Obes Surg. 2010;20 (8):1149–53. ( ) 3. Perez AR, Moncure AC, Rattner DW. Obesity adversely affects the outcome of antireflux operations. Surg Endosc. 2001;15(9):986–9. The two patients in this series with persistent symptoms of GERD both had a large HH on preoperative endoscopy and a small HH on postoperative endoscopy. This raises the question of the need for using mesh in such cases, as has been suggested in the literature [18–20]. The development of improved biological meshes might facilitate their use in this situation. There was a surprising difference in the diag- nosis of HH on preoperative endoscopy compared with intraoperative findings. One plausible explanation for this is that small hernias may have been reduced during dissec- tion and pulling of the fundus to free it from the left crus of the diaphragm. 4. Perez AR, Moncure AC, Rattner DW. Obesity is a major cause of failure for both abdominal and transthoracic antireflux operations. Gastroenterology. 1999;116:A1343. 5. Fraser J, Watson DI, O’Boyle CJ, et al. Obesity and its effect on outcome of laparoscopic Nissen fundoplication. Dis Esoph. 2001;14(1):50–3. 6. Braghetto I, Csendes A, Korn O, et al. Gastroesophageal reflux disease after sleeve gastrectomy. Surg Laparosc Endosc Percutan Tech. 2010;20(3):148–53. 7. Cottam D, Qureshi FG, Mattar SG, et al. Laparoscopic sleeve gastrectomy as an initial weight-loss procedure for high-risk patients with morbid obesity. Surg Endosc. 2006;20(6):859–63. 8. Hamoui N, Anthone GJ, Kaufman HS, et al. Sleeve gastrectomy in the high-risk patient. Obes Surg. 2006;16(11):1445–9. Our study has several limitations. First, postoperative endoscopy was not performed routinely, but only in patients with symptoms of reflux. Discussion Bariatric surgery is the most effective treatment for GERD in obese patients, with the Roux-en-Y gastric bypass being the preferred technique by most surgeons [3–5]. Our Table 4 Characteristics of patients with GERD at follow-up, com- pared with all other patients in the study cohort Table 4 Characteristics of patients with GERD at follow-up, com- pared with all other patients in the study cohort Characteristic GERD (patient 1) GERD (patient 2) No GERD (n0132) Age (years) 53 27 37.3 Sex Female Female 87M, 47F BMI (kg/m2) 47 34 38 Hiatal hernia Yes Yes 32 (24 %) Hiatal hernia, large Yes Yes 10 (7.5 %) Reduction of excess weight 58.4 % 81 % 73.5 % We identified three technical errors that explain most of our cases of GERD after sleeve gastrectomy: relative nar- rowing at the junction of the vertical and horizontal parts of the sleeve, dilation of the fundus, and persistence of the HH or a patulous cardia. OBES SURG (2012) 22:1874–1879 1878 We have performed over 1,300 sleeve gastrectomies since 2006. Early in our experience, 18 patients developed severe reflux symptoms, food intolerance, and vomiting or nausea postoperatively. Endoscopy revealed narrowing of the sleeve in these patients, and all responded to several endoscopic balloon dilatations and medical therapy. Some of these patients had prolonged symptoms, but none re- quired additional surgery. We believe that the narrowing was due to a technical error resulting from excessive tension on the lesser curvature of the stomach when attempting to place the bougie closer to the lesser curvature during the first or second firing of the stapler. Another cause of partial obstruction in the distal part of the stomach was torsion of the sleeve, which can be prevented by maintaining the same distance between the lesser curvature and the entire staple line during division of the stomach. Conclusions Our study confirms a substantial prevalence of GERD symptoms and HH in obese patients who have undergone LSH. Our results show that careful attention to surgical techniques can significantly reduce the frequency of post- operative symptoms of GERD up to 12 months after sur- gery, compared with previous reports of LSG. Conflicts of interest The authors: Jorge Daes, Rodolfo J Dennis, Manuel E Jimenez, Nadin Said, and Juan C Daza declare no financial or other conflicts of interest related to this publication. Open Access This article is distributed under the terms of the Crea- tive Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. Later in our experience, four patients developed symp- toms of GERD after LSG. All these patients had HH, dilation of the fundus, and weight regain. We performed remedial operations in all these patients, including repair of the HH and repeat formation of the sleeve, with excellent symptomatic relief and no complications. References The study was designed to eval- uate symptomatic GERD, but routine postoperative endos- copy can detect recurrent HH. Second, we did not have a concurrent control cohort of patients who underwent a dif- ferent surgical procedure, so our results can only be com- pared with those in the literature. However, the results of this study may be used as a basis for such an investigation. Third, our surgical team has substantial experience over time with LSG, and results may differ in other cohorts. Finally, we still need to evaluate longer term outcomes (more than 12 months postoperatively). 9. Nocca D, Krawczykowsky D, Bomans B, et al. A prospective multicenter study of 163 sleeve gastrectomies: results at 1 and 2 years. Obes Surg. 2008;18(5):560–5. 10. Gagner M, Deitel M, Kalberer TL, et al. The second international consensus summit for sleeve gastrectomy. Surg Obes Relat Dis. 2009;5:476–85. 11. Deitel M,Gagner M,EricksonA,et al. Thirdinternational summit: current status of sleeve gastrectomy. Surg Obes Relat Dis. 2011;7:749–59. 12. Chiu S, Birch DW, Shi X, et al. Effect of sleeve gastrectomy on gastroesophageal reflux disease: a systematic review. Surg Obes Relat Dis. 2011;7(4):510–5. 13. Lazoura O, Zacharoulis D, Triantafyllidis G, et al. Symptoms of gastroesophageal reflux following laparoscopic sleeve gastrectomy are related to the final shape of the sleeve as depicted by radiology. Obes Surg. 2010;21(3):295–9. OBES SURG (2012) 22:1874–1879 1879 18. Soricelli E, Basso N, Genco A, et al. Long-term results of hiatal hernia mesh repair and antireflux laparoscopic surgery. Surg Endosc. 2009;23:2499–509. 14. Keidar A, Appelbaum L, Schweiger C, et al. Dilated upper sleeve can be associated with severe postoperative gastroesophageal dys- motility and reflux. Obes Surg. 2010;20(2):140–7. 15. Prachand V, Alverdy JC. Gastroesophageal reflux disease and severe obesity: fundoplication or bariatric surgery? World J Gas- troenterol. 2010;16(30):3757–61. 19. Frantzides CT, Madan AK, Carlson MA, et al. A prospective, randomized trial of laparoscopic polytetraflouroethylene (PTFE) patch repair vs. simple cruroplasty for large hiatal hernia. Arch Surg. 2002;137(6):649–53. 16. Braghetto I, Lanzarini E, Korn O, et al. Manometric changes of the lower esophageal sphincter after sleeve gastrectomy in obese patients. Obes Surg. 2010;20(3):357–62. 20. Granderath FA, Schweiger UM, Kamolz T, et al. Laparoscopic Nissen fundoplication with prosthetic hiatal closure reduces postoperative in- trathoracic wrap herniation: preliminary results of a prospective ran- domized functional and clinical study. Arch Surg. 2005;140(1):40–8. 17. Melissas J, Koukouraki S, Askoxylakis J, et al. Sleeve gastrectomy: a restrictive procedure? Obes Surg. 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https://openalex.org/W4384204173	https://zenodo.org/records/8149554/files/BDJ_article_105863.pdf	English	null	Genetic diversity and relatedness amongst captive saker falcons (Falco cherrug) in the Green Balkans’ Wildlife Rehabilitation and Breeding Centre in Bulgaria	Biodiversity data jurnal	2,023	cc-by	7,230	© Petrov R et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Biodiversity Data Journal 11: e105863 doi: 10.3897/BDJ.11.e105863 Research Article Genetic diversity and relatedness amongst captive saker falcons (Falco cherrug) in the Green Balkans’ Wildlife Rehabilitation and Breeding Centre in Bulgaria Rusko Petrov , Thierry Hoareau , Loic Lesobre , Yana Andonova , Dobry Yarkov , Nayden Chakarov ‡ Green Balkans - Stara Zagora NGo, Stara Zagora, Bulgaria § Trakia University, Stara Zagora, Bulgaria \| Reneco International Wildlife Consultants LLC, Abu Dhabi, United Arab Emirates ¶ Green Balkans - Stara Zagora NGO, Stara Zagora, Bulgaria # University of Bielefeld, Bielefeld, Germany Corresponding author: Rusko Petrov (rpetrov@greenbalkans.org) Academic editor: Emilian Stoynov Received: 03 May 2023 \| Accepted: 23 Jun 2023 \| Published: 13 Jul 2023 Citation: Petrov R, Hoareau T, Lesobre L, Andonova Y, Yarkov D, Chakarov N (2023) Genetic diversity and relatedness amongst captive saker falcons (Falco cherrug) in the Green Balkans’ Wildlife Rehabilitation and Breeding Centre in Bulgaria. Biodiversity Data Journal 11: e105863. https://doi.org/10.3897/BDJ.11.e105863 Abstract The globally endangered saker falcon (Falco cherrug) is currently being re-introduced in ‡,§ \| \| ¶ § # Biodiversity Data Journal 11: e105863 doi: 10.3897/BDJ.11.e105863 Research Article Biodiversity Data Journal 11: e105863 doi: 10.3897/BDJ.11.e105863 Research Article Genetic diversity and relatedness amongst captive saker falcons (Falco cherrug) in the Green Balkans’ Wildlife Rehabilitation and Breeding Centre in Bulgaria Rusko Petrov , Thierry Hoareau , Loic Lesobre , Yana Andonova , Dobry Yarkov , Nayden Chakarov ‡ Green Balkans - Stara Zagora NGo, Stara Zagora, Bulgaria § Trakia University, Stara Zagora, Bulgaria \| Reneco International Wildlife Consultants LLC, Abu Dhabi, United Arab Emirates ¶ Green Balkans - Stara Zagora NGO, Stara Zagora, Bulgaria # University of Bielefeld, Bielefeld, Germany Corresponding author: Rusko Petrov (rpetrov@greenbalkans.org) Academic editor: Emilian Stoynov Received: 03 May 2023 \| Accepted: 23 Jun 2023 \| Published: 13 Jul 2023 Citation: Petrov R, Hoareau T, Lesobre L, Andonova Y, Yarkov D, Chakarov N (2023) Genetic diversity and relatedness amongst captive saker falcons (Falco cherrug) in the Green Balkans’ Wildlife Rehabilitation and Breeding Centre in Bulgaria. Biodiversity Data Journal 11: e105863. https://doi.org/10.3897/BDJ.11.e105863 ‡,§ \| \| ¶ § # Biodiversity Data Journal 11: e105863 doi: 10.3897/BDJ.11.e105863 Research Article Research Article Genetic diversity and relatedness amongst captive saker falcons (Falco cherrug) in the Green Balkans’ Wildlife Rehabilitation and Breeding Centre in Bulgaria Rusko Petrov , Thierry Hoareau , Loic Lesobre , Yana Andonova , Dobry Yarkov , Nayden Chakarov ‡,§ \| \| ¶ § # ‡ Green Balkans - Stara Zagora NGo, Stara Zagora, Bulgaria § Trakia University, Stara Zagora, Bulgaria \| Reneco International Wildlife Consultants LLC, Abu Dhabi, United Arab Emirates ¶ Green Balkans - Stara Zagora NGO, Stara Zagora, Bulgaria # University of Bielefeld, Bielefeld, Germany Corresponding author: Rusko Petrov (rpetrov@greenbalkans.org) Academic editor: Emilian Stoynov Received: 03 May 2023 \| Accepted: 23 Jun 2023 \| Published: 13 Jul 2023 Citation: Petrov R, Hoareau T, Lesobre L, Andonova Y, Yarkov D, Chakarov N (2023) Genetic diversity and relatedness amongst captive saker falcons (Falco cherrug) in the Green Balkans’ Wildlife Rehabilitation and Breeding Centre in Bulgaria. Biodiversity Data Journal 11: e105863. https://doi.org/10.3897/BDJ.11.e105863 Corresponding author: Rusko Petrov (rpetrov@greenbalkans.org) Academic editor: Emilian Stoynov Received: 03 May 2023 \| Accepted: 23 Jun 2023 \| Published: 13 Jul 2023 Citation: Petrov R, Hoareau T, Lesobre L, Andonova Y, Yarkov D, Chakarov N (2023) Genetic diversity and relatedness amongst captive saker falcons (Falco cherrug) in the Green Balkans’ Wildlife Rehabilitation and Breeding Centre in Bulgaria. Biodiversity Data Journal 11: e105863. https://doi.org/10.3897/BDJ.11.e105863 Introduction The saker falcon (Falco cherrug) is a globally endangered species by the categorisation of the International Union for Conservation of Nature (BirdLife International 2021). In Bulgaria, at the south-eastern edge of its range in Europe, the saker falcon is marked as critically endangered in the Red Data Book of the Republic of Bulgaria (Domuschiev et al. 2015). Currently, the only known successfully breeding pairs of saker falcons in the country are formed of re-introduced birds, part of the ongoing programme to restore the local breeding population. Since 2011, there has been a captive breeding group performed by the Wildlife Rehabilitation and Breeding Centre - Green Balkans (WRBC), part of Green Balkans - Stara Zagora NGO. These sakers’ offspring are being released through the hacking method (Sherrod et al. 1987) in the Upper Thracian Plain in Bulgaria. The objective of the current stage of the species restoration project is to release 100 juvenile sakers over a five- year period (2020-2024). They are retained in the hack sites until independence through food provision in order to imprint on the area and return to it when they reach breeding age at around 3-years-old (Dixon et al. 2020, Lazarova et al. 2021, Petrov et al. 2021, Petrov et al. 2022). Captive breeding with the objective of population restocking or re-introduction is increasingly relied upon to prevent the local or global extinction of species (Conde et al. 2011, Pritchard et al. 2011, Ralls and Ballou 2013, McGowan et al. 2016). When breeding animals in captivity, importance should be placed on the genetic relatedness and diversity of the group, especially when the aim is re-introduction. In order for rearing healthy offspring which will be able to form a future self-sustaining population, the founding individuals should be carefully chosen and paired. There are certain risks associated with maintaining a captive breeding group of animals - too-small gene pool can lead to reduced genetic diversity in the long run and increased prevalence of genetic disorders and inbreeding depression (IUCN/SSC 2013). Captive breeding programmes typically use pedigrees to manage genetic diversity and avoid inbreeding by analysis of stud books which contain data on births, deaths and parental information; however, they can often be incomplete and overestimated (Rabier et al. 2022). Managing the captive group can be a challenge if there are such deficiencies. Keywords genomics, birds of prey, saker falcon, captive breeding, re-introduction, biodiversity Petrov R et al Petrov R et al 2 than expected. The research could be beneficial to other raptor captive breeding programmes dealing with a similar group composition. than expected. The research could be beneficial to other raptor captive breeding programmes dealing with a similar group composition. Abstract The globally endangered saker falcon (Falco cherrug) is currently being re-introduced in Bulgaria, where the falcons are bred in captivity and released through the hacking method. We relied on the birds’ pedigree when forming the breeding pairs from 2011. In 2021-2022, we had the opportunity to evaluate our captive population via DNA tests. We performed the first genetic assessment of the sakers in the WRBC through a genome evaluation of the most important founders (n = 12) and, in 2022, we executed a microsatellite analysis on 30 saker falcons from the programme. We compared the results with the known pedigree and history of the saker falcons. The genetic tests helped to assign relatedness to some birds with missing or incomplete pedigrees, indicating the test can complement that information and lead to better management of the captive group. One pair was separated as a precaution as it was indicated by one the tests that the two birds are more closely related Breeding programme Between 2011 and 2022, from the start of the captive breeding efforts to date, there have been, on average, 21 saker falcons in the WRBC captive breeding programme (range 4-29) (Table 1). There were, on average, 10 formed pairs each year (range 2-13), on average eight of them exhibited breeding behaviour (range 2-12), an average of five laid eggs in one or two clutches (range 0-12) and out of them, an average of four pairs per year reared chicks (range 0-8). The founding individuals were obtained from breeders from Central Europe and the UK. Season Birds in the breeding programme Formed pairs Pairs with breeding behaviour Pairs which laid eggs Pairs which reared chicks 2011 4 2 2 0 0 2012 18 8 7 2 1 2013 21 10 8 3 3 2014 20 10 8 5 4 2015 24 10 9 5 5 2016 22 9 8 5 2 2017 22 11 9 5 5 2018 22 11 10 6 6 2019 21 10 9 5 4 2020 25 10 9 9 3 2021 29 12 12 12 8 2022 28 13 9 5 5 Table 1. Breeding saker falcons in the WRBC for the period 2011-2022. Breeding saker falcons in the WRBC for the period 2011-2022. Introduction DNA analysis can help fill in these gaps and aid the genetic management of the breeding stock (Allendorf et al. 2010, Frankham et al. 2010). The aim of this research was to gain further knowledge of the individuals in the WRBC saker falcon captive breeding group, beyond the history of arrival and pedigree which were Genetic diversity and relatedness amongst captive saker falcons (Falco ... 3 3 incomplete for some of the birds. In our saker falcon breed-and-release programme in Bulgaria, we relied on the birds’ pedigree when forming the breeding pairs from 2011. Recently, in 2021-2022, we had the opportunity to evaluate our captive population via DNA tests. We performed the first genetic assessment of the sakers in the WRBC through a genome evaluation of the most important founders (n = 12) and a microsatellite analysis on 30 saker falcons from the programme. Samples In 2021, a total of 12 samples, representing the most important founding individuals from the captive population of the WRBC with unclear background, were analysed through Petrov R et al Petrov R et al 4 4 whole genome sequencing. Some of the saker falcons had been wild-caught and there were no data on them, others needed to be tested to clear relations in order to form breeding pairs of not closely-related birds. Seven of these 12 birds subsequently bred successfully (excluding Frodo, Arnold, Luna, Barir, Ariel). One individual - Bilbo, was known to be the offspring of Thomas and Lucia. Some of the other founders were not alive at the time of sampling and the other untested saker falcons were with known pedigree. Sample details of the 12 tested individuals are listed in Table 2. Individual ID Ring Name 184 CZ151592 Frodo 319 6-98569 Arnold 320 0621 Hedy 0155 084544 Luna 0092 6-98611 Bilbo 1546 CZ126556 Lucia 1544 SKVG3601 Thomas 1543 CZ120480 Eurydice 1542 0286 /G13 11 Orpheus 0103 1153 Barir 1754 IBRUK77684W Ariel 0149 А1300Е898 Romeo Table 2. Sample details of 12 saker individuals used as founders of the captive population of the WRBC. Sample details of 12 saker individuals used as founders of the captive population of the WRBC. In 2022, 30 samples of sakers from the breeding group were analysed at nine microsatellite loci - SSR11, SSR15, SSR45, SSR48, SSR53 SSR57, SSR63, SSR82 (Hou et al. 2018), Fp92 (Nesje et al. 2000). Details of these individuals are listed in alphabetical order in Table 3. Microsatellite genotyping was performed as described by Chakarov et al. (2013). M13-tagged primers for the corresponding loci were used in a 10 μl polymerase chain reaction (PCR) volume with 20–200 ng DNA, which was amplified for 35 cycles using a Type-it microsatellite PCR kit (QIAGEN), following the manufacturer's standard protocols and using an annealing temperature of 56°C. Diluted amplification products (1 μl; 1:20 dilution) were then resolved on an ABI 3730 Automated DNA Analyser (Applied Biosystems). Fragment lengths were scored for all individuals using Genemarker 1.95 (SoftGenetics LCC). Genotypes were analysed with Colony 2.0.6.6 (Wang 2013) with polygamy allowed for both sexes (option chosen given the possibility of re-pairing of individuals). All birds were examined by a veterinary physician upon blood collection and were determined to be clinically healthy. Surfaces were disinfected with Desclean solution. We disinfected the area and collected 0.1 ml of whole blood from either left or right basilic vein (Vena cutanea ulnaris superficialis) of all specimens tested. Genomic data For the 12 founders, the original genomic data were first analysed to estimate genetic parameters (genomic coverage, number of informative variants, genetic diversity, relatedness and inbreeding coefficient) and were applied to a clustering analysis (principal component analysis: PCA). The genomic data has been generated by the Beijing Genomic Institute (China) and included DNA extraction, library construction and whole genome resequencing at an expected coverage of 10×. As a reference for read mapping, we have used a chromosome-level genome assembly available for gyrfalcon on the NCBI portal (BioProject ID PRJNA561988; https://www.ncbi.nlm.nih.gov/assembly/GCF_015220075.1). The quality of this assembly is higher than the reference saker genome assembly. Moreover, gyrfalcons and sakers are closely-related species that are sometimes difficult to genetically differentiate - a recent study has shown that saker and gyrfalcon have the same genome organisation (Joseph et al. 2018). Petrov R et al We immediately placed the Genetic diversity and relatedness amongst captive saker falcons (Falco ... 5 5 blood into Eppendorf collection tubes of 1.5 ml volume containing 1 ml 90% alcohol. We used 3 ml syringes with 23G needles. Hatched Ring Name 2014 IRBUK77684W Ariel 2006 6-98569 Arnold 2015 CZ151595 Arwen 2003 8201823783300W Bandit N/A 1153 Barir 2015 6-98611 Bilbo 2014 6-60051 Boryana 2015 6-60020 DJ 2013 CZ147598 Dobry 2019 6-60012 Dracarys 2011 CZ120480 Eurydice 2015 CZ151592 Frodo 2012 6-98519 Gogo 2013 6-98808 Grum 2009 0621 Hedy 2015 CZ151594 Lobelia 2010 CZ126556 Lucia 2016 CZ147613 Maul 2010 ZG110286/130 Orpheus 2009 Z288 Penda 2008 SK074503 Pizho 2010 6-98568 Plamena 2019 5-105915 Pluto 2019 5-105916 Rhaegal 1994 А1300Е898 Romeo 2020 6-98573 Shira 2010 SKVG3601 Thomas 2014 BG11CAAWRBC007 Thomas II 2010 6-98779 Vulna 2016 BG613C2016PLAM008 Willow Table 3. Sample details of the 30 saker individuals from the captive population of the WRBC. Eleven of them were the same from the previous sample (in bold). Petrov R et al 6 Read mapping, SNP calling and filtering The quality of the Illumina paired-end reads (~ 21.7 GB of FASTQ files per falcon specimen) were first checked using the programme FASTP (v.0.20.0; https://github.com/ opengene/fastp). These reads were then mapped on the gyrfalcon reference genome (1.12 GB of FASTA file) using BWA-MEM (Li and Durbin 2009). The obtained SAM files were then compressed and merged into a single BAM file (~ 21.7 GB per individual), which was then sorted and indexed using SAMTOOLS (Li et al. 2009). Duplicates were marked and removed from the BAM files using the PICARD tool. Variant sites2 were then identified using SAMTOOLS mpileup (Li et al. 2009) and called using BCFTOOLS (v.1.6.33) calls function (http://samtools.github.io/bcftools/bcftools.html). For the mapping, the identification and call of the variants were performed on each chromosome in parallel, which helped speed up the analyses. These analyses produced multiple VCF compressed files that contained all the information on the variant sites. To select high quality variants, we used functions available in VCFTOOLS and BCFTOOLS that include a selection of filters: • Genotype phred-quality score of 30: Q30 indicates an error rate that represents one incorrect base call in 1000 times. • Genotype phred-quality score of 30: Q30 indicates an error rate that represents one incorrect base call in 1000 times. • a maximum of two alleles per single nucleotide polymorphism (hereafter SNP). • a maximum of 50% missing data per site. • a maximum of 50% missing data per site. Reference genome A single reference assembly was selected. It is the chromosome-level assembly obtained for gyrfalcon from the Vertebrate Genomes Project initiative (Bioproject PRJNA561988, release 03/11/2020; https://www.ncbi.nlm.nih.gov/assembly/GCF_015220075.1). It represents 25 haploid chromosomes and plasmids for a total sequence length of 1.2 Gbases and an average size of 49.8 million bp for the chromosomes, ranging from 126.9 to 0.4 million bp. The assembly counts a total of 108 scaffolds (unplaced sequences) that represent a total of 4.6 million bp. Analysis of genomic diversity The basic genomic statistics were obtained per individual using the whole genomic dataset. Three genetic parameters that include observed heterozygosity (Ho), inbreeding coefficient (F) and genetic relatedness (r) were calculated using BCFTOOLS (Danecek et al. 2011) from polymorphic biallelic variants. Finally, we used the plink v.1.90 package to generate a PCA. This programme starts by pruning variants to avoid linkage disequilibrium Genetic diversity and relatedness amongst captive saker falcons (Falco ... 7 before performing the multidimensional analysis, which results in a lower number of variants suitable for the analysis. before performing the multidimensional analysis, which results in a lower number of variants suitable for the analysis. Read mapping and coverage The coverage is 28.1× on average across samples and ranges from 19× to 44×. Distribution of genome sequence coverage for individuals of saker falcons from the captive population. The coverage is 28.1× on average across samples and ranges from 19× to 44×. Read mapping and coverage The whole genome sequencing for the 12 gyrfalcon samples produced a total of 260 GB of clean data (average of 21.7 GB per individual) that provided an average of 231 million reads per individual, ranging from 165 to 355 million reads per individuals (Table 4). The duplication rate is low with 1.0% on average, ranging from 0.3 to 1.7%. When considering the full dataset, the sequencing depth is around 28.1× coverage on average for each individual with 95% of the reference bases covered more than nine times in most cases (Table 5; Fig. 1). Individual ID Raw reads Size Filtered reads Filtered reads (%) Duplication rate Insert size peak 0092A 355,290,258 33.4 GB 355,160,964 3.64E-04 0.0152282 269 0103A 174,086,976 16.5 GB 174,024,596 3.58E-04 0.0038 269 0149A 208,120,172 19.8 GB 208,001,018 5.73E-04 0.0062 269 0155A 255,429,480 24.3 GB 255,306,192 4.83E-04 0.0118 269 1542A 174,721,138 16.1 GB 174,663,876 3.28E-04 0.0073 269 1543A 254,042,750 23.1 GB 253,942,168 3.96E-04 0.0155 269 1544A 185,516,958 17.1 GB 185,467,582 2.66E-04 0.0067 269 1546A 241,417,820 22.7 GB 241,318,934 4.10E-04 0.0072 269 1754A 164,998,882 15.7 GB 164,885,096 6.90E-04 0.0035 269 Table 4. Sequence quality for the 12 samples of saker falcon before and after trimming and filtering using the programme FASTP v.0.20.0 (https://github.com/opengene/fastp). Sequence quality for the 12 samples of saker falcon before and after trimming and filtering us the programme FASTP v.0.20.0 (https://github.com/opengene/fastp). Petrov R et al 8 Individual ID Raw reads Size Filtered reads Filtered reads (%) Duplication rate Insert size peak 184A 241,030,674 22.9 GB 240,921,062 4.55E-04 0.0153 269 319A 237,942,550 22.3 GB 237,832,056 4.64E-04 0.0114 269 320A 278,550,182 26.2 GB 278,419,226 4.70E-04 0.0174 269 Total 2,771,147,840 260.1 GB 2,769,942,770 Figure 1. Distribution of genome sequence coverage for individuals of saker falcons from the captive population. The coverage is 28.1× on average across samples and ranges from 19× to 44×. 8 Petrov R et al Individual ID Raw reads Size Filtered reads Filtered reads (%) Duplication rate Insert size peak 184A 241,030,674 22.9 GB 240,921,062 4.55E-04 0.0153 269 319A 237,942,550 22.3 GB 237,832,056 4.64E-04 0.0114 269 320A 278,550,182 26.2 GB 278,419,226 4.70E-04 0.0174 269 Total 2,771,147,840 260.1 GB 2,769,942,770 Figure 1. Distribution of genome sequence coverage for individuals of saker falcons from the captive population. The coverage is 28.1× on average across samples and ranges from 19× to 44×. Distribution of genome sequence coverage for individuals of saker falcons from the captiv population. Table 5. Mapping statistics obtained after aligning the sakers’ reads to the chromosome-level genome assembly of gyrfalcons. “Mapped reads” indicates the number and percentage of reads that are mapped to the genome; “Mapped paired-end” indicates the number and percentage of paired-end reads that properly mapped to the reference genome. Individual ID Total reads Mapped reads % Mapped reads Mapped paired-end % Mapped paired-end 0092A 355,635,683 354,311,958 99.63% 349,903,098 98.52% 0103A 174,292,918 173,545,178 99.57% 171,874,088 98.76% 0149A 208,354,172 207,173,630 99.43% 204,734,796 98.43% 0155A 255,673,112 254,723,148 99.63% 251,812,422 98.63% 1542A 174,935,440 174,149,259 99.55% 171,854,942 98.39% Individual ID Total reads Mapped reads % Mapped reads Mapped paired-end % Mapped paired-end 1543A 254,349,330 253,383,348 99.62% 249,725,366 98.34% 1544A 185,748,451 185,000,530 99.60% 182,659,534 98.49% 1546A 241,724,700 240,849,092 99.64% 237,734,840 98.51% 1754A 165,204,993 164,557,856 99.61% 162,506,184 98.56% 184A 241,253,321 240,250,110 99.58% 237,704,994 98.67% 319A 238,206,967 237,297,258 99.62% 234,424,374 98.57% 320A 278,908,043 277,847,380 99.62% 274,189,792 98.48% Average 231,190,594 230,257,396 99.59% 227,427,036 98.58% Genetic diversity and relatedness amongst captive saker falcons (Falco ... 9 Genetic diversity and relatedness amongst captive saker falcons (Falco ... 9 Figure 3. Frequency distribution of genetic relatedness performed on the 12 samples. Note that several comparisons of individual pairs show higher relatedness than expected amongst the founder individuals if they were unrelated. These pairs involve individuals 092, 1544 and 1546 and the individuals 1543, 184 and 319, all characterised by relatedness values around 0.2 and above. Genetic diversity and genetic relatedness amongst individuals The overall genetic diversity of the samples are 21.89×10 ± 6.77×10 for nucleotide diversity (π) and 0.293 ± 0.042 for observed heterozygosity. Some individuals like 0103 and 1754 show lower genetic diversity while one, 092, show higher genetic diversity when compared to the rest of the samples (Fig. 2). -5 -5 Figure 2. Genetic diversity of the different individuals as measured by the observed heterozygosity. The average observed heterozygosity (and standard deviation) calculated across samples (Ho = 0.293 ± 0.042) are represented by the horizontal lines and blue shading. g Genetic diversity of the different individuals as measured by the observed heterozygosity. The average observed heterozygosity (and standard deviation) calculated across samples (Ho = 0.293 ± 0.042) are represented by the horizontal lines and blue shading. g Genetic diversity of the different individuals as measured by the observed heterozygosity. The average observed heterozygosity (and standard deviation) calculated across samples (Ho = 0.293 ± 0.042) are represented by the horizontal lines and blue shading. Petrov R et al Petrov R et al 10 The frequency distribution of the genetic relatedness coefficient (r) shows that some saker pairs were more highly related than the sample average, i.e. when individuals were unrelated (Fig. 3). The average value of the relatedness coefficient for all samples is 0.126 ± 0.089 and around 94% of the pairwise comparisons are below a relatedness value of 0.2. Individuals involved in these higher values of relatedness involve individual 092 that show a higher value of relatedness when compared to 1544 (r = 0.243) and 1546 (r = 0.239). The individual, 1543, also has high relatedness values when compared to individuals 0184 (r = 0.218) and 0319 (r = 0.205). Figure 3. Frequency distribution of genetic relatedness performed on the 12 samples. Note that several comparisons of individual pairs show higher relatedness than expected amongst the founder individuals if they were unrelated. These pairs involve individuals 092, 1544 and 1546 and the individuals 1543, 184 and 319, all characterised by relatedness values around 0.2 and above. Figure 3. Figure 4. PCA illustrating the genetic difference amongst founding individuals of saker falcons from the captive population. Three clusters named 1, 2 and 3 can be observed and the colour shadings highlight these clusters. PCA-based genetic clustering Applying the PCA to the current variant dataset, several groupings can be observed (Fig. 4 ). On this figure, each point represents the genetic sample of an individual with its identifier and the distances between points are correlated with genetic distances. When individuals are close on the figure, this means that they are closer genetically than other individuals that are found more distant on the figure. There are three groups that can be observed. The most distinct is group 3 that comprises 0184, 0319 and 1543 and is separated from the two other groups along the first axis. Groups 1 and 2 are differentiated along the second axis. Group 2 is composed of parents and an offspring (092). Genetic diversity and relatedness amongst captive saker falcons (Falco ... Genetic diversity and relatedness amongst captive saker falcons (Falco ... 11 11 Figure 4. PCA illustrating the genetic difference amongst founding individuals of saker falcons from the captive population. Three clusters named 1, 2 and 3 can be observed and the colour shadings highlight these clusters. Quality of the genomic data The blood tissues provided good quality of genomic data with high number of reads and low level of duplication. The sequencing depth ranges from 19× to 44× with 95% of the reference bases covered more than nine times. Genetic diversity and relatedness of founder individuals The analyses of genetic diversity of the different individuals indicated that some individuals have lower genetic diversity than the rest of samples (Barir & Ariel), which may suggest a higher level of inbreeding. Moreover, one individual showed higher genetic diversity than the rest (Bilbo). The relatedness analyses indicated several individuals that seem to be more closely related than expected in the overall samples. These samples included the individual Eurydice that seems to be related to Frodo and Arnold and the individual Bilbo that is found to be kin related to both Thomas and Lucia. Microsatellite analysis The Colony analysis of microsatellite data arranged the sampled individuals as family/ cluster members - the 30 birds were grouped into 17 clusters - 11 of which were represented by only one saker and the other six by two or more which are putatively related. These results are presented in Table 6. Prob (Inc.) Prob (Exc.) Member 1 Member 2 Member 3 Member 4 Member 5 1 1 0.5134 Barir 2 0.9843 0.984 Ariel 3 0.9969 0.5424 Pluto 4 0.9844 0.0963 Orpheus 5 0.7411 0.141 Thomas Table 6. Microsatellite analysis showing the probability that the sampled WRBC saker falcons are part of a family/cluster. Prob (Inc.) gives the probability that all members of the cluster have r = 0.5. Prob (Exc.) indicates the probability that no further members belong to the corresponding category r = 0.5 or full sibling cluster. Microsatellite analysis showing the probability that the sampled WRBC saker falcons are part of a family/cluster. Prob (Inc.) gives the probability that all members of the cluster have r = 0.5. Prob (Exc.) indicates the probability that no further members belong to the corresponding category r = 0.5 or full sibling cluster. Prob (Inc.) Prob (Exc.) Member 1 Member 2 Member 3 Member 4 Member 5 1 1 0.5134 Barir 2 0.9843 0.984 Ariel 3 0.9969 0.5424 Pluto 4 0.9844 0.0963 Orpheus 5 0.7411 0.141 Thomas Petrov R et al 12 Prob (Inc.) Prob (Exc.) Member 1 Member 2 Member 3 Member 4 Member 5 6 0.9328 0.9328 Bilbo Willow Shira Thomas II 7 1 0.9115 Dobry Dracarys 8 1 0.5252 Vulna Penda Plamena Gogo Arnold 9 1 0.0937 Grum 10 0.9464 0.0953 Lucia Boryana 11 1 0.548 Pizho DJ 12 1 0.8856 Lobelia Frodo Eurydice Arwen 13 1 0.1665 Hedy 14 1 0.1037 Bandit 15 1 0.0475 Rhaegal 16 1 0.1086 Maul 17 1 0.2344 Romeo Genetic diversity and relatedness amongst captive saker falcons (Falco ... Genetic diversity and relatedness amongst captive saker falcons (Falco ... 13 Genetic clustering amongst founder individuals The search for genetic structure within the founder individuals of the saker population revealed genetic differences between individuals. It is unclear whether these differences underlie genetic differences between wild individuals or differences related to genetic drift associated with breeding strategies in the captive populations over multiple generations. It is important to note that two of the tree genetic clusters observed on the multidimensional analysis involved the individuals that are suspected to be kin related from the relatedness analysis. These results are in support of a genetic structure potentially arising from the breeding strategy rather than the underlying genetic structure in the wild. Comparison between stud book and microsatellite data There were a number of close-kin relations that were known from our pedigree data, but did not show up in the microsatellite analysis, indicating it could not replace the pedigree records, but can complement them. The test correctly determined certain male parent- offspring relationships (cluster 11: Pizho & DJ; cluster 7: Dobry & Dracarys) and certain sibling relationships (cluster 6: Bilbo, Willow & Thomas II; cluster 8: Vulna, Penda, Plamena & Gogo; cluster 12: Lobelia, Frodo & Arwen). However, Thomas and Lucis’s offspring were not in a cluster with any of them nor was Rhaegal - he was not in either DJ’s or Willow’s (his parents). Nevertheless, he also had a very low probability of not having close relatives in the sample. Revelations stemming from the microsatellite results included three of the sakers - Shira, Arnold and Eurydice. Shira was confiscated in Bulgaria in 2020, far away from the single wild saker breeding territory known at the time - assumed to be an unrelated wild bird from a different line. She was included in the breeding programme. Being genetically associated with T&L’s progeny, it appears most likely that she was instead hatched in captivity in the Breeding Centre for Birds of Prey in Burgas, Bulgaria (breeding saker falcons for commercial purposes, where Thomas and Lucia resided that year), sold to a falconer, escaped and then taken in by a private home in 2020. By the same logic, Arnold may be an offspring of Adam and Eve, as are the others from cluster 8. It could have hatched before we obtained the pair. However this is not confirmed from the genomic study, placing it close to Frodo and Euridice. Eurydice came from the Czech Republic with incomplete pedigree, together with Lobelia, Frodo and Arwen. Both tests in this case clarified the origin of this individual - she seemed to descend from the same parents, however from an earlier clutch as she is older than the other three birds which were known to be from one clutch. WRBC saker stud book Due to the lack of wild saker falcons in Bulgaria, birds from the western population of the species (Falco cherrug cherrug) were obtained from breeding sources in Central Europe and the UK. The saker falcon breeding group at the WRBC consisted of three founding pairs - Adam & Eve (A&E), Orpheus & Eurydice (O&E) and Thomas & Lucia (T&L) (Fig. 5). The offspring of A&E and T&L have formed the rest of the breeding pairs. Five of A&E’s offspring (Juliet, Penda, Plamena, Vulna, Gogo) have formed pairs with imported unrelated individuals. Two of T&L’s offspring (Bilbo and Willow) have formed pairs with A&E’s ‘grandchildren’ (Boryana and DJ). From the date when the saker falcons are paired in the WRBC, the family ties are recorded in a breed registry. However, not all prior relations were known, as it is often the case with pedigrees according to Rabier et al. (2022). Four new relations were uncovered through the tests, one of them required a pair to be separated as a precaution (T&L). Some of the findings were inconsistent between the two DNA tests made, so more research is required for more definitive results. Figure 5. Family tree of the founding pairs and their unreleased offspring, left in the WRBC breeding programme. Family tree of the founding pairs and their unreleased offspring, left in the WRBC breeding programme. Petrov R et al 14 Conclusion Since 2011, in the WRBC in Stara Zagora, Bulgaria, there is a captive breeding group of the globally-endangered saker falcons. The pairs were formed, based on their known pedigree in order for their offspring to be as genetically diverse as possible. The progeny are being released in the wild in the country with the aim to restore the population of the species. Ten years later, in 2021 and 2022, the WRBC team had the opportunity to conduct genomic evaluation of blood samples obtained from 12 founding birds and undertake microsatellite analysis of 30 sakers from the programme. The genomic analysis indicated that two of the individuals in the WRBC breeding group may be kin-related and precautionary measures were taken to avoid breeding them. The rest of the results confirmed the prior information and, in addition, revealed unknown connections between some of the sakers with missing or incomplete pedigrees, indicating they can and should be used together for a better genetic management of these and other species bred in captivity, especially if performed in a timely manner. The development of a greater number of microsatellite loci for sakers and other large falcons through new genomic techniques will greatly enhance this process. Genetic diversity and relatedness amongst captive saker falcons (Falco ... Genetic diversity and relatedness amongst captive saker falcons (Falco ... 15 Acknowledgements The saker falcon captive group is part of project Saker Falcon Reintroduction in Bulgaria, funded by the Mohamed Bin Zayed Raptor Conservation Fund (UAE). Support funding is provided by Armeec JSC (Bulgaria). The project activities are implemented by the Wildlife Rehabilitation and Breeding Centre, part of Green Balkans - Stara Zagora NGO. The authors thank Andreana Dicheva, Andrew Dixon, Ivaylo Klisurov, Stefka Dimitrova and Elke Hippauf. Conflicts of interest The authors have declared that no competing interests exist. Comparison between stud book and genomic data Through the test, it was discovered that birds which were previously thought to be unrelated - Thomas, with a ring from Slovakia and Lucia, with a Czech ring, are closely related. They had been paired before that, in 2014, as 4-year-old birds and had proved to be a very successful pair, raising a total of 81 chicks since then. The results indicated that their offspring Bilbo has the highest genetic diversity of the other 12 tested falcons. Most of their other offspring were reared when the pair were in the Breeding Centre for Birds of Prey, so the chicks were sold for the purposes of falconry. 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Agricultural Science and Technology 14 (4): 23‑29. https://doi.org/10.15547/ast.2022.04.045 • Petrov R, Andonova Y, Yarkov D, Dicheva A (2022) Diets for captive breeding and hacking of saker falcons (Falco cherrug) in Bulgaria. Agricultural Science and Technology 14 (4): 23‑29. https://doi.org/10.15547/ast.2022.04.045 • Pritchard D, Fa J, Oldfield S, Harrop S (2011) Bring the captive closer to the wild: redefining the role of ex situ conservation. Oryx 46 (1): 18‑23. Genetic diversity and relatedness amongst captive saker falcons (Falco ... https://doi.org/10.1017/ s0030605310001766 • Pritchard D, Fa J, Oldfield S, Harrop S (2011) Bring the captive closer to the wild: redefining the role of ex situ conservation. Oryx 46 (1): 18‑23. https://doi.org/10.1017/ s0030605310001766 • Rabier R, Erlichman A, Lesobre L, Robert A (2022) The necessity of considering founder kinships in conservation breeding programs. 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Peregrine Fund • Wang J (2013) A simulation module in the computer program colony for sibship and parentage analysis. Molecular Ecology Resources 13 (4): 734‑739. https://doi.org/ 10.1111/1755-0998.12106 method for releasing peregrine falcons and other birds of prey. Peregrine Fund • Wang J (2013) A simulation module in the computer program colony for sibship and parentage analysis. Molecular Ecology Resources 13 (4): 734‑739. https://doi.org/ 10.1111/1755-0998.12106
https://openalex.org/W2888276131	https://ejb.springeropen.com/track/pdf/10.4103/ejb.ejb_11_18	English	null	Evaluation of health-related quality of life in patients with chronic obstructive pulmonary disease	The Egyptian Journal of Bronchology	2,018	cc-by	5,293	Introduction allitems.Lowscoresindicateabetter HRQoL[10].The current management lines for COPD did not include assessment of HRQoL as a routine assessment. This study aimed to evaluate the QoL in COPD patients and its relationship to the severity of COPD. Health-related quality of life (HRQoL) is composed of various domains that involves physical, mental, and social wellbeing; which greatly varies for each patient [1]. Impaired quality of life (QoL) is closely related to shortness of breath, impaired physical activities, and mental problems including; depression, anxiety, and psychological disorders [2,3]. Chronic obstructive pulmonary disease (COPD) is linked with impaired daily activities (DAs), and reduced HRQoL [4]. Numerous studies [5–7] identified that lower education level, more breathlessness, fatigue, lower BMI, and depressive symptoms adversely affect the HRQoL. QoL in COPD can be measured in nearly 30 min through St George’s Respiratory Questionnaire (SGRQ) [8]. The Egyptian–Arabic version of SGRQ is validated, reliable, and easy to understand [9]. The SGRQ is a self-administered questionnaire that examines three domains through 50 items with 76 weighted responses. The questionnaire has two parts; part I produce the ‘symptoms score’, which examines respiratory manifestations, frequency, and severity; and part 2 ‘the activity score’, which examines the current conditions of a patient that lead to or are affected by dyspnea; and ‘impact scores’ that measures social function and psychosocial disorders resulted from the respiratory illness. A total score is calculated, including Keywords: chronic obstructive pulmonary disease, quality of life, St George’, s respiratory questionnaire Keywords: chronic obstructive pulmonary disease, quality of life, St George’, s respiratory questionnaire aChest Diseases Department, Faculty of Medicine for Girls, Al-Azhar University, Cairo, bTanta Chest Hospital, Tanta, Egypt aChest Diseases Department, Faculty of Medicine for Girls, Al-Azhar University, Cairo, bTanta Chest Hospital, Tanta, Egypt Results There was male predominance (72%); in most of the cases (79%), there were more than 50 years old. COPD patients who had significantly reduced HRQoL. Age, frequency of exacerbations per year, comorbidity, particularly hypertension and ischemic heart disease, modified Medical Council Research Dyspnea scale dyspnea scale, BODE index, GOLD spirometric staging, and New GOLD stage showed significant positive correlation with St George’s Correspondence to Eman S.M. Sobh, MD, Chest Diseases Department, Al-Zahraa University Hospital, Al-Abbassia, Cairo 11517, Egypt. Tel: +201005824279; fax: +20222638359; e-mail: emansobh@azhar.edu.eg Correspondence to Eman S.M. Sobh, MD, Chest Diseases Department, Al-Zahraa University Hospital, Al-Abbassia, Cairo 11517, Egypt. Tel: +201005824279; fax: +20222638359; e-mail: emansobh@azhar.edu.eg Received 15 February 2018 Accepted 20 March 2018 This is an open access journal, and articles are distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms. © 2018 Egyptian Journal of Bronchology \| Published by Wolters Kluwer - Medknow Original article 288 Original article 288 Egyptian Journal of Bronchology 2018 12:288–294 Egyptian Journal of Bronchology 2018 12:288–294 p y Taghreed S. Faraga, Eman S.M. Sobha, Sawsan B. Elsawya, b Respiratory Questionnaire. Lower spirometric parameters, 6- min walk tests, and SpO2%, were correlated with impaired HRQoL. Introduction The quality of life plays an important role in chronic disease management, including chronic obstructive pulmonary disease (COPD). Conclusion Patients with COPD had reduced HRQoL. Frequent exacerbations, advanced airway obstruction, and dyspnea severity had negative impact on HRQoL. Egypt J Bronchol 2018 12:288–294 Objective To assess the quality of life in COPD patients and its relationship to disease severity. Patients and methods Two hundred Egyptian COPD patients were included in the study. We collected demographic data, comorbidities, dyspnea score, and other symptoms. Spirometry, 6-min walk tests were also conducted. Health-related quality of life (HRQoL) assessment was done with the Arabic version of St George’s Respiratory Questionnaire. © 2018 Egyptian Journal of Bronchology © 2018 Egyptian Journal of Bronchology Patients and methods Study design A cross-sectional observational multicentre study (Al- Zahraa University Hospital & Tanta Chest Hospital) involved 200 stable COPD patients from January 2016 to January 2018. All patients had symptoms of chronic airflow limitation and fulfilled GOLD [11] criteria for the diagnosis of COPD.AllhadpostbronchodilatorFEV1/FVClessthan 70% of predicted value, and an increase in FEV1 less than 200 ml or less than 12% of baseline value, 15–20 min after 400 mg of inhaled salbutamol [12]. Demographic data, medical history, and examination data were recorded for each patient. The following tests were done: This is an open access journal, and articles are distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms. DOI: 10.4103/ejb.ejb_11_18 Health-related quality of life in COPD Farag et al. 289 289 found in (56.5%), hypertension was the most common comorbidity detected (34%), followed by diabetes mellitus (18.5%). The majority of patients (83%) had mMRC dyspnea scale, grades 4 and 3, in addition; the majority of patients (93.5%) ranging from moderate to very severe airway obstruction (GOLD grades 2, 3, and 4), mild obstruction (GOLD 1) recorded only in 6.5% of the patients. Concerning new GOLD, ABCD risk classification, most of them lie in grade D, then grade B (53.5%, 45.0%), respectively. Table 2 indicates worsening of airflow limitations, more COPD symptoms with frequent exacerbations. We found reduced HRQoL across all SGRQ domains (symptom, activity, impact, and total scores). The activity score was the highest, indicating that the dyspnea is the most worrying symptom of COPD affecting the HRQoL (Table 3). Multivariate regression analysis was done to test different factors affecting COPD HRQoL to identify the key domains of HRQoL for patients with COPD (Table 4). The results of regression analysis show that the variables are linearly dependent. The major factors that have significant effect on the QoL in patients with COPD (P<0.05) are FEV1%, New GOLD, cardiac diseases, 6MWD, mMRC dyspnea scale, and (1) Spirometry: was carried out using (Care Fusion Spirometry; Hoechberg, Germany) according to ERS/ATS guidelines. The best of three technically acceptable trials was selected [13]. The following indices were recorded FEV1, FVC, and FEV1/ FVC, FEF25–75%, PEF. Airflow limitations were classified according to postbronchodilator FEV1% and to new GOLD, ABCD assessment tool [11]. Statistical analysis Data analyses were performed by using the mean, SD, and χ2 by using the (SPSS), version 20 (IBM Corp., Armonk, New York, USA). Qualitative data were expressed as number and percentage of the total, whereas parametric data were expressed as mean±SD. Multivariate linear regression was used for identification of different factors affecting SGRQ score. Significance level is considered at P-value less than 0.05. Patients and methods Study design (2) BODE index assessment: it includes BMI, FEV1%, modified Medical Council Research Dyspnea scale (mMRC), and 6-minute walking distance (6MWD) [14–16]. (3) Assessment of HRQoL: using Arabic for Egypt version of SGRQ [10]. The questionnaire was self- administered during an interview when the patients were sitting in a calm area, free from interruption, after obtaining consent and a full explanation of the purpose. We explained the questionnaire and stated that no ideal answers are expected, and requested simply to answer what they felt reflects their condition, without any directions from the researchers. Three SGRQ domains (symptom, activity, and impact scores) and the total score were calculated. The scores vary from 0 (no disability) to 100 (maximum disability indicating poorer QoL). (3) Assessment of HRQoL: using Arabic for Egypt version of SGRQ [10]. The questionnaire was self- administered during an interview when the patients were sitting in a calm area, free from interruption, after obtaining consent and a full explanation of the purpose. We explained the questionnaire and stated that no ideal answers are expected, and requested simply to answer what they felt reflects their condition, without any directions from the researchers. Three SGRQ domains (symptom, activity, and impact scores) and the total score were calculated. The scores vary from 0 (no disability) to 100 (maximum disability indicating poorer QoL). Table 1 Demographic and clinical data of the studied chronic obstructive pulmonary disease patients Table 1 Demographic and clinical data of the studied chronic obstructive pulmonary disease patients Variables Mean±SD/n (%) Age (years) 58.30±9.12 <50 41 (20.5) 50–70 147 (73.5) >70 12 (6) Sex Male 144 (72.0) BMI (minimum–maximum) (kg/m2) 24.33±2.79 (18.29–36.89) Smoking habits Nonsmoker 69 (34.5) Current smoker 73 (36.5) Ex-smoker 58 (29.0) Smoking pack/year 37.29±25.18 COPD: duration/year 11.02±7.46 Frequency of exacerbation (minimum–maximum) (year) 3.53±1.98 (1.0–10.0) Comorbidity 113 (56.5) Type of comorbidity HTN 68 (34) DM 37 (18.5) IHD 21 (10.5) Liver diseases 20 (10) GERD 15 (7.5) Renal diseases 7 (3.5) CVA 1 (0.5) COPD, chronic obstructive pulmonary disease; CVA, cerebrovascular attack; DM, diabetes mellitus; GERD, Gastero-esophageal reflux; HTN, hypertension; IHD, ischemic heart diseases. Ethical statement The study was approved by the Institutional Ethical Review Board and Ethics Committee of the Faculty of Medicine for Girls, Al-Azhar University. All patients gave informed consent. Written permission to use St George’s Respiratory Questionnaire in this study was obtained before starting the study through e-mail contact with the developers. Results We selected 200 COPD patients, men were predominant (72%) and most of them (79%) were more than 50 years old. Table 1 shows demographic and clinical characteristics of the studied COPD patients, patients had at least one exacerbation within the previous year. Comorbid illness were 0 Egyptian Journal of Bronchology, Vol. 12 No. 3, July-September 2018 290 exacerbations per year respectively. This model of regression can achieve accurate results by about 80% (adjusted R2=0.806). It is statistically significant and accepted (F=37.176). significant positive correlation with the activity and impact scores (r=0.208 and 0.228, respectively), also, ischemic heart disease had a significant positive correlation with activity, impact, and total scores (r=0.302, 0.329, and 0.325, respectively). mMRC dyspnea scale, BODE index, and Gold stages had a significant positive correlation with SGRQ scores. However, 6-min walk tests, SpO2%, and spirometric indices had a significant negative correlation with all SGRQ scores (Figs 1–3). significant positive correlation with the activity and impact scores (r=0.208 and 0.228, respectively), also, ischemic heart disease had a significant positive correlation with activity, impact, and total scores (r=0.302, 0.329, and 0.325, respectively). mMRC dyspnea scale, BODE index, and Gold stages had a significant positive correlation with SGRQ scores. However, 6-min walk tests, SpO2%, and spirometric indices had a significant negative correlation with all SGRQ scores (Figs 1–3). In the current study, age, frequency of exacerbations per year, and comorbidity had a significant positive correlation with SGRQ total score and scores of all domains. Among the comorbidities hypertension had a Table 2 Spirometric indices, BODE index and its parameters and St George’s Respiratory Questionnaire score for the studied population (n=200) Variables Mean±SD/n (%) FEV1% 50.76±19.80 FVC% 71.53±24.17 FEV1/FVC (%) 58.82±10.82 FEF25–75% 27.28±18.99 GOLD stage GOLD 1 mild 13 (6.5) GOLD 2 moderate 84 (42.0) GOLD 3 severe 74 (37.0) GOLD 4 very severe 29 (14.5) New GOLD Grade A 0 (0.0) Grade B 90 (45.0) Grade C 3 (1.5) Grade D 107 (53.5) BODE index 6.14±2.28 6MWD 158.57±80.16 mMRC dyspnea scale 3.26±0.74 Grade 1 1 (0.5) Grade 2 33 (16.5) Grade 3 80 (40.0) Grade 4 86 (43.0) BODE, body mass index, obstruction (airway), dyspnea, exercise; mMRC, modified Medical Council Research Dyspnea scale; 6MWD, 6 minute walk distance. Table 2 Spirometric indices, BODE index and its parameters and St George’s Respiratory Questionnaire score for the studied population (n=200) B, unstandardized coefficients; IHD, ischemic heart diseases; mMRC, modified Medical Council Research Dyspnea scale; R, coefficient of regression; t, t-test of significance. P≤0.05, statistically significant. Discussion Correlation between 6 minute walk distance and St George’s Respi- ratory Questionnaire. [26], reported that HRQoL was more impaired in younger patients. Correlation between age and St George’s Respiratory Questionnaire. g g p y Figure 2 Correlation between FEV1% and St George’s Respiratory Question- naire. Concerning BMI, the current study demonstrated no correlations between BMI and HRQoL. Various studies [27,28] showed that the correlation between the BMI and total SGRQ scores was weak. Moy et al. [26], have shown that the overweight patients have worse HRQoL. Sundh et al. [29], concluded that comorbidity and BMI are associated with impaired HRQoL in COPD patients. Katsura et al. [30], added that the patients with low BMI had poor HRQoL and suggested that the introduction of nutritional intervention might be beneficial to improve the QoL and dyspnea in those patients. Correlation between FEV1% and St George’s Respiratory Question- naire. In terms of smoking habit, the majority of studied patients (65.5%) are current and ex-smokers, no correlation is there between smoking and HRQoL by SGRQ score. The possible explanation may be that if once the impairment occurs, further exposure to smoking does not affect the overall HRQoL. Our results coincide with Katsura and colleagues [30–32]. They reported no significant adverse effect of smoking on SGRQ. However, Zamzam et al. [33], have shown that there was a statistically significant positive correlation between smoking index and both symptoms score and impact score. Whereas, the correlations between both total score and activity score and smoking pack/year were nonsignificant. Obaseki et al. [25], found that the heavy smokers had worse HRQoL symptom scores compared with nonsmokers. Also, Ahmed et al. [34] and Shavro et al. [35], showed a significant inverse correlation between smoking pack/year and HRQoL scores. On the other, a study carried out by Wijnhoven et al. [36], reported better HRQoL in current smokers. activity score was the highest, indicating that the dyspnea is the most distressing COPD symptom affecting HRQoL. The same results were reported in previous studies [5,6,20]. The effects of age on HRQoL have been researched in numerous clinical studies, and their results are controversial. In the current study, the HRQoL in COPD patients was adversely affected by age, there was a statistically significant positive correlation between age and SGRQ score with a strong linear relationship across all domains (Fig. Discussion In the current study; the mean age of Egyptian COPD patients was (58.30±9.12), most of them (79%) were more than 50 years old with majority as men (72%); every patient had one or more exacerbations in the previous year. COPD is a male-dominant disease with a higher incidence in fifth and sixth decades of life [17,18]. This may be attributed to the difference in tobacco smoking/consumption in both sexes [19]. The current study demonstrates that COPD patient had significantly impaired HRQoL based on high SGRQ scores across (symptom, activity, impact, and total scores). Among all aspects affecting COPD, the Table 3 St George’s Respiratory Questionnaire scores in the studied group Variables Mean±SD/n (%) SGRQ symptoms score 65.42±17.42 SGRQ activity score 68.85±21.38 SGRQ impact score 56.47±19.97 SGRQ total score 61.77±18.63 SGRO, St George’s Respiratory Questionnaire. BODE, body mass index, obstruction (airway), dyspnea, exercise; mMRC, modified Medical Council Research Dyspnea scale; 6MWD, 6 minute walk distance. BODE, body mass index, obstruction (airway), dyspnea, exercise; mMRC, modified Medical Council Research Dyspnea scale; 6MWD, 6 minute walk distance. Table 4 Multivariate linear regression for factors affecting St George’s Respiratory Questionnaire B SE β t P FEV1 0.155 0.061 0.164 2.526 0.013* New GOLD 4.361 1.768 0.239 2.467* 0.015* IHD −7.757 3.277 −0.107 2.367* 0.020* 6MWD −0.039 0.018 −0.187 2.158* 0.034* mMRC dyspnea scale 4.809 2.218 0.193 2.168* 0.033* Exacerbations/year 0.972 0.468 0.124 2.076* 0.041* Renal diseases 4.107 2.272 0.092 1.808 0.074 Age −0.137 0.116 −0.062 1.183 0.240 FEF25–75% −0.294 0.176 −0.315 1.672 0.098 FVC% 0.075 0.076 0.103 0.985 0.327 FVC/l 1.505 1.094 0.085 1.376 0.172 FEV1/FVC (%) 0.040 0.100 0.026 0.405 0.687 BODE index 1.375 1.267 0.169 1.086 0.280 GOLD stage −1.481 3.278 −0.066 0.452 0.652 Comorbidity −10.905 10.080 −0.059 1.082 0.282 R2=0.850, adjusted R2=0.816, SE=7.48, F=24.905, P<0.001 B, unstandardized coefficients; IHD, ischemic heart diseases; mMRC, modified Medical Council Research Dyspnea scale; R, coefficient of regression; t, t-test of significance. *P≤0.05, statistically significant. Table 4 Multivariate linear regression for factors affecting St George’s Respiratory Questionnaire Health-related quality of life in COPD Farag et al. 29 291 Figure 3 Correlation between 6 minute walk distance and St George’s Respi- ratory Questionnaire. Figure 3 Correlation between 6 minute walk distance and St George’s Respi- ratory Questionnaire. Figure 1 Figure 3 Correlation between age and St George’s Respiratory Questionnaire. Correlation between age and St George’s Respiratory Questionnaire. Discussion The previous data indicate that BODE index and its parameters have excellent discriminative capabilities, to detect the deterioration of HRQoL earlier and predicts future mortality risk to take appropriate and effective actions to relieve patients’ symptoms, improve patients’ exercise tolerance, and slow down disease progression. Numerous studies reported the same results, Nonato et al. [44] and Sarkar et al. [27], concluded that BODE index showed very strong correlation with the total SGRQ score, activity and impact domain of SGRQ score, but showedmoderatecorrelationwiththesymptomdomain. Otherstudies[45–47] reported that the6-minwalktests correlatedwithalldomainsandwiththetotalscoreofthe SGRQ. Many researchers [27,28,45] concluded that mMRC scale for dyspnea showed a positive correlation with the QoL in variable degrees. They explained that patients who are still smoking might be those with a less advanced stage of the disease. In this study, COPD patients experienced at least one exacerbation within the previous year. The frequency of exacerbations illustrates strong linear relationship across all SGRQ domains (P<0.001) suggesting that worse HRQoL is related to COPD exacerbations (Tables 1–4). Ekici et al. [37], reported that poor HRQoL was related to severity of COPD, dyspnea grade, and exacerbation frequency. Seemungal et al. [38], reported worse impact scores in patients with frequent past exacerbations. In addition, this study noted that 56.5% of the patients with COPD had comorbidities. Hypertension, diabetes mellitus, and ischemic heart diseases (IHD), are the most recorded comorbidities. All domains of SGRQ scores are adversely affected by comorbidities (P<0.001). IHD and hypertension were statistically correlated with activity and impact score, indicating that impaired DAs, breathlessness, fatigue, psycho- social disturbances, and anxiety are more common in COPD with cardiovascular disorders. IHD is one of the most significant factors affecting the QoL of patients with COPD (P<0.02). Huber et al. [39], concluded that there was significant negative association between comorbidities and HRQoL in COPD. Concerning lung function assessment most of our studied patients have advanced airflow limitations, more COPD symptoms with frequent exacerbations. The SGRQ score worsens considerably with increasing disease severity. The worst scores were detected among advanced stages of COPD (Table 2). Using multiple regression analysis, it was demonstrated that FEV1, new GOLD stage, IHD, mMRC, exacerbations per year, and 6MWD were the best predictor of impaired HRQoL assessed by the SGRQ score. Although, age, other spirometry parameters, BODE index, and GOLD stage have significant correlations with the SGRQ. In the multiple regression analysis; all variables were ruled out as predictive variables. Discussion 1), but the impact score is the highest (P<0.002) indicating more worsening of social function and psychosocial disturbances with aging, this could be clarified by the fact that aging is usually linked with impaired activity, depressive symptoms, and anxiety. Similar results have been reported by many authors [20–23]. Conversely, others [24,25] found a nonsignificant correlation between age and HRQoL. However; Moy et al. 292 Egyptian Journal of Bronchology, Vol. 12 No. 3, July-September 2018 292 COPD had impaired physical activity determined by advanced grades of dyspnea, limited 6MWD, and high BODE index score (Table 2) affecting their HRQoL. The majority of the patients (83%) had mMRC dyspnea grades 4 and 3, indicating high subjective dyspnea with a higher degree of disability, it also predicts future mortality risk. Also, we found a significant positive correlation between SGRQ and both BODE index and mMRC dyspnea with a strong linear relationship across all domains (P<0.001). However; there was a significantnegativecorrelationbetweenthe6MWDand all SGRQ domains. The 6MWD and mMRC are good predictors of HRQoL in patients with COPD (P<0.05) (Table 4). The previous data indicate that BODE index and its parameters have excellent discriminative capabilities, to detect the deterioration of HRQoL earlier and predicts future mortality risk to take appropriate and effective actions to relieve patients’ symptoms, improve patients’ exercise tolerance, and slow down disease progression. Numerous studies reported the same results, Nonato et al. [44] and Sarkar et al. [27], concluded that BODE index showed very strong correlation with the total SGRQ score, activity and impact domain of SGRQ score, but showedmoderatecorrelationwiththesymptomdomain. Otherstudies[45–47] reported that the6-minwalktests correlatedwithalldomainsandwiththetotalscoreofthe SGRQ. Many researchers [27,28,45] concluded that mMRC scale for dyspnea showed a positive correlation with the QoL in variable degrees. COPD had impaired physical activity determined by advanced grades of dyspnea, limited 6MWD, and high BODE index score (Table 2) affecting their HRQoL. The majority of the patients (83%) had mMRC dyspnea grades 4 and 3, indicating high subjective dyspnea with a higher degree of disability, it also predicts future mortality risk. Also, we found a significant positive correlation between SGRQ and both BODE index and mMRC dyspnea with a strong linear relationship across all domains (P<0.001). However; there was a significantnegativecorrelationbetweenthe6MWDand all SGRQ domains. The 6MWD and mMRC are good predictors of HRQoL in patients with COPD (P<0.05) (Table 4). References Health-related quality of life in patients with chronic obstructive pulmonary disease in North India. J Postgrad Med 2014; 60:7. 32 Garrido PC, de Miguel Díez J, Gutiérrez JR, Centeno AM, Vázquez EG, de Miguel ÁG, et al. Negative impact of chronic obstructive pulmonary disease on the health-related quality of life of patients. Results of the EPIDEPOC study. Health Qual Life Outcomes 2006; 4:31. 8 Pereira ED, Pinto R, Alcantara M, Medeiros M, Mota RM. 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Determinants of health related quality of life in a sample of patients with chronic obstructive pulmonary disease in Nigeria using the St. George’s respiratory questionnaire. Afr Health Sci 2013; 13:694–702. Financial support and sponsorship 23 Hesselink AE, van Der Windt DA, Penninx BW, Wijnhoven HA, Twisk JW, Bouter LM, et al. What predicts change in pulmonary function and quality of life in asthma or COPD?. J Asthma 2006; 43:513–519. 24 Burgel PR, Escamilla R, Perez T, Carré P, Caillaud D, Chanez P, et al. Impact of comorbidities on COPD-specific health-related quality of life. Respir Med 2013; 107:233–241. Discussion Using multiple regression analysis, it was demonstrated that FEV1, new GOLD stage, IHD, mMRC, exacerbations per year, and 6MWD were the best predictor of impaired HRQoL assessed by the SGRQ score. Although, age, other spirometry parameters, BODE index, and GOLD stage have significant correlations with the SGRQ. In the multiple regression analysis; all variables were ruled out as predictive variables. Miyazaki et al. [40] and Burgel et al. [24], documented a significant correlation between COPD severity and SGRQ. As the degree of severity increases, the more impaired is the SGRQ. Zamzam et al. [33], concluded that there was a statistically significant negative correlation between FEV1, FEV1/FVC, PEFR, FEF25–75%, and SGRQ score. In addition, Jones et al. [41], reported that HRQoL impairment varied extensively within each GOLD stage of severity, and marked HRQoL impairment takes place even in the early stages of COPD with little variability between patients in GOLD stage I and II. However, Ong et al. [42], concluded that COPD stage according to GOLD had a mild correlation with SGRQ. This study has some strengths including large sample size, different modalities to examine functional status (spirometry, 6MWD, and BODE index). But we still have some limitations; the most important is the small number of female participant, reflecting the fact that the number of women with COPD seen in our clinical practice is low. However, this has been seen in previous studies. Also, the number of participants in GOLD stage 1 are significantly low. So; nearly our results measured HRQoL in moderate to very severe cases. This is because most patients with COPD in our community seek medical assistance only when they have significant symptoms and advanced airway obstruction. Previous studies documented impaired physical activity in COPD patients [43]. In this study, patients with Conclusion 19 Aryal S, Diaz-Guzman E, Mannino DM. Influence of sex on chronic obstructive pulmonary disease risk and treatment outcomes. Int J Chron Obstruct Pulmon Dis 2014; 9:1145. This study suggests that the SGRQ is of great value for evaluating the impact of HRQoL on COPD. 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A study of health related quality of life in COPD patients in relation to severity of disease. Assam J Int Med 2015; 5:22–25. 41 Jones PW, Brusselle G, Dal Negro RW, Ferrer M, Kardos P, Levy M, et al. Health-related quality of life in patients by COPD severity within primary care in Europe. Respir Med 2011; 105:57–66. 42 Ong KC, Lu SJ, Soh CS. Does the multidimensional grading system (BODE) correspond to differences in health status of patients with COPD?. Int J Chron Obstruct Pulmon Dis 2006; 1:91. 46 Santos KD, Karloh M, Araujo CL, D’Aquino AB, Mayer AF. Relationship between the functional status constructs and quality of life in COPD. Fisioter Mov 2014; 27:361–369. 43 Galal-Eldin MM, Ahmad SE, Hafez RM, Sobh E, Alrayes HM. Telomere length in chronic obstructive pulmonary disease. Egypt J Bronchol 2015; 9:20–26. 47 Busby AK. Sex Differences in Inflammation, Psychological Functioning, and Disease Outcomes Among COPD Patients Participating in Pulmonary Exercise Rehabilitation. Electronic Thesis or Dissertation. Ohio State University, 2010. OhioLINK Electronic Theses and Dissertations Center. [Last accessed 15 February 2018]. 44 Nonato NL, Díaz O, Nascimento OA, Dreyse J, Jardim JR, Lisboa C. 45 Konwar G, Sarma J. A study of health related quality of life in COPD patients in relation to severity of disease. Assam J Int Med 2015; 5:22–25. 294 Egyptian Journal of Bronchology, Vol. 12 No. 3, July-September 2018 References Behavior of quality of life (SGRQ) in COPD patients according to BODE scores. Arch Bronconeumol 2015; 51:315–321.
https://openalex.org/W1543091857	https://ccforum.biomedcentral.com/counter/pdf/10.1186/s13054-015-1001-y	English	null	Erratum to: Effect of insertion route on risk of central line-associated bloodstream infection in critically ill patients	Critical care	2,015	cc-by	580	ERRATUM Open Access Reference 1. Hassan N, Hammodi A, Alhubail R, Ali Alazem, Rayyan N. Effect of insertion route on risk of central line-associated bloodstream infection in critically ill patients. Crit Care. 2015;19:76. Erratum After publication of our recent Poster presentation [1], we noticed that three authors were inadvertently omitted as co-authors. The author list is now complete and reads follows: N. Hassan, A. Hammodi, R. Alhubail, Ali Alazem, N. Rayyan. Erratum to: Effect of insertion route on risk of central line-associated bloodstream infection in critically ill patients N. Hassan, A. Hammodi, R. Alhubail, Ali Alazem and N. Rayyan N. Hassan, A. Hammodi, R. Alhubail, Ali Alazem and N. Rayyan Hassan et al. Critical Care (2015) 19:315 DOI 10.1186/s13054-015-1001-y Hassan et al. Critical Care (2015) 19:315 DOI 10.1186/s13054-015-1001-y Reference Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit .med.sa Hospital-Dammam, 6830 Ammar Bin mmam, Saudi Arabia © 2015 Hassan et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit his article is distributed under the terms of the Creative Commons Attribution 4.0 commons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and ded you give appropriate credit to the original author(s) and the source, provide a e, and indicate if changes were made. The Creative Commons Public Domain mmons.org/publicdomain/zero/1.0/) applies to the data made available in this Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit • Convenient online submission • Thorough peer review Correspondence: Rasheed.Hubail@kfsh.med.sa ICU department, King Fahad Specialist Hospital-Dammam, 6830 Ammar Bin Thabit St, Al Muraikabat, 32253-3202 Dammam, Saudi Arabia * Correspondence: Rasheed.Hubail@kfsh.med.sa ICU department, King Fahad Specialist Hospital-Dammam, 6830 Ammar Bin Thabit St, Al Muraikabat, 32253-3202 Dammam, Saudi Arabia © 2015 Hassan et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
https://openalex.org/W2910182884	https://europepmc.org/articles/pmc6752325?pdf=render	English	null	Deep-intronic ABCA4 variants explain missing heritability in Stargardt disease and allow correction of splice defects by antisense oligonucleotides	Genetics in medicine	2,019	cc-by	10,221	Deep-intronic ABCA4 variants explain missing heritability in Stargardt disease and allow correction of splice defects by antisense oligonucleotides Riccardo Sangermano, PhD1,2, Alejandro Garanto, PhD1,3, Mubeen Khan, MSc1,3, Esmee H. Runhart, MD3,4, Miriam Bauwens, PhD5, Nathalie M. Bax, MD3,4, L. Ingeborgh van den Born, MD, PhD6, Muhammad Imran Khan, PhD1,2, Stéphanie S. Cornelis, MSc1,3, Joke B. G. M. Verheij, MD7, Jan-Willem R. Pott, MD, PhD8, Alberta A. H. J. Thiadens, MD, PhD9, Caroline C. W. Klaver, MD, PhD4,9, Bernard Puech, MD10, Isabelle Meunier, PhD11,12, Sarah Naessens, MSc5, Gavin Arno, PhD13,14, Ana Fakin, PhD13,14, Keren J. Carss, PhD15,16, F. Lucy Raymond, MD, PhD16,17, Andrew R. Webster, MD, PhD13,14, Claire-Marie Dhaenens, MD, PhD18, Heidi Stöhr, PhD19, Felix Grassmann, PhD19,20, Bernhard H. F. Weber, PhD19, Carel B. Hoyng, MD, PhD3,4, Elfride De Baere, MD, PhD5, Silvia Albert, PhD1,3, Rob W. J. Collin, PhD1,3 and Frans P. M. Cremers, PhD1,3 ARTICLE Deep-intronic ABCA4 variants explain missing heritability in Stargardt disease and allow correction of splice defects by antisense oligonucleotides Riccardo Sangermano, PhD1,2, Alejandro Garanto, PhD1,3, Mubeen Khan, MSc1,3, Esmee H. Runhart, MD3,4, Miriam Bauwens, PhD5, Nathalie M. Bax, MD3,4, L. Ingeborgh van den Born, MD, PhD6, Muhammad Imran Khan, PhD1,2, Stéphanie S. Cornelis, MSc1,3, Joke B. G. M. Verheij, MD7, Jan-Willem R. Pott, MD, PhD8, Alberta A. H. J. Thiadens, MD, PhD9, Caroline C. W. Klaver, MD, PhD4,9, Bernard Puech, MD10, Isabelle Meunier, PhD11,12, Sarah Naessens, MSc5, Gavin Arno, PhD13,14, Ana Fakin, PhD13,14, Keren J. Carss, PhD15,16, F. Lucy Raymond, MD, PhD16,17, Andrew R. Webster, MD, PhD13,14, Claire-Marie Dhaenens, MD, PhD18, Heidi Stöhr, PhD19, Felix Grassmann, PhD19,20, Bernhard H. F. Weber, PhD19, Carel B. Hoyng, MD, PhD3,4, Elfride De Baere, MD, PhD5, Silvia Albert, PhD1,3, Rob W. J. Collin, PhD1,3 and Frans P. M. Cremers, PhD1,3 ARTICLE Deep-intronic ABCA4 variants explain missing heritability in Stargardt disease and allow correction of splice defects by antisense oligonucleotides Riccardo Sangermano, PhD1,2, Alejandro Garanto, PhD1,3, Mubeen Khan, MSc1,3, Esmee H. Runhart, MD3,4, Miriam Bauwens, PhD5, Nathalie M. Bax, MD3,4, L. Ingeborgh van den Born, MD, PhD6, Muhammad Imran Khan, PhD1,2, Stéphanie S. Cornelis, MSc1,3, Joke B. G. M. Verheij, MD7, Jan-Willem R. Pott, MD, PhD8, Alberta A. H. J. Thiadens, MD, PhD9, Caroline C. W. Klaver, MD, PhD4,9, Bernard Puech, MD10, Isabelle Meunier, PhD11,12, Sarah Naessens, MSc5, Gavin Arno, PhD13,14, Ana Fakin, PhD13,14, Keren J. Carss, PhD15,16, F. Lucy Raymond, MD, PhD16,17, Andrew R. Webster, MD, PhD13,14, Claire-Marie Dhaenens, MD, PhD18, Heidi Stöhr, PhD19, Felix Grassmann, PhD19,20, Bernhard H. F. Weber, PhD19, Carel B. Hoyng, MD, PhD3,4, Elfride De Baere, MD, PhD5, Silvia Albert, PhD1,3, Rob W. J. Collin, PhD1,3 and Frans P. M. Cremers, PhD1,3 Purpose: Using exome sequencing, the underlying variants in many persons with autosomal recessive diseases remain undetected. We explored autosomal recessive Stargardt disease (STGD1) as a model to identify the missing heritability. found in cis with c.5603A>T (p.Asn1868Ile), so its causal role could not be fully established. Variant c.4253+43G>A resulted in partial skipping of exon 28. Remarkably, antisense oligonucleotides targeting the aberrant splice processes resulted in (partial) correc- tion of all splicing defects. Methods: Sequencing of ABCA4 was performed in 8 STGD1 cases with one variant and p.Asn1868Ile in trans, 25 cases with one variant, and 3 cases with no ABCA4 variant. The effect of intronic variants was analyzed using in vitro splice assays in HEK293T cells and patient-derived fibroblasts. Antisense oligonucleotides were used to correct splice defects. Conclusion: Our data demonstrate the importance of assessing noncoding variants in genetic diseases, and show the great potential of splice modulation therapy for deep-intronic variants. Genetics in Medicine (2019) 21:1751–1760; https://doi.org/10.1038/s41436- 018-0414-9 Results: In 24 of the probands (67%), one known and five novel deep-intronic variants were found. The five novel variants resulted in messenger RNA pseudoexon inclusions, due to strengthening of cryptic splice sites or by disrupting a splicing silencer motif. Variant c.769-784C>T showed partial insertion of a pseudoexon and was Keywords: ABCA4; antisense oligonucleotide; deep-intronic variant; missing heritability; Stargardt disease Keywords: ABCA4; antisense oligonucleotide; deep-intronic variant; missing heritability; Stargardt disease Submitted 19 April 2018; accepted: 7 December 2018 Published online: 15 January 2019 Correspondence: Rob W. J. Collin (rob.collin@radboudumc.nl) or Frans P. M. Cremers (frans.cremers@radboudumc.nl). #Affiliations are listed at the end of the paper. Shared first authors: Riccardo Sangermano, Alejandro Garanto, Mubeen Khan Shared senior: Rob W.J. Collin and Frans P.M. Cremers GENETICS in MEDICINE \| Volume 21 \| Number 8 \| August 2019 Correspondence: Rob W. J. Collin (rob.collin@radboudumc.nl) or Frans P. M. Cremers (frans.cremers@radboudumc.nl). #Affiliations are listed at the end of the paper. Shared first authors: Riccardo Sangermano Alejandro Garanto Mubeen Khan Rescue studies using antisense oligonucleotides in fibroblasts Fibroblast cells were transfected with each of the antisense oligonucleotides, or transfected with empty liposomes (NT). Forty-four hours posttransfection, medium was removed and medium containing cycloheximide (CHX) was added to block nonsense-mediated decay (NMD). Four hours later, cells were harvested and subjected to RNA analysis by RT-PCR. All experiments were performed in two independent replicates. Primer sequences are indicated in Table S1. Samples Written informed consent was obtained prior to participation in the study, which adhered to the Declaration of Helsinki. This study was approved by the Medical Ethical Committee 2010-359 (Protocol nr. 2009-32; NL nr. 34152.078.10) and the Commissie Mensgebonden Onderzoek Arnhem-Nijmegen (Dossier no. 2015-1543; dossier code sRP4h). More informa- tion regarding the proband cohort, and details of the materials and methods used, are provided in the Supplementary Material. Antisense oligonucleotides For each deep-intronic variant that caused PE inclusion, three AONs were designed (Table S2). The design of the therapeutic molecules was performed as described elsewhere.32 Oligonu- cleotides had a phosphorothioate backbone with a 2-O- methyl sugar modification (2OMe/PS) and were synthesized by Eurogentec. The oligonucleotides were resuspended in phosphate-buffered saline (PBS) and used at a concentration of 0.5 µM. In this study, we employed STGD1 as a model to assess the challenges to identify pathogenic noncoding variants. We sequenced the ABCA4 locus in 36 STGD1 individuals with one ABCA4 variant and p.Asn1868Ile in trans (n = 8), one ABCA4 variant (n = 25), or no (n = 3) ABCA4 variants. Selected noncoding variants were tested by in vitro splice assays or using STGD1-derived fibroblasts. Subsequently, several AONs were designed and successfully applied to redirect erroneous splicing. ARTICLE SANGERMANO et al (MIM 601691)10 as biallelic variants can be found in the majority of the cases11 and in a smaller proportion of other IRD subtypes.12–14 In ~30% of STGD1 cases, however, the expected second ABCA4 variant was lacking. A frequent coding variant, p.Asn1868Ile, was present in ~10% of STGD1 cases who had a severe pathogenic variant in trans and who generally showed late-onset disease.15,16 The remaining pathogenic variants in ABCA4 rarely consist of heterozygous deletions,17–19 whereas deep-intronic ABCA4 variants also account for some of the missing heritability in STGD1.11,17,20,21 By employing STGD1-derived keratinocytes, it was shown that some of these variants strengthen cryptic splice sites or create new splice sites, ultimately resulting in pseudoexon (PE) inclusions.21 Using STGD1-derived photo- receptor precursor cells (PPCs), we recently showed that two intronic variants in ABCA4 (c.4539+2001G>A and c.4539 +2028C>T) strengthen exonic splice enhancer elements (ESEs), resulting in the inclusion of a 345-nt PE.22 (MIM 601691)10 as biallelic variants can be found in the majority of the cases11 and in a smaller proportion of other IRD subtypes.12–14 In ~30% of STGD1 cases, however, the expected second ABCA4 variant was lacking. A frequent coding variant, p.Asn1868Ile, was present in ~10% of STGD1 cases who had a severe pathogenic variant in trans and who generally showed late-onset disease.15,16 The remaining pathogenic variants in ABCA4 rarely consist of heterozygous deletions,17–19 whereas deep-intronic ABCA4 variants also account for some of the missing heritability in STGD1.11,17,20,21 By employing STGD1-derived keratinocytes, it was shown that some of these variants strengthen cryptic splice sites or create new splice sites, ultimately resulting in pseudoexon (PE) inclusions.21 Using STGD1-derived photo- receptor precursor cells (PPCs), we recently showed that two intronic variants in ABCA4 (c.4539+2001G>A and c.4539 +2028C>T) strengthen exonic splice enhancer elements (ESEs), resulting in the inclusion of a 345-nt PE.22 clones described previously30 and the newly designed BA30. WT and mutant constructs were transfected in HEK293T cells and the extracted total RNA was subjected to reverse transcription polymerase chain reaction (RT-PCR) (Table S1) as described previously.30 Capillary analysis To quantify the ratios between correct and aberrant transcripts we loaded the RT-PCR samples onto a Fragment Analyzer Auto Capillary Electrophoresis System (Advanced Analytical Technologies, Inc.). Analysis of the peaks was In vitro rescue studies in HEK293T cells using midigenes and AONs HEK293T cells were transfected with either the wild-type or the mutant construct. Twenty-four hours posttransfection, each well was subdivided in five wells to be transfected with the respective AON, the sense oligonucleotide (SON), or left untransfected (NT). Forty-eight hours post-AON delivery, cells were harvested and RNA analysis was performed by RT- PCR. All experiments were performed in two independent replicates. Primer sequences can be found in Table S1. INTRODUCTION the majority of noncoding sequence variants,1 but it will be very challenging to pinpoint the causal variants in the absence of assays that reveal the resulting messenger RNA (mRNA) and/or protein defects. In the past decade, next-generation sequencing (NGS) has enabled a rapid identification of novel disease genes,1–3 and transformed molecular diagnostic testing.4–6 Thus far, the emphasis was on coding and flanking splice site sequences that harbor the majority of genetic defects. Herewith, the underlying defects could be identified in ~50% of individuals with genetically heterogeneous diseases, including inherited retinal diseases (IRDs).7–9 Genome sequencing can identify For diseases that are due to variants in a single gene, the yield of variants generally exceeds 50%. This is true for Stargardt disease (STGD1) (MIM 248200), which is caused by pathogenic variants in the gene encoding the adenosine triphosphate (ATP) binding cassette type A4 (ABCA4) 1751 GENETICS in MEDICINE \| Volume 21 \| Number 8 \| August 2019 GENETICS in MEDICINE \| Volume 21 \| Number 8 \| August 2019 GENETICS in MEDICINE Single-molecule molecular inversion probe–based sequence analysis of intronic ABCA4 regions To test the occurrence of five intronic ABCA4 variants (c.769- 784C>T, c.859-506G>C, c.4253+43G>A, c.4539+1100A>G, c.4539+1106C>T) in 412 genetically unsolved STGD1 cases from France (n = 224) or Germany (n = 188), we designed single-molecule molecular inversion probes (smMIPs) based on a previously developed cost-effective sequencing proto- col.31 The variant calling and annotation was performed using an in-house pipeline. The number of single-molecule consensus reads ranged from 14 to 1587, with an average coverage of 489x. PE inclusions due to deep-intronic variants are attractive targets for antisense oligonucleotide (AON)-based splicing correction.23 In the field of IRDs, these molecules have shown therapeutic potential for deep-intronic pathogenic variants in CEP290,24–27 USH2A,28 or OPA1,29 and a phase 1/2 clinical trial using AONs to treat CEP290-associated Leber congenital amaurosis is ongoing (NCT03140969). ABCA4 sequence analysis and selection of candidate splice variants Detailed information of the selection of candidate splice variants and inclusion criteria is provided in the Supplemen- tary Material. RESULTS Haloplex sequencing of eight STGD1 cases carrying one causal ABCA4 variant and p.Asn1868Ile in trans, 22 STGD1 cases with only one, and two cases carrying no ABCA4 variant, yielded 220 rare ABCA4 variants (allele frequency [AF] ≤0.01 in control individuals), consisting of 85 indepen- dent novel variants (Table S3). In the four British STGD1 cases with one (n = 3) or no (n = 1) ABCA4 variant analyzed by genome sequencing, eight novel rare (AF ≤0.01) independent ABCA4 variants remained (Table S3). The deep-intronic variant c.859-506G>C was predicted to significantly strengthen a cryptic intronic splice acceptor site and is accompanied by a downstream splice donor site. This variant resulted in a 56-nt PE insertion (Fig. 1b, Figure S2), which accounted for 75.5% of total splice products (Table S7), indicating the highly deleterious effect of this variant at the RNA and (predicted) protein level (r.[858_859ins(56), =]; p.[Phe287Thrfs32, =]). Eleven noncoding variants were selected for splice assays (Table S4). Seven of these (c.768+7329A>G, c.858+526T>G, c.859-506G>C, c.1555-5008C>T, c.4539+1100A>G, c.4539 +1106C>T, c.6148-421T>C) were selected as they were located at cryptic splice sites or created a new putative splice site with a relative strength of at least 75% of the maximal score in two of five algorithms, and showed an increased splice prediction score of at least 2% (Table S4). Two variants, c.769-784C>T and c.4253+43G>A, belonging to three alleles (c.[769-784C>T; 5603A>T], c.[4253+43G>A; 6006-609T>A], c.[4253+43G>A; 5603A>T]), were enriched among the monoallelic cases, i.e., in 7 and 9 of 36 probands, respectively. The c.4253+43G>A variant was reported previously.33 Finally, the c.1937+435C>G variant was selected because it was located in the genomic sequence of an alternate exon in intron 13 (Chr1: 94,527,737–94,527,644).21 Deep-intronic variants c.4539+1100A>G and c.4539 +1106C>T were predicted to alter the same cryptic splice donor site located at position g.94,493,901. A very strong splice acceptor site was located at g.94,493,968. Besides the expected 68-nt PE fragment (PE30.1) due to the splice acceptor site at position c.4539+1033 and the splice donor site at position c.4539+1100, an additional 112-nt PE (PE30.2) carrying the same splice donor but another acceptor site located at g.94,494,012 was observed (Fig. 1b, Figure S2). Interestingly, the relative ratios of these two PEs significantly differed between the midigene constructs carrying c.4539 +1100A>G or c.4539+1106C>T. Four novel deep-intronic variants result in PE generation through the strengthening of splice sites g g g p To test the functional effect of the 11 selected noncoding variants, seven ABCA4 wild-type midigene constructs pre- viously described as BA4, BA6, BA7, BA9, BA11, BA19, and BA2130 and the newly designed BA30 (Table S5) were mutagenized. Upon RT-PCR and Sanger sequence validation, four variants had no effect (Figure S1). Variant c.858 +526T>G did not show a PE insertion, but did yield four weak smaller fragments (Figure S1), the relevance of which is unknown in the absence of sequence results. This variant was found in cis with a complex allele containing a protein- truncating pathogenic variant (p.[Trp273; Asn1868Ile]) in individual E-II:1, as determined by segregation analysis. The remaining six variants resulted in clear splice defects (Fig. 1, Figure S2) that allowed us to detect the second variant in 15/ 26 monoallelic probands, two deep-intronic variants in 2/3 cases without ABCA4 variants, and a complex allele (c.[769- 784C>T; 5603A>T]) in seven STGD1 cases carrying one deep-intronic variant. Considering that these variants or complex alleles are causal, segregation was as expected (Table 1). The remaining 12 unsolved STGD1 cases are listed in Table S6. RESULTS PE30.1 accounted for 60.6% and 93.1% of the total transcript in c.4539+1100A>G or c.4539+1106C>T respectively, while PE30.2 was present in 20.1% and 3.8% of the complementary DNA (cDNA) products, respectively (Table S7). Because both c.4539 +1100A>G and c.4539+1106C>T showed only a minor fraction of correctly spliced products (19.2% and 3.0%) and because PE30.1 and PE30.2 are both predicted to lead to a frameshift, these variants were both deemed to have a severe effect (r.[4539_4540ins(68), 4539_4540ins(112), =]; p.[Arg1514Glyfs3, Arg1514Valfs31, =]) (Table 1, Table S8). ARTICLE SANGERMANO et al performed using the corrected peak area parameter and only the peaks corresponding to the expected bands were taken into account. The deep-intronic variant c.769-784C>T, present in 7/36 STGD1 cases, is located in intron 6, and was predicted to increase the strength of an intronic splice acceptor site at position g.94,549,775. In addition, a strong splice donor site was situated 161 nt downstream. RT-PCR analysis using wild- type and mutant minigene constructs revealed that, compared with the wild-type construct, the mutant c.769-784C>T construct showed an additional band of 296 bp, containing a 162-nt PE (Fig. 1b, Figure S2). Quantification of mutant mRNA in transfected HEK293T cells revealed that the PE fragment only accounted for ~8% of the total ABCA4 pre- mRNA (Table S7), suggesting a mild effect at the RNA and thus protein level (r.[=, 768_769ins(162)]; p.[=, Leu257Aspfs3]). Midigene-based splice assay The effect of 11 noncoding variants was assessed by midigene- based splicing assays employing seven wild-type (WT) BA 1752 Volume 21 \| Number 8 \| August 2019 \| GENETICS in MEDICINE Volume 21 \| Number 8 \| August 2019 \| GENETICS in MEDICINE ARTICLE The c.4253+43G>A mutant yielded bands of 495 and 370 bp, the first corresponding to the correctly spliced product, and the second to a product in which exon 28 was Although the splice defect caused by this variant disrupted the reading frame (r.[=, 1937_1938ins(134)]; p.[=, Ser646- Serfs25]), the fraction of mutant transcript was low (8.6%) (Table S7). Although the splice defect caused by this variant disrupted the reading frame (r.[=, 1937_1938ins(134)]; p.[=, Ser646- Serfs25]), the fraction of mutant transcript was low (8.6%) (Table S7). ARTICLE ARTICLE SANGERMANO et al c.769-784C>T 7 11 10 9 8 7 12 26 30 31 32 27 28 29 13 14 15 BA6 BA7 BA11 BA19 BA21 c.859-506G>C 1 kb c.1937+435C>G c.4253+43G>A c.4539+1106C>T c.4539+1100A>G a a 84C>T 7 1 kb RHO exon 5 RHO exon 3 pCI-Neo-RHO Genomic fragment of interest 1 kb RHO exon 3 c.769-784C>T MQ WT MUT RHO ex3 - ABCA4 ex7 RHO exon 5 200 100 100 200 300 400 Pseudoexon Plasmid artifact Correct transcript 500 b b Pseudoexons Correct transcript MQ WT MUT RHO exon 5 ABCA4 ex30 - ex32 c.4539+1106C>T ABCA4 ex30 - ex32 MQ WT MUT c.4539+1100A>G RHO exon 5 Pseudoexons Correct transcript ABCA4 ex26 - ex29 c.4253+43G>A MQ WT MUT Exon skipping (Δ28) Exon skipping (Δ27/28) Correct transcript RHO exon 5 200 100 200 300 400 500 200 100 200 300 400 500 200 100 100 200 300 400 500 600 ABCA4 ex7 - ex8 RHO exon 5 200 100 200 300 400 500 Pseudoexon Correct transcript c.859-506G>C MQ WT MUT Pseudoexon Correct transcript RHO exon 5 MQ WT MUT ABCA4 ex13 - ex14 c.1937+435C>G 200 100 200 300 400 500 Fig. 1 Generation and assessment of splicing defects using midigenes. a Schematic representation of the five wild-type midigenes used that were cloned between exons 3 and 5 of Rhodopsin in pCI-Neo-RHO. Positions of the variants present in six mutant midigenes are indicated. b Assessment of the splicing defects upon midigene transfection in HEK293T cells. Five pseudoexon (PE) inclusions and one exon skipping event were detected in the mutant (MUT) constructs compared to the wild-type (WT). MQ stands for the negative control of the polymerase chain reaction (PCR). Rhodopsin (RHO) amplifi- cation was used as a transfection and loading control. variant and p.Asn1868Ile in trans. It does not weaken the splice donor site of exon 28, nor does it affect any other cryptic splice sites in silico. It does affect the strength of several splice enhancer or silencer motifs (Figure S4). The c.4253+43G>A variant was found in cis with the previously reported c.6006-609T>A11 in eight of nine probands, but c.6006-609T>A showed no effect in an in vitro splice assay (Figure S2). ARTICLE ARTICLE SANGERMANO et al c.769-784C>T 7 11 10 9 8 7 12 26 30 31 32 27 28 29 13 14 15 BA6 BA7 BA11 BA19 BA21 c.859-506G>C 1 kb c.1937+435C>G c.4253+43G>A c.4539+1106C>T c.4539+1100A>G RHO exon 5 RHO exon 3 pCI-Neo-RHO Pseudoexons Correct transcript MQ WT MUT RHO exon 5 ABCA4 ex30 - ex32 c.4539+1106C>T ABCA4 ex30 - ex32 MQ WT MUT c.4539+1100A>G RHO exon 5 Pseudoexons Correct transcript ABCA4 ex26 - ex29 c.4253+43G>A MQ WT MUT Exon skipping (Δ28) Exon skipping (Δ27/28) Correct transcript RHO exon 5 Pseudoexon Correct transcript RHO exon 5 MQ WT MUT ABCA4 ex13 - ex14 c.1937+435C>G ABCA4 ex7 - ex8 RHO exon 5 200 100 200 300 400 500 200 100 200 300 400 500 200 100 200 300 400 500 200 100 200 300 400 500 Pseudoexon Correct transcript c.859-506G>C MQ WT MUT c.769-784C>T MQ WT MUT RHO ex3 - ABCA4 ex7 RHO exon 5 200 100 100 200 300 400 Pseudoexon Plasmid artifact Correct transcript 500 200 100 100 200 300 400 500 600 a b Genomic fragment of interest Fig. 1 Generation and assessment of splicing defects using midigenes. a Schematic representation of the five wild-type midigenes used that were cloned between exons 3 and 5 of Rhodopsin in pCI-Neo-RHO. Positions of the variants present in six mutant midigenes are indicated. b Assessment of the splicing defects upon midigene transfection in HEK293T cells. Five pseudoexon (PE) inclusions and one exon skipping event were detected in the mutant (MUT) constructs compared to the wild-type (WT). MQ stands for the negative control of the polymerase chain reaction (PCR). Rhodopsin (RHO) amplifi- cation was used as a transfection and loading control. Volume 21 \| Number 8 \| August 2019 \| GENETICS in MEDICINE c.1937+435C>G results in PE generation through the disruption of putative splice silencers The c.1937+435C>G variant did not alter the strength of a cryptic splice site. Instead, it was predicted to disrupt an ESE (SC35) motif, and, more importantly, to inactivate two splice silencer motifs and to lower the predicted strength of a third one (Figure S3). This variant was located within the genomic sequence of a low-abundance ABCA4 transcript that contains intron 13 sequences.21 RT-PCR analysis revealed a 134-nt PE insertion between positions c.1937+396 and c.1937+529 in mutant BA11 compared with WT (Fig. 1b, Figure S2). 1753 GENETICS in MEDICINE \| Volume 21 \| Number 8 \| August 2019 Variant c.4253+43G>A results in exon skipping through the disruption of putative splice silencers The c.4253+43G>A variant was present in eight monoallelic STGD1 individuals and in one proband carrying a causal Volume 21 \| Number 8 \| August 2019 \| GENETICS in MEDICINE 1754 ARTICLE SANGERMANO et al derived cells, we generated fibroblast cell lines from a proband carrying c.[769-784C>T; 5603A>T] and c.1822T>A (p.Phe608Ile) (patient A-I:2), and from a STGD1 proband carrying c.859-506G>C and a putative deletion on the other allele encompassing intron 7 (case DNA14-33085; not listed in Table 1). For both cases, the same PEs were detected as in the midigene assays (Fig. 3), yet a larger amount of PE- containing mRNA was detected in fibroblasts from A-I:2 grown under NMD-suppressing conditions (18.2%) com- pared with HEK293T-transfected cells (8.6%). Considering that this STGD1 individual carries a missense variant (p.Phe608Ile), the amount of PE-containing mRNA from the c.769-784C>T allele likely is much higher than 18%. The same AONs that restored splicing in the midigenes also showed rescue in fibroblasts (Fig. 3). For c.769-784C>T, AON2 was the most effective, correcting 100% of the transcripts, whereas AON3 also showed a strong rescue (Fig. 3, Figure S6, Table S10). AON1 also resulted in PE skipping but induced an alternative splicing event. Interest- ingly, the insertion of this partial PE was also detected in control fibroblasts. Sequence analysis revealed that the upper band of the artifact corresponded to the partial PE, while the lower was a heteroduplex consisting of the WT and partial PE-containing transcripts. ABCA4 mRNA was also barely detected in the fibroblasts carrying c.859-506G>C when they were not subjected to CHX treatment (Fig. 3). After CHX incubation, a 56-nt PE was detected that, upon delivery of AON1 and AON3, was almost completely absent from the transcripts. skipped (Fig. 1b, Figure S2). This 125-nt deletion is predicted to lead to a frameshift, and although both WT and mutant midigenes showed some skipping of exon 28, in the mutant construct this was significantly higher compared with WT (26.1% vs. 2.8%, Table S7). The fact that in all nine probands, a severe variant was found in trans, in combination with a late age of onset, suggests a mild effect of this variant (r.[=, 4129_4253del(125)]; p.[=, Ile1377Hisfs3]). An overview of the allele frequencies and of the splice defects caused by all six variants can be found in Tables S8 and S9. AON-based exon inclusion For c.769-784C>T, AON2 completely restored correct splicing, whereas AON3 had a partial effect (Fig. 2b). When using AON1, also some splice redirection was observed; an additional faint band was present both in WT and mutant. Cloning and sequence analysis of this band revealed that the 5’ 68 nt of the PE were skipped, while the 3’ 94 nt remained. Given the position of AON1, this AON most likely blocks the splice acceptor site of the PE, triggering the use of a downstream splice acceptor site. For c.859-506G>C, two AONs (AON1 and AON3) corrected the splicing defect (Fig. 2b). For c.1937+435C>G, all three AONs restored normal splicing (Fig. 2b). In addition, we observed that the 134-nt PE could also naturally be included in the WT construct, as the band was also present in the nontreated and the SON-treated samples. Since the PE is also inserted when transfecting the WT construct, the variant apparently enhances PE recognition. Finally, for the two neighboring variants c.4539+1100A>G and c.4539+1106C>T, AON1 and AON2 correctly restored splicing, while AON3 did not show any rescue (Fig. 2b). To correct the splice defect of c.4253+43G>A, AONs were designed to promote exon 28 inclusion. Two AONs were delivered to the midigene-transfected HEK293T cells. To assess whether the AON was inducing the inclusion of exon 28, the ratio between correct and Δexon 28 transcript was calculated with Fiji, or using the corrected peak area obtained in the capillary analysis (Figure S5). Using both methods, AON2 was shown to induce ~10% extra inclusion of exon 28 (Fig. 2), whereas for AON1, splice redirection was less evident (Table S7). ARTICLE SANGERMANO et al Table 1 Persons with Stargardt disease (STGD1) carrying two pathogenic ABCA4 alleles Patient_ID Gender Age at onset (years) Segregation confirmed Allele1 DNA Allele1 protein Allele 2 DNA Allele 2 protein A-I:2 F 38 Yes c.1822T>A p.(Phe608Ile) c.[769-784C>T; 5603A>T] p.[=, Leu257Aspfs3; Asn1868Ile]b B-I:2 F 10 Yes c.768G>T p.(Leu257Valfs17)c c.859-506G>C p.[Phe287Thrfs32, =]b C-I:2 F 48 Yes c.768G>T p.(Leu257Valfs17)c c.[769-784C>T; 5603A>T] p.[=, Leu257Aspfs3; Asn1868Ile]b D-III:1 M 45 Yes c.4363T>C p.(Cys1455Arg) c.[4253+43G>A; 6006-609T>A] p.[=, Ile1377Hisfs3]b E-II:1 F 7 Yes c.[818G>A; 5603A>T] p.[Trp273; Asn1868Ile] c.4539+1100A>G p.[Arg1514Valfs31, Arg1514Glyfs3, =]b F-II:2 F 20 Yes c.1822T>A p.(Phe608Ile) c.[4253+43G>A; 6006-609T>A] p.[=, Ile1377Hisfs3]b G-I:2 F 51 Yes c.1822T>A p.(Phe608Ile) c.[4253+43G>A; 6006-609T>A] p.[=, Ile1377Hisfs3]b H-I:2 F 53 Yes c.[5461-10T>C; 5603A>T] p.[Thr1821Valfs13, Thr1821Aspfs6; Asn1868Ile] c.[4253+43G>A; 6006-609T>A] p.[=, Ile1377Hisfs3]b I-II:3 F 44 Yes c.4577C>T p.(Thr1526Met) c.[769-784C>T; 5603A>T] p.[=, Leu257Aspfs3; Asn1868Ile]b J-II:3 M 61 Yes c.768G>T p.(Leu257Valfs17)c c.[4253+43G>A; 6006-609T>A] p.[=, Ile1377Hisfs3]b K-II:1 M 62 Yes c.6155del p.(Asn2052Thrfs9) c.[769-784C>T; 5603A>T] p.[=, Leu257Aspfs; Asn1868Ile]b L-II:1 M 61 Yes c.[5461-10T>C; 5603A>T] p.[Thr1821Valfs13, Thr1821Aspfs6; Asn1868Ile] c.[769-784C>T; 5603A>T] p.[=, Leu257Aspfs; Asn1868Ile]b M-II:1 M 18 Yes c.4539+2001G>A p.[=, Arg1514Leufs36]d c.[4253+43G>A; 5603A>T] p.[=, Ile1377Hisfs3; Asn1868Ile]b N-II:3 M 31 Yes c.4773+1G>A p.(?) c.[4253+43G>A; 6006-609T>A] p.[=, Ile1377Hisfs3]b O-I:1 M 49 Yes c.3113C>T p.(Ala1038Val) c.859-506G>C p.[Phe287Thrfs32, =]b P-II:3 F 4 Yes c.5196+1137G>A p.[Met1733Glufs78, =]e c.859-506G>C p.[Phe287Thrfs32, =]b Q-II:1 M 69 Yes c.4539+1G>T p.(?) c.[769-784C>T; 5603A>T] p.[=, Leu257Aspfs3; Asn1868Ile]b R-II:1 M 52 n.t. c.4539+1G>T p.(?) c.[4253+43G>A; 6006-609T>A] p.[=, Ile1377Hisfs3]b S-II:1 F 64 n.t. c.768G>T p.(Leu257Valfs17)c c.[769-784C>T; 5603A>T] p.[=, Leu257Aspfs3; Asn1868Ile]b T-II:1 F 35 Yes c.[2588G>C; 5603A>T] p.[Gly863Ala, Gly863del; Asn1868Ile] c.1937+435C>G p.[=, Ser646Serfs25]b U-II:1 M 9 n.t. c.768G>T p.(Leu257Valfs17)c c.1937+435C>G p.[=, Ser646Serfs25]b V-II:1 F 15 n.t. c.[2588G>C; 5603A>T] p.[Gly863Ala, Gly863del; Asn1868Ile] c.4539+1100A>G p.[Arg1514Valfs31, Arg1514Glyfs3, =]b W-II:1 n.a. 11a n.t. c.[1622T>C; 3113C>T] p.[Leu541Pro; Ala1038Val] c.4539+1106C>T p.[Arg1514Valfs31, Arg1514Glyfs3]b X-II:1 n.a. 52a n.t. c.4469G>A p.(Cys1490Tyr) c.[4253+43G>A; 6006-609T>A] p.[=, Ile1377Hisfs3]b n.t., not tested aUnknown age of onset. Current age bPredicted based on densitometry percentages in this study based on the method described in Sangermano et al.30 cEffect based on Sangermano et al.30 dEffect based on Albert et al 22 1755 DISCUSSION To identify missing noncoding variants in STGD1 cases, we sequenced the ABCA4 locus in 36 probands and identified one known and five novel intronic variants in 24 (67%) probands. Four novel deep-intronic variants (c.769-784C>T, c.859-506G>C, c.4539+1100A>G, c.4539+1106C>T) strengthen cryptic splice sites at noncanonical nucleotide positions and thereby result in PE insertions, contrary to most of the published deep-intronic splice site variants that create canonical splice sequences.34,35 Variant c.1937+435C>G disrupted a splicing silencer motif that is located within a low-expressed alternate exon, and resulted in a 134-nt PE formation.21 This PE contains a proper splice acceptor site but, similar to the alternate exon, no consensus splice donor To identify missing noncoding variants in STGD1 cases, we sequenced the ABCA4 locus in 36 probands and identified one known and five novel intronic variants in 24 (67%) probands. Four novel deep-intronic variants (c.769-784C>T, c.859-506G>C, c.4539+1100A>G, c.4539+1106C>T) ARTICLE smMIP-based sequence analysis of intronic ABCA4 regions Five of six intronic ABCA4 variants were analyzed for their recurrence in 412 genetically unsolved STGD1 cases from France (n = 224) or Germany (n = 188), by employing smMIPs.31 Three variants were found in a heterozygous state in this cohort: c.769-784C>T (n = 4), c.4253+43G>A (n = 29), and c.4539+ 1106C>T (n = 1) (Table S8 and Table S9), whereas variants c.859-506G>C and c.4539+1100A>G were not detected. Antisense oligonucleotide-based exclusion of PEs g For five variants (c.769-784C>T, c.859-506G>C, c.1937 +435C>G, c.4539+1100A>G, and c.4539+1106C>T), we designed three different AONs each that aim to result in PE exclusion. For c.4253+43G>A, only two AONs were designed, aiming to block splice silencer motifs to reinforce inclusion of exon 28. A summary of the sequences and characteristics of the AONs is provided in Table S2. The effect of the AONs at the RNA level was assessed by RT-PCR and subsequently semiquantified using capillary analysis (Fig- ure S5, Table S7). For five variants (c.769-784C>T, c.859-506G>C, c.1937 +435C>G, c.4539+1100A>G, and c.4539+1106C>T), we designed three different AONs each that aim to result in PE exclusion. For c.4253+43G>A, only two AONs were designed, aiming to block splice silencer motifs to reinforce inclusion of exon 28. A summary of the sequences and characteristics of the AONs is provided in Table S2. The effect of the AONs at the RNA level was assessed by RT-PCR and subsequently semiquantified using capillary analysis (Fig- ure S5, Table S7). AON-based PE exclusion using STGD1-derived fibroblast cells To investigate whether the AON-based splice corrections observed in the midigene assays would also occur in patient- Volume 21 \| Number 8 \| August 2019 \| GENETICS in MEDICINE 1756 ARTICLE SANGERMANO et al RHO ex3 - ABCA4 ex7 100 200 300 400 RHO exon 5 200 100 c.769-784C>T MQ HEK NT AON1 AON2 WT MUT AON3 SON NT AON1 AON2 AON3 SON Pseudoexon Plasmid artifact Correct transcript MQ HEK NT AON1 AON2 WT MUT AON3 SON NT AON1 AON2 AON3 SON c.859-506G>C ABCA4 ex7 - ex8 RHO exon 5 200 200 300 400 100 Pseudoexon Correct transcript Pseudoexon Correct transcript RHO exon 5 200 200 100 300 400 100 ABCA4 ex13 - ex14 MQ HEK NT AON1 AON2 WT MUT AON3 SON NT AON1 AON2 AON3 SON c.1937+435C>G 100 200 300 400 500 600 MQ HEK NT AON1 AON2 WT MUT SON NT AON1 AON2 SON RHO exon 5 200 100 ABCA4 ex26- ex29 c.4253+43G>A Exon skipping (Δ28) Exon skipping (Δ27/28) Correct transcript RHO exon 5 200 100 200 300 400 500 ABCA4 ex30- ex32 Correct transcript MQ HEK NT AON1 AON2 WT MUT AON3 SON NT AON1 AON2 AON3 SON c.4539+1106C>T Pseudoexon 2 Pseudoexon 1 RHO exon 5 200 100 200 300 400 ABCA4 ex30- ex32 Pseudoexon 2 Pseudoexon 1 Correct transcript MQ HEK NT AON1 AON2 WT MUT AON3 SON NT AON1 AON2 AON3 SON c.4539+1100A>G 10 bp 162 bp C>T AON1 AON2 AON3 c.769-784C>T c.859-506G>C 56 bp G>C AON1 AON2 AON3 134 bp C>G AON1 AON2 AON3 c.1937+435C>G 68 bp 112 bp A>G C>T AON1 Pseudoexon 1 Pseudoexon 2 AON2 AON3 c.4539+1100A>G & c.4539+1106C>T Pseudoexon exclusion Pseudoexon exclusion 10 bp Exon 28 (125 bp) G>A AON1 AON2 c.4253+43G>A Exon inclusion b a Fig. 2 Antisense oligonucleotide (AON) rescue using midigenes in HEK293T cells. a Position of the AONs and sizes of the pseudoexons introduced by the variants. b Assessment of the splicing correction by reverse transcription polymerase chain reaction (RT-PCR) upon AON delivery. Wild-type (WT) midigenes and mutant (MUT) midigenes were cotransfected with different AONs and a SON, except the nontransfected cells lane (NT). For each variant introducing a pseudoexon, at least one AON was able to redirect splicing. In the case of exon 28 skipping, a 10% inclusion was achieved with AON2. MQ denotes the negative control of the PCR reaction and HEK for the untransfected HEK293T. AON-based PE exclusion using STGD1-derived fibroblast cells a Position of the AONs and sizes of the pseudoexons introduced by the variants. b Assessment of the splicing correction by reverse transcription polymerase chain reaction (RT-PCR) upon AON delivery. Wild-type (WT) midigenes and mutant (MUT) midigenes were cotransfected with different AONs and a SON, except the nontransfected cells lane (NT). For each variant introducing a pseudoexon, at least one AON was able to redirect splicing. In the case of exon 28 skipping, a 10% inclusion was achieved with AON2. MQ denotes the negative control of the PCR reaction and HEK for the untransfected HEK293T. Rhodopsin (RHO) amplification was used as a transfection and loading control. CON MQ 600 500 400 300 500 400 300 200 400 300 200 500 400 300 200 –CHX +CHX AON1 AON2 AON3 SON –CHX +CHX AON1 AON2 AON3 SON Correct transcript Correct transcript AON-artifacts Pseudoexon Pseudoexon MQ –CHX +CHX AON1 AON2 AON3 SON –CHX +CHX AON1 AON2 AON3 SON c.769-784C>T ABCA4 ex6-7 ACTB ex3-4 ABCA4 ex7-8 ACTB ex3-4 c.859-506G>C STGD1 CON STGD1 Fig. 3 Antisense oligonucleotide (AON) rescue in fibroblasts derived from Stargardt disease (STGD1)-affected individuals carrying c.769- 784C>T or c.859-506G>C. Fibroblast cells from a control (CON) and a STGD1 individual were transfected with three AONs and a SON. To detect the pseudoexon, cells were subjected to cycloheximide (+CHX) treatment. Only the nontransfected cells were not subjected to CHX treatment (-CHX). The MQ lane contains the negative control of the PCR reaction. Actin (ACTB) was used as a loading control. CON MQ 600 500 400 300 500 400 300 200 400 300 200 500 400 300 200 –CHX +CHX AON1 AON2 AON3 SON –CHX +CHX AON1 AON2 AON3 SON Correct transcript Correct transcript AON-artifacts Pseudoexon Pseudoexon MQ –CHX +CHX AON1 AON2 AON3 SON –CHX +CHX AON1 AON2 AON3 SON c.769-784C>T ABCA4 ex6-7 ACTB ex3-4 ABCA4 ex7-8 ACTB ex3-4 c.859-506G>C STGD1 CON STGD1 site sequence. The c.4253+43G>A variant disrupted predicted splice silencers and created an ESE, and led to partial skipping of the upstream exon 28. Interestingly, we identified two probands (M-II:1 and P-II:3) that carried deep-intronic variants on both alleles. All novel variants were predicted to result in protein truncation, but two variants, c.769-784C>T and c.4253 +43G>A, found in seven and nine probands, respectively, only affect a small proportion of the mRNA. Testing of 412 other genetically unexplained STGD1 cases revealed 29 persons to carry c.4253+43G>A and four cases to carry c.769-784C>T. AON-based PE exclusion using STGD1-derived fibroblast cells Rhodopsin (RHO) amplification was used as a transfection and loading control. a 10 bp 162 bp C>T AON1 AON2 AON3 c.769-784C>T c.859-506G>C 56 bp G>C AON1 AON2 AON3 134 bp C>G AON1 AON2 AON3 c.1937+435C>G Pseudoexon exclusion a 68 bp 112 bp A>G C>T AON1 Pseudoexon 1 Pseudoexon 2 AON2 AON3 c.4539+1100A>G & c.4539+1106C>T Pseudoexon exclusion 10 bp Exon 28 (125 bp) G>A AON1 AON2 c.4253+43G>A Exon inclusion pt pt pt 100 200 300 400 500 600 MQ HEK NT AON1 AON2 WT MUT SON NT AON1 AON2 SON RHO exon 5 200 100 ABCA4 ex26- ex29 c.4253+43G>A Exon skipping (Δ28) Exon skipping (Δ27/28) Correct transcript RHO exon 5 200 100 200 300 400 500 ABCA4 ex30- ex32 Correct transcript MQ HEK NT AON1 AON2 WT MUT AON3 SON NT AON1 AON2 AON3 SON c.4539+1106C>T Pseudoexon 2 Pseudoexon 1 RHO exon 5 200 100 200 300 400 ABCA4 ex30- ex32 Pseudoexon 2 Pseudoexon 1 Correct transcript MQ HEK NT AON1 AON2 WT MUT AON3 SON NT AON1 AON2 AON3 SON c.4539+1100A>G 68 bp 112 bp A>G C>T AON1 Pseudoexon 1 Pseudoexon 2 AON2 AON3 c.4539+1100A>G & c.4539+1106C>T Pseudoexon exclusion 10 bp Exon 28 (125 bp) G>A AON1 AON2 c.4253+43G>A Exon inclusion K293T ll P iti f th AON d i f th d i t d d b RHO ex3 - ABCA4 ex7 100 200 300 400 RHO exon 5 200 100 c.769-784C>T MQ HEK NT AON1 AON2 WT MUT AON3 SON NT AON1 AON2 AON3 SON Pseudoexon Plasmid artifact Correct transcript MQ HEK NT AON1 AON2 WT MUT AON3 SON NT AON1 AON2 AON3 SON c.859-506G>C ABCA4 ex7 - ex8 RHO exon 5 200 200 300 400 100 Pseudoexon Correct transcript Pseudoexon Correct transcript RHO exon 5 200 200 100 300 400 100 ABCA4 ex13 - ex14 MQ HEK NT AON1 AON2 WT MUT AON3 SON NT AON1 AON2 AON3 SON c.1937+435C>G b b Pseudoexon 2 Pseudoexon 1 Correct transcript HEK NT AON1 AON2 WT MUT AON3 SON NT AON1 AON2 AON3 SON Correct transcript HEK NT AON1 AON2 WT MUT AON3 SON NT AON1 AON2 AON3 SON Pseudoexon 2 Pseudoexon 1 t 100 200 300 400 500 600 MQ HEK NT AON1 AON2 WT MUT SON NT AON1 AON2 SON RHO exon 5 200 100 ABCA4 ex26- ex29 c.4253+43G>A Exon skipping (Δ28) Exon skipping (Δ27/28) Correct transcript Fig. 2 Antisense oligonucleotide (AON) rescue using midigenes in HEK293T cells. GENETICS in MEDICINE \| Volume 21 \| Number 8 \| August 2019 ARTICLE In STGD1-derived photo- receptor precursor cells however, we observed up to 15% (c.4539+2028C>T) and 25% (c.4539+2001G>A) of PE- containing mRNA.22 Based on their heterozygous presence in the corresponding STGD1 cases and their moderate-to- severe (c.4539+2028C>T) or severe (c.4539+2001G>A) nature, about two times these percentages were expected. We hypothesize that the photoreceptor precursor cells had not yet reached a differentiation stage that closely mimics the human retina situation. It is also of interest that, while c.1937 +435C>G is predicted to disrupt ESEs, c.4539+2001G>A and c.4539+2028C>T are predicted to activate ESEs. Possibly, the action of these splicing motifs requires specific factors that are exclusively present in mature photoreceptors, while being absent in HEK293T cell and explain the small effect for c.1937 +435C>G. Patient-derived photoreceptor-like cells may reveal a more complete splice defect. y g Interestingly, c.4253+43G>A also shows a relatively high non-Finnish European AF (0.00598 in gnomAD). Although c.4253+43G>A, with one exception (M-II:1), was found in cis with c.6006-609T>A, we did not observe a splice defect for the latter variant in vitro. This could be due to the lack of retina- specific splice factors in the tested cells or due to another missed deep-intronic variant that acts in concert with c.4253 +43G>A. In the one proband with c.4253+43G>A who lacked c.6006-609T>A, the c.5603A>T (p.Asn1868Ile) variant was found in cis. The age of onset of the cases carrying c.4253 +43G>A ranged from 18 to 61 years (average 41 years). Interestingly, the earliest age at onset (18 years) was observed in the proband M-II:1 who carried c.[4253+43G>A; 5603A>T]. The use of AONs to exclude PEs has been used for several IRD-associated genes.24–29 However, in this study, several technical challenges were observed. For the c.859-506G>C variant, the design of the AONs was limited to very small parts of the PE due to the repetitive nature of parts of the PE. In case of the c.4539+1100A>G and c.4539+1106C>T variants, we aimed to find effective AONs that would target the PE for both variants. In addition, using midigenes for c.769-784C>T, we observed variability between replicates, probably due to differences in the transfection efficiencies or passage of the cells. ARTICLE Nevertheless, for each variant, at least one effective AON was discovered.17 p p Recently, it was determined that c.2588G>C (p.[Gly863Ala, Gly863del]) only acts as a penetrant pathogenic variant when present in cis with c.5603A>T (p.Asn1868Ile).15 The common p.(Asn1868Ile) variant was found as a second ABCA4 allele in up to 50% of monoallelic STGD1 cases and is strongly associated with late-onset STGD1.15 Upon Haloplex-based ABCA4 gene sequencing, we found p.Asn1868Ile as a single variant in trans with other (potentially) causal variants in 25/ 65 (38%) of probands, who had an average age at onset of 42 years. In addition, we identified four biallelic but unaffected persons and calculated that this variant, when present in trans with a loss-of-function ABCA4 variant, shows a penetrance <5%.16 Based on the relatively high frequency of c.769- 784C>T in non-Finnish European controls (AF 0.00426 in gnomAD [http://gnomad.broadinstitute.org/]), its partial effect on splicing, and the very late age of onset observed in compound heterozygotes carrying c.[769-784C>T; 5603A>T] (61.0 years; range: 38–69 years), we consider that c.769- 784C>T without c.5603A>T in cis may not be causative. We therefore investigated the MAFs of the c.769-784C>T and c.5603A>T variants (single and in combination) in 250 control Dutch families (GoNL: http://www.nlgenome.nl/).38 The single c.769-784C>T allele was found more often (6/998 alleles) than the c.[769-784C>T; 5603A>T] allele (2/998 alleles). The allele frequency of c.[769-784C>T; 5603A>T] in our STGD1 patient cohort (n = 250), 0.014, is significantly higher than in our general population (Chi-square test: p = 0.023; Bonferroni corrected). In two previous studies, the mean ages at onset for STGD1 patients carrying c.5603A>T as a single second allele in trans with severe ABCA4 variants Despite the significant yield of novel ABCA4 variants in this study, a second ABCA4 variant was not detected in 11 monoallelic probands and there was one case with no ABCA4 variants, which could be due to (1) the presence of heterozygous copy-number variations (CNVs), as only three were tested using CNV analysis; (2) missed noncoding variants residing in noncovered sequences; (3) too-stringent selection criteria when using splicing algorithms; (4) incom- plete sensitivity of the midigene in vitro splice assay; and (5) genocopies in view of the high AF of ABCA4 variants (5–10%) in the general population.18,36,39 Additional studies will be needed to further unravel the missing heritability of STGD1. AON-based PE exclusion using STGD1-derived fibroblast cells AONs were able to (partially) correct the observed splice defects for all variants in HEK293T cells and, for two variants, in STGD1-derived fibroblasts. Fig. 3 Antisense oligonucleotide (AON) rescue in fibroblasts derived from Stargardt disease (STGD1)-affected individuals carrying c.769- 784C>T or c.859-506G>C. Fibroblast cells from a control (CON) and a STGD1 individual were transfected with three AONs and a SON. To detect the pseudoexon, cells were subjected to cycloheximide (+CHX) treatment. Only the nontransfected cells were not subjected to CHX treatment (-CHX). The MQ lane contains the negative control of the PCR reaction. Actin (ACTB) was used as a loading control. Fourteen first alleles in the 24 probands carry protein- truncating variants and are thus assumed to have a severe effect (Table 1). For other missense or complex alleles, the severity is less clear. Based on previous studies,15,36,37 our mRNA analysis of the deep-intronic variants, and the ages at onset of the STGD1 probands described here, we can hypothesize on the severity of the intronic variants. The c.859-506G>C, c.4539+1100A>G, and c.4539+1106C>T variants all show PE insertion in the majority of ABCA4 transcripts and can thus be considered severe variants. This correlates very well with an age at onset ≤10 years when found in trans with a severe variant and between 15 and 49 years when in trans with a mild variant (Table 1). For c.1937+435C>G, the picture is more complicated. The splice defect was mild, and the two probands carrying this 1757 GENETICS in MEDICINE \| Volume 21 \| Number 8 \| August 2019 Volume 21 \| Number 8 \| August 2019 \| GENETICS in MEDICINE ARTICLE SANGERMANO et al variant carry either a severe variant p.(Leu257Valfs17) (U- II:1) or a mild allele p.[Gly863Ala, Gly863del; Asn1868Ile] (T- II:1) in trans. The ages of onset were 9 and 35 years, respectively, which argues for a severe nature of c.1937 +435C>G in U-II:1 and a moderate-to-severe nature in T- II:1. This is discordant with the observed partial PE insertion in HEK293T cells. A similar observation was recently made while studying the effect of c.4539+2001G>A and c.4539 +2028C>T.21 In STGD1-derived fibroblasts, mRNA analysis did not show any PE insertion. In STGD1-derived photo- receptor precursor cells however, we observed up to 15% (c.4539+2028C>T) and 25% (c.4539+2001G>A) of PE- containing mRNA.22 Based on their heterozygous presence in the corresponding STGD1 cases and their moderate-to- severe (c.4539+2028C>T) or severe (c.4539+2001G>A) nature, about two times these percentages were expected. We hypothesize that the photoreceptor precursor cells had not yet reached a differentiation stage that closely mimics the human retina situation. It is also of interest that, while c.1937 +435C>G is predicted to disrupt ESEs, c.4539+2001G>A and c.4539+2028C>T are predicted to activate ESEs. Possibly, the action of these splicing motifs requires specific factors that are exclusively present in mature photoreceptors, while being absent in HEK293T cell and explain the small effect for c.1937 +435C>G. Patient-derived photoreceptor-like cells may reveal a more complete splice defect. were 36 years15 and 42 years.16 The mean age of onset for STGD1 patients carrying c.[769-784C>T; 5603A>T] is higher than the age of onset of patients carrying the single c.5603A>T variant (Mann–Whitney U; p<0.05). Given its relatively small effect on mRNA splicing (18.2% PE inclusion in mRNA from patient carrying this variant in a heterozygous manner) and its invariable cis-configuration with c.5603A>T, we cannot unequivocally assign pathogenicity to c.769- 784C>T. Possibly, patient-derived photoreceptor progenitor cells may shed further light on this matter. variant carry either a severe variant p.(Leu257Valfs17) (U- II:1) or a mild allele p.[Gly863Ala, Gly863del; Asn1868Ile] (T- II:1) in trans. The ages of onset were 9 and 35 years, respectively, which argues for a severe nature of c.1937 +435C>G in U-II:1 and a moderate-to-severe nature in T- II:1. This is discordant with the observed partial PE insertion in HEK293T cells. A similar observation was recently made while studying the effect of c.4539+2001G>A and c.4539 +2028C>T.21 In STGD1-derived fibroblasts, mRNA analysis did not show any PE insertion. SUPPLEMENTARY INFORMATION The online version of this article (https://doi.org/10.1038/s41436- 018-0414-9) contains supplementary material, which is available to authorized users. ARTICLE SANGERMANO et al and A.W.), NIHR for the NIHR BioResource (RG65966) (to F.L.R.) by grants from the Federal Ministry of Education and Research (BMBF) (ref. IDs 01GM0851 and 01GM1108B) (to B.H.F.W.). G.A. is supported by a Fight for Sight UK Early Career Investigator Award. The funding organizations had no role in the design or conduct of this research, and provided unrestricted grants. This study made use of data generated by the Genome of the Netherlands Project. Funding for the project was provided by the Netherlands Organization for Scientific Research under award number 184021007, dated 9 July 2009 and made available as a Rainbow Project of the Biobanking and Biomolecular Research Infrastructure Netherlands (BBMRI-NL). Samples where contrib- uted by LifeLines (http://lifelines.nl/lifelines-research/general), the Leiden Longevity Study (http://www.healthy-ageing.nl; http:// www.langleven.net), the Netherlands Twin Registry (NTR: http:// www.tweelingenregister.org), the Rotterdam studies (http:// www.erasmus-epidemiology.nl/rotterdamstudy), and the Genetic Research in Isolated Populations program (http://www.epib.nl/ research/geneticepi/research.html#gip). The sequencing was car- ried out in collaboration with the Beijing Institute for Genomics (BGI). could not establish causality for c.769-784C>T. Interestingly, all the observed splice defects could be rescued with at least one of the tested AONs, which provides a basis for the development of new therapeutic strategies for individuals with STGD1 carrying these variants. ACKNOWLEDGEMENTS We thank Ellen Blokland, Lonneke Duijkers, Duaa Elmelik, Anita Hoogendoorn, Marlie Jacobs-Camps, Saskia van der Velde-Visser, and Marijke Zonneveld-Vrieling for technical assistance. We thank Sabine Defoort, Hélène Dollfus, Isabelle Drumare, Christian P. Hamel, Karsten Hufendiek, Cord Huchzermeyer, Herbert Jägle, Ulrich Kellner, Philipp Rating, Klaus Rüther, Eric Souied, Georg Spital, and Xavier Zanlonghi for their cooperation and ascertain- ing STGD1 cases. This work was supported by the FP7-PEOPLE- 2012-ITN programme EyeTN, agreement 317472 (to F.P.M.C.); the Macula Vision Research Foundation (to F.P.M.C.); the Foundation Fighting Blindness USA, grant no. PPA-0517-0717- RAD (to A.G., C.B.H., F.P.M.C., R.W.J.C., and S.A.); the RP Fighting Blindness UK (RetinaUK), grant no. GR591 (to F.P.M.C. and S.A.); the Rotterdamse Stichting Blindenbelangen, the Stichting Blindenhulp, and the Stichting tot Verbetering van het Lot der Blinden (to F.P.M.C. and S.A.); and by the Landelijke Stichting voor Blinden en Slechtzienden, Macula Degeneratie fonds, and the Stichting Blinden-Penning, which contributed through Uitzicht 2016-12 (to F.P.M.C. and S.A.). This work was also supported by the Algemene Nederlandse Vereniging ter Voorkoming van Blindheid, Stichting Blinden-Penning, Landelijke Stichting voor Blinden en Slechtzienden, Stichting Oogfonds Nederland, Stichting Macula Degeneratie Fonds, and Stichting Retina Nederland Fonds, which contributed through UitZicht 2015-31, together with the Rotterdamse Stichting Blindenbelan- gen, Stichting Blindenhulp, Stichting tot Verbetering van het Lot der Blinden, Stichting voor Ooglijders, and Stichting Dowilvo (to A.G. and R.W.J.C.); the Stichting Macula Degeneratie Fonds; and the Stichting A.F. Deutman Researchfonds Oogheelkunde (to C.B. H.). This work was also supported by the Algemene Nederlandse Vereniging ter Voorkoming van Blindheid and Landelijke Stichting voor Blinden en Slechtzienden, which contributed through UitZicht 2014-13, together with the Rotterdamse Stichting Blindenbelangen, Stichting Blindenhulp, and the Stichting tot Verbetering van het Lot der Blinden (to F.P.M.C.), the Ghent University Research Fund (BOF15/GOA/011), the Research Foun- dation Flanders (FVO) G0C6715N, and the Hercules Foundation AUGE/13/023 and JED Foundation to E.D.B. M.B. was PhD fellow of the FWO and recipient of a grant of the funds for Research in Ophthalmology (FRO). E.D.B. is Senior Clinical Investigator of the FWO (1802215N). This work was also supported by the National Institute for Health Research (NIHR) Biomedical Centre at Moorfields and UCL Institute of Ophthalmology (to A.W.), UK NIHR Rare Disease Translational Research Consortium (to G.A. DISCLOSURE R.W.J.C., A.G., F.P.M.C., and S.A. are inventors on one or two filed patents (nos. 16203864.0 and 18184432.5) that are related to the contents of this manuscript. The other authors declare no conflicts of interest. Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ARTICLE In conclusion, we identified deep-intronic variants in 24/36 (67%) of STGD1 cases with no ABCA4 variants, one variant, or one causal variant in trans with c.5603A>T. Two alleles (c. [769-784C>T; 5603A>T] and c.4253+43G>A) are frequent in our STGD1 cohort and appear to be associated with late-onset STGD1. Due to its small effect on ABCA4 mRNA in HEK293T cells and patient-derived fibroblasts, and its invariable presence on the same allele as c.5603A>T, we 1758 Volume 21 \| Number 8 \| August 2019 \| GENETICS in MEDICINE REFERENCES 1. Carss KJ, Arno G, Erwood M, et al. 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GENETICS in MEDICINE \| Volume 21 \| Number 8 \| August 2019 1759 1759 1Department of Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands. 2Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands. 3Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, Nijmegen, The Netherlands. 4Department of Ophthalmology, Radboud University Medical Center, Nijmegen, The Netherlands. 5Center for Medical Genetics, Ghent University and Ghent University Hospital, Ghent, Belgium. 6The Rotterdam Eye Hospital and the Rotterdam Ophthalmic Institute, Rotterdam, The Netherlands. 7Department of Medical Genetics, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands. 8Department of Ophthalmology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands. 9Department of Ophthalmology, Erasmus Medical Center, Rotterdam, The Netherlands. 10Service d′Exploration de la Vision CHU, Lille, France. 11Institute for Neurosciences of Montpellier INSERM U1051, University of Montpellier, Montpellier, France. 12Centre d′ Etude du Polymorphisme Humain, Fondation Jean Dausset, Paris, France. 13UCL Institute of Ophthalmology, London, UK. 14Moorfields Eye Hospital, London, UK. 15Department of Haematology, University of Cambridge, Cambridge, UK. 16NIHR BioResource, Cambridge University Hospitals NHS Foundation Trust, Cambridge Biomedical Campus, Cambridge, UK. 17Department of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK. 18Univ. Lille, Inserm UMR-S 1172, CHU Lille, Biochemistry and Molecular Biology Department - UF Génopathies, Lille, France. 19Institut für Humangenetik, Universität Regensburg, Regensburg, Germany. 20Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden Volume 21 \| Number 8 \| August 2019 \| GENETICS in MEDICINE ARTICLE Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/ by/4.0/. ARTICLE SANGERMANO et al 12. Maugeri A, Klevering BJ, Rohrschneider K, et al. Mutations in the ABCA4 (ABCR) gene are the major cause of autosomal recessive cone-rod dystrophy. Am J Hum Genet. 2000;67:960–966. 28. Slijkerman RW, Vache C, Dona M, et al. Antisense oligonucleotide-based splice correction for USH2A-associated retinal degeneration caused by a frequent deep-intronic mutation. Mol Ther Nucleic Acids. 2016;5:e381. y p y 13. Cremers FP, van de Pol DJ, van Driel M, et al. Autosomal recessive retinitis pigmentosa and cone-rod dystrophy caused by splice site mutations in the Stargardt's disease gene ABCR. Hum Mol Genet. 1998;7:355–362. 29. Bonifert T, Gonzalez Menendez I, Battke F, et al. Antisense oligonucleotide mediated splice correction of a deep intronic mutation in OPA1. Mol Ther Nucleic Acids. 2016;5:e390. 14. Fakin A, Robson AG, Fujinami K, et al. Phenotype and progression of retinal degeneration associated with nullizigosity of ABCA4. Invest Ophthalmol Vis Sci. 2016;57:4668–4678. 30. Sangermano R, Khan M, Cornelis SS, et al. ABCA4 midigenes reveal the full splice spectrum of all reported noncanonical splice site variants in Stargardt disease. Genome Res. 2018;28:100–110. 31. Hiatt JB, Pritchard CC, Salipante SJ, O'Roak BJ, Shendure J. Single molecule molecular inversion probes for targeted, high-accuracy detection of low-frequency variation. Genome Res. 2013;23:843–854. 15. Zernant J, Lee W, Collison FT, et al. Frequent hypomorphic alleles account for a significant fraction of ABCA4 disease and distinguish it from age- related macular degeneration. J Med Genet. 2017;54:404–412. 32. Garanto A, Collin RWJ. Design and in vitro use of antisense oligonucleotides to correct pre-mRNA splicing defects in inherited retinal dystrophies. Methods Mol Biol. 2018;1715:61–78. 16. Runhart EH, Sangermano R, Cornelis SS, et al. The common ABCA4 variant p.Asn1868Ile shows nonpenetrance and variable expression of Stargardt disease when present in trans with severe variants. Invest Ophthalmol Vis Sci. 2018;59:3220–3231. 33. Zernant J, Schubert C, Im KM, et al. Analysis of the ABCA4 gene by next- generation sequencing. Invest Ophthalmol Vis Sci. 2011;52:8479–8487. p 17. Bax NM, Sangermano R, Roosing S, et al. Heterozygous deep-intronic variants and deletions in ABCA4 in persons with retinal dystrophies and one exonic ABCA4 variant. Hum Mutat. 2015;36:43–47. 34. Liquori A, Vache C, Baux D, et al. Whole USH2A gene sequencing identifies several new deep intronic mutations. Hum Mutat. 2016;37:184–193. 18. Maugeri A, van Driel MA, van de Pol DJ, et al. 24. Collin RW, den Hollander AI, van der Velde-Visser SD, Bennicelli J, Bennett J, Cremers FP. Antisense oligonucleotide (AON)-based therapy for Leber congenital amaurosis caused by a frequent mutation in CEP290. Mol Ther Nucleic Acids. 2012;1:e14. 25. Garanto A, Chung DC, Duijkers L, et al. In vitro and in vivo rescue of aberrant splicing in CEP290-associated LCA by antisense oligonucleotide delivery. Hum Mol Genet. 2016;25:2552–2563. y 26. Gerard X, Perrault I, Hanein S, et al. AON-mediated exon skipping restores ciliation in fibroblasts harboring the common Leber congenital amaurosis CEP290 mutation. Mol Ther Nucleic Acids. 2012;1:e29. 27. Parfitt DA, Lane A, Ramsden CM, et al. Identification and correction of mechanisms underlying inherited blindness in human iPSC-derived optic cups. Cell Stem Cell. 2016;18:769–781. © The Author(s) 2019 1Department of Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands. 2Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands. 3Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, Nijmegen, The Netherlands. 4Department of Ophthalmology, Radboud University Medical Center, Nijmegen, The Netherlands. 5Center for Medical Genetics, Ghent University and Ghent University Hospital, Ghent, Belgium. 6The Rotterdam Eye Hospital and the Rotterdam Ophthalmic Institute, Rotterdam, The Netherlands. 7Department of Medical Genetics, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands. 8Department of Ophthalmology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands. 9Department of Ophthalmology, Erasmus Medical Center, Rotterdam, The Netherlands. 10Service d′Exploration de la Vision CHU, Lille, France. 11Institute for Neurosciences of Montpellier INSERM U1051, University of Montpellier, Montpellier, France. 12Centre d′ Etude du Polymorphisme Humain, Fondation Jean Dausset, Paris, France. 13UCL Institute of Ophthalmology, London, UK. 14Moorfields Eye Hospital, London, UK. 15Department of Haematology, University of Cambridge, Cambridge, UK. 16NIHR BioResource, Cambridge University Hospitals NHS Foundation Trust, Cambridge Biomedical Campus, Cambridge, UK. 17Department of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK. 18Univ. Lille, Inserm UMR-S 1172, CHU Lille, Biochemistry and Molecular Biology Department - UF Génopathies, Lille, France. 19Institut für Humangenetik, Universität Regensburg, Regensburg, Germany. 20Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden Volume 21 \| Number 8 \| August 2019 \| GENETICS in MEDICINE 1760
https://openalex.org/W2889289499	https://jdc.jefferson.edu/cgi/viewcontent.cgi?article=1085&context=medoncfp	English	null	Metformin as a Therapeutic Target in Endometrial Cancers	Frontiers in oncology	2,018	cc-by	14,292	Thomas Jefferson University Thomas Jefferson University Jefferson Digital Commons Jefferson Digital Commons Thomas Jefferson University Thomas Jefferson University Jefferson Digital Commons Jefferson Digital Commons Department of Medical Oncology Faculty Papers Department of Medical Oncology 8-28-2018 Scott D. Richard Thomas Jefferson University See next page for additional authors Follow this and additional works at: https://jdc.jefferson.edu/medoncfp Part of the Obstetrics and Gynecology Commons, and the Oncology Commons Let us know how access to this document benefits you See next page for additional authors Follow this and additional works at: https://jdc.jefferson.edu/medoncfp Part of the Obstetrics and Gynecology Commons, and the Oncology Commons Let us know how access to this document benefits you Authors Authors Teresa Lee, Ubaldo E. Martinez-Outshoorn, Russell J. Schilder, Christine H. Kim, Scott D. Richard, Norman G. Rosenblum, and Jennifer Johnson Recommended Citation Recommended Citation Lee, Teresa; Martinez-Outshoorn, Ubaldo E.; Schilder, Russell J.; Kim, Christine H.; Richard, Scott D.; Rosenblum, Norman G.; and Johnson, Jennifer, "Metformin as a Therapeutic Target in Endometrial Cancers." (2018). Department of Medical Oncology Faculty Papers. Paper 85. https://jdc.jefferson.edu/medoncfp/85 This Article is brought to you for free and open access by the Jefferson Digital Commons. The Jefferson Digital Commons is a service of Thomas Jefferson University's Center for Teaching and Learning (CTL). The Commons is a showcase for Jefferson books and journals, peer-reviewed scholarly publications, unique historical collections from the University archives, and teaching tools. The Jefferson Digital Commons allows researchers and interested readers anywhere in the world to learn about and keep up to date with Jefferson scholarship. This article has been accepted for inclusion in Department of Medical Oncology Faculty Papers by an authorized administrator of the Jefferson Digital Commons. For more information, please contact: JeffersonDigitalCommons@jefferson.edu. This article is available at Jefferson Digital Commons: https://jdc.jefferson.edu/medoncfp/85 Metformin as a Therapeutic Target in Endometrial Cancers Teresa Y. Lee 1, Ubaldo E. Martinez-Outschoorn 1, Russell J. Schilder 1, Christine H. Kim 2, Scott D. Richard 2, Norman G. Rosenblum 2 and Jennifer M. Johnson 1* 1 Department of Medical Oncology, Thomas Jefferson University, Philadelphia, PA, United States, 2 Department of Obstetrics and Gynecology, Thomas Jefferson University, Philadelphia, PA, United States Endometrial cancer is the most common gynecologic malignancy in developed countries. Its increasing incidence is thought to be related in part to the rise of metabolic syndrome, which has been shown to be a risk factor for the development of hyperestrogenic and hyperinsulinemic states. This has consequently lead to an increase in other hormone-responsive cancers as well e.g., breast and ovarian cancer. The correlation between obesity, hyperglycemia, and endometrial cancer has highlighted the important role of metabolism in cancer establishment and persistence. Tumor-mediated reprogramming of the microenvironment and macroenvironment can range from induction of cytokines and growth factors to stimulation of surrounding stromal cells to produce energy-rich catabolites, fueling the growth, and survival of cancer cells. Such mechanisms raise the prospect of the metabolic microenvironment itself as a viable target for treatment of malignancies. Metformin is a biguanide drug that is a ﬁrst-line treatment for type 2 diabetes that has beneﬁcial effects on various markers of the metabolic syndrome. Many studies suggest that metformin shows potential as an adjuvant treatment for uterine and other cancers. Here, we review the evidence for metformin as a treatment for cancers of the endometrium. We discuss the available clinical data and the molecular mechanisms by which it may exert its effects, with a focus on how it may alter the tumor microenvironment. The pleiotropic effects of metformin on cellular energy production and usage as well as intercellular and hormone-based interactions make it a promising candidate for reprogramming of the cancer ecosystem. This, along with other treatments aimed at targeting tumor metabolic pathways, may lead to novel treatment strategies for endometrial cancer. Authors Authors REVIEW REVIEW published: 28 August 2018 doi: 10.3389/fonc.2018.00341 published: 28 August 2018 doi: 10.3389/fonc.2018.00341 Edited by: Simona Pisanti, Edited by: Simona Pisanti, Università degli Studi di Salerno, Italy Reviewed by: Ronca Roberto, Università degli Studi di Brescia, Italy Raquel Aloyz, Lady Davis Institute (LDI), Canada Correspondence: Jennifer M. Johnson jennifer.m.johnson@jefferson.edu Specialty section: This article was submitted to Molecular and Cellular Oncology, a section of the journal Frontiers in Oncology ENDOMETRIAL CANCER Received: 31 May 2018 Accepted: 06 August 2018 Published: 28 August 2018 Cancer of the endometrium is the ﬁfth most common malignancy in women worldwide, with 455,000 new cases diagnosed worldwide in 2015 (1). The incidence is rising, and is noted to be much higher in developed than developing countries (1, 2). The American Cancer Society estimates that 63,230 new cases will be diagnosed in the United States in 2018 (representing 7% of cancer diagnoses in women), with 11,350 predicted deaths (3). The majority of cases arise in the post-menopausal period, but up to 14% of cases occur in women age 40 or younger (4). The principal risk factor for development of endometrial cancer is exposure to endogenous and exogenous estrogens, which is inﬂuenced by factors such as age at menarche and menopause, parity, Keywords: tumor microenvironment, metabolism, metformin, endometrial cancer, reverse Warburg Edited by: Simona Pisanti, Università degli Studi di Salerno, Italy Reviewed by: Ronca Roberto, Università degli Studi di Brescia, Italy Raquel Aloyz, Lady Davis Institute (LDI), Canada Correspondence: Jennifer M. Johnson jennifer.m.johnson@jefferson.edu ASSOCIATIONS WITH METABOLIC SYNDROME, OBESITY, AND METABOLISM Among the risk factors associated with endometrial cancer, metabolic syndrome (a constellation of obesity, hyperglycemia, hypertension, and hyperlipidemia) has attracted a large amount of interest in recent years. Multiple associative studies have suggested that the metabolic syndrome is a risk factor for development of many diﬀerent types of cancers (16–18), including endometrial cancer (19–24). A meta-analysis of 6 studies from North America, Europe, and China estimated a relative risk (RR) for endometrial cancer of 1.89 in patients with metabolic syndrome (95% conﬁdence interval [CI] 1.34–2.67, p = 0.001) (25). Another meta-analysis of 7 European cohorts reported a 56% increase in endometrial cancer risk per increase of one standard deviation in a composite metabolic risk score derived from sex- and cohort-speciﬁc means in body mass index (BMI), blood pressure, plasma cholesterol, triglycerides, and glucose (18). Apart from incidence, Ni and colleagues reported increased endometrial cancer stage, grade, vascular invasion, tumor size, and lymphatic metastasis in patients with metabolic syndrome, as well as decreased overall survival (26). g Most cases are diagnosed at an early stage due to the early detection sign of abnormal bleeding. Standard treatment for apparent stage I endometrial cancer consists of surgical resection (primary hysterectomy with bilateral salpingo-oophorectomy with possible lymph node mapping). Disease that is conﬁrmed to be uterine-conﬁned with low risk features, can be treated with surgery only and has a >90% relapse-free survival rate at 5 years (8). Radiation decreases local relapse rates but does not aﬀect relapse at distant sites or increase overall survival (9, 10). Trials of adjuvant chemotherapy alone with cyclophosphamide, doxorubicin, and cisplatin demonstrated no signiﬁcant improvement in progression-free survival, overall survival, or relapse (11). Combined adjuvant chemoradiation has shown a slight increase in progression-free survival but not overall survival (11). For metastatic or recurrent disease, management may include surgery or radiation (if localized to a single site); those with unresectable disease may sometimes receive primary chemotherapy followed by cytoreductive surgery. For disease not amenable to local therapy, a carboplatin-paclitaxel combination is increasingly used as a ﬁrst-line alternative to the traditional cisplatin, paclitaxel, and doxorubicin (7). With respect to hormonal therapy in advanced disease, a 33% response rate was noted after alternating tamoxifen and medroxyprogesterone (11– 13). In recurrent or metastatic disease, progestogens, tamoxifen alternated with megestrol, gonadotropin-releasing hormone analogues, selective estrogen receptor modulators, and aromatase inhibitors have been used with response rates ranging from 11 to 56% (13, 14). Citation: Lee TY, Martinez-Outschoorn UE, Schilder RJ, Kim CH, Richard SD, Rosenblum NG and Johnson JM (2018) Metformin as a Therapeutic Target in Endometrial Cancers. Front. Oncol. 8:341. doi: 10.3389/fonc.2018.00341 August 2018 \| Volume 8 \| Article 341 Frontiers in Oncology \| www.frontiersin.org Lee et al. Metformin in Endometrial Cancer use of unopposed estrogen therapy or other hormonal therapies (e.g., tamoxifen), and a host of metabolic factors including obesity. 5–25% of cases are also associated with high risk germline mutations, particularly those aﬀecting DNA mismatch repair pathways, leading to early onset of disease (5). (11). The results of single-agent mTOR inhibitor treatment, or EGFR and HER2 inhibitors have been disappointing, with response rates of 0–12%. Anti-angiogenic drugs such as bevacizumab, sunitinib, brivanib, and lenvatinib have resulted in slightly higher objective response rates of 14–19%. Studies of additional targets, including ﬁbroblast growth factor receptor (FGFR), luteinizing hormone releasing hormone (LHRH), poly ADP-ribose polymerase (PARP), and Programmed Death- 1/Programmed Death Ligand-1 (PD-1/PD-L1) are underway. At this point, no targeted therapies have been approved. Therefore, further interest has been focused on other factors that could contribute to development and progression of endometrial cancer. The most common classiﬁcation system divides endometrial cancers into two subtypes (6). Type I cancers are low- grade, diploid, endometrioid, and hormone-receptor positive, carrying a better prognosis. They frequently display mutations in phosphate and tensin homolog (PTEN). Type II cancers (which include the serous, clear cell, mixed cell, undiﬀerentiated, and carcinosarcoma histologies) are high grade, non-endometrioid, aneuploid, and hormone-negative, with higher rates of metastasis and worse prognosis. They tend to occur in older patients and are more likely to have mutations in the tumor suppressor p53. Type II cancers make up only 10% of endometrial cancers but account for almost 50% of relapses and deaths (7), suggesting a fundamental biologic diﬀerence between the two subsets. Frontiers in Oncology \| www.frontiersin.org ASSOCIATIONS WITH METABOLIC SYNDROME, OBESITY, AND METABOLISM When stratiﬁed by duration of use or cumulative doses, the decrease in incidence demonstrated a dose-response eﬀect. Additionally, a meta-analysis by Tang and colleagues found that metformin use was associated with a decreased risk of endometrial cancer incidence (RR 0.87, 95% CI 0.80–0.95) (54). 29 cohort studies reporting a summary relative risk of 1.89 [95% CI, 1.46–2.45, p < 0.001] (32). This study also noted a small increased risk of disease-speciﬁc mortality in diabetic patients with endometrial cancer (RR 1.32, 95% CI, 1.10–1.60; p = 0.003). The major driver of increased risk of endometrial and other hormone-responsive cancers in obesity is thought to be the generation of a hyper-estrogenic state caused by the presence of the aromatase enzyme in adipose tissue (35). This enzyme catalyzes conversion of androgens to estrogens, making adipose tissue a key source of estrogens in post-menopausal women. In addition, adiposity has been associated with other factors that may drive tumorigenesis in general, including increased inﬂammation, depressed immune function, and chronic insulin resistance and hyperinsulinemia. Endometrial cancer patients have been shown to have increased markers of insulin resistance, including higher fasting insulin levels and elevated non-fasting and fasting C-peptide levels (36, 37). Supporting this link between abnormal glucose metabolism and cancer risk is the observation that better diabetic control is associated with decreased endometrial cancer risk (21). Ultimately, these data suggest that abnormal metabolism, including insulin resistance and hyperglycemia, may play a role in the development of endometrial cancer and thus represent a possible therapeutic target. Other associative studies have focused instead on the relationship between metformin exposure and endometrial cancer outcomes (Table 2). Metformin use in diabetic patients with endometrial cancer was associated with improved overall survival compared to those not taking metformin in two separate studies, including one involving patients with stage III–IV or recurrent endometrial cancer receiving chemotherapy (56, 59). The study by Ko also found improved recurrence-free survival in patients taking metformin. In contrast, some did not ﬁnd any eﬀect of metformin exposure on survival parameters (57, 58, 61). Still others have reported eﬀects only on certain subgroups of patients. For example, Nevadunsky found increased survival for metformin users only among patients with non-endometrioid but not endometrioid forms of endometrial cancer (55), while Hall reported a signiﬁcantly lower recurrence rate of only endometrioid endometrial cancers among metformin users (60). As with the incidence research, these studies are limited by heterogeneity and sample size. ASSOCIATIONS WITH METABOLIC SYNDROME, OBESITY, AND METABOLISM Ultimately, the response rates for recurrent advanced disease are low, and there are no standard second line therapies (13, 15). The individual components of the metabolic syndrome have also been studied in relation to endometrial cancer risk, but it is unknown if their contribution is additive or synergistic. In particular, obesity has been noted to be strongly associated with risk of endometrial cancer in several case-control studies and meta-analyses (21–25, 27, 28). Multiple measures of adiposity, including BMI, waist circumference, waist-to-hip-ratio, and hip circumference, have been found to be directly associated with endometrial cancer incidence. Increased waist circumference and BMI have also been shown to be signiﬁcantly associated with increased risk of overall mortality from endometrial cancer (29, 30). Other studies have demonstrated positive albeit less robust association between endometrial cancer and the other components of the metabolic syndrome: hypertension (21–24), hyperlipidemia (21–24), and hyperglycemia or diabetes mellitus (19, 21–25, 31, 32). The association between diabetes and endometrial cancer appears to be partially confounded by co- existing overweight/obesity (33, 34). However, elevated risk of endometrial cancer in patients with diabetes has been reported even after adjustment for BMI, with one meta-analysis including This lack of eﬀective treatment for advanced stage endometrial cancer has led to exploration of alternative therapeutic modalities. In particular, numerous studies have examined the eﬀectiveness of targeted therapies acting on the phosphoinositide 3-Kinase (PI3K)/Protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), and vascular endothelial growth factor (VEGF), reviewed elsewhere August 2018 \| Volume 8 \| Article 341 2 Metformin in Endometrial Cancer Lee et al. overall cancer incidence in metformin users, reviewed by several groups (47–53). Studies evaluating the relationship between metformin use and endometrial cancer incidence speciﬁcally have yielded more conﬂicting results. Three cohort studies and two case-control studies found no decrease in the risk of endometrial cancer in metformin users compared to nonusers (34, 42, 43, 45, 46). However, these studies show considerable heterogeneity in factors such as study size, indication for metformin use, and duration and method of measurement of metformin exposure (e.g., prescriptions vs. self-report). Notably, a large study of 478,921 Taiwanese women with diabetes showed a signiﬁcantly decreased incidence of endometrial cancer (hazard ratio [HR] 0.675, 95% CI 0.614–0.742) in metformin users compared to never users (44). ASSOCIATIONS WITH METABOLIC SYNDROME, OBESITY, AND METABOLISM However, a 2017 meta-analysis including 6 of the above studies supports a higher overall survival rate in metformin-users with endometrial cancer compared to non-metformin users and non-diabetic patients (HR 0.82, 95% CI 0.70–0.95, I2 = 40%) (62). Finally, a meta-analysis of 28 studies reported that metformin use was associated with decreased all-cause mortality in patients with concurrent diabetes for several cancer types, including endometrial (RR 0.49, 95% CI 0.32, 0.73, p < 0.001) (63). METFORMIN REPURPOSING AND EPIDEMIOLOGIC DATA FROM ENDOMETRIAL CANCER In recent years there has been growing interest in drug repurposing or repositioning, a process which seeks to identify new pharmacologic properties (e.g., anti-tumorigenic) of existing medications for use as primary or adjuvant treatments for other conditions (38, 39). These drugs are already well-studied in terms of tolerability and side eﬀects, often inexpensive, and amenable to retrospective and associative studies as many patients are already taking them for other indications. The association between obesity, diabetes, hyperinsulinemia, and endometrial cancer has led to the hypothesis that medications which target glucose metabolism such as metformin may be eﬀective in preventing or treating such malignancies. One drug that has received a signiﬁcant amount of attention in this arena has been metformin [1,1-dimethylbiguanide] which is a ﬁrst line oral antihyperglycemic agent used in the treatment of type 2 diabetes (40). Broadly, its eﬀects include lowering of blood glucose concentrations, increasing insulin sensitization, and reducing plasma fasting insulin levels. Furthermore, unlike with some oral hypoglycemic medications and insulin, metformin users show a tendency toward sustained weight loss (41). The low toxicity of metformin makes it especially interesting as a potential adjunctive therapy, or even as monotherapy for patients with contraindications to chemotherapy or considerations such as the desire to preserve fertility. Frontiers in Oncology \| www.frontiersin.org CELLULAR AND MOLECULAR MECHANISMS OF METFORMIN INHIBITION OF ENDOMETRIAL CANCER • Ever-use of metformin not associated with risk of endometrial cancer (OR 0.86, 95% CI 0.63–1.18) • Ever-use of metformin not associated with risk of endometrial cancer (OR 0.86, 95% CI 0.63–1.18) • Self-reported metformin use at study baseline not associated with risk of endometrial cancer (HR 1.64, 95% CI 0.92–2.91) • Self-reported metformin use at study baseline not associated with risk of endometrial cancer (HR 1.64, 95% CI 0.92–2.91) p p Multiple studies have demonstrated the ability of metformin to inhibit proliferation of both type I and type II human endometrial cancer cell lines in culture (66–73). Metformin treatment of endometrial cancer cell lines upregulates markers of cell cycle arrest (66, 68, 69, 74), apoptosis (66, 67, 69, 72, 73, 75, 76), and autophagy (69, 77), while decreasing markers associated with senescence (66, 74) and inhibiting cell migration (68, 71, 76). The anticancer eﬀects of metformin treatment may not be limited to direct eﬀects on endometrial cancer cells, but may also result from changes to the systemic milieu. Polycystic ovary syndrome (PCOS) is a condition which is associated with endometrial hyperplasia and predisposition to endometrial cancer (78). Endometrial cancer cell lines incubated with sera from PCOS patients showed increased migration and markers of invasiveness such as activity of matrix metalloproteinases (MMP)−2 and −9 compared to cells incubated with sera from healthy controls. In contrast, sera from PCOS patients treated with metformin for 6 months showed attenuation of this eﬀect, with decreased migration and MMP-2/9 activity compared to cells treated with sera from PCOS patients not on metformin (79). • Metformin use not associated with endometrial cancer risk compared to sulfonylurea use (HR 1.09, 95% CI 0.88–1.35) • Metformin use not associated with endometrial cancer risk compared to sulfonylurea use (HR 1.09, 95% CI 0.88–1.35) • Ever-use of metformin associated with decreased incidence of endometrial cancer (HR 0.675, 95% CI 0.614–0.742) • Dose-response was observed when adjusted for duration of metformin use or cumulative metformin doses (45) (Italy) Case-control 376 cases, 7,485 controls • Ever-use of metformin use not associated with endometrial cancer risk (OR 1.07, 95% CI 0.82–1.41) • Metformin ever-use associated with increased risk of endometrial cancer in full cohort (OR 1.23, 95% CI 1.03–1.48). ( ) The molecular mechanisms of metformin’s eﬀects in endometrial cancer cells are diverse and continue to be an active area of investigation (Figure 1). CELLULAR AND MOLECULAR MECHANISMS OF METFORMIN INHIBITION OF ENDOMETRIAL CANCER Its general mechanisms are complex and multifactorial and are reviewed in detail elsewhere (80). Multiple groups have demonstrated that metformin’s ability to inhibit oxidative phosphorylation (OXPHOS) at the mitochondrial level is an important mediator of its biologic activity (81, 82). The end result is a decrease in proton gradient across the inner mitochondrial membrane, ultimately leading to reduction in proton-driven synthesis of adenosine triphosphate (ATP) and an increase in the ratio of cellular adenosine monophosphate (AMP) to ATP, caused by imbalance in the rate of ATP production vs. consumption. The decrease in ATP is theorized to be responsible for a key eﬀect of metformin treatment, namely, phosphorylation and activation of the serine/threonine AMP-activated protein kinase (AMPK), a regulatory protein which plays a role in sensing and energy status of the cell and regulating cellular function under conditions of energy restriction (83, 84). This leads to AMP binding to AMPK and a conformational change that allows for phosphorylation/activation of AMPK by liver kinase B1 (LKB1) (85). Activation of AMPK switches cells to a catabolic state via AMPK-mediated phosphorylation and inhibition of key enzymes and transcription factors involved • Metformin use associated with increased risk of endometrial cancer in nested case-control (OR 1.24, 95% CI 1.02–1.51) • Metformin use associated with increased risk of endometrial cancer in nested case-control (OR 1.24, 95% CI 1.02–1.51) • Metformin use associated with increased risk of endometrial OR, odds ratio; CI, conﬁdence interval; HR, hazard ratio. compared to normal endometrium (64). They also reported that multiple human endometrial cancer cell lines showed strong upregulation of the glucose transporter GLUT6 as well as activation of AKT compared to nonmalignant cells. In vitro metabolic proﬁling demonstrated that these changes were associated with upregulation of glycolysis, decreased glucose oxidation, and increased de novo lipogenesis. Finally, the authors demonstrated that endometrial cancer cell cultures experience cytotoxicity when exposed to a variety of inhibitors targeting metabolic pathways, including the glycolysis inhibitors 2-deoxy- D-glucose [2-DG] and 3-bromopyruvate (BrPA), the lipogenesis inhibitor 5-(tetradecyloxy)-2-furoic acid (TOFA), the fatty acid oxidation inhibitor etomixir, and the pleiotropic metabolic inhibitor metformin. Further support for the importance of glucose metabolism on endometrial cancer cell growth comes from Han and colleagues, who studied the growth of two endometrial cancer cell lines (ECC-1 and Ishikawa cells) under compared to normal endometrium (64). Frontiers in Oncology \| www.frontiersin.org CELLULAR AND MOLECULAR MECHANISMS OF METFORMIN INHIBITION OF ENDOMETRIAL CANCER References Design Results (42) (UK) Case-control 2,554 cases, 15,324 controls • Ever-use of metformin not associated with risk of endometrial cancer (OR 0.86, 95% CI 0.63–1.18) • Long-term use of metformin (>25 prescriptions) not associated with risk of endometrial cancer (OR 0.79, 95% CI 0.54–1.17) (34) (USA) Retrospective cohort 88,107 postmenopausal women (age 50–79) • Self-reported metformin use at study baseline not associated with risk of endometrial cancer (HR 1.64, 95% CI 0.92–2.91) (43) (USA) Retrospective cohort 541,128 women (new prescription of metformin or sulfonylurea, any indication) • Metformin use not associated with endometrial cancer risk compared to sulfonylurea use (HR 1.09, 95% CI 0.88–1.35) (44) (Taiwan) Retrospective cohort 478,921 women (new diagnosis of type 2 diabetes) • Ever-use of metformin associated with decreased incidence of endometrial cancer (HR 0.675, 95% CI 0.614–0.742) • Dose-response was observed when adjusted for duration of metformin use or cumulative metformin doses (45) (Italy) Case-control 376 cases, 7,485 controls • Ever-use of metformin use not associated with endometrial cancer risk (OR 1.07, 95% CI 0.82–1.41) (46) (Finland) Retrospective cohort 92,366 women (new diagnosis of type 2 diabetes) Nested case-control 590 cases (endometrioid), 11,792 controls • Metformin ever-use associated with increased risk of endometrial cancer in full cohort (OR 1.23, 95% CI 1.03–1.48). • Metformin use associated with increased risk of endometrial cancer in nested case-control (OR 1.24, 95% CI 1.02–1.51) OR, odds ratio; CI, conﬁdence interval; HR, hazard ratio. low, normal, or high glucose conditions (65). High glucose conditions (corresponding to physiologic hyperglycemia) led to increased cell proliferation, in vitro colony formation, and increased expression of the GLUT1 glucose transporter along with increased glucose uptake. High glucose also increased phosphorylation of lactate dehydrogenase A (LDHA) and decreased levels of pyruvate dehydrogenase (PDH), suggesting an increase in glycolytic activity. Conversely, low glucose conditions led to increased cell apoptosis, cell cycle arrest, decreased adhesion, and invasion. All of these data support the idea that the metabolic vulnerabilities of endometrial cancer may make it susceptible to therapies such as metformin. (45) (Italy) Case-control 376 cases, 7,485 controls CELLULAR AND MOLECULAR MECHANISMS OF METFORMIN INHIBITION OF ENDOMETRIAL CANCER Metabolic alterations in endometrial cancer have been described not only on a systemic but also on a cellular and molecular level. For example, Byrne and colleagues examined microarray data from women with women with type I endometrial cancer and demonstrated that tumor-derived endometrium showed enrichment of genes related to glycolysis and lipogenesis Many investigators have sought to examine the eﬀect of metformin exposure on the development of endometrial cancer (Table 1). Multiple epidemiologic studies have reported lower August 2018 \| Volume 8 \| Article 341 Frontiers in Oncology \| www.frontiersin.org 3 Metformin in Endometrial Cancer Lee et al. TABLE 1 \| Studies of metformin use and incidence of endometrial cancer. References Design Results (42) (UK) Case-control 2,554 cases, 15,324 controls • Ever-use of metformin not associated with risk of endometrial cancer (OR 0.86, 95% CI 0.63–1.18) • Long-term use of metformin (>25 prescriptions) not associated with risk of endometrial cancer (OR 0.79, 95% CI 0.54–1.17) (34) (USA) Retrospective cohort 88,107 postmenopausal women (age 50–79) • Self-reported metformin use at study baseline not associated with risk of endometrial cancer (HR 1.64, 95% CI 0.92–2.91) (43) (USA) Retrospective cohort 541,128 women (new prescription of metformin or sulfonylurea, any indication) • Metformin use not associated with endometrial cancer risk compared to sulfonylurea use (HR 1.09, 95% CI 0.88–1.35) (44) (Taiwan) Retrospective cohort 478,921 women (new diagnosis of type 2 diabetes) • Ever-use of metformin associated with decreased incidence of endometrial cancer (HR 0.675, 95% CI 0.614–0.742) • Dose-response was observed when adjusted for duration of metformin use or cumulative metformin doses (45) (Italy) Case-control 376 cases, 7,485 controls • Ever-use of metformin use not associated with endometrial cancer risk (OR 1.07, 95% CI 0.82–1.41) (46) (Finland) Retrospective cohort 92,366 women (new diagnosis of type 2 diabetes) Nested case-control 590 cases (endometrioid), 11,792 controls • Metformin ever-use associated with increased risk of endometrial cancer in full cohort (OR 1.23, 95% CI 1.03–1.48). • Metformin use associated with increased risk of endometrial cancer in nested case-control (OR 1.24, 95% CI 1.02–1.51) OR, odds ratio; CI, conﬁdence interval; HR, hazard ratio. TABLE 1 \| Studies of metformin use and incidence of endometrial cancer. CELLULAR AND MOLECULAR MECHANISMS OF METFORMIN INHIBITION OF ENDOMETRIAL CANCER References Design Results (42) (UK) Case-control 2,554 cases, 15,324 controls • Ever-use of metformin not associated with risk of endometrial cancer (OR 0.86, 95% CI 0.63–1.18) • Long-term use of metformin (>25 prescriptions) not associated with risk of endometrial cancer (OR 0.79, 95% CI 0.54–1.17) (34) (USA) Retrospective cohort 88,107 postmenopausal women (age 50–79) • Self-reported metformin use at study baseline not associated with risk of endometrial cancer (HR 1.64, 95% CI 0.92–2.91) (43) (USA) Retrospective cohort 541,128 women (new prescription of metformin or sulfonylurea, any indication) • Metformin use not associated with endometrial cancer risk compared to sulfonylurea use (HR 1.09, 95% CI 0.88–1.35) (44) (Taiwan) Retrospective cohort 478,921 women (new diagnosis of type 2 diabetes) • Ever-use of metformin associated with decreased incidence of endometrial cancer (HR 0.675, 95% CI 0.614–0.742) • Dose-response was observed when adjusted for duration of metformin use or cumulative metformin doses (45) (Italy) Case-control 376 cases, 7,485 controls • Ever-use of metformin use not associated with endometrial cancer risk (OR 1.07, 95% CI 0.82–1.41) (46) (Finland) Retrospective cohort 92,366 women (new diagnosis of type 2 diabetes) Nested case-control 590 cases (endometrioid), 11,792 controls • Metformin ever-use associated with increased risk of endometrial cancer in full cohort (OR 1.23, 95% CI 1.03–1.48). • Metformin use associated with increased risk of endometrial cancer in nested case-control (OR 1.24, 95% CI 1.02–1.51) OR, odds ratio; CI, conﬁdence interval; HR, hazard ratio. TABLE 1 \| Studies of metformin use and incidence of endometrial cancer. CELLULAR AND MOLECULAR MECHANISMS OF METFORMIN INHIBITION OF ENDOMETRIAL CANCER They also reported that multiple human endometrial cancer cell lines showed strong upregulation of the glucose transporter GLUT6 as well as activation of AKT compared to nonmalignant cells. In vitro metabolic proﬁling demonstrated that these changes were associated with upregulation of glycolysis, decreased glucose oxidation, and increased de novo lipogenesis. Finally, the authors demonstrated that endometrial cancer cell cultures experience cytotoxicity when exposed to a variety of inhibitors targeting metabolic pathways, including the glycolysis inhibitors 2-deoxy- D-glucose [2-DG] and 3-bromopyruvate (BrPA), the lipogenesis inhibitor 5-(tetradecyloxy)-2-furoic acid (TOFA), the fatty acid oxidation inhibitor etomixir, and the pleiotropic metabolic inhibitor metformin. Further support for the importance of glucose metabolism on endometrial cancer cell growth comes from Han and colleagues, who studied the growth of two endometrial cancer cell lines (ECC-1 and Ishikawa cells) under August 2018 \| Volume 8 \| Article 341 4 Metformin in Endometrial Cancer Lee et al. TABLE 2 \| Observational studies of metformin exposure in endometrial cancer. Number DM No DM References Total MFM No MFM No MFM Results (55) (USA) 985 114 136 735 • Non-endometrioid endometrioid cancer patients had greater OS in metformin vs. non- metformin users (HR 0.54, 95% CI 0.30–0.97) • No association between metformin use and endometrioid endometrial cancer (HR 0.79, 95% CI 0.31–2.0) (56) (USA) 1,495 196 167 1,132 • RFS 1.8 times worse in patients not on metformin compared to metformin users (95% CI 1.1–2.9) • OS 2.3 times worse in patients not on metformin compared to metformin users (95% CI 1.3–4.2) • TTR not associated with metformin use (HR 1.12, 95% CI 0.6–2.2) (57) (Poland) 107 30 38 39 • No difference in OS between metformin users vs. non-users (p = 0.86) (58) (USA) 1,303 116 161 1,026 • OS not signiﬁcantly different between diabetic metformin-users and diabetic non-metformin users (HR 0.61; 95% CI 0.30–1.23) or non-diabetic patients (HR 1.03; 95% CI 0.57–1.85). • PFS not signiﬁcantly different between diabetic metformin-users compared to diabetic non-metformin users (HR 1.06; 95% CI 0.34–3.30) or non-diabetic patients (HR 1.14; 95% CI 0.46–2.62) (59) (USA) 349 31 27 291 • OS greater in diabetic metformin-users compared to diabetic patients not taking metformin (HR 0.42, 95% CI 0.23–0.78) but not compared to non-diabetic patients (HR 0.65, 95% CI 0.41–1.05) (60) (USA) 351 64 287 • Recurrence rate for all metformin-users vs. CELLULAR AND MOLECULAR MECHANISMS OF METFORMIN INHIBITION OF ENDOMETRIAL CANCER non-metformin users not statistically different, but recurrence of type I endometrial cancers was signiﬁcantly lower for metformin users (1.9%) compared to non-metformin users (10.3%), p = 0.05 (61) (Austria) 465 46 41 378 • Metformin use not associated with OS (HR 0.9, 95% CI 0.69–1.2) or RFS (HR 1.2, 95% CI 0.8–1.70) MFM, metformin; OS, overall survival; HR, hazard ratio; CI, conﬁdence interval; RFS, recurrence-free survival; TTR, time to regression; PFS, progression-free survival. TABLE 2 \| Observational studies of metformin exposure in endometrial cancer. • OS greater in diabetic metformin-users compared to diabetic patients not taking metformin (HR 0.42, 95% CI 0.23–0.78) but not compared to non-diabetic patients (HR 0.65, 95% CI 0.41–1.05) • Recurrence rate for all metformin-users vs. non-metformin users not statistically different, but recurrence of type I endometrial cancers was signiﬁcantly lower for metformin users (1.9%) compared to non-metformin users (10.3%), p = 0.05 • Metformin use not associated with OS (HR 0.9, 95% CI 0.69–1.2) or RFS (HR 1.2, 95% CI 0.8–1.70) MFM, metformin; OS, overall survival; HR, hazard ratio; CI, conﬁdence interval; RFS, recurrence-free survival; TTR, time to regression; PFS, progression-free survival. d ratio; CI, conﬁdence interval; RFS, recurrence-free survival; TTR, tim in ATP-consuming synthetic pathways (e.g., glucose, lipid and protein). mitogen-activated protein-kinase/extracellular signal-regulated kinase (MAPK/ERK) pathways (92–94). Inhibition of PI3K/Akt signaling has been observed in metformin-treated endometrial cancer cells (70), and both Akt and ERK1/2 are inhibited in endometrial cancer cells incubated with serum from women with PCOS who are receiving metformin treatment (79). The cumulative result is inhibition of individual cell growth and proliferation, decreased synthesis of proteins and fatty acids, as well as decreased paracrine and endocrine release of pro- proliferative systemic factors. Among the known downstream eﬀects of AMPK activation is decreased protein synthesis due to inhibition of the mTOR pathway, resulting in the inhibition of translation by the eukaryotic initiation factor 4E-binding protein-1 [4E-BP1] complex and decreased activity of the S6 kinase 1 (S6K1) responsible for phosphorylation of the ribosomal S6 protein (rpS6) (86, 87). Metformin inhibition of mTOR signaling in endometrial cancer cells has been conﬁrmed by multiple groups (66, 74, 88–90). Other well-described eﬀects of AMPK activation include phosphorylation and inactivation of Acetyl- CoA carboxylase (ACC) leading to downregulation of fatty acid synthesis (83, 91) as well as inhibition of signaling via the insulin-like growth factor-1 receptor (IGF-1R). Frontiers in Oncology \| www.frontiersin.org CELLULAR AND MOLECULAR MECHANISMS OF METFORMIN INHIBITION OF ENDOMETRIAL CANCER Metformin inhibition of ACC has not been reported in endometrial cancer cell lines, but Wallbillich did observe decreased expression of fatty acid synthetase (FAS) in metformin-treated tumor tissue from a xenograft model (73). In endometrial cancer cell cultures, metformin treatment lowers secretion of insulin-like growth factor (IGF-1) (70), downregulates expression of insulin receptor (68) and IGF-1R (70, 75), inhibits phosphorylation of IGF-1R (68), and increases expression of insulin-like growth factor-binding protein 1 (IGFBP-1) (75). In other cancer types, this is associated with inhibitory phosphorylation of the signaling adapter, insulin receptor substrate-1 (IRS- 1) and inhibition of the downstream PI3K/Akt/mTOR and In addition to IGF-1-related signaling pathways, metformin treatment of endometrial cancer cells has been reported to aﬀect the activity of the transcription factor signal transducer and activator of transcription 3 (STAT3), which is usually activated via signaling by various growth factors and cytokines to dimerize, translocate to the nucleus, and induce transcription of multiple pro-survival and pro-proliferative genes (95). STAT3 levels are elevated in endometrial cancer cells, in particular the serine- phosphorylated form, phospho-STAT3 Ser727 (96). High glucose concentrations induces transcription of STAT3 as well as its upstream regulators Janus kinases 1 and 2 (JAK1/2), while metformin treatment reduces total STAT3 protein as well as phospho-STAT3 Ser727 (73). This is associated with signiﬁcantly decreased expression of multiple pro-survival downstream targets of STAT3, including c-Myc and B-cell lymphoma (Bcl)-2 and –XL, providing another possible mechanism for metformin’s anti-cancer activity. The authors also examined the eﬀect of August 2018 \| Volume 8 \| Article 341 Frontiers in Oncology \| www.frontiersin.org 5 Lee et al. Metformin in Endometrial Cancer Lee et al. Lee et al. Metformin in Endometrial Cancer FIGURE 1 \| (A) Mechanisms of action of metformin within the endometrial cancer cell. (B) Downstream molecular targets of metformin showing differential expression or activity in endometrial cancer. FIGURE 1 \| (A) Mechanisms of action of metformin within the endometrial cancer cell. (B) Downstream molecular targets of metformin showing differential expression or activity in endometrial cancer. s of action of metformin within the endometrial cancer cell. (B) Downstream molecular targets of metformin showing differential ometrial cancer. FOXO1 is negatively regulated via inhibitory phosphorylation by Akt, which causes it to translocate out of the nucleus to the cytoplasm where it is degraded (98). Conversely, AMPK activation leads to FOXO1 nuclear localization and activation (99). Frontiers in Oncology \| www.frontiersin.org CELLULAR AND MOLECULAR MECHANISMS OF METFORMIN INHIBITION OF ENDOMETRIAL CANCER Metformin treatment has also been shown to inhibit epithelial-to-mesenchymal transition (EMT) in endometrial cancer cell lines. Metformin increases epithelial markers such as E-cadherin and pan-keratin in endometrial cancer cells in vitro (76, 100) and decreases mesenchymal markers (e.g., N- cadherin, ﬁbronectin, vimentin) (76, 100) and transcriptional drivers of EMT (e.g., Twist-1, snail-1, zinc ﬁnger E-box-binding homeobox-1 [ZEB-1]) (100). In cultured cells, metformin was able to attenuate the molecular and morphologic changes induced by EMT-inducing stimuli such as 17β-estradiol and transforming-growth factor-β (TGF-β) (76). Correspondingly, histologic staining for E-cadherin was signiﬁcantly higher in endometrial carcinomas taken from patients with a history of metformin use (100). Attention is now also being paid to the impact of metformin on other cellular components of the tumor microenvironment (TME) beyond the cancer cells themselves as described in Rivadeneira and Delgoﬀe (107). Metformin is capable of decreasing the rate of tumor cell oxygen consumption and thus is able to reduce hypoxia levels within the tumor. The reduction of hypoxia can enhance the activity of agents aimed at stimulating anti-tumor T-cells (108). Further eﬀects of metformin on the immune microenvironment are hypothesized to be mediated through tumor-associated macrophage reprogramming from an M2 to M1-like phenotype (109). A review on the eﬀects of metformin on other tumor cells is beyond the scope of this work. In sum, the pre-clinical data provide suﬃcient evidence for continued evaluation of metformin’s antineoplastic potential. Nonetheless, they also highlight the likely complex interaction between tumor- and patient-speciﬁc factors dictating the eﬃcacy of metformin as an anticancer treatment and underscore the need for ongoing human studies. Metformin has demonstrated the ability to synergize with other endometrial cancer therapies in vitro, leading to enhanced apoptosis of cultured endometrial cell lines in the presence of paclitaxel (74, 101), cisplatin (101, 102), or progestin (89). For the latter two, this eﬀect is dependent on downregulation of glycoxylase I (GloI), a mediator of chemotherapy resistance (89, 101). Metformin treatment was also able to increase expression of the progesterone receptor in endometrial cancer cells (88) and sensitize progestin-resistant endometrial carcinoma cells to medroxyprogesterone-induced apoptosis (89). Conversely, metformin alters expression of the estrogen receptor (ER) in endometrial cancer cells, decreasing the ERα isoform while increasing expression of ERβ, with overall inhibition of estradiol- induced proliferation (90). CELLULAR AND MOLECULAR MECHANISMS OF METFORMIN INHIBITION OF ENDOMETRIAL CANCER Zou demonstrated that endometrial cancer cells show decreased levels of phospho-AMPK and total FOXO1 protein, and endometrial cancer tissues show signiﬁcantly less staining of activated AMPK and higher levels of phospho-Akt compared to controls (72). This is associated with a shift toward cytoplasmic (inactive) rather than nuclear (active) FOXO1 staining. In vitro, metformin in a xenograft model of endometrioid endometrial cancer cells. While not statistically signiﬁcant, metformin exposure was associated with a trend toward decreased tumor size, and analysis of tumor tissues demonstrated decreased expression of STAT3 and its targets (73). Another recently-reported target of metformin in endometrial cancer is the transcription factor forkhead box protein 1 (FOXO1), which plays numerous roles in cellular function, including regulation of gluconeogenesis, adipogenesis, protection from oxidative stress, and tumor suppression (97). August 2018 \| Volume 8 \| Article 341 Frontiers in Oncology \| www.frontiersin.org 6 Lee et al. Metformin in Endometrial Cancer patient biopsies for culture and inoculation into nude mice. The authors noted that one sample contained a K-Ras mutation while the other was wild-type; neither tumor was inhibited by metformin treatment, either alone or in combination with cisplatin (104). It should be noted that the dosage of metformin used in this study was lower than in the study by Iglesias (250 mg/kg/day for 21 days vs. 1 g/kg/day for 29-64 days). However, in both these studies, the tumors that displayed no susceptibility to metformin treatment also showed no changes in levels of activated AMPK or downstream mediators, emphasizing the likely importance of this pathway for mediating metformin- induced tumor suppression. Several studies have now shown that higher doses of metformin are required in mice to have antitumor eﬀects than those administered in clinical trials and this may be a reﬂection of pharmacokinetic diﬀerences between rodents and humans (86, 105, 106). they reported that metformin treatment increased FOXO1 protein levels, decreased inhibitory FOXO1 phosphorylation, and increased FOXO1 nuclear accumulation in an AMPK- dependent manner, resulting in inhibition of endometrial cancer cell proliferation. Conversely, knockdown of FOXO1 expression using siRNA partially attenuates the antiproliferative eﬀect of metformin on endometrial cancer cells. Metformin treatment inhibited growth of tumors in a xenograft mouse model, and tumor staining also showed increased phospho-AMPK and nuclear localization of FOXO1 as well as decreased staining of the proliferative marker Ki-67 (72). Decreased FOXO1 expression during metformin treatment has been reported by others as well (68). CELLULAR AND MOLECULAR MECHANISMS OF METFORMIN INHIBITION OF ENDOMETRIAL CANCER In endometrial cancer patients with type 2 diabetes, metformin leads to decreased expression of the estrogen receptor in tumor tissue compared to insulin treatment (103). Frontiers in Oncology \| www.frontiersin.org METFORMIN IN PRESURGICAL AND OTHER CLINICAL TRIALS IN ENDOMETRIAL CANCER The preponderance of preclinical data has prompted several early phase clinical trials of metformin in human endometrial cancers (Table 3). Many groups have utilized the pre-surgical window approach, in which patients with a biopsy-based histologic diagnosis of endometrial cancer receive metformin treatment during the period prior to hysterectomy. Compared to baseline levels or control patients, patients receiving metformin (between 850 and 2,250 mg daily) showed a post-treatment reduction in markers of DNA replication (topoisomerase IIα) (112) and cellular proliferation (Ki-67) (111, 114, 115, 119). No change in Ki-67 staining was observed by Soliman and colleagues, though notably the dose and duration of metformin exposure was the lowest among these studies (117). Metformin treatment was associated with histologic evidence for inhibition of key signaling pathways including PI3K/Akt/mTOR (111, 112, 114, 116, 117, 119) and MAPK/ERK (112, 117). Tumor immunohistochemistry in one trial also revealed decreased expression of estrogen Possible mutation-speciﬁc eﬀects of metformin have also been explored in preclinical in vivo models of endometrial cancer. Oral metformin is capable of reducing in vitro cell proliferation as well as tumor size in xenograft models of multiple human and mouse endometrial cancer cell lines. (67). This eﬀect occurred only in cell lines with activating K-Ras mutations but not wild- type K-Ras and could be partially attenuated by siRNA-based inhibition of K-Ras expression. Moreover, metformin treatment was shown to cause mislocalization of K-Ras to the cytoplasm in a protein kinase C (PKC)-dependent manner. No association was seen between metformin-responsiveness and PTEN mutations. Another study utilized a primary endometrioid endometrial carcinoma xenograft model using cells taken directly from August 2018 \| Volume 8 \| Article 341 Frontiers in Oncology \| www.frontiersin.org 7 Lee et al. Metformin in Endometrial Cancer References Design Assessment Treatment No MFM MFM Results (110) (Iran) Non-blinded randomized controlled trial Endometrial histology after metformin vs. METFORMIN IN PRESURGICAL AND OTHER CLINICAL TRIALS IN ENDOMETRIAL CANCER progesterone treatment for dysfunctional uterine bleeding Metformin 500 mg twice daily or megestrol 40 mg daily for 3 months 21 22 • Metformin induced endometrial atrophy in 95.5% (21/22) of patients (including 2 with low grade EEC) compared to 61.9% (13/21) receiving megestrol (111) (Canada) Single arm Effect of metformin on serum and tumor biomarkers during window from biopsy-proven EC diagnosis to resection in non-diabetic patients Metformin 500 mg three times daily from enrollment to surgery (21–50 days, mean 36.6 days, median 38 days) 10 (banked tissues) 11 (8 EEC, 3 NEEC) • Metformin reduced plasma insulin, IGF-1 and IGFBP-7 and decreased tumor Ki-67 and phospho-rpS6. • Metformin treatment led to non-signiﬁcant increase in plasma IGFBP-1 (112) (Japan) Single arm Effect of metformin on serum and tumor biomarkers during window from biopsy-proven EC diagnosis to surgical resection in non-diabetic patients Metformin starting dose 750 mg daily, increased weekly as tolerated to 1,500–2,250 mg daily (divided) from enrollment to surgery (4–6 weeks) 10 (banked tissues) 31 • Metformin reduced tumor topoisomerase IIα, Ki-67, phospho-rpS6, and phospho-ERK1/2 and increased tumor phospho-AMPK • Metformin decreased serum insulin, glucose, and IGF-1 • metformin decreased ability of sera to stimulate DNA synthesis in cultured cells (113) (China) Single arm Effect of metformin plus estrogen/progesterone combination on early stage EC (Ia) in women with PCOS Cyproterone acetate 2 mg daily, ethinyl estradiol 35 µg daily, and metformin 1,000 mg daily for 6 months N/A 5 • Estrogen/progesterone treatment combined with metformin led to reversion to normal endometrial epithelium in all patients (114) (USA) Single arm Effect of metformin on tumor biomarkers during window from biopsy-proven EEC diagnosis to surgical resection in obese women Metformin 850 mg daily from enrollment to surgery (7–28 days, mean 14.65 days) N/A 20 • Metformin decreased tumor Ki-67, phospho-AMPK, phospho-Akt, phospho-rpS6, phospho-4E-BP-1 and ER but did not change PR level • Responders had increased serum free fatty acids and tumor staining for markers of fatty acid oxidation and glycogen synthesis (115) (UK) Non- randomized controlled trial Effect of metformin on tumor biomarkers during window from biopsy-proven diagnosis of AEH or EEC to surgical resection Metformin 850 mg twice daily from enrollment to surgery (7–34 days, median 20 days) vs. August 2018 \| Volume 8 \| Article 341 Frontiers in Oncology \| www.frontiersin.org METFORMIN IN PRESURGICAL AND OTHER CLINICAL TRIALS IN ENDOMETRIAL CANCER no treatment 12 (2 AEH, 10 EEC) 28 (0 AEH, 28 EEC) • Metformin decreased tumor Ki-67 and phospho-4E-BP1 but did not change levels of phospho-Akt, phospho-ACC, phospho-rpS6, ER, PR, or caspase-3 (116) (China) Non- randomized controlled trial Effect of metformin on serum and tumor biomarkers during window from biopsy-proven diagnosis of EC and surgical resection in non-diabetic women Metformin 500 mg three times daily from enrollment to surgery (3–4 weeks) vs. no treatment 30 30 • EC patients had higher serum IGF-1, lower tumor phospho-AMPK, and higher tumor phospho-mTOR at baseline than non-EC patients • Metformin led to lower serum IGF-1, higher tumor phospho-AMPK and lower tumor phospho-mTOR in EC patients (117) (USA) Single arm Effect of metformin on serum and tumor biomarkers during window from biopsy-proven diagnosis of EC to surgical resection Metformin 850 mg daily from enrollment to surgery (7–24 days, median 9.5 days) N/A 20 • Metformin decreased serum IGF-1, omentin, insulin, C-peptide, and leptin • Metformin decreased tumor phospho-Akt, phospho-rpS6, phospho-ERK1/2 but did not change levels of Ki-67, phospho-ACC or caspase-3 (118) (Japan) Single arm Efﬁcacy of metformin in preventing recurrence after progestin therapy for AEH or early stage EC (stage Ia) Metformin starting dose 750 mg daily, increased weekly as tolerated to 2,250 mg daily (divided, concurrent with medroxyprogesterone acetate-based protocol for 24–36 weeks, continued alone in complete responders until conception or recurrence) N/A 16 AEH, 13 EC • Metformin maintenance associated with 3-year recurrence-free survival of 89%, compared to expected baseline of 52% recurrence-free survival at 2 years (119) (China) Non- randomized controlled trial Effect of metformin on tumor biomarkers during window from biopsy-proven diagnosis of EC to surgical resection Metformin 500 mg three times daily for 3–4 weeks 32 33 • EC patients had higher tumor Ki-67, PI3K, phospho-Akt, phospho- S6K1, and phospho-4E-BP1 at baseline than non-EC patients • Metformin decreased tumor Ki-67, PI3K, phospho-Akt, phospho-S6K1, and phospho-4E-BP1 in EC patients MFM, metformin; EC, endometrial cancer; EEC, endometrioid endometrial cancer; NEEC, non-endometrioid endometrial cancer; IGF-1, insulin-like growth factor-1; IGFBP-1/7, insulin-like growth factor binding protein-1/7; rpS6, ribosomal protein S6; ERK1/2, extracellular signal-regulated kinase-1/2; AMPK, AMP-activated kinase; PCOS, polycystic ovary syndrome; Akt, protein kinase B; 4E-BP1, eukaryotic initiation factor 4E-binding protein-1; ER, estrogen receptor; PR, progesterone receptor; AEH, atypical endometrial hyperplasia; ACC, acetyl-CoA carboxylase; mTOR, mammalian target of rapamycin; PI3K, phosphoinositide 3-kinase; S6K1, S6 kinase 1. THE METABOLIC MICROENVIRONMENT AS A THERAPEUTIC TARGET IN ENDOMETRIAL CANCER Aside from lactate, Sousa and colleagues have described a similar two-compartment metabolic system in pancreatic ductal adenocarcinoma in which stroma-associated pancreatic stellate cells are stimulated by contact with pancreatic cancer cells to undergo autophagy and secrete primarily alanine which fuels the tricarboxylic acid (TCA) cycle and biosynthesis in the cancer cells themselves (133). In terms of clinical outcomes, a randomized study by Tabrizi examined the ability of metformin to reverse endometrial hyperplasia or disordered proliferative endometrium in patients with abnormal uterine bleeding compared to megestrol. Metformin was able to induce endometrial atrophy/restore endometrial histology in 95.5% of patients compared to 61.9% in the megestrol group. Signiﬁcantly, this study included two patients with low grade endometrial carcinomas (stage Ia) who received metformin. After 3 months of treatment, repeat biopsy showed conversion to atrophic endometrium (110). A study of 5 PCOS patients with early stage endometrial carcinoma showed that co-treatment with the oral contraceptive Diane-35 (cyproterone and ethinyl estradiol) and metformin for 6 months led to reversion to normal epithelia on repeat biopsy in all patients (113). This included three patients who had previously been on megestrol treatment for 3 months with documented progesterone resistance. Mitsuhashi also used a combination approach of metformin with medroxyprogesterone to induce remission in patients with atypical endometrial hyperplasia (AEH) or early stage endometrial cancer, and further studied the use of maintenance metformin to prevent relapse. Metformin treatment led to a relapse-free survival of 89% at 3 years, which was higher than their projected baseline of 52% (118). These studies were also notable for their stated goal of fertility preservation, an important consideration for some endometrial cancer patients. Larger trials are underway, including a phase 3 trial of metformin monotherapy as chemoprevention for endometrial cancer compared to placebo and lifestyle interventions in non-diabetic obese women (NCT01697566) and a phase 2/3 trial by the Gynecologic Oncology Group for advanced (stage III, IVA, IVB) or recurrent endometrial cancer that will compare the addition of metformin vs. placebo to combination paclitaxel/carboplatin as ﬁrst-line therapy (NCT02065687). Several phase 2 or earlier studies will utilize metformin in combination with hormonal treatments such as megestrol acetate (NCT01968317) or levonorgestrel (as an intrauterine device) for early stage endometrial cancer or complex atypical hyperplasia in young women with the goal of fertility preservation or contraindications to surgery (NCT02990728, NCT02035787, NCT01686126). THE METABOLIC MICROENVIRONMENT AS A THERAPEUTIC TARGET IN ENDOMETRIAL CANCER receptor after metformin treatment (114). Plasma measures of a hyperinsulinemic state, including insulin, IGF-1, glucose, and leptin were signiﬁcantly reduced post-metformin (111, 112, 116, 117). Furthermore, serum from metformin-treated patients showed decreased ability to stimulate DNA synthesis in cultured endometrial cancer cells (112), suggesting that systemic eﬀects of metformin also play a role in its antiproliferative activity. The role of glucose metabolism in endometrial cancer is also still being explored. Malignant tissues in general have been known to have high levels of glycolytic metabolism, even in the presence of oxygen, a phenomenon known as the Warburg eﬀect after its discoverer, Otto Warburg (120). One theory was that cancer cells are less dependent on oxidative phosphorylation, allowing them to survive in the often relatively hypoxic tumor microenvironment. However, in recent years, a more nuanced model has emerged for some tumor types, known as the reverse Warburg theory (121). This is based on the recognition of heterogeneity in tumor composition; malignant tissues are composed of cancer cells surrounded by diverse types of stromal cells and adipocytes, each of which make contributions to the tumor microenvironment (122, 123). Our research in breast and prostate cancer cell models among others have demonstrated that glycolysis occurs not in the tumor cells themselves, but in the surrounding stromal cells (124– 127). This relationship is thought to occur via oxidative stress in the cancer associated stroma (CAS), driven by tumor cell generation of reactive oxygen species and stromal cell loss of caveolin-1 (CAV1), an inhibitor of nitric oxide production (124– 129). CAS cells undergo metabolic reprogramming associated with mitophagy (selective degradation of mitochondria by autophagy) and cell autophagy, and they generate high levels of energy-rich metabolites (including lactate and ketones) through glycolysis, which is then shuttled to cancer cells as substrates for oxidative phosphorylation. We have reported that lactate shuttling to tumor epithelial cells is dependent on transporters of the monocarboxylate transporter (MCT) family, with MCT4 being important for lactate eﬄux from stromal cells and MCT1 for lactate uptake in tumor epithelial cells in a breast cancer cell co-culture system (130). Others have observed similar lactate shuttling in prostate cancer and sarcoma models and have reported that lactate upload promotes tumor cell proliferation and angiogenesis (131, 132). METFORMIN IN PRESURGICAL AND OTHER CLINICAL TRIALS IN ENDOMETRIAL CANCER MFM, metformin; EC, endometrial cancer; EEC, endometrioid endometrial cancer; NEEC, non-endometrioid endometrial cancer; IGF-1, insulin-like growth factor-1; IGFBP-1/7, insulin-like growth factor binding protein-1/7; rpS ribosomal protein S6; ERK1/2, extracellular signal-regulated kinase-1/2; AMPK, AMP-activated kinase; PCOS, polycystic ovary syndrome; Akt, protein kinase B; 4E-BP1, eukaryotic initiation factor 4E-binding protein-1; ER, estroge receptor; PR, progesterone receptor; AEH, atypical endometrial hyperplasia; ACC, acetyl-CoA carboxylase; mTOR, mammalian target of rapamycin; PI3K, phosphoinositide 3-kinase; S6K1, S6 kinase 1. 8 Lee et al. Metformin in Endometrial Cancer August 2018 \| Volume 8 \| Article 341 CONCLUSIONS The existence of such symbiotic metabolic reprogramming has not yet been investigated closely in endometrial cancer, but supportive evidence comes from a study by Latif et al. that showed diﬀerential histologic localization for MCT1 (tumor) vs. MCT4 (stroma) for some though not all endometrial cancer samples analyzed (134). High MCT1 expression was also a poor prognostic indicator for recurrence-free, cancer- free, and overall survival in the same study. Moreover, Zhao studied endometrial stromal-epithelial cell interactions in a non-cancer primary cell culture model and showed that epithelial cell proliferation and migration was enhanced when cultured with conditioned media from CAV1 depleted stromal cells (135). A phase 2 study currently underway at our institution utilizes a combination of metformin and doxycycline for the treatment of breast and uterine cancers, with outcomes including the measurement of biomarkers of tumor-stromal metabolic compartmentalization, such as stromal CAV1 and MCT4 and tumor MCT1 (NCT02874430). This will provide new insight into the eﬀects of metformin treatment on the metabolic microenvironment in endometrial cancer. Endometrial cancer is a disease with few eﬀective treatments for advanced and metastatic disease. In addition, the need for fertility-sparing options for patients with early stage disease means there is a need for more primary or adjunctive treatment approaches. A large body of evidence links endometrial cancer incidence to metabolic conditions such as obesity and hyperglycemic states. Increasing rates of the latter has been mirrored by a rise in the former, particularly in developing countries, highlighting the need for a better understanding of the contribution of the metabolic microenvironment to endometrial cancer tumorigenesis. There is substantial evidence that the mechanisms of nutrient utilization and synthesis are signiﬁcantly dysregulated in malignancy on both an intracellular and intercellular level. Models such as the reverse Warburg eﬀect especially emphasize the importance of considering the interplay between cancer epithelial cells and their surrounding stroma. Dysregulation of metabolic pathways may represent adaptations that facilitate survival and proliferation in some scenarios (e.g., the idea of the parasitic cancer cell), but can also become liabilities for cancer cells particularly in times of nutrient or energy deprivation. Drugs such as metformin may be uniquely poised to exploit these defects, possibly in conjunction with other therapies that target glucose utilization. Indeed, preclinical and early clinical studies have shown promise for metformin as an adjunctive treatment for endometrial cancer, with eﬀects on both cancer-speciﬁc as well as patient-speciﬁc metrics. THE METABOLIC MICROENVIRONMENT AS A THERAPEUTIC TARGET IN ENDOMETRIAL CANCER Also ongoing are a phase 2 study of metformin combined with letrozole and everolimus for advanced or recurrent endometrial cancer (NCT01797523) and a phase 1/2 study of metformin plus metronomic cyclophosphamide and olaparib for advanced or recurrent endometrial cancer (NCT02755844). These eﬀorts will give valuable insight into the preventative and therapeutic value of metformin in endometrial malignancy. In this model, metformin treatment may play an important part in disrupting cancer cell metabolism via its direct inhibition of mitochondrial respiration in the cancer cells. In agreement with this, our previous results suggest that metformin treatment of cultured breast cancer cells inhibits their ability to induce loss of CAV1 (a marker of tumor-stromal metabolic coupling) in co- cultured ﬁbroblasts (126). We have also observed that a short course of metformin was also able to increase stromal CAV1 expression in vivo in patients with head and neck squamous cell carcinoma in a presurgical window of opportunity trial (106). August 2018 \| Volume 8 \| Article 341 Frontiers in Oncology \| www.frontiersin.org 9 Metformin in Endometrial Cancer Lee et al. CONCLUSIONS Further study is needed to elucidate the role of metformin as a therapy for endometrial cancer, and several clinical trials are underway that will greatly expand our understanding of its potential beneﬁts. The Reverse Warburg framework also opens up the possibility of multi-targeted therapies that simultaneously act on aberrant glucose metabolism in both cancer and stromal cells, such as metformin combined with inhibitors of glycolysis, autophagy, or transport of lactate and other energetic substrates. Combined metformin and glycolytic inhibitors have been utilized in xenograft models of breast cancer, gastric cancer, and glioblastoma with synergistic inhibition of tumor growth or prolongation of survival occurring at doses where each agent is ineﬀective alone (136, 137). Such approaches would exploit the vulnerabilities of tumor- stroma metabolic reprogramming that typically allow for cancer cell survival under a variety of energetic conditions. Overall, the metabolic microenvironment of endometrial cancer represents a promising therapeutic target, one which metformin and other biguanides may be uniquely poised to act upon. 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Cell Cycle (2010) 9:1960–71. doi: 10.4161/cc.9.10.11601 Conﬂict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. 129. Ko YH, Lin Z, Flomenberg N, Pestell RG, Howell A, Sotgia F, et al. Glutamine fuels a vicious cycle of autophagy in the tumor stroma and oxidative mitochondrial metabolism in epithelial cancer cells: implications for preventing chemotherapy resistance. Cancer Biol Ther. (2011) 12:1085–97. doi: 10.4161/cbt.12.12.18671 Copyright © 2018 Lee, Martinez-Outschoorn, Schilder, Kim, Richard, Rosenblum and Johnson. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 130. Whitaker-Menezes D, Martinez-Outschoorn UE, Lin Z, Ertel A, Flomenberg N, Witkiewicz AK, et al. Evidence for a stromal-epithelial “lactate shuttle” in August 2018 \| Volume 8 \| Article 341 Frontiers in Oncology \| www.frontiersin.org 14
https://openalex.org/W4213027995	https://journals.dut.ac.za/index.php/ajims/article/download/967/735	English	null	My Trepidation: Personal Ethical Pitfalls and Dilemmas in the Scholarship of Teaching and Learning	African journal of inter-multidisciplinary studies	2,021	cc-by-sa	8,434	My Trepidation: Personal Ethical Pitfalls and Dilemmas in the Scholarship of Teaching and Learning Berrington Ntombela University of Limpopo Berrington Ntombela University of Limpopo berrington.ntombela@ul.ac.za DOI: https://doi.org/10.51415/ajims.v3i1.967 African Journal of Inter/Multidisciplinary Studies Volume 3 (2021a Special Issue), 13-25 My Trepidation: Personal Ethical Pitfalls and Dilemmas in the Scholarship of Teaching and Learning African Journal of Inter/Multidisciplinary Studies Volume 3 (2021a Special Issue), 13-25 My Trepidation: Personal Ethical Pitfalls and Dilemmas in the Scholarship of Teaching and Learning African Journal of Inter/Multidisciplinary Studies Volume 3 (2021a Special Issue), 13-25 African Journal of Inter/Multidisciplinary Studies Volume 3 (2021a Special Issue), 13-25 My Trepidation: Personal Ethical Pitfalls and Dilemmas in the Scholarship of Teaching and Learning Abstract Located within the broad framework of critical approach and critical pedagogy, this paper reflects on personal experiences of being caught within the complexities of research ethics in the scholarship of teaching and learning. The paper presents multiplicities in the interpretation and application of ethical procedures in the drive to maintain rigour and transformation in teaching and learning research. Whilst advocating for adherence to research ethics, the paper problematises resistance to change often mounted by those in power in the name of ethics. By implication, the paper exposes the technocratic utility of research ethics at the expense of emancipation and at the same time argues for contextual interpretation and application of ethical procedures for the emancipation of the marginalised. The paper is conceptualised and presented in the tradition of autoethnographic thick description. The findings reveal differences of opinion about the adopted ethical procedures. Whilst on the one hand, many participants identify with the ethical spirit adopted by the researcher, on the other hand, some participants rejected the ethical stance of the researcher. The conclusions suggest that the adoption of covert ethical approaches coupled with critical pedagogy in the context of the scholarship of teaching and learning are most likely to attract pitfalls in research ethics. Keywords: research ethics; critical approach; critical pedagogy; scholarship of teaching and learning Research Ethics According to the Policy and Procedures on Research Ethics of the University of Zulula Research ethics refers to the principles and practices that guide the ethical conduct of research. These should embody respect for the rights of others who are directly or indirectly affected by the research. Such rights include rights of privacy and confidentiality, protection from harm, giving informed consent, access to information pre- and post-research and due acknowledgement. Ethical conduct in research also includes the avoidance of inflicting animal suffering of any kind and protection of the environment. Scott and Morrison (2007: 87) identified three forms of ethical approaches: covert, open autocratic and open democratic. By covert approach, they mean that the researcher conceals the nature of his/her activities from the participants. This is to avoid participants from behaving in a particular way once they are aware of the nature of the research (Scott and Usher 2004: 129). In this approach, participants could be said to behave in the most natural way as they would not be influenced by what the researcher is looking for. In fact, the researcher could be one of the participants. One example that captures such an approach, is presented by Yancey (2014: 77-78) where he discusses Welch’s research among a fundamentalist group of the Baptist church. Welch did not reveal her project until she had collected enough data to author a book. Because she had become one of them, she was well- received when she finally revealed her identity and her reason for joining the congregation. However, the overriding desire for researchers is to obtain informed consent (Silverman 2011: 93), in which case, the researcher would be operating on either an open autocratic or an open democratic approach. Scott and Morrison (2007: 88) argue that the researcher would want to protect the participants by unilaterally deciding on how to conceal certain identifiers, one of which would include the setting of the research project. In other words, the researcher identifies what could potentially harm the participants and, sometimes through deception, hides that information. This represents the open autocratic approach. The open democratic approach represents negotiating and renegotiating with participants about what information and perhaps how the information should be reported (see for instance Ntombela 2020: 20). Introduction Higher education teaching and learning is built on the tradition of research. In South Africa, universities are categorised into universities of technology, which are the former Technikons that trained students for the job market as opposed to theoretical orientation offered by the traditional universities (De Jager, Monapula-Mapasela and Ntshoe 2017:244); comprehensive universities which are supposed to offer a combination of Technikon-type of qualifications and traditional university qualifications and lastly, and arguably the most prestigious category of universities, the research universities which have all the characteristics of traditional universities as research institutions (De Jager et al. 2017: 244). Notwithstanding these various categories, it is important to highlight that the different categories of universities are generally built on and have in common three ‘legs’ that form the core business of the institution of higher learning which are Teaching and Learning, Research, and Community Engagement (Darmalaksana, Ramdhani, Cahyana and Amin 2018:788). Whilst only the research leg may appear to focus on research, the two other legs are also built on research tradition. For instance, Teaching and Learning has the scholarship of teaching and learning which represents the research aspect; similarly, Community Engagement has scholarship of community engagement for the same purpose. earch is the foundation upon which the institutions of higher learning build their Therefore, research is the foundation upon which the institutions of higher learni 13 activities. This is because universities are taken as epitomes of knowledge construction. Since epistemological issues are admittedly a contested terrain, there is a need to validate the constructed knowledge in terms of the methodological procedures and ethics. Ethical issues are more critical in research that deals with human and animal subjects. This paper deals with some ethical issues in the context of the scholarship of teaching and learning. Obviously, the principal players in the scholarship of teaching and learning are the human subjects – the teachers and the learners. Although issues of ethics in such a context may seem straightforward, there are pitfalls when it comes to research that seeks to emancipate the vulnerable and transform the institutions. This is because change by itself is prone to resistance. In this paper, such resistance, as it shall become apparent later, is mounted on the grounds of ethics. Critical Approach and Critical Pedagogy The critical approach to research is consistent with critical theory (with small letters ‘c’ and ‘t’) and Critical Theory (with capital ‘C’ and ‘T’). The latter refers to the theory that was founded by the Frankfurt School, which could be said to be a reaction to the ‘meaning of Enlightenment in the [context] of Marxism and Freudianism’ (Scott and Usher 2004: 23). The former constitutes a theory that critiques the positivist view of research. In that respect, critical theory would constitute elements of Critical Theory but will also draw from other approaches that are critical not only of positivism but also of ‘scientism and technical-rationality’ (Scott and Usher 2004: 24). Emancipation is foregrounded in many aspects of critical theory; among its tenets are dialectical reflexivity and praxis with the educational values of empowerment and transformative form of life (Scott and Morrison 2007: 49; Scott and Usher 2004: 35). Action research has been included as part of critical approach as it critiques practices and by its dependence on action, influences curricular changes and institutional traditions (Scott and Usher 2004: 35). Notably, action research fuses the notion of ethics and epistemology and further acknowledges the fact that practitioners are knowledgeable about their actions in a manner that they can analyse and reflect on them (Scott and Usher 2004: 36). Similarly, a feminist approach is part of critical theory on account of its emancipatory and transformative agenda (Baum 2017: 83). In this case, the dominant discourses embedded on patriarchy and therefore oppressive, are interrogated with the principal aim of dismantling them and thus emancipating those who are oppressed by them. The feminist approach seeks to critique and disrupt patriarchal dominance in order to facilitate social inclusion and transformation (Baum 2017: 97). Linked to the critical approach theory is critical pedagogy. Writing in the context of Africa, Bunting (2019: 142) posits that critical pedagogy ‘is substantiated and informed by an emancipatory purpose intended to foster the philosophical disposition … to realise … the objectives and the social, political and economic transformation of Africa.’ According to Abdollhzadah and Narafshan (2016: 198), there are three tenets of critical pedagogy: reflection upon the individual’s culture; development of voice and transforming of society. They argue that these tenets should find themselves into the school curriculum as part of the adoption of critical pedagogy. Critical Approach and Critical Pedagogy Earlier, Giroux (1985: 40) suggested that critical pedagogy would allow teachers to be transformative intellectuals and develop schools into democratic public spheres. The critical approach theory and critical pedagogy are intertwined in that they both have emancipatory ends. The critical approach theory on the one hand, finds its expression in research whilst critical pedagogy on the other hand is realised in praxis. This means criticality is at the centre of intellectual work, be it the passing or construction of knowledge. In that respect, reflexivity becomes central as researchers and lecturers reflect on their praxis and epistemological orientations (Ntombela 2017: 62-63). Research Ethics The open democratic approach underscores the interconnectedness of epistemology and ethics (Scott and Morrison 2007: 88; Scott and Usher 2004: 134) where through the process of negotiating what is to be collected as data and how to report it, the participants, and the researchers’ become co-creators of knowledge. 14 Autoethnography Autoethnography is research that looks at and analyses individual experiences with an aim of understanding cultural experiences (Ellis, Adams and Bochner 2010: par 1). Ellis et al. (2010: par 3) regard autoethnography as ‘one of the approaches that acknowledges and accommodates subjectivity, emotionality, and the researcher’s influence on research, rather than hiding from these matters or assuming they don’t exist.’ It is important to note that the protagonist in autoethnography is the ‘self’ of the researcher. This research is therefore framed within the autoethnographic thick description which I shall now turn to. The participants’ names have been coded in keeping with the ethical consideration of anonymity. Any direct reference to identifiable institutions has been altered for the sake of confidentiality. I joined the historically black university in February 2013. It was wonderful to be in the institution that contributed to my academic stature. Although I was thrilled to be the member of the academic department, I had a slight sense of uneasiness because some of my colleagues were my former lecturers. I did not find it immediately easy to forget that I had always considered them my superiors and then I had to relate with them at a collegial level. I must say that this relation was easy with some of my former lecturers but not with others. Some made it a point that I recognise my subordinate status and not rise to the level where they already were. This sometimes happened not only to me but also to my other colleagues who were at the same level with me. Unfortunately, because of my independence of mind, some of my contributions were publicly bastardised but I endured. As I established my footing in the department, I began to understand some of the things I went through as an undergraduate student. I also realised that little had changed in terms of how the department was viewed by either the current or the former students – it was still very much viewed as a ‘no go area’ and an obstruction to student attainment. Like some of my colleagues, I was bothered by this image. I had always wished to contribute to my fellow Africans’ wellbeing and to humanity in general. I then started to interrogate some of the challenges that were responsible for the image the department was projecting. Scholarship of Teaching and Learning It should be acknowledged that the scholarship of teaching and learning is often misunderstood and misused; however, Mathany, Clow and Aspenlieder (2017: 6) define it as hypothesis-driven research that involves one’s critical reflection about own practices in order ‘to understand the relationship between teaching strategies and learning outcomes. The other definition is that it is ‘a systematic, evidence-based way’ of evaluating as well as doing research on one’s teaching (Mathany et al., 2017: 6). De Jager et al. (2017: 245) add that ‘[a]n activity has to meet … these conditions in order to be considered an SoTL [scholarship of teaching and learning] activity’: systematic study of some aspects of teacher’s teaching; must be based on validated criteria of scholarship; making transparent how teaching makes learning possible and making outcomes of such an activity publicly available. More importantly is the place of reflection in the scholarship of teaching and learning which must cover 15 content of what is taught, processes of how such content is taught and why things are done the way they are (i.e., why teaching proceeds the way it does) (De Jager et al. 2017: 245). The common theme that seems to run through critical theory, critical pedagogy and the scholarship of teaching and learning is reflection. The practitioners in all instances must reflect upon their own practices. In critical theory and critical pedagogy, reflection happens in order to adopt an emancipatory ethos. In the scholarship of teaching and learning it is necessary in order to improve one’s own practices. This seems to overlap with tenets of action research located within critical theory. Autoethnography There were different issues that I began to research with an aim of contributing the necessary change to the department. My findings were often communicated at various local conferences in the presence of my colleagues. Most of my research had been informed by my following the critical approach, which I began to see made some of my colleagues uncomfortable. My adoption of this approach was mainly to highlight the issues at hand. In May 2016, in the 1st Teaching and Learning conference, where the central issue was assessment, I presented a paper that investigated linguistic imperialism. Even though the presentation was well-received, I did not develop the paper further. In the following year, the dean of the faculty, probably captivated by the abstract of that paper, located a journal where he felt the paper could be published. I welcomed the idea because it was also going to help me effectively communicate my findings to my colleagues. I anticipated that my findings would kick-start debates and possible adjustments in the departmental assessment scheme, and I almost achieved that. In April 2018, the article was published by an accredited peer-reviewed journal. 16 The article was also circulated by the then dean of the faculty, which was a common practice that encouraged knowledge sharing among academics. It was immediately met with congratulatory messages, but my euphoria was to be short-lived as there were a few negative messages. Most of the initial messages were positive. For example: Congrats to 214. Way to go for the department in 2018! (11 – 8 May) And, Congratulations 214 on a very interesting article! Especially since the article is based on practice within the department, I think we should have a presentation and discussion on it soon – 214? 11? (163 – 8 May) Below is an example of a negative response: I think it would have been polite for you to advise us you were writing about us, 214. And even, perhaps have invited responses. (138 – 8 May) To this message I responded: Yes, I did wrestle with that part for the greater part of the writing and ultimately ended up going it solo. I am always struggling with ethical issues when writing articles of similar nature and try by all means to be on the objective side as possible, but I must admit that it is not always that easy. Autoethnography I did discuss ethical considerations with journal editors, and they gave me a green light. In my mind I thought I would appreciate responses for the work already out there, but I’m aware that that may not have been ideal for most. Whilst I’m still very much open to responses, the article reflects my personal opinion which has not been informed by any other’s input and that’s how I wanted it to be. The second round might be collaborative where the interpretation takes various points of view. Thank you all and apologies to those who feel offended by the article. To this message I responded: Yes, I did wrestle with that part for the greater part of the writing and ultimately ended up going it solo. I am always struggling with ethical issues when writing articles of similar nature and try by all means to be on the objective side as possible, but I must admit that it is not always that easy. I did discuss ethical considerations with journal editors, and they gave me a green light. In my mind I thought I would appreciate responses for the work already out there, but I’m aware that that may not have been ideal for most. Whilst I’m still very much open to responses, the article reflects my personal opinion which has not been informed by any other’s input and that’s how I wanted it to be. The second round might be collaborative where the interpretation takes various points of view. Thank you all and apologies to those who feel offended by the article. f Thank you all and apologies to those who feel offended by the article. And then there was a constructive message suggesting a way-forward to responding to the issues raised in the article: And then there was a constructive message suggesting a way-forward to responding to the issues raised in the article: Perhaps we (as a department) will be prevailed upon to discuss some of the issues underpinning the arguments made by 214. For me, one of the important issues include: • To what extent do we ‘penalise’ perceived language deficiencies? • What ‘rules’ will influence us? • What ‘rules’ will influence us? My suggestion is that we hold a series of meetings to thrash these issues. Autoethnography (11 – 8 May) The other message was positive but raised the same concern highlighted by 138: My suggestion is that we hold a series of meetings to thrash these issues. (11 – 8 May) The other message was positive but raised the same concern highlighted by 138: This is in fact an issue we do discuss. Formalizing it too much is impractical because of the real fluidity of the language/content boundary. We indeed ought to keep the conversation going, and current, and a departmental policy document might be an idea – although with the caveat about fluidity. This is in fact an issue we do discuss. Formalizing it too much is impractical because of the real fluidity of the language/content boundary. We indeed ought to keep the conversation going, and current, and a departmental policy document might be an idea – although with the caveat about fluidity. But I, too am I [sic] interested in the ethical underpinnings of this paper. Were consent forms issued? Did it – or its underlying project – go through the Research Committee? (31 – 8 May) Whilst the drive so far seemed to have been that of engaging with the contents of the article academically by seeing the extent to which we could converse around the arguments and perhaps be able to inform our practice, some colleagues took a different turn that amounted to personal attacks which angered other colleagues in the department. The first of such comments went like this: 17 I suggest that we read the article before sending out unqualified congratulations … (138 – 8 May) [emphasis in the original] This comment was countered by some colleagues: I think your sarcasm is unwarranted and offensive. Publications in accredited journals are always cause for congratulations, surely? We can and often do congratulate our colleagues before we have read their work – as you yourself congratulated 214 earlier today, seemingly before reading his article. However, my and other’s reading of it has brought its contents into our conversation. To suggest that colleagues who have subsequently chosen to send unqualified congratulations have done so in a state of ignorance or naivety – my reading of the implications of your comment below – seems to me to be an intellectual and personal slight that I find hurtful. I think you should send an apology. Autoethnography (163 – 8 May) [emphasis in the original] These kinds of exchanges were never intended and even this one below seems to deviate from the spirit earlier taken by other colleagues: These kinds of exchanges were never intended and even this one below seems to deviate from the spirit earlier taken by other colleagues: I have read 214’s article and am surprised that while participating in a departmental process of selecting potential students, members of the department and the concerned students became subjects of a research. Apart from not complying with ethical issues requiring the researcher to get the informed consent of his/her subjects, the article makes many incredible assumptions and innuendos about archival sources/foundations of the lecturers impacting on their interpretation of pedagogy. So, a Zimbabwean trained academic, even when two of his degrees are obtained from South African universities, is by some inexplicit definition/criteria - British oriented and a peddler of British linguistic imperialism? How do we exercise our academic functions without feeling that we are being pried upon and preyed on, or being academically ambushed? Would some of us be wrong if we refuse to participate in future activities that produce literature (Scoresheets) that become sources of research. That is why ethical considerations in research are essential, and in my view of this violation, 138’s point is valid. I suppose 163 is aware of the informed consent requirement when using human subjects for research purposes. There is nothing sarcastic or hypocritical about making a point in defence of research ethics, and in defence of the integrity of the department, whose skirts have now been blown above our heads. (1213 – 8 May) There was an attempt to bring back the rationale for the article and to approach it from the positive spirit in which it was written. The comment that follows is a case in point: I really like 31’s idea of formulating a departmental policy on some of these things. And I actually think this will help when we start marking and moderating. Even though we need not aim to produce formulate [sic] a concrete policy immediately, I would like to suggest that we consider having those discussions right after sending semester marks in. Any seconder, please? (11 – 10 May) The attempt was effectively squashed once and for all. The writing was on the wall and no further comment on the article was to be made after this one: I appreciate your trying to ameliorate this debate, but perhaps pouring oil on it is likely to muddy the waters … if you’ll forgive the mixed metaphor! I think the ethical issues raised by 214’s article and how it was written are rather more urgent than a discussion of assessment practices, which I think is what you’re proposing. I hope 131 will call a meeting in which we can all voice our opinions. In the meanwhile, here’s why I think this is urgent and concerns us all as members of the department. So, colleagues: , g 1. The processes described in the article were for a specific purpose, namely, to select applicants to a programme. The assessment undertaken was by definition normative not developmental. It is not representative of assessments made by students already admitted within the department. 1. The processes described in the article were for a specific purpose, namely, to select applicants to a programme. The assessment undertaken was by definition normative not developmental. It is not representative of assessments made by students already admitted within the department. 2. The processes described were internal to the department, which all of us took for granted were confidential. Comments we made on applicants were made on the understanding of privacy. 214 participated in these processes and made no attempt at the time to question or contest them. 3. There was no informed consent. As colleagues we were not asked if we were willing to have our information turned into an ‘archive for research’ – or to be published. Applicants to the department were not asked this either. 3. There was no informed consent. As colleagues we were not asked if we were willing to have our information turned into an ‘archive for research’ – or to be published. There was an attempt to bring back the rationale for the article and to approach it from the positive spirit in which it was written. The comment that follows is a case in point: There was an attempt to bring back the rationale for the article and to approach it from the positive spirit in which it was written. The comment that follows is a case in point: Others can take up the issue about whether 214’s paper may have infringed upon any institutional rules in terms of committees, ethical clearance etc. But I think it would be very naïve to assume that such bureaucratic processes are merely there to fulfil their ostensible function – to protect against ethical violations in research – and do not also function as machinery of power and gatekeeping, to police and discipline knowledge production. Nonetheless, I am not disputing their necessity. However, in this case, I feel that the research is ethical in its spirit, whether or not the letter of the law has been breached in any way. The actual comments published are innocuous in themselves and I’m sure similar ones have been made in assessment by academics in the field from across the globe. I can’t see that there is anything there that would embarrass any individual personally, even if he or she could be identified. What the article does is highlight the way a particular kind of assessment functions in a collegial setting and I think it makes an important contribution in that. As scholars we are free to challenge 214’s conclusions but we should first engage with them on their merits. 18 It is brave and tricky to research one’s own academic environment – yet such research is vital in order for our work to engage with social challenges and become a force for positive change. 214’s careful and responsible use of anthropological techniques of participant observation within our department seems to me to be within the spirit of Bourdieu’s Homo Academicus – about which there is a nice summary/overview by Jessica Poulin at the following link : https://newanthro.wordpress.com/2015/03/18/pierre-bourdieus-homo-academicus- objectifying-the-academic-object/ Just my personal views and I look forward to further discussion and questions raised by the article, such as those that 11 has highlighted. (163 – 8 May) There was another attempt to resurrect the discussions so that the department would benefit from the arguments raised in the article: Thanks prof. I reckon this will help us set a productive tone for discussions on some of the issues that we ought not to gloss over. There was an attempt to bring back the rationale for the article and to approach it from the positive spirit in which it was written. The comment that follows is a case in point: Applicants to the department were not asked this either. p 4. Public disclosure, without consultation, is a drastic breach of trust. How can we work co- operatively and collegially with one another if we cannot trust one another, and if we do not share respect for one another’s privacy? p 4. Public disclosure, without consultation, is a drastic breach of trust. How can we work co- operatively and collegially with one another if we cannot trust one another, and if we do not share respect for one another’s privacy? 5. The information that was drawn on was not set up for research purposes, nor was it designed for public dissemination. This information was generated by a colleague whose work was not credited or acknowledged. Student records – and documents to do with departmental processes – are the intellectual property of the department and the university, and permission should be sought to use them. 19 6. There was no clear research methodology. The ethnic and national categorisation seem irrelevant to the central argument, that criterion-based assessment is preferable to norm- referenced assessment. 7. These categorisations clearly identify particular applicants, some of whom have become our students. These categorisations are distasteful and reductive. In what ways is it useful for research purposes to describe us as ‘Zambian black male’, ‘Zimbabwean black male’, ‘South African Indian male, ‘South African coloured male’, ‘Nigerian black female’, ‘South African white male’ etc.? Is this really how we see each other? male etc.? Is this really how we see each other? 8. The journal is online and open access. And the academy to which we belong is small. So, this disclosure makes very public the processes and the persons who’ve been turned into ‘subjects’ in the study. This may well impact on prospective employment or study opportunities. In a nutshell, the research is not ethical – and this makes it reflect badly on our university, let alone our faculty and our department. (138 – 10 May) In a nutshell, the research is not ethical – and this makes it reflect badly on our university, let alone our faculty and our department. (138 – 10 May) After this, there was no further engagement about this article on the virtual platform. Under the dictates of the last respondent, the meeting was called in the department to have everyone voice their feelings. There was an attempt to bring back the rationale for the article and to approach it from the positive spirit in which it was written. The comment that follows is a case in point: The matter was further escalated to the chairperson of the University Ethics Committee. Some colleagues in the department were not happy about this arrangement and had wanted to write a counter report to the chairperson of the Ethics Committee but, not to create further rift among colleagues, this was not pursued. There was also concern that the colleague who reported the matter acted as the spokesperson of the department on the matter on which no mandate was given. The complaining colleague continued to use plural pronouns as if representing the concerns of other members and not personal ones. The reporter then indicated that the decision to report was based on the provisions given to whistle-blowers in which no one owed the department any explanation. I decided to retract the paper from the journal. I even deleted the article which I had uploaded on ResearchGate. The matter unfortunately seemed not to go away easily. Although the article was removed from the journal to which it was initially sent, it still appeared on another platform which indexed the journal. Further complaints were sent to the chairperson of the University Ethics Committee, and I was asked to make sure that the article was removed from the Internet. I wrote to the editor of the journal again and asked if it would be possible to have the article removed from that indexer. Although I would not want to comment on the timing at which both complaints were launched with the chairperson of the Ethics Committee, the coincidence left a lot to be desired. I believe that as a department, we lost an opportunity to refine some of our crucial practices that impact heavily on the department’s throughput and success rate. To date, there has not been any open talk about assessment in the department. Any mention of that subject seems to be an anathema and seems to carry the same amount of accursed omen as my article. It is unfortunate that what should be for the benefit of academia at large is safeguarded as a sacred space that must be perpetually kept away from the public. For the department, the status quo must be maintained at all costs! It is this very status quo that I initially wanted to challenge, because to me, it had long been an inglorious one. In terms of performance, our department has constantly remained in the red. There was an attempt to bring back the rationale for the article and to approach it from the positive spirit in which it was written. The comment that follows is a case in point: And I think maybe that is the reason why…like…they are using their own formulas that I do not even know. (Gazu 2017: XV) The debate was going to help us discuss some of these issues and would have afforded us the opportunity to improve the department’s assessment practices, unfortunately it was not to be so. It was even more unfortunate that those who opposed such debates happened to be the most senior members in the faculty who used their seniority to hinder progress. Although I of my own accord decided to retract the article, it does concern me that only the opinion and voice of the senior member who took the matter up with the chairperson of the Ethics Committee was taken. There was no consideration of the voices of other members. It is believed that the department at large would have benefitted from deliberations on matters of this nature. And I feel that it is this state of affairs that stands in the way of change and transformation in the department. There was an attempt to bring back the rationale for the article and to approach it from the positive spirit in which it was written. The comment that follows is a case in point: The way in which some colleagues interpreted my article indicated that assessment has a lot to contribute to the improvement of our performance. In order to understand how students, feel about assessment, I will quote a few interview responses from a study conducted in the department on a similar issue. I must also highlight that the same former colleague was denied permission to use some departmental records such as an external examiner’s report in his study. Here is what his students said as extracted from his thesis: 20 Interviewer: Ok. Are there any other experiences surrounding the study of […]? What other factors do you think we should be aware of? Anything. Interviewer: Ok. Are there any other experiences surrounding the study of […]? What other factors do you think we should be aware of? Anything. Respondent: Regarding performance, I do not know why – I’m not saying I’m intelligent, but then I don’t know why we are…I mean…I am getting low mark when it comes to […] because… I think maybe the way they mark, the way they… I mean…ja…the way they give us a lot of assignments or a lot of works to do. That also like affects the way…the way I perform. Interviewer: So, if you compare your marks in the other modules and your marks in […], would you say there is a vast difference? Respondent: Of course. You don’t wanna know. I mean if I can refer you to my last semester modules, I got two distinctions, and one merit and a pass…a pass…when I say a pass I mean a pass for […]. Amandle’ ndoda [Man’s strength], that’s what they say. Interviewer: Do you feel that as a student you have an understanding of how the marking process is happening? Respondent: I was concerned why I’m getting – I mean low marks, and I decided to go to Dr 712 and to consult and ask him how they mark. And he showed a long process. And I think maybe that is the reason why…like…they are using their own formulas that I do not even know. (Gazu 2017: XV) Respondent: I was concerned why I’m getting – I mean low marks, and I decided to go to Dr 712 and to consult and ask him how they mark. And he showed a long process. Discussion First and foremost, it is important to locate these experiences within the broader framework of teaching and learning in higher education. Whilst teaching and learning might appear as apolitical, instrumental and a simple cognitive process, critical pedagogy informs us that teaching and learning is never neutral, but is personal, cultural and situated within ideological and negotiated knowledge (Canagarajah 1999:15-16). Therefore, traversing a teaching and learning terrain is similar to negotiating a political journey where it is easy to be met with opponents along the way. The adoption of critical pedagogy by the researcher immediately exposed the differing orientations of colleagues. Similarly, it might seem that whilst the scholarship of teaching and learning expects practitioners to reflect on their practices, the notion of reflexivity is a ‘no go area’ for some academics. This is seen in the comment made by 138 that departmental practices are confidential and by implication should not be subjected to scholarly interrogation which might be perceived as negative. It is disturbing that it is regarded as unethical if departmental practices are subjected to interrogation in the tradition of academic rigour. However, it is important to note that not all colleagues held the same notion of the sanctity of departmental practices. This is shown by the eagerness of some colleagues to engage with the 21 findings of the research by attempting to influence some change that is implied by the article. But the fact that higher education is filled with power dynamics where, among other things, seniority, which often oppresses, is used to maintain the status quo, critical pedagogy becomes a viable option in order to disrupt such resistance. The negative reactions to the article were quite revealing about institutional sub-cultures. It showed that some ideological orientations were resistant to different voices. For instance, the unhappiness about the contents of the article invoked discourses of ethics and further sought to dictate how representations ought to have been carried out. The University of Zululand (2013) Research and Ethics policy indicates that differences of opinion in interpretive matters of research do not constitute ethical violation. Invoking ethical violations in such matters should therefore be understood as a mechanism for blocking the necessary change and transformation which is envisaged in any critical approach. Nonetheless, the question of informed consent is a valid one within open autocratic and open democratic approaches. Discussion This requirement was invoked by the colleague who was not happy about the arguments raised in the paper. Without doubt, had the consent been sought, it would have been highly unlikely that it would have been given. Thus, opting for the covert approach was one way of interacting with a ‘difficult’ decision to publish or not to publish. Other colleagues did not see any ethical violation that informed consent was never sought; on the contrary, as one colleague responded, the research demonstrated another way of critiquing the system that did not always work in the best interest of free expression. This was further demonstrated by the willingness of some colleagues to interact with the findings in a manner that would advance and propose changes implied in the paper. Unfortunately, the dissenting voices proved too powerful for a positive response. The decision to retract the paper from the journal and all the other internet platforms was a way of fostering collegiality. It was also another way of acknowledging that those who did not give consent might have been genuinely hurt. The initial reaction by 138 was that some input could have been sought. This would also border on epistemological assumption that I was not in a position to write with authority on the matter that concerned the department. The authority to do so might either lie in the collective or with others. That I had projected my voice solo on the matter regarded as sacred was almost tantamount to sacrilege. If this be the case, then the great offence might have been the feelings of exclusion. Even the fact that standard protocols of anonymity and confidentiality in the way the information was presented were followed such that none was identifiable, informed consent remained the biggest violation. This was even though none could identify information that pointed to oneself. This experience revealed to the researcher a few realities in the ethics of research. One of them is that ethical decisions are not as straightforward as they might seem. Even though the covert approach was adopted, not all participants approved such an approach. Another reality is that the taken-for- granted ethical decisions sometimes have dire consequences. In this case, the envisaged outcome of having colleagues engage in a conversation on the subject that was regarded as being of common interest and importance turned out to be a political contest. Conclusion This paper set out to highlight the pitfalls that surround ethical decisions in the context of the scholarship of teaching and learning. The experiences of the researcher are presented in the tradition of autoethnographic thick description. These experiences highlight critical pedagogy in teaching and learning adopted by the researcher, which resonates with emancipation. Alongside critical pedagogy is critical approach to research which also interrogates power dynamics and oppressive regimes with an aim of emancipating the marginalised. The experiences of the researcher confirmed that power dynamics do complicate the dissemination of research findings within the scholarship of teaching and learning. Rather than ethics being used to safeguard and protect the vulnerable from harm caused by researchers, the experiences of this researcher confirmed that ethics can also be used as a gatekeeping mechanism. In this case, the disapproval of some colleagues about how the author of the article decided to formulate the text and frame the discourse compelled him to relegate such decisions as they were ethical violations. The analysis of the experiences shows that researching one’s place of work with an aim of fostering change and dialogue is not favourably received among all colleagues. The subject of interrogation of teaching and learning practices can be an ethical minefield. To balance between the outcomes of research and research protocols is not always straightforward. In that respect, ethical procedures are sometimes invoked by those who are in position of power to make sure that certain forms of research are not carried out while they are in control. More importantly, the experiences of the researcher highlighted the place of informed consent in research that deals with human subjects. Even though there could be perceived urgency in the research, which often is the case when dealing with the marginalised, there must be careful navigation on the issues of informed consent. One must be prepared to accept that some research might never be carried out as consent might never be obtained. This is more so in the context where consent is sought from those who feel threatened by the changes. In the case of the experiences of the researcher, the colleagues were expected to emancipate students, but some resisted that challenge by citing technical rationality. Notwithstanding the centrality of informed consent in research that involves human subjects, there is also an issue of conscience in the context of continued repression. Discussion But it also revealed that dissemination of findings could be treated as an exposé. This is despite the fact that institutions of higher learning as research centres thrive on research output. However, research on teaching and learning, in the case of these experiences, does not seem to be expected to meet the public by some academics. This ambivalence is also reported by Mathany et al. (2017: 245). Whilst the scholarship of teaching and learning promotes reflections on one’s engagement with teaching, the reactions of some colleagues indicate that such reflection should be kept private and should not suggest changes to be made by other stakeholders, especially peers. Those who had proposed that we meet as a department and discuss how we could address some of the pertinent issues raised in the article were probably driven by the urgency of change that characterises action 22 research. As noted earlier, action research is dependent on action (Scott and Usher 2004: 36). It differs from traditional research where academics simply visit such for the sake of citations. Some of my colleagues therefore saw the urgency of action arising from the findings and arguments of the article. research. As noted earlier, action research is dependent on action (Scott and Usher 2004: 36). It differs from traditional research where academics simply visit such for the sake of citations. Some of my colleagues therefore saw the urgency of action arising from the findings and arguments of the article. Also, it seems the colleagues who resisted the publication of the article failed to see the emancipatory ethos underlying the research. Seemingly, they failed to appreciate the extent at which the actions of academics affect students. For them, it seemed the processes should never be disturbed but should be shrouded in secrecy so that the outside world would never get a glimpse of what was happening. This is confirmed by the fact that the extracted interview that one colleague conducted with students showed how frustrated they were with the issues of assessment being kept unclear to them. The operating ethos seems to be one of keeping others in the dark. This is no different from any oppressive regime to which critical approaches are set to unsettle. Conclusion One reason why there are not enough progressive changes in favour of vulnerable students (and sometimes lecturers) in many academic regimes is the bureaucratic red tape, sometimes in the form of ethical procedures even though such procedures were put in place for the protection of the vulnerable. The result is that research that would assist those who are disadvantaged is often abandoned and the status quo remains unchanged. Those who challenge the system are most likely to encounter many obstacles 23 such as those encountered by this researcher. There is therefore a need to contextualise ethical procedures especially in the light of the envisaged outcome. The example could be taken from colleagues who appreciated the urgency of the matters presented in the article and tried to forge the way forward in a manner that would improve the life experiences of students. But also, it would be important not to confuse ethical issues with epistemology. The issues for instance regarding how the author chose to carry out the presentation of arguments are epistemological and not necessarily ethical. Similarly, the initial suggestion by one colleague to wish to have been invited is epistemological as it seems to authenticate knowledge that is co-created. If there is one lesson that came out of these experiences is that serious research in the context of teaching and learning where critical pedagogy has been adopted and research is approached through the lens of critical theory, ethical pitfalls are not far away, and the terrain needs to be navigated with extra boldness and extra caution. Ntombela, B. 2017. Perspective on becoming a supervisor: Towards a critical approach. In: McKenna, B. 2017. Perspective on becoming a supervisor: Towards a critical approach. In: McKen References Abdollhzadeh, H. and Narafshan, M. H. 2016. The practicality of critical pedagogy: A case study of Iranian EFL learners’ motivation. 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https://openalex.org/W2011887959	https://europepmc.org/articles/pmc3642991?pdf=render	English	null	Urinary chitinase 3-like 1, a novel biomarker for acute kidney injury in adult cardiac intensive care patients: a pilot study	Critical care	2,013	cc-by	263,567	P1 Protective role of autophagy in mouse cecal ligation and puncture-induced sepsis model Methods After profound anesthesia with pentothal, the Wistar rats were killed by exsanguination. After sternotomy, the heart was taken and connected to the Langendorf column. The apex of the heart was hooked to a strength sensor. Biopac student laboratory software was used to record and analyse heart contractions. Contractions were recorded every 5 minutes during periods of 20 minutes. Control measurements were fi rst recorded. We measured four parameters: heart rate, contraction force, speeds of contraction and relaxation for control, during TNFα (20 ng/ml) exposure and after removal of TNFα. We express the variations of parameters as percentage of the control ± SEM. A paired t test was used to compare heart rate, contraction amplitude, speeds of contraction and relaxation with TNFα and control measurements and after removal of TNFα. pu ctu e duced seps s ode W Takahashi1, H Hatano2, H Hirasawa1, S Oda1 1Graduate School of Medicine, Chiba University, Chiba, Japan; 2Biomedical Research Center, Chiba University, Chiba, Japan Critical Care 2013, 17(Suppl 2):P1 (doi: 10.1186/cc11939) Introduction Autophagy is well known as one of the biogenic responses against various stresses, which possesses the benefi cial roles for survival, but little is known about the dynamics and its signifi cance during the septic condition. We hypothesized that autophagy is induced during the septic condition, and contributes to protect from tissue damage which subsequently leads to organ dysfunction. We confi rm whether the autophagic process is accelerated or sustained in an acute phase of sepsis and we also determine its physiological role. Results Eight rat hearts Wistar (weight = 325 ± 23 g) were studied. See Table 1. y g Methods Sepsis was induced by cecal ligation and puncture (CLP) in mice. We examined the kinetics of autophagosome and auto lysosome formation which may explain the status of autophagy by western blotting, immunohistochemistry, and electron microscopy. To investigate a precise role of autophagy in CLP-induced sepsis, chloroquine, an autophagy inhibitor, was administered to the CLP-operated mice, and blood chemistry, pathology of the liver and survival were evaluated. Table 1 (abstract P2) TNFα Removal of TNFα Heart rate 78 ± 6* 91 ± 5 Contractile 62 ± 8* 91 ± 4 Speed of contraction 72 ± 6* 93 ± 2 Speed of relaxation 53 ± 10* 89 ± 4 Results expressed as percentage of control ± SEM. P <0.05. MEETING ABSTRACTS MEETING ABSTRACTS P1 Protective role of autophagy in mouse cecal ligation and puncture-induced sepsis model y p gy Results Autophagy demonstrated by the ratio of LC3-II/LC3-I was induced over the time course up to 24 hours after CLP. The ratio was particularly increased in the liver, heart and spleen. Autophagosome formation became maximal at 6 hours and declined by 24 hours after CLP. Autolysosome formation as evaluated by both fusion of GFP-LC3 dots with LAMP1 immunohistochemistry and electron microscopy was also increased after the procedure. Furthermore, inhibition of autophagy by chloroquine during the CLP procedure resulted in elevation of serum AST levels, and signifi cantly increased mortality in mice. Conclusion TNFα decreases signifi cantly the heart rate, contractile force, speeds of contraction and relaxation on isolated perfused rat heart. TNFα probably plays a role in the pathophysiology of cardiomyopathy during septic shock. The partial reversibility of these eff ects could explain why left ventricular hypokinesia in patients with septic shock is reversible. Conclusion Autophagy was induced in several organs over the time course of the CLP sepsis model and then the process was gradually completed to degradation of the components. Our data suggest autophagy plays a protective role in organ dysfunction in sepsis. Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 P1 Protective role of autophagy in mouse cecal ligation and puncture-induced sepsis model W Takahashi1, H Hatano2, H Hirasawa1, S Oda1 1Graduate School of Medicine, Chiba University, Chiba, Japan; 2Biomedical Research Center, Chiba University, Chiba, Japan Critical Care 2013, 17(Suppl 2):P1 (doi: 10.1186/cc11939) P1 Protective role of autophagy in mouse cecal ligation and puncture-induced sepsis model W Takahashi1, H Hatano2, H Hirasawa1, S Oda1 1Graduate School of Medicine, Chiba University, Chiba, Japan; 2Biomedical Research Center, Chiba University, Chiba, Japan Critical Care 2013, 17(Suppl 2):P1 (doi: 10.1186/cc11939) References References 1. Vieillard-Baron A, et al.: Actual incidence of global left ventricular hypokinesia in adult septic shock. Crit Care Med 2008, 36:1701-1706. 2. Hunter JD, et al.: Sepsis and the heart. Br J Anaesth 2010, 104:3-11. 1. Vieillard-Baron A, et al.: Actual incidence of global left ventricular hypokinesia in adult septic shock. Crit Care Med 2008, 36:1701-1706. yp p 2. Hunter JD, et al.: Sepsis and the heart. Br J Anaesth 2010, 104:3-11. 2. Hunter JD, et al.: Sepsis and the heart. Br J Anaesth 2010, 104:3-11. 33rd International Symposium on Intensive Care and Emergency Medicine Brussels, Belgium, 19-22 March 2013 Published: 19 March 2013 The aim of our study was to observe the eff ects of TNFα on the model of perfused rat heart. P3 Eff ect of cdp-choline on microcirculatory alterations during endotoxemia BV Nguyen1, M Guillouet2, MA Giroux-Metges2, G Gueret3, M Ould-Ahmed1, JP Pennec2 1Hôpital d’Instruction des Armées Clermont Tonnerre, Brest, France; 2Laboratory of Physiology, Faculty of Medicine, Brest, France; 3University Hospital, Brest, France Critical Care 2013, 17(Suppl 2):P2 (doi: 10.1186/cc11940) K Schmidt1, M Doerr1, T Brenner1, S Hofer1, A Walther2 1Universitätsklinikum Heidelberg, Germany; 2Klinikum Stuttgart, Klinik für Anästhesiologie u. operative Intensivmedizin, Stuttgart, Germany Critical Care 2013, 17(Suppl 2):P3 (doi: 10.1186/cc11941) K Schmidt1, M Doerr1, T Brenner1, S Hofer1, A Walther2 1Universitätsklinikum Heidelberg, Germany; 2Klinikum Stuttgart, Klinik für Anästhesiologie u. operative Intensivmedizin, Stuttgart, Germany Critical Care 2013, 17(Suppl 2):P3 (doi: 10.1186/cc11941) Introduction Acute myocardial depression in septic shock is common [1]. Myocardial depression is mediated by circulating depressant substances, which until now have been incompletely characterized [2]. Introduction The cholinergic anti-infl ammatory pathway (CAP) is a physiological mechanism that inhibits cytokine production and minimizes tissue injury during infl ammation. CAP-mediated anti- infl ammatory signals in vagal eff erent nerve fi bers result in the release of acetylcholine, which interacts with innate immune cells that express the nicotinic acetylcholine receptor subunit α7 (α7nAChR). Introduction The cholinergic anti-infl ammatory pathway (CAP) is a physiological mechanism that inhibits cytokine production and minimizes tissue injury during infl ammation. CAP-mediated anti- infl ammatory signals in vagal eff erent nerve fi bers result in the release of acetylcholine, which interacts with innate immune cells that express the nicotinic acetylcholine receptor subunit α7 (α7nAChR). © 2010 BioMed Central Ltd © 2013 BioMed Central Ltd Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Endothelial dysfunction during sepsis is responsible for increased endothelial permeability, leukocyte–endothelial interaction and functional breakdown of microvascular perfusion. Endotoxemia- induced endothelial dysfunction can be reduced by cholinergic CAP activation [1]. The aim of this study was to determine the eff ects of the α7nAChR-agonist cdp-choline on microcirculatory alterations during experimental endotoxemia. response (a fi vefold to ninefold rise compared with other groups who had an increase of 14-fold to 36-fold). Conclusion Severely ill patients and secondarily hemodialysis and diabetic patients are in a proinfl ammatory state. The response of all examined groups to provocation by LTA was suffi cient, with a diff erential expression of severely ill patients, a fact that refl ects their diff erent immunologic status. P5 Correlation of the oxygen radical activity and antioxidants and severity in critically ill surgical patients: preliminary report J Lee1, H Shim2, JY Jang1 , , g 1Yonsei University College of Medicine, Seoul, South Korea; 2Wonju Severance Christian Hospital, Yonsei University Wonju College of Medicine, Wonju, Korea Critical Care 2013, 17(Suppl 2):P5 (doi: 10.1186/cc11943) Introduction In septic patients, the oxygen radical (OR) showed toxic eff ect to induce infl ammation and antioxidant activity could aff ect organ dysfunction. This study was designed to determine the relationship between antioxidant level and severity of organ dysfunction. Results There were no signifi cant diff erences in venular wall shear rate between the groups after 120 minutes. There was no signifi cant diff erence in the number of adhering leukocytes between the cdp- choline/LPS groups (iii, iv) and the LPS group after 120 minutes. Macromolecular effl ux signifi cantly increased in all groups over 120 minutes. All groups (i, ii, iii, iv) showed a signifi cantly reduced macromolecular effl ux compared with the LPS group after 120 minutes. Conclusion Cdp-choline has no eff ect on leukocyte–endothelial interaction and microhemodynamic alterations during endotoxemia. By activating the CAP, cdp-choline reduces capillary leakage. Thus cdp- choline might have a prophylactic and therapeutic anti-infl ammatory eff ect on LPS-induced endothelial permeability. These fi ndings identify the endothelium as a target of anti-infl ammatory cholinergic mediators and cdp-choline as a potential therapeutic substance in sepsis treatment. Methods The medical records of adult patients managed in a surgical ICU from August 2012 to December 2012 were reviewed prospectively. Abstracted data included age, body weight (with BMI), APACHE II scores, SOFA scores, MODS scores, fl uid intake, fl uid output, nutritional support, shock, antioxidant levels, OR activities, zinc and selenium levels, complication and mortality. In addition, length of stay (LOS) in the ICU and in hospital, and in-hospital mortality were collected. These data were investigated on the fi rst, the third and the seventh day, respectively. p y Results A total of 13 patients were enrolled. The in-hospital mortality rate was 7.7% and mean LOS in the ICU and hospital was 6.5 and 27.6, respectively. Mean APACHE II score was 20.2. On the fi rst day of ICU, the mean antioxidant level and OR were 1.5 (± 0.5) mmol/l and 1.6 (± 0.5) mmol/l, respectively. At the same time, SOFA and MODS scores were 7.3 and 5.0, respectively, and zinc and selenium were 32.6 μg/dl and 68.4 ng/ml. P5 Correlation of the oxygen radical activity and antioxidants and severity in critically ill surgical patients: preliminary report J Lee1, H Shim2, JY Jang1 On the third day, mean antioxidant and OR were 1.5 (± 0.4) and 1.8 (± 0.7) respectively (SOFA 6.6, MODS 4.9, zinc 50.0, selenium 70.7). On the seventh day, mean antioxidant and OR were 1.4 (± 0.5) and 1.9 (± 0.7), respectively (SOFA 4.3, MODS 3.1, zinc 62.8, selenium 77.3). In the correlation analysis, MODS scores and antioxidant level had signifi cant correlations on the fi rst and seventh days of ICU (P = 0.001, P = 0.009). p Reference 1. Peter C, et al.: Shock 2010, 33:405-411. References 1. Noveanu M, Mebazaa A, Mueller C: Cardiovascular biomarkers in the ICU. Curr Opin Crit Care 2009, 15:377-383. y Methods Blood sample was obtained from 10 healthy volunteers, 10 hemodialysis patients with end-stage chronic renal failure (CRF), 10 patients with type II diabetes mellitus (DM) and 10 ICU patients on the second day of hospitalization, who suff ered nonseptic SIRS and had an APACHE II score >25. After suitable treatment the samples were incubated with 1 mg LTA for 8 hours and maintained at –20°C until the measurement of cytokines TNFα, IL-6, IL-1β, and IL-10, using the ELISA method. The results are presented as mean values ± SEM. Graph Pad 4.0 was used, applying a t test to test the variation of each cytokine in each group, and ANOVA to assess the diff erences between the four groups. Methods Blood sample was obtained from 10 healthy volunteers, 10 hemodialysis patients with end-stage chronic renal failure (CRF), 10 patients with type II diabetes mellitus (DM) and 10 ICU patients on the second day of hospitalization, who suff ered nonseptic SIRS and had an APACHE II score >25. After suitable treatment the samples were incubated with 1 mg LTA for 8 hours and maintained at –20°C until the measurement of cytokines TNFα, IL-6, IL-1β, and IL-10, using the ELISA method. The results are presented as mean values ± SEM. Graph Pad 4.0 was used, applying a t test to test the variation of each cytokine in each group, and ANOVA to assess the diff erences between the four groups. Results Baseline cytokine values in the three groups were increased compared with the control group, but the diff erence was signifi cant only for the ICU group (Table 1, data only for IL-6 and IL-10). The quotient IL-10/IL-6 of baseline values was between 0.23 and 0.96 among healthy, ESRD and DM persons, and 1.32 among ICU patients. In all examined groups the levels of cytokines increased signifi cantly after stimulation with LTA, although ICU patients showed a diff erential 2. Piechota M, Banach M, Irzmanski R, Barylski M, Piechota-Urbanska M, Kowalski J, et al.: Plasma endothelin-1 levels in septic patients. J Intensive Care Med 2007, 22:232-239. 3. Kotsovolis G, Kallaras K: The role of endothelium and endogenous vasoactive substances in sepsis. Hippokratia 2010, 14:88-93. P3 Eff ect of cdp-choline on microcirculatory alterations during endotoxemia Methods Using fl uorescent intravital microscopy, we determined venular wall shear rate, macromolecular effl ux and leukocyte adhesion in mesenteric postcapillary venules of male Wistar rats. Endotoxemia was induced over 120 minutes by intravenous infusion of lipopolysaccharide (LPS). Control groups received an equivalent volume of saline. Cdp-choline was applied as an i.v. bolus in treatment groups. Animals received either (i) saline alone, (ii) cdp-choline 10 minutes prior to saline administration, (iii) cdp-choline 10 minutes prior to LPS administration, (iv) cdp-choline 30 minutes after LPS administration or (v) LPS alone.if Immune response after stimulation with wall components of Gram-positive bacteria S Aloizos1, E Tsigou2, P Myrianthefs2, S Gourgiotis1, A Tsakris3, G Baltopoulos2 1NIMTS Hospital, Athens, Greece; 2A. Anargiroi Hospital, Athens, Greece; 3M di l S h l Ath G S Aloizos1, E Tsigou2, P Myrianthefs2, S Gourgiotis1, A Tsakris3, G Baltopoulos2 1NIMTS Hospital, Athens, Greece; 2A. Anargiroi Hospital, Athens, Greece; 3Medical School Athens Greece S Aloizos1, E Tsigou2, P Myrianthefs2, S Gourgiotis1, A Tsakris3, G Baltopoulos2 1NIMTS Hospital, Athens, Greece; 2A. Anargiroi Hospital, Athens, Greece; 3Medical School, Athens, Greece Critical Care 2013, 17(Suppl 2):P4 (doi: 10.1186/cc11942) Critical Care 2013, 17(Suppl 2):P4 (doi: 10.1186/cc11942) Critical Care 2013, 17(Suppl 2):P4 (doi: 10.1186/cc11942) Conclusion Antioxidant level had a correlation with organ dysfunction which might be used as a prognostic factor in critically septic patients. To prove this, large-scale data collection is required. R f Introduction The purpose of this study was to evaluate the immune response of patients susceptible to infection by Gram-positive bacteria after ex vivo provocation with lipoteichoic acid (LTA) and to compare the reaction with the one of healthy adults. Prevention of sepsis by correcting apoptosis M Puhtinskaya, V Estrin y Research Institute of Obstetrics and Pediatrics, Rostov-on-Don, Russia Critical Care 2013, 17(Suppl 2):P8 (doi: 10.1186/cc11946) Research Institute of Obstetrics and Pediatrics, Rostov-on-Don, Russia Critical Care 2013, 17(Suppl 2):P8 (doi: 10.1186/cc11946) Research Institute of Obstetrics and Pediatrics, Rostov-on-Don, Russ Critical Care 2013, 17(Suppl 2):P8 (doi: 10.1186/cc11946) Introduction Activation of apoptosis in lymphocytes determines the development of neutropenia and of sepsis [1,2]. We investigated prevention of sepsis and correction of lymphocyte apoptosis by recombinant human granulocyte colony-stimulating factor (hr-GCSF, fi lgrastim) [1,2]. Methods With the permission of the ethics committee, a controlled, randomized, blind clinical trial included 69 term newborns on mechanical ventilation, without neutropenia and clinical signs of infection, with a content of lymphocytes in early apoptosis (AnnexinV– FITC+PI–) of >9.59%, and in late (AnnexinV–FITC+PI+) of 0.56%. Lymphocytes in apoptosis were detected using antibodies to AnnexinV and propidium iodide staining method of immunophenotyping (fl ow cytometry; Beckman Coulter Epics XL, USA). The survey was conducted at admission, at 3 to 5 days, and 20 days. The method of random numbers in Group I included 39 newborns who on admission (with written parental consent) received an intravenous infusion of hr-GCSF dose of 10 μg/kg, 3 days. Newborns of Group II (n = 30) did not receive hr-GCSF. Power of the study was 80% (α ≤0.05).i Conclusion In this study we noticed that during sepsis some signifi cant diff erences in the status of activation of immune cells exist between peripheral blood and lung resident cells. The lung milieu seems to promote activation of monocytes while neutrophil activation is more dependent on the site of infection. However, these observations require further studies in a larger group of patients. Acknowledgements This study was supported by the Centre of Postgraduate Medical Education grant no 501-01-02-012 and by the sources of the Medical University of Warsaw. P7 Anti-infl ammatory eff ects of Kupff er cells through α7-nicotinic acetylcholine receptors Y Li, X Shi Changzheng Hospital, Second Military Medical University, Shanghai, China Critical Care 2013, 17(Suppl 2):P7 (doi: 10.1186/cc141945) Results For 3 to 5 days, Group I signifi cantly decreased apoptosis of lymphocytes in the early from 16.1% to 7.8%, and in late from 1.3% to 0.1%. The development of sepsis and neutropenia have been reported. We observed no clinical or laboratory signs of adverse eff ects of the drug. Fatal outcomes (n = 4) are not associated with hr-GCSF, which was confi rmed postmortem. Prevention of sepsis by correcting apoptosis M Puhtinskaya, V Estrin Decreased duration of mechanical ventilation (P <0.05). In Group II, 27 patients at 3 to 5 days developed neutropenia and increased lymphocytes in apoptosis (P <0.05). Sepsis was diagnosed in 19 children; eight fatal outcomes. Introduction Nicotine exerts anti-infl ammatory eff ects in several cell types. α7-nicotinic acetylcholine receptor (α7-nAChR), which has high permeability to calcium, is believed to contribute signifi cantly to nicotinic anti-infl ammatory eff ects. However, the molecular mechanism is largely unknown. Kupff er cells in the liver play an important role in infl ammatory response to pathogens invading, but whether there is α7-nAChR expression in Kupff er cells or cholinergic anti-infl ammatory pathway involved in this process remains unclear.f Conclusion hr-GCSF reduces the incidence of septic complications and one of the mechanisms of its clinical eff ectiveness is the reduction of apoptotic factors aff ecting the development of neutropenia. References 1. Gillan ER, Christensen RD, Suen Y, et al.: A randomized, placebo-controlled trial of recombinant human granulocyte colony-stimulating factor administration in newborn infants with presumed sepsis: signifi cant induction of peripheral and bone marrow neutrophilia. Blood 1994, 84:1427-1433. 1. Gillan ER, Christensen RD, Suen Y, et al.: A randomized, placebo-controlled trial of recombinant human granulocyte colony-stimulating factor administration in newborn infants with presumed sepsis: signifi cant induction of peripheral and bone marrow neutrophilia. Blood 1994, 84:1427-1433. Methods (1) Kupff er cells, isolated by collagenase digestion and diff erential centrifugation from mice and labeled with FITC-aBGT, were observed under laser scanning confocal microscope to test the expression of α7-nAChR. Protein level was also tested by western blotting, with RAW264.7 as positive control; (2) 100 nM LPS was given to Kupff er cells, with or without 1 mM nicotine. TNFα, IL-10 and HMGB-1 were tested at 4 hours, 12 hours or 24 hours, respectively; (3) 100 BALB/c mice were randomly divided into four group: Group I (only lethal dose of LPS was given), Group II (nicotine and LPS were given), Group III (LPS, nicotine and GdCl3 were given), and Group IV (LPS and nicotine were given and the left cervical vagus nerve was cut off ). The mortality of mice was observed for 72 hours.fi 2. Pukhtinskaya MG, Estrin VV, Gulova ES: Clinical and diagnostic value of apoptosis markers in the pathogenesis of neutropenia and bacterial complications in newborns with respiratory distress syndrome. Cytokines Infl amm 2011, 10:66-69. Prevention of sepsis by correcting apoptosis M P h k V E g y Results The mortality of sepsis reached 70%. Nonsurvivors had signifi cantly (P = 0.001) elevated expression of CD64 on neutrophils. Expression of HLA-DR was higher in monocytes from BAL than PB GMF (1,032 vs. 342; P = 0.02) and this tendency was present in sepsis originating from both pneumonia and peritonitis. Percentage of HLA- DR-positive T cells was lower in PB than in BAL (2.9% vs. 6.5%; P = 0.07), but the GMF values for HLA-DR were higher in the circulating T cells (1,904 vs. 1,346; P = 0.004). The expression of CD64 on neutrophils was not signifi cantly diff erent in PB and BAL, but there was a trend towards its higher expression in BAL from patients with pneumonia while its expression was higher in PB of patients with peritonitis.i Simultaneous analysis of the expression of CD64 and HLA-DR in the peripheral blood and bronchoalveolar lavage fl uid in sepsis It was verifi ed that left cervical vagus nerve was essential for the anti- infl ammatory eff ect of nicotine and α7 acetylcholine receptors might play a critical role. anti-infl ammatory compensatory response. However, controversies exist regarding the status of the immune system in local tissue compartments during sepsis. The aim of this study was to compare selected markers of activation between the systemic circulation and local lung environment. Methods Twenty patients with severe sepsis were included into this study. Peripheral blood (PB) samples and bronchoalveolar lavage fl uid (BALF) samples were obtained on the day of diagnosis (D1). BALF was collected from 11 patients. Samples were stained with antibodies: CD15/CD64 and CD3/CD14/HLA-DR and isotypic control. Cells were analysed by fl ow cytometry. Expression of markers of activation was analysed as the geometric median of fl uorescence (GMF). All values are expressed as median values. Comparisons between groups were performed using Mann–Whitney and Wilcoxon tests. References 1. Wang H, et al.: Nicotinic acetylcholine receptor α7 subunit is an essential regulator of infl ammation. Nature 2003, 421:384-388. 1. Wang H, et al.: Nicotinic acetylcholine receptor α7 subunit is an essential regulator of infl ammation. Nature 2003, 421:384-388. 2. Wang H, et al.: Cholinergic agonists inhibit HMGB1 release and improve survival in experimental sepsis. Nat Med 2004, 10:1216-1221. 2. Wang H, et al.: Cholinergic agonists inhibit HMGB1 release and improve survival in experimental sepsis. Nat Med 2004, 10:1216-1221. Simultaneous analysis of the expression of CD64 and HLA-DR in the peripheral blood and bronchoalveolar lavage fl uid in sepsis f Results Baseline cytokine values in the three groups were increased compared with the control group, but the diff erence was signifi cant only for the ICU group (Table 1, data only for IL-6 and IL-10). The quotient IL-10/IL-6 of baseline values was between 0.23 and 0.96 among healthy, ESRD and DM persons, and 1.32 among ICU patients. In all examined groups the levels of cytokines increased signifi cantly after stimulation with LTA, although ICU patients showed a diff erential l T Skirecki1, M Mikaszewska-Sokolewicz2, G Hoser1, U Zielińska-Borkowska1 1The Centre of Postgraduate Medical Education, Warsaw, Poland; 2 Medical University of Warsaw, Poland T Skirecki1, M Mikaszewska-Sokolewicz2, G Hoser1, U Zielińska-Borkowska1 1The Centre of Postgraduate Medical Education, Warsaw, Poland; 2 Medical University of Warsaw, Poland y Critical Care 2013, 17(Suppl 2):P6 (doi: 10.1186/cc11944) Introduction The core pathophysiological changes in sepsis involve systemic activation of the immune system followed by the Table 1 (abstract P4). Levels of cytokines before and after stimulation with LTA Control baseline LTA ESRD baseline LTA DM baseline LTA ICU baseline LTA IL-6 8.90 ± 0.76, 245.30 ± 26.68 86.60 ± 45.55, 1,310.00 ± 154.80 15.90 ± 1.89, 252.00 ± 35.52 372.40 ± 120.60, 3,659.00 ± 485.20 IL-10 3.00 ± 1.08, 40.90 ± 7.45 19.20 ± 7.14, 273.10 ± 126.50 15.30 ± 2.08, 350.50 ± 89.42 492.60 ± 66.72, 2,822.00 ± 432.70 Values in pg/ml. Table 1 (abstract P4). Levels of cytokines before and after stimulation with LTA Table 1 (abstract P4). Levels of cytokines before and after stimulation with LTA Control baseline LTA ESRD baseline LTA S3 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Conclusion Kupff er cells played a crucial rule in modulating infl ammation and the anti-infl ammatory eff ect of nicotine was partially weakened after left cervical vagus nerve cut off or aBGT was given. It was verifi ed that left cervical vagus nerve was essential for the anti- infl ammatory eff ect of nicotine and α7 acetylcholine receptors might play a critical role. Conclusion Kupff er cells played a crucial rule in modulating infl ammation and the anti-infl ammatory eff ect of nicotine was partially weakened after left cervical vagus nerve cut off or aBGT was given. Immune paralysis in trauma patients; implications for prehospital interventionf Results (1) Expression of α7-nAChR in Kupff er cells was confi rmed by confocal microscope and western blotting; (2) after nicotine was administered, the level of TNFα and HMGB-1 increased and the level of IL-10 decreased. Given left cervical vagus nerve cut off or aBGT, the eff ect of nicotine was weakened; (3) Group I had the highest mortality rate, while in Group II nicotine did reduce the mortality rate dramatically. After the left cervical vagus nerve was cut off or aBGT was given, the eff ects of nicotine were weakened. Diff erence for the mortality rate between Group III and Group IV was not signifi cant. M Kox, K Timmermans, M Vaneker, GJ Scheff er, P Pickkers Radboud University Nijmegen Medical Center, Nijmegen, the Netherlands Critical Care 2013, 17(Suppl 2):P9 (doi: 10.1186/cc11947) Introduction Multi-trauma is one of the major indications for intensive care admission. Recovery is frequently complicated by post-injury immunological complications, caused by a dysfunctional immune system; for example, sepsis and multiple organ failure. In order to treat S4 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 hematologic or metastasized malignancies, cardiac arrest, and use of bone marrow suppressive drugs. Laboratory data were collected from blood samples, prior to in-hospital i.v. fl uid therapy. In order to detect a diff erence in Hb concentration of 0.2 mmol/l, we anticipated a sample size of 283 per group, based on a standard deviation (SD) of 1.2, α = 0.05 and β = 0.8. Data are expressed as mean ± SD. or prevent this immune paralysis, knowledge on the time course of immune paralysis in vivo and the pathophysiological mechanisms of immune paralysis is essential. The aim of this study is to determine factors that could predict and/or induce immunological complications in these patients to ultimately fi nd a suitable target and timeframe for intervention. hematologic or metastasized malignancies, cardiac arrest, and use of bone marrow suppressive drugs. Laboratory data were collected from blood samples, prior to in-hospital i.v. fl uid therapy. In order to detect a diff erence in Hb concentration of 0.2 mmol/l, we anticipated a sample size of 283 per group, based on a standard deviation (SD) of 1.2, α = 0.05 and β = 0.8. Data are expressed as mean ± SD. β p Results We included 296 patients in the S-group and 320 in the C-group. Do changes in red blood cell deformability in patients with septic shock correlate with changes in SOFA scores? T Clark1, S Jewell2, M Sair1, P Petrov2, P Winlove2 T Clark1, S Jewell2, M Sair1, P Petrov2, P Winlove2 1Derriford Hospital, Plymouth, UK; 2University of Exeter, UK C l C (S l ) P (d / ) Derriford Hospital, Plymouth, UK; 2University of Exeter, UK Critical Care 2013 17(Suppl 2):P11 (doi: 10 1186/cc11949) Conclusion Immune paralysis can be established within hours after trauma. Production of anti-infl ammatory IL-10 in the prehospital phase could play a crucial role in the pathogenesis. Patients with a higher injury severity score are more prone to produce excessive IL-10 in this phase. Immune stimulatory strategies applied by the HEMS or early after hospital admission could form a potential future approach to prevent immune paralysis in multitrauma patients in the intensive care ward. p , y , ; y , Critical Care 2013, 17(Suppl 2):P11 (doi: 10.1186/cc11949) Introduction Traditional whole blood experiments suggest that sepsis causes abnormal red blood cell (RBC) deformability. To investigate this at the cellular level, we employed a novel biophysical method to observe individual RBC membrane mechanics in patients with septic shock. Methods We collected blood samples from patients with septic shock until either death or day 5 of admission. Thermal fl uctuations of individual RBCs were recorded allowing a complete analysis of RBC shape variation over time. Mean elasticity of the cell membrane was then quantifi ed for each sample collected. Is hemoglobin concentration aff ected by sepsis in the acute phase? G Jansma, H Buter, RT Gerritsen, EC Boerma Medical Centre Leeuwarden, the Netherlands Critical Care 2013, 17(Suppl 2):P10 (doi: 10.1186/cc11948) Immune paralysis in trauma patients; implications for prehospital interventionf The diff erence in Hb between the S-group and C-group was not signifi cant (8.76 ± 1.18 mmol/l vs. 8.93 ± 1.16 mmol/l, P = 0.07). After correction for a number of confounders, using a multivariate regression analysis, we observed a signifi cant diff erence in Hb of –0.23 mmol/l in the S-group in comparison with the C-group (P = 0.01).il Methods Blood was drawn from adult multi-trauma patients (n = 94) admitted to the emergency room (ER) of the Radboud University Nijmegen Medical Center. Blood was drawn at the trauma scene by the helicopter emergency medical services (HEMS), at arrival in the ER and at days 1, 3, 5, 7, 10 and 14 after trauma. Plasma concentrations of TNFα, IL-6, IL-10, IFNγ, IL-8 and MCP-1 were determined by Luminex. Ex vivo 24-hour whole blood stimulations with LPS or pam3cys were performed and produced TNFα, IL-6 and IL-10 was measured using ELISA to determine the level of immune paralysis. Clinical data – for example, Injury Severity Scores, trauma mechanism, medication and survival – were collected from electronic patient fi les. g p p g p Conclusion At fi rst presentation, prior to in-hospital i.v. fl uid therapy, Hb concentration in patients with sepsis is signifi cantly lower in comparison with controls; however, the diff erence is very small, without the existence of anemia. R f Is hemoglobin concentration aff ected by sepsis in the acute phase? G Jansma, H Buter, RT Gerritsen, EC Boerma Medical Centre Leeuwarden, the Netherlands Critical Care 2013, 17(Suppl 2):P10 (doi: 10.1186/cc11948) i Results We recruited nine patients with septic shock. Table 1 shows mean RBC thermal fl uctuation and SOFA scores.l Introduction In the acute phase of sepsis several potential mechanisms may change the hemoglobin (Hb) concentration. On the one hand, endothelial activation may lead to increased vascular permeability and fl uid sequestration to the interstitium, leading to hemoconcentration. On the other hand, degradation of the glycocalyx has been reported [1]. Shedding of this carbohydrate-rich layer with an estimated thickness of 0.2 to 0.5 μm may lead to a substantial increase of the intravascular space, and thus to decrease of Hb concentration [2]. The aim of this study is to determine whether there is a decrease in Hb in the acute phase of sepsis.i l Conclusion RBC thermal fl uctuation analysis allows variations in RBC elasticity during sepsis to be quantifi ed at a cellular level. We could not identify any specifi c trend between sepsis severity and erythrocyte elasticity. Cells demonstrated both increases and decreases in fl uctua- tion independent of SOFA score. This is contrary to current evidence that suggests RBC deformability is reduced during sepsis. Reference 1. Piagnerelli M, et al.: Intensive Care Med 2003, 29:1052-1061. References i Results The plasma IL-10 concentration at ER was 16.5-fold increased in comparison with time-point HEMS (P <0.01). Similar but less pronounced eff ects were found for IL-8 and MCP-1. A signifi cant correlation (P = 0.03, R = 0.53) was found between injury severity scores and IL-10 plasma concentration at time-point ER. Time-courses of ex vivo produced cytokines suggest that LPS-induced IL-6 and TNFα production is already decreased in the fi rst few hours after trauma and recovering from day 5. Ex vivo IL-10 production shows an inverse pattern. 1. Steppan J, et al.: Sepsis and major abdominal surgery lead to fl aking of the endothelial glycocalyx. J Surg Res 2011, 165:136-141. 2. van den Berg BM, et al.: The endothelial glycocalyx protects against myocardial edema. Circ Res 2003, 92:592-594. 2. van den Berg BM, et al.: The endothelial glycocalyx protects against myocardial edema. Circ Res 2003, 92:592-594. Reference Reference 1. Strauss R, et al.: Thrombocytopenia in patients in the medical intensive care unit: bleeding prevalence, transfusion requirements, and outcome. Crit Care Med 2002, 30:1765-1771. Methods We collected blood samples from elective cardiac surgery patients pre (at induction) and post (immediately, each day until CICU discharge) CPB. Thermal fl uctuations of individual RBCs were recorded using a high-frame-rate camera allowing a complete analysis of RBC shape variation over time. Mean elasticity of the cell membrane was then quantifi ed for each sample collected. P14 N t i Results Fifteen patients were recruited. Table 1 displays the results. RBC thermal fl uctuation is measured relative to pre-bypass values. An increase in RBC fl uctuation marks a decrease in stiff ness. CPB caused two distinct changes in RBC elasticity; pre-fi x A indicates samples where stiff ness increases or shows no change, B those where stiff ness decreases. Data on day 2 were not collected in patients discharged from the CICU. CPB type or time had no apparent impact on RBC response to CPB. Introduction Neutrophil gelatinase-associated lipocalin (NGAL)/ lipocalin2, known as a sensitive biomarker of acute kidney injury, prevents bacterial iron uptake, resulting in the inhibition of its overgrowth [1]. We previously demonstrated that this protein was discharged into gut lumen from crypt cells in septic conditions, and inhibited the growth of Escherichia coli [2]. However, it remains unclear which pathway is associated with the upregulation of NGAL. We therefore designed the present study to reveal whether the pattern- recognition receptor of bacteria, the Toll-like receptor (TLR) family, plays a pivotal role for NGAL secretion from gut crypt cells. Conclusion RBC thermal fl uctuation analysis quantifi es the impact of CPB on erythrocyte membrane elasticity. We clearly identifi ed two separate RBC elasticity responses to CPB. This fi nding is contrary to traditional fl ow measurement techniques that suggest CPB impairs whole blood fl ow and reduces RBC deformability. Reference 1. Lindmark et al.: J Thoracic Cardiovasc Surg 2002, 123:381-383. p y p g yp Methods With our institutional approval, the ileum and colon of male C57BL/6J mice (6 to 7 weeks) were everted and washed by Ca2+ and Mg2+ free PBS buff er fi ve times. Tissues were incubated with Ca2+ and Mg2+ free PBS containing 30 mM EDTA for 1 hour to isolate crypt cells of gut. Platelet-associated oxidative stress and ADAMTS-13 levels are inversely associated with a poor prognosis in septic shock Introduction Sepsis causes widespread microvascular injury and thrombosis. Some hemostatic factors mediate the mechanisms involved in sepsis-related organ ischemia and failure. Oxidative stress is also increased in sepsis and reactive oxygen species (ROS) favor secretion of von Willebrand factor (vWF) multimers from endothelium and inhibit vWF proteolysis by ADAMTS-13. Moreover, the enzyme indoleamine-2,3-dioxygenase, an important immune regulator, is activated in sepsis and, through generation of kynurenins, promotes antioxidative and anti-infective activities. This study evaluated the relative role of ADAMTS-13, vWF and fi brinogen in the morbidity and mortality of patients with septic shock (SS). The above hemostatic factors were measured together with kynurenine and plasma protein carbonyls, marker of oxidative stress. Results More than 70 to 80% of collected cells were stained by amido black. LPS signifi cantly upregulated the expression of NGAL and TLR4 mRNA in ileum and colon crypt cells (P <0.05). Although the CpG-DNA did not upregulate NGAL and TLR9 mRNA in ileum crypt cells, the apparent expression of NGAL and TLR9 mRNA was found in colon crypt cells (P <0.05). Conclusion Bacterial stimulation of TLR4 and TLR9 pathways plays a pivotal role in the expression of NGAL mRNA in gut, suggesting that NGAL, derived from gut crypt cells, could contribute to the regulation of the intraluminal microfl ora in the critically ill. y Methods One group of 12 patients with SS, defi ned using standard criteria, was enrolled in the ICU of the ‘A. Gemelli’ Hospital (Rome, Italy). Biochemical, hematologic and hemodynamic parameters were measured on days 1 to 4, 7, 14 and 21. A group of 12 age-matched and gender-matched healthy subjects was used as controls. References References 1. Nature 2004, 432:917. 2. Crit Care Med 2011, 39:46. 1. Nature 2004, 432:917. 2. Crit Care Med 2011, 39:46. Reference The cell suspension was fi ltered through a cell strainer (40 μm) twice, and deposited the crypt cells by centrifugation at 700×g. The isolated crypt cells were resuspended in PBS and stained with 0.25% amido black for labeling paneth cells. The 5×105 crypt cells were resuspended in 50 ml HBSS containing 2.5% fetal bovine serum and 1% penicillin–streptomycin. The crypt cells were incubated at 37°C with or without TLR ligands: lipopolysaccharide (TLR4 ligand, 10 μg/ml) and CpG-DNA (TLR9 ligand, 8 μg/ml). After a 2-hour incubation period, the crypt cells were deposited and eluted mRNA to measure the expression of both NGAL and TLR mRNA using real-time PCR. P13 Platelet-associated oxidative stress and ADAMTS-13 levels are inversely associated with a poor prognosis in septic shock L Montini1, G De Pascale1, MA Pennisi1, ES Tanzarella1, SL Cutuli1, A Occhionero1, R De Cristofaro2, M Antonelli1 1Catholic University School of Medicine, Rome, Italy; 2Haemostasis and Thrombosis Center, Catholic University School of Medicine, Rome, Italy Critical Care 2013, 17(Suppl 2):P13 (doi: 10.1186/cc11951) P15 y Results Low ADAMTS-13 activity was observed in SS patients (268 ± 123 ng/ml vs. 760 ± 80 ng/ml in controls). vWF levels (antigen and activity) were increased ~3-fold compared with controls. Likewise, plasma protein carbonyls and kynurenine were globally increased in patients (2.1 ± 1.5 nmol/mg vs. 0.3 ± 02 nmol/mg and 14.4 ± 9.7 μM vs. 2.3 ± 1.3 μM, respectively). Intra-ICU mortality (3 of 15) was strongly and inversely correlated with carbonyl levels (P = 0.04) and platelets (P = 0.022). Lethal infl uenza virus A H1N1 infection in two relatives with autosomal dominant GATA-2 defi ciency J Sole-Violan1, I Sologuren1, E Betancor2, S Zhang3, C Pérez1, E Herrera-Ramos1, M Martínez-Saavedra1, M López-Rodríguez1, J Pestano2, J Ruiz-Hernández1, J Ferrer1, F Rodríguez de Castro1, J Casanova3, C Rodríguez-Gallego1 1Hospital GC Dr Negrín, Las Palmas de GC, Spain; 2Universidad Las Palmas de GC, Spain; 3The Rockefeller University, New York, NY, USA Critical Care 2013, 17(Suppl 2):P15 (doi: 10.1186/cc11953) Lethal infl uenza virus A H1N1 infection in two relatives with autosomal dominant GATA-2 defi ciency P12 Do erythrocytes subjected to cardiopulmonary bypass exhibit changes in their membrane mechanical properties? T Cl k1 S J ll2 M S i 1 P P 2 P Wi l 2 p p Methods In this single-center retrospective analysis we identifi ed patients with sepsis as the primary reason for non-elective ICU admission from a standard patient database. Patients who fulfi lled the international criteria of sepsis and organ failure during ICU admission were included in the sepsis group (S-group). The control group was formed by patients with other non-elective reasons for ICU admission (C-group). Exclusion criteria were (recent) bleeding, surgery in the last 6 weeks, chronic renal failure (creat >177 μmol/l, or hemodialysis), untreated chronic anemia, pregnancy, polytrauma, age <18, T Clark , S Jewell , M Sair , P Petrov , P Winlove 1Derriford Hospital, Plymouth, UK; 2University of Exeter, UK Critical Care 2013, 17(Suppl 2):P12 (doi: 10.1186/cc11950) Introduction Whole blood experiments suggest that cardiopulmonary bypass (CPB) causes red blood cell (RBC) trauma and changes in deformability that may contribute to postoperative microcirculatory Table 1 (abstract P11). Mean RBC fl uctuation (daily SOFA score) Day A B C D E F G H I 1 4.8 (10) 5.2 (9) – 4.8 (12) 4.6 (16) 4.9 (13) 5.1 (16) 4.6 (18) 5.1 (15) 2 4.9 (9) 5.0 (10) 4.6 (13) 5.1 (11) 4.8 (17) 5.1 (13) 5.0 (16) 4.9 (19) – (16) 3 4.0 (6) 5.1 (9) 4.7 (12) 4.8 (11) 4.9 (18) 4.8 (13) 5.0 (16) 4.7 (21) 5.3 (16) 4 – 4.8 (7) 4.6 (11) 5.0 (9) 5.9 (19) – – (18) – 5.0 (15) 5 – – – 5.1 (6) 5.2 (18) – – (19) – 5.0 (10) Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S5 Table 1 (abstract P12). Change in RBC thermal fl uctuation relative to baseline: two distinct groups seen Patient A1 A2 A3 A4 A5 A6 A7 B8 B9 B10 B11 B12 B13 B14 B15 Post CPB +0.1 –0.4 –0.1 –0.5 –0.1 –0.2 –0.1 +0.5 0 +0.4 +0.1 +0.2 0 +0.2 +0.1 Day 1 0 0 +0.1 –0.1 –0.1 +0.1 +0.1 0 +0.5 +0.4 +0.7 +0.4 +0.6 +0.5 +0.7 Day 2 –0.2 +0.1 0 NA NA 0 NA +0.5 –0.1 +0.4 NA +0.1 NA NA NA Table 1 (abstract P12). Change in RBC thermal fl uctuation relative to baseline: two distinct groups seen dysfunction. We used a novel fl uctuation microscopy technique to quantify the eff ects of CPB on RBC elasticity at a cellular level. Lethal infl uenza virus A H1N1 infection in two relatives with autosomal dominant GATA-2 defi ciency Conclusion Hence, we hypothesize that, in the SS setting, platelets contribute to oxidative stress that counteracts the organ failure- associated mortality. Thus, low platelet count, irrespective of bleedings, may favor mortality in SS patients by generating lower ROS amounts. Introduction Most individuals infected with the 2009 pandemic H1N1 infl uenza A virus (IAV) (H1N1pdm) experienced uncomplicated fl u. Introduction Most individuals infected with the 2009 pandemic H1N1 infl uenza A virus (IAV) (H1N1pdm) experienced uncomplicated fl u. S6 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 isolated and incubated for 24 hours in the presence of 10 ng/gl LPS of Escherichia coli O55:B5 and of 5 μg/ml phytohemagglutin (PHA); TNFα was measured in supernatants by a bioassay on L929 fi broblasts.i isolated and incubated for 24 hours in the presence of 10 ng/gl LPS of Escherichia coli O55:B5 and of 5 μg/ml phytohemagglutin (PHA); TNFα was measured in supernatants by a bioassay on L929 fi broblasts. Results Fifty percent of rabbits died early; that is, within the fi rst 48 hours. Mean ± SE log10 bacteria in the liver and lung of animals that died early was 2.27 ± 0.62 and 3.16 ± 0.78 cfu/g; respective values of rabbits that started dying late (that is, after 72 hours) were below the limit of detection. Mean circulating LPS at 24 hours was 2.09 EU/ ml and 1.99 EU/ml respectively (P = NS). Mean LPS of the portal vein of the sham and of the injury groups were 1.25 and 5.62 EU/ml (P = 0.047). Concentrations of TNFα in splenocyte supernatants are shown in Figure 1. However, in a small subset of patients the infection rapidly progressed to primary viral pneumonia (PVP) and a minority of them developed ARDS. Inherited and acquired variability in host immune responses may infl uence susceptibility and outcome of IAV infection. However, the molecular nature of such human factors remains largely elusive. p y yi Results Fifty percent of rabbits died early; that is, within the fi rst 48 hours. Mean ± SE log10 bacteria in the liver and lung of animals that died early was 2.27 ± 0.62 and 3.16 ± 0.78 cfu/g; respective values of rabbits that started dying late (that is, after 72 hours) were below the limit of detection. Lethal infl uenza virus A H1N1 infection in two relatives with autosomal dominant GATA-2 defi ciency Mean circulating LPS at 24 hours was 2.09 EU/ ml and 1.99 EU/ml respectively (P = NS). Mean LPS of the portal vein of the sham and of the injury groups were 1.25 and 5.62 EU/ml (P = 0.047). Concentrations of TNFα in splenocyte supernatants are shown in Figure 1. y Methods We report three adult relatives with the autosomal dominant GATA-2 defi ciency. P1 and his son P2 had a history of myelodysplastic syndrome and a few episodes of mild respiratory infections. They developed PVP by H1N1pdm which rapidly evolved to ARDS. They died at the age of 54 and 31, respectively. y Results Patients were heterozygous for a novel R396L mutation in GATA2. Like other patients with GATA-2 defi ciency, the three relatives had absence of peripheral NK and B cells and monocytopenia. However a high number of plasma cells, which were found to be pauciclonal, were observed in peripheral blood from P1 during H1N1pdm infection. P1 and P2 had normal levels of immunoglobulins and IgG antibodies against common viruses. Microneutralization test showed that P1 produced normal titers of neutralizing antibodies against H1N1pdm and against the previous annual H1N1 strain. Our results suggest that a few clones of long-living memory B cells against IAV expanded in P1; and that these cells produced cross-reactive antibodies against H1N1pdm, similar to those recently described. During the fl u episode P1 had a strong increase of IFNγ-producing T cells and of IFNγ production. The Th1-related chemokines CXCL10 and CXCL9, as well as IFNγ, MCP-1 and IL-8, were strongly elevated in serum from P1 and P2 in the course of H1N1pdm infection. g Conclusion Early death after injury is not related to peripheral endotoxemia and sepsis; bacterial translocation priming for enhanced proinfl ammatory responses is a likely explanation. Eff ects of the common 34C>T variant of the AMPD1 gene on immune function, multiorgan dysfunction and mortality in patients with sepsis Eff ects of the common 34C>T variant of the AMPD1 gene on immune function, multiorgan dysfunction and mortality in patients with sepsis B Ramakers1, E Giamarellos-Bourboulis2, M Coenen1, M Kox1, J Van der Hoeven1, C Routsi2, A Savva2, I Perdios3, F Diamantea4, D Sinapidis5, P Smits1, N Riksen1, P Pickkers1 1Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands; 2University of Athens, Medical School, Athens, Greece; 3Nafplion General Hospital, Nafplio, Greece; 4‘G.Gennimatas’ General Hospital, Athens, Greece; 5Alexandra General Hospital, Athens, Greece Critical Care 2013, 17(Suppl 2):P17 (doi: 10.1186/cc11955) Conclusion GATA-2 defi ciency is the fi rst described Mendelian inborn error of immunity underlying severe IAV infection. Primary immunodefi ciencies predisposing to severe IAV infections may debut, even in adults without a history of previous severe infections. The massive IFNγ-mediated cytokine storm may explain the fatal course of H1N1pdm infection in our patients. Introduction Adenosine exerts anti-infl ammatory and tissue protective eff ects during systemic infl ammation. While the anti-infl ammatory properties may induce immunoparalysis and impede bacterial clearance, the tissue protective eff ects might limit organ damage. The eff ects of a common loss-of-function variant of the adenosine monophosphate deaminase 1 gene (AMPD1), which is associated with increased adenosine formation, in patients with sepsis are unknown. Methods In a prospective cohort, genetic-association study, the eff ects of the presence of the AMPD1 gene on immune function, multiorgan dysfunction and mortality in septic patients was studied. Pneumosepsis patients (n = 402) and controls without infection (n = 101) were enrolled. P16 P16 Bacterial translocation primes proinfl ammatory responses and is connected to early death in an experimental model of lethal injury N Baxevanos, T Tsaganos, A Pistiki, D Droggiti, A Spyridaki, E Giamarellos-Bourboulis University of Athens, Medical School, Athens, Greece Critical Care 2013, 17(Suppl 2):P16 (doi: 10.1186/cc11954) P18 P18 Exploring the translational disconnect between the murine and human infl ammatory response: in vitro analysis of the dose– response relationship of LPS and NFκB activation in murine and human immune cells EP McCarron, I Welters, D Williams, D Antoine, A Kipar University of Liverpool, UK Critical Care 2013, 17(Suppl 2):P18 (doi: 10.1186/cc11956) Introduction Infl ammation, as seen in sepsis and systemic infl ammatory response, is dependent on the activation of the NFκB pathway through Toll-like receptors (TLRs) [1]. Recreating an infl ammatory response using lipopolysaccharide (LPS) can provide results that are diff erent to clinical sepsis [2]. By examining NFκB activation in murine and human cells, a species comparison can be made to investigate diff erences at the cell level that may contribute to the translational disconnect seen in vivo. Methods In the prospective, noninterventional study, we assessed the correlation between the capillary leakage marker angiopoetin-2 and serum levels of IgG and IgM in 41 patients with community-acquired severe sepsis or septic shock on admission. Blood samples were obtained during the fi rst 12 hours after admission to hospital. Results Mean age of patients (17 females) was 70 years. Median APACHE II and SOFA scores at admission were 24 and 11, respectively. The mortality rate was 45%. Thirty-four percent of all patients had level of IgG <650 mg/dl. The median concentration of angiopoietin-2 in the hypo-IgG group was 11,958 pg/ml, which was not statistically diff erent (Mann–Whitney; P >0.05) than in the rest of patients with normal levels of IgG (15,688 pg/ml). The concentration of IgM <40 mg/dl was found in only four patients (10%) and all died. Pearson’s correlation test showed that the correlation between the concentrations of angiopoietin-2 and IgG (correlation coeffi cient 0.191) or IgM (correlation coeffi cient 0.0408), respectively, were not statistically signifi cant (P <0.05). Methods THP1 human monocytes (passages 9 to 11) and RAW 264.7 murine macrophages (passages 15 to 20) were cultured in RPMI-1640 and DMEM respectively and then challenged with LPS. After settling for 24 hours, cells were dosed with six or seven doses of LPS. After 1 hour, nuclear extraction and proteins were separated by acrylamide gel electrophoresis. Membranes where then immunoblotted for actin and p65, followed by densitometric analysis in order to quantify the amount of p65 that had translocated from the cytoplasm to the nucleus (by subtraction from consistent nuclear actin). Bacterial translocation primes proinfl ammatory responses and is connected to early death in an experimental model of lethal injury N Baxevanos, T Tsaganos, A Pistiki, D Droggiti, A Spyridaki, y , , , itical Care 2013, 17(Suppl 2):P16 (doi: 10.1186/cc11954) Introduction Some cases of multiple trauma are rapidly deteriorating; the mechanism was investigated. Introduction Some cases of multiple trauma are rapidly deteriorating; the mechanism was investigated. Results In pneumosepsis patients and controls, a similar prevalence of the 34C>T (rs17602729) mutation in the AMPD1 gene was found. Univariate logistic regression analysis showed a tendency of increased mortality in patients with the CT genotype, compared with patients with the CC genotype (OR 1.53; 95% CI 0.95 to 2.5). Moreover, carriers of the CT genotype tended to suff er more from multiorgan dysfunction, OR 1.4 (0.84 to 2.3) and 3.0 (0.66 to 13.8), for CT and TT, respectively (P = 0.07). In septic carriers of the CT genotype, the ex vivo production of TNFα by LPS-stimulated monocytes was attenuated (P = 0.005), Methods Forty-one rabbits were assigned into two groups; sham- operated and subject to crush of the right femur. Survival was recorded; peripheral blood was sampled for LPS measurement by the kinetic QCL-1000 LAL assay; quantitative tissue growth was assessed after death. Some rabbits were sacrifi ced at 48 hours; blood was sampled from the portal vein for LPS measurement; splenocytes were Figure 1 (abstract P16). Stimulation of TNFα from isolated splenocytes. y y Figure 1 (abstract P16). Stimulation of TNFα from isolated splenocytes. Figure 1 (abstract P17). Kaplan–Meier curve for the 402 sepsis patients. Figure 1 (abstract P17). Kaplan–Meier curve for the 402 sepsis patients. Figure 1 (abstract P17). Kaplan–Meier curve for the 402 sepsis patients. Figure 1 (abstract P17). Kaplan–Meier curve for the 402 sepsis patients. S7 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 indicative for more pronounced immunoparalysis in these patients. See Figure 1. P18 i Conclusion At present the hypothesis that increased microvascular leakage is responsible for hypogammaglobulinemia in septic patients could not be accepted. Studies on larger number of patients are needed. In addition, it is necessary to further explore other possible mechanisms, such as increased catabolism and consumption of antibodies or inadequate synthesis of immunoglobulins, which could also be responsible for hypogammaglobulinemia in sepsis. References y Results Murine cells required higher doses of LPS compared with human cells in order to detect p65 (human, 1 pg/ml to 100 ng/ml; murine, 30 pg/ml to 1,000 ng/ml). THP1 cells showed a greater fold increase in the p65:actin ratio compared with RAW 264.7 cells. Human cells responded to lower concentrations of LPS. Murine cells appeared to show a molecular resistance to lower doses, but their response was very sensitive at higher doses. A dose–response relationship of LPS dosing and NFκB activation was observed in both cell lines. P20 P20 Hypogammaglobulinemia in sepsis is not correlated to high circulating angiopoietin-2 levels Conclusion The presence the 34C>T variant of the AMPD1gene is not related to infection susceptibility; however, it is associated with more pronounced immunoparalysis in patients with sepsis, and shows a tendency towards increased mortality. Mechanistically, the anti- infl ammatory eff ects of adenosine may account for this and apparently overrule its tissue protective eff ects. U Kovačič1, F Starič2, M Kmet2, M Godnič2, R Kapš2 1Faculty of Medicine, University of Ljubljana, Slovenia; 2General Hospital Novo Mesto, Slovenia Critical Care 2013, 17(Suppl 2):P19 (doi: 10.1186/cc11957) Introduction Hypogammaglobulinemia has been frequently found in adult patients with severe sepsis and septic shock. Furthermore, it seems that at least a low serum level of IgM is correlated with higher mortality in sepsis. The mechanisms of hypogammaglobulinemia in septic shock have not yet been explained. It has been hypothesized that outfl ow of immunoglobulins into the extravascular space due to increased capillary permeability could reduce immunoglobulin serum concentrations. Angiopoietin-2, which directly disrupts the endothelial barrier, is markedly elevated in sepsis and other infl ammatory states and its serum level has been correlated with microvascular leakage, end-organ dysfunction and death in sepsis. TREM-1 levels are elevated in patients with liver cirrhosis SD G 1 CR G h 2 P K ll h 2 N S i1 M F t 1 S Si h 1Chelsea and Westminster Hospital, London, UK; 2Imperial College, London, UK Critical Care 2013, 17(Suppl 2):P20 (doi: 10.1186/cc11958) 1Chelsea and Westminster Hospital, London, UK; 2Imperial College, London, UK Critical Care 2013, 17(Suppl 2):P20 (doi: 10.1186/cc11958) References 1. Taccone FS, et al.: Gamma-globulin levels in patients with community- acquired septic shock. Shock 2009, 32:379-385. 1. Taccone FS, et al.: Gamma-globulin levels in patients with community- acquired septic shock. Shock 2009, 32:379-385. 2. Werdan K, et al.: Score-based immunoglobulin G therapy of patients with sepsis: the SBITS study. Crit Care Med 2007, 35:2693-2701. g Conclusion Immunoblotting for p65 is a reliable and reproducible method to determine NFκB activation in cultured cells. Macrophages are more responsive to LPS than monocytes [3] so diff erences between cell lines would have been expected to be the reverse of what was observed. The species diff erence in response to LPS may contribute to the apparent disconnect between human and murine responses to LPS and may partially explain the diffi culties of translating therapeutic interventions into clinical human sepsis. 2. Werdan K, et al.: Score-based immunoglobulin G therapy of patients with sepsis: the SBITS study. Crit Care Med 2007, 35:2693-2701. P22 Introduction Septic encephalopathy is a frequent complication in severe sepsis but its pathogenesis and mechanisms are not fully understood. Oxygen supply and utilization are critical for organ function, especially for the brain, a tissue extremely dependent on oxygen and glucose. Disturbances in oxygen utilization are common in sepsis and a number of mitochondrial dysfunctions have been described in diff erent tissues in septic animals as well as in septic patients. Our group described mitochondrial dysfunctions in the brain during experimental sepsis. l Results TREM-1 expression is signifi cantly higher in the CA group compared with HC across all monocyte subsets but not neutrophils, even in the absence of sepsis (see Figure 1). There is no correlation between cell surface TREM-1 expression and severity of cirrhosis by Child–Pugh or MELD score. sTREM and sCD14 levels were elevated in the CA group compared with HC (P = 0.0010 and 0.0016 respectively). In addition, plasma sTREM-1 levels correlated with disease severity according to MELD score (R = 0.71, CI = 0.22 to 0.92 P = 0.012) and serum bilirubin (R = 0.78,CI = 0.36 to 0.94, P = 0.004). There was no correlation with either form of TREM-1 with sCD14 levels. There was no diff erence in cell surface or soluble TREM-1 expression between blood and ascitic fl uid monocytes in culture-negative, non-neutrophilic ascites. Methods Experimental sepsis was induced by endotoxemia (LPS 10 mg/ kg i.p.) in Sprague–Dawley rats and by polymicrobial fecal peritonitis in Swiss mice. Brain glucose uptake was observed in vivo in endotoxemic rats using positron emission tomography with [18F]fl uorodeoxyglucose and autoradiography with 2-deoxy-14C-glucose. Results Mice with polymicrobial sepsis present hypoglycemia, hyperlactatemia and long-term cognitive impairment. We observed a rapid increase in the uptake of fl uorescent glucose analog 2-deoxy- 2-((7-nitro-2,1,3-benzoxadiazol-4-yl)amino)-D-glucose in brain slices from septic mice in vitro. A similar increase in brain glucose uptake was observed in vivo in endotoxemic rats. Remarkably, the increase in glucose uptake started 2 hours after LPS injection, earlier than other organs. The brains of mice with experimental sepsis presented neuroinfl ammation, mitochondrial dysfunctions and oxidative stress, but mitochondria isolated from septic brains generated less ROS in vitro in the fi rst 24 hours. This led us to investigate the role of NADPH oxidase, an enzyme induced during innate immune response, as a potential source of reactive oxygen species in experimental sepsis. 1. Olson JC, et al.: Intensive care of the patient with cirrhosis. Hepatology 2011, 54:1864-1872. P22 Inhibiting NADPH oxidase with apocynin acutely after sepsis prevented cognitive impairment in mice. l Conclusion Blood monocyte and soluble TREM-1 are elevated in cirrhotic patients even in the absence of sepsis. Soluble TREM-1 levels correlate with disease severity in cirrhosis. Further studies are ongoing to ascertain the utility of TREM-1 as a biomarker in cirrhosis. Reference 1. Olson JC, et al.: Intensive care of the patient with cirrhosis. Hepatology 2011, 54:1864-1872. g p Conclusion Our data indicate that a bioenergetic imbalance and oxidative stress is associated with the pathophysiology of septic encephalopathy. We are observing a new metabolic phenotype in the brain during sepsis, characterized by a rapid increase in glucose uptake and mitochondrial dysfunctions that may be secondary to infl ammation and hypoxia. P21 Eff ects of diff erent doses and serotypes of LPS on blood–brain barrier permeability in Sprague–Dawley rats E Senturk, F Esen, P Ergin Ozcan, G Orhun, N Orhan, N Arican, M Kucuk, M Kaya University of Istanbul, Turkey Critical Care 2013, 17(Suppl 2):P21 (doi: 10.1186/cc11959) University of Istanbul, Turkey Introduction The blood–brain barrier (BBB) is highly restrictive of the transport of substances between blood and the central nervous system. Lipopolysaccharide (LPS) from Gram-negative bacteria was reported to aff ect the permeability of the BBB. BBB disruption using a LPS is used as a model of septic encephalopathy in mice. The present study was designed to investigate the eff ects of diff erent doses and serotypes of LPS on BBB integrity in Sprague–Dawley rats. Pathophysiology of sepsis-associated brain dysfunction: an experimental study of cerebral microdialysis and mitochondrial function P Kurtz1, C Vargas-Lopes1, C Madeira1, I Mello1, R Panizzutti1, L C Azevedo2, F A Bozza1 1Fiocruz, Rio de Janeiro, Brazil; 2Hospital Sirio e Libanes, São Paulo, Brazil Critical Care 2013, 17(Suppl 2):P23 (doi: 10.1186/cc11961) P22 to the ICU have an in-hospital mortality of up to 50% [1]. Microbial translocation (MT) is the pathogenic mechanism implicated in SBP. The triggering receptor expressed by myelocytes-1 (TREM-1) modulates the immune response with resultant production of proinfl ammatory cytokines and has been used as a biomarker in the diagnosis of bacterial infection. We wish to evaluate the role of TREM-1 as a biomarker in cirrhosis. Bioenergetic imbalance and oxidative stress in the pathophysiology of septic encephalopathy J D’Avila1, R Rodrigues2, H Castro-Faria-Neto1, M Oliveira3, F Bozza4 1Oswaldo Cruz Foundation – FIOCRUZ, Rio de Janeiro, Brazil; 2Federal University of Rio de Janeiro (UFRJ) and D’Or Institute for Research and Education (IDOR), Rio de Janeiro, Brazil; 3UFRJ, Rio de Janeiro, Brazil; 4Oswaldo Cruz Foundation – FIOCRUZ and IDOR, Rio de Janeiro, Brazil Critical Care 2013, 17(Suppl 2):P22 (doi: 10.1186/cc11960) Bioenergetic imbalance and oxidative stress in the pathophysiology of septic encephalopathy J D’Avila1, R Rodrigues2, H Castro-Faria-Neto1, M Oliveira3, F Bozza4 1Oswaldo Cruz Foundation – FIOCRUZ, Rio de Janeiro, Brazil; 2Federal University of Rio de Janeiro (UFRJ) and D’Or Institute for Research and Education (IDOR), Rio de Janeiro, Brazil; 3UFRJ, Rio de Janeiro, Brazil; 4Oswaldo Cruz Foundation – FIOCRUZ and IDOR, Rio de Janeiro, Brazil Critical Care 2013, 17(Suppl 2):P22 (doi: 10.1186/cc11960) Methods Blood samples were obtained from 18 healthy controls (HC) and 29 cirrhotic patients (CA) as defi ned by clinicoradiological criteria. Disease severity was graded according to Child–Pugh class (median 10, range 5 to 13) and modifi ed end-stage liver disease (MELD) score (median 14, range 6 to 21). Simultaneous ascitic fl uid samples were taken from 10 patients in the CA group. Soluble TREM-1 and CD14 levels (a surrogate marker of MT) were measured by ELISA. Flow cytometry was used to quantify the expression of TREM-1 on monocytes and neutrophils in blood and ascitic fl uid.i Introduction Septic encephalopathy is a frequent complication in severe sepsis but its pathogenesis and mechanisms are not fully understood. Oxygen supply and utilization are critical for organ function, especially for the brain, a tissue extremely dependent on oxygen and glucose. Disturbances in oxygen utilization are common in sepsis and a number of mitochondrial dysfunctions have been described in diff erent tissues in septic animals as well as in septic patients. Our group described mitochondrial dysfunctions in the brain during experimental sepsis. References 1. Bonizzi G, Karin M: Trends Immunol 2004, 25:280-288. 1. Bonizzi G, Karin M: Trends Immunol 2004, 25:280-288. Introduction Sepsis and spontaneous bacterial peritonitis (SBP) are common sequelae in patients with cirrhosis. Cirrhotics admitted 2. Remick DG, Ward PA: Shock 2005, 24(Suppl 1):7-11. pp 3. Takashiba S, et al.: Infect Immun 1999, 67(11):5573-5578. Figure 1 (abstract P20). TREM-1 expression in healthy controls compared with cirrhotic patients. cirrhotic patients. Figure 1 (abstract P20). TREM-1 expression in healthy controls compared with cirrhotic patients. Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S8 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Cholinergic modulation of hippocampal activity during septic encephalopathy 2 2 Cholinergic modulation of hippocampal activity during septic encephalopathy A Zivkovic1, CP Bengtson2, O Sedlaczek1, R Von Haken1, H Bading2, S Hofer1 1Universitätsklinikum Heidelberg, Germany; 2Universität Heidelberg, Germany Critical Care 2013, 17(Suppl 2):P24 (doi: 10.1186/cc11962) A Zivkovic1, CP Bengtson2, O Sedlaczek1, R Von Haken1, H Bading2, S Hofer1 1Universitätsklinikum Heidelberg, Germany; 2Universität Heidelberg, Germany Critical Care 2013, 17(Suppl 2):P24 (doi: 10.1186/cc11962) Introduction Septic encephalopathy is a sepsis-related brain dys- function with a deterioration of cortical functions. The experimental studies in the rat brain revealed a deranged neurotransmitter profi le during septic encephalopathy. Glutamatergic synapses, essential in learning and memory, undergo use-dependent changes in synaptic strength, referred to as plasticity. Permanent strengthening of synapses after a brief stimulus, termed long-term potentiation (LTP), was discovered in the hippocampus, and here it has been most thoroughly studied. Cholinergic neurotransmission plays an important role in regulating the cognitive functions of the brain. It acts as a signal-to- noise ratio modulator of sensory and cognitive inputs. The irregularities in brain functions give rise to the symptoms of delirium, including disorganized thinking and disturbances of attention and consciousness, which in turn might aff ect learning and memory. Possible mechanisms for cholinergic defi ciency include impairment of synaptic functions of acetylcholine. Imbalances in the cholinergic system during sepsis might therefore play an extensive role in the septic delirium. Results Of the 1,600 patients who gave consent to participate in the study, 205 had an ECI. Sixty-four had a positive blood culture for E. coli. Fifty of the patients met one or more criteria for severe sepsis or septic shock. The NLCR was signifi cantly higher (P <0.001) within the severe sepsis group (median = 21.1 with quartiles 11.1 to 42.4) compared with the group with no severe sepsis (median = 11.6 with quartiles 7.6 to 18.9). Conclusion The NLCR can be used as a biomarker of disease severity even in ECIs. The biomarker reacts rapidly, is cheap and needs no extra sampling. The higher the value, the higher the probability for severe sepsis. A high value can even precede the development of severe sepsis or septic shock. However, a low value never excludes neither bacteremia nor severe sepsis. The method cannot be used in patients with disturbances in neutrophil or lymphocyte levels due to other causes than sepsis. Methods By using MRI imaging we identifi ed functional changes in the hippocampal region of patients with severe sepsis. Pathophysiology of sepsis-associated brain dysfunction: an experimental study of cerebral microdialysis and mitochondrial function P Kurtz1, C Vargas-Lopes1, C Madeira1, I Mello1, R Panizzutti1, L C Azevedo2, F A Bozza1 1Fiocruz, Rio de Janeiro, Brazil; 2Hospital Sirio e Libanes, São Paulo, Brazil Critical Care 2013, 17(Suppl 2):P23 (doi: 10.1186/cc11961) y y y Methods Male Sprague–Dawley rats weighing 200 to 250 g were used in the study. Rats were given two diff erent types of LPS (026:B6- L5543 and 026:B6-L2762) in diff erent doses (3, 5, and 10 mg/kg; i.v.). Rectal temperature and arterial blood pressure measurements were recorded for sepsis severity. The changes in the BBB permeability were measured using the Evans blue (EB) and sodium fl uorescein (NaFl) dye extravasation techniques 24 hours after LPS administration. Introduction Pathophysiology of brain dysfunction associated with sepsis is still poorly understood. Potential mechanisms involve oxidative stress, neuroinfl ammation and blood–brain barrier altera- tions. Our purpose was to study the metabolic alterations and markers of mitochondrial dysfunction in a clinically relevant model of septic shock. Results Both LPS serotypes showed comparable arterial blood pressure and rectal temperature recordings and the severity of the disease increased with the increasing doses (5 mg/kg and 10 mg/kg) of LPS and the mortality rates were found to be 29% and 63% respectively. The extravasated contents of EB and NaFl tracers did not signifi cantly increase in brain parenchyma following the administration of diff erent doses of LPS with diff erent serotypes. Methods Twelve anesthetized (midazolam/fentanyl/pancuronium), invasively monitored, and mechanically ventilated pigs were allocated to a sham procedure (n = 5) or sepsis (n = 7), in which peritonitis was induced by intra-abdominal injection of autologous feces. Animals were studied until spontaneous death or for a maximum of 24 hours. In addition to global hemodynamic and laboratory assessment, intracranial pressure and cerebral microdialysis were assessed at baseline, 6, 12, 18 and 24 hours after sepsis induction. After death, brains were removed and brain homogenates were studied to assess markers of mitochondrial dysfunction. f Conclusion Our results showed no disruption to BBB by two diff erent serotypes of LPS even administered in increasing doses. These result indicate that the BBB integrity of Sprague–Dawley rats are resistant to the eff ects of two diff erent serotypes of LPS. Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S9 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Figure 1 (abstract P23). Pathophysiology of sepsis-associated brain dysfunction: an experimental study of cerebral microdialysis and mitochondrial function Results Critically ill ICU patients diagnosed with septic delirium using the CAM-ICU method underwent diagnostic MRI scans. The serial MRI analysis revealed increased signal intensity in the hippocampal region in diff usion-weighted MRI (DWI). We used endotoxemia model to induce sepsis in the rats. Electrophysiological analysis of the hippocampal neurons in LPS-treated rats showed impaired LTP in the excitatory synapses, as compared with controls. Application of physostigmine, a blood–brain barrier permeable cholinesterase inhibitor, resulted in a partial recovery of LTP in the hippocampal synapses of LPS-treated rats. Conclusion The patients with septic delirium show functional changes in the hippocampus. Furthermore, we show that endotoxemia aff ects synaptic plasticity in the rat hippocampus, suggesting the involvement of this brain region in the pathophysiology of septic delirium. Moreover, the eff ect of the cholinergic neurotransmission onto the induction and maintenance of synaptic plasticity in the rat hippocampus during endotoxemia suggests that cholinergic neurotransmission might play a critical role in septic encephalopathy. Results All septic animals developed a hyperdynamic state associated with lower arterial pressure, fever and organ dysfunction in comparison with control animals. In the septic animals, we observed increased brain dialysate glutamin levels at 12, 18 and 24 hours after sepsis induction, as compared with control animals. Moreover, after analyzing homogenates from the frontal cortex, we found higher concentrations of glutamin and glutamate in septic as compared with control animals (85.67 ± 14.98 vs. 28.77 ± 7.0; P = 0.01 and 132.1 ± 19.72 vs. 53.33 ± 16.83; P = 0.02, respectively). See Figure 1. Results All septic animals developed a hyperdynamic state associated with lower arterial pressure, fever and organ dysfunction in comparison with control animals. In the septic animals, we observed increased brain dialysate glutamin levels at 12, 18 and 24 hours after sepsis induction, as compared with control animals. Moreover, after analyzing homogenates from the frontal cortex, we found higher concentrations of glutamin and glutamate in septic as compared with control animals (85.67 ± 14.98 vs. 28.77 ± 7.0; P = 0.01 and 132.1 ± 19.72 vs. 53.33 ± 16.83; P = 0.02, respectively). See Figure 1. P25 Cholinergic modulation of hippocampal activity during septic encephalopathy A Zivkovic1, CP Bengtson2, O Sedlaczek1, R Von Haken1, H Bading2, S Hofer 1Universitätsklinikum Heidelberg, Germany; 2Universität Heidelberg, Germany Critical Care 2013, 17(Suppl 2):P24 (doi: 10.1186/cc11962) de Jager C, et al.: The neutrophil–lymphocyte count ratio in patients with community acquired pneumonia. PLoS ONE 2012, 7:e46561. Cholinergic modulation of hippocampal activity during septic encephalopathy 2 2 This fi nding was further supported by the experimental recordings in the rat brains of lipopolysaccharide (LPS)-treated rats using the electrophysiological patch clamp technique. P25 P25 Neutrophil–lymphocyte count ratio as a biomarker of severe sepsis in Escherichia coli infections in adults LR Ljungstrom1, G Jacobsson1, R Andersson2 1Skaraborgs Sjukhus, Skövde, Sweden; 2Gothenburg University, Gothenburg, Sweden Critical Care 2013, 17(Suppl 2):P25 (doi: 10.1186/cc11963) P25 Neutrophil–lymphocyte count ratio as a biomarker of severe sepsis in Escherichia coli infections in adults LR Ljungstrom1, G Jacobsson1, R Andersson2 1Skaraborgs Sjukhus, Skövde, Sweden; 2Gothenburg University, Gothenburg, Sweden Critical Care 2013, 17(Suppl 2):P25 (doi: 10.1186/cc11963) Neutrophil–lymphocyte count ratio as a biomarker of severe sepsis in Escherichia coli infections in adults LR Ljungstrom1, G Jacobsson1, R Andersson2 Critical Care 2013, 17(Suppl 2):P25 (doi: 10.1186/cc11963) Introduction The neutrophil–lymphocyte count ratio (NLCR) is an easy to analyse biomarker reacting very early in the course of acute infl ammation. It has previously been reported to correspond to bacteremia and recently to disease severity in community-acquired pneumonia [1]. We have looked at 205 consecutive patients with Escherichia coli infections (ECI) and found the same to be true for ECIs. This may be of great clinical importance since E. coli is the most frequently isolated pathogen in patients with infections requiring in hospital care. Conclusion We found higher concentrations of glutamate and glutamin in brain tissues of septic animals as compared with control. Furthermore, glutamin concentrations increased over time in the extracellular space as measured by cerebral microdialysis. These fi ndings suggest an increased excitatory state that is potentially associated with high energy expenditure. However, associations with neuronal injury need further study. Methods This study is part of a 9-month consecutive study of community-acquired severe sepsis and septic shock in adults at Skaraborg Hospital in the western region of Sweden. The hospital serves a population of 256,000 inhabitants and has approximately 60,000 annual visits to the ED. All patients admitted to the hospital receiving intravenous antibiotic treatment within the fi rst 48 hours of admission were evaluated for severe sepsis and septic shock. Upon admission, two sets of blood cultures and other relevant cultures were obtained from each patient as well as sampling for NLCR and venous plasma lactate. The patient records were evaluated by one infectious diseases specialist. Approximately 2,300 patients were diagnosed as having a bacterial infection. From those, an informed consent to participate in the study could be obtained from approximately 1,600 patients. Reference de Jager C, et al.: The neutrophil–lymphocyte count ratio in patients with community acquired pneumonia. PLoS ONE 2012, 7:e46561. de Jager C, et al.: The neutrophil–lymphocyte count ratio in patients with community acquired pneumonia. PLoS ONE 2012, 7:e46561. S10 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Figure 1 (abstract P26). P26 Obesity and infl ammatory markers in severe sepsis P Simon1, D Thomas-Rüddel2, T Nemes1, K Reinhart2, F Bloos2, UX Kaisers1 1University Hospital of Leipzig, Germany; 2Center for Sepsis Control and Care, Jena, Germany Critical Care 2013, 17(Suppl 2):P26 (doi: 10.1186/cc11964) Introduction Chronic infl ammation has recently been recognized as an important factor in the pathophysiology of obesity and associated morbidities [1]. In this clinical study we aimed at identifying possible eff ects of obesity on infl ammatory markers in severe sepsis. fl Methods With institutional ethical committee approval, 243 consecutive patients treated for severe sepsis or septic shock in the ICUs of two university hospitals over a period of 5 months were studied. Six patients were excluded due to cachexia, syndromal disorders or missing clinical data. Diagnosis of sepsis was made according to SCCM criteria. Serum levels of C-reactive protein (CRP, mg/l) and procalcitonin (PCT, ng/ml) on day 1 of sepsis were compared among fi ve body mass index (BMI) strata according to WHO defi nitions. Two groups (BMI <30, normal weight, and BMI ≥30, obesity) were formed for further analysis, and PCT was logarithmically transformed (LogPCT), resulting in normal distribution. Statistical analysis was performed using a t test. y Results Patients with BMI ≥30 had higher values of PCT and CRP (Table 1). The diff erence in LogPCT was of borderline signifi cance (P = 0.052). However, patients with positive blood cultures had signifi cantly higher LogPCT values (P = 0.017) (Figure 1). Diff erence in CRP was not signifi cant (P = 0.09). The trends over all fi ve BMI strata (Table 1) were not signifi cant. P27 Cytokine gene expression can predict infectious complications following severe trauma HD Torrance, K Brohi, CJ Hinds, MJ O’Dwyer Bart’s Health NHS Trust, London, UK Critical Care 2013, 17(Suppl 2):P27 (doi: 10.1186/cc11965) Cytokine gene expression can predict infectious complications following severe trauma HD Torrance, K Brohi, CJ Hinds, MJ O’Dwyer Bart’s Health NHS Trust, London, UK Critical Care 2013, 17(Suppl 2):P27 (doi: 10.1186/cc11965) gi Conclusion Obesity with BMI ≥30 seems to be associated with an increase in infl ammatory markers in patients with severe sepsis, particularly in bacteraemia. The role of adipose tissue in severe sepsis should therefore be studied in more detail. 1. Wellen K, et al.: Infl ammation, stress, and diabetes. J Clin Invest 2005, 115:1111-1119. 1. Wellen K, et al.: Infl ammation, stress, and diabetes. J Clin Invest 2005, 115:1111-1119. Introduction Identifying a group of patients at high risk of developing infectious complications is the fi rst step in the introduction of eff ective pre-emptive therapies in specifi c patient groups. Quantifying cytokine gene expression also furthers our understanding of trauma-induced immunosuppression. Our group has already demonstrated that a predictive immunological signature derived from mRNA expression in elective thoracic surgical patients accurately predicts pneumonia risk [1]. Methods In total, 121 ventilated polytrauma patients were recruited. mRNA was extracted from PaxGene tubes collected within 2 hours of the initial insult, at 24 and 72 hours. T-helper cell subtype specifi c cytokines and transcription factors mRNA was quantifi ed using qPCR. Ten healthy controls served as a comparator. Table 1 (abstract P26). PCT and CRP as median (IQR) BMI n PCT (ng/ml) CRP (mg/l) 18.5 to 24.9 196 4.8 (11.9) 153 (189) ≥30.0 47 7.3 (24.6) 228 (254) 18.5 to 24.9 101 4.9 (11.7) 147 (175) 25 to 29.9 95 4.4 (12.0) 157 (218) 30 to 34.9 32 6.9 (22.6) 212 (118) 35 to 39.9 7 7.4 (38.3) 241 (264) ≥40.0 8 11.4 (32) 278 (272) y p Results The Median Injury Severity Score (ISS) was 29. Time 0 bloods demonstrated a reduction in TNFα†, IL-12§, IL-23‡, RORγT and T bet§, and an increase in IL-10* and IL-4† mRNA levels in comparison with the control group (P <0.0001, †P <0.001 to 0.0001, ‡P <0.01 to 0.001, §P <0.05 to 0.01). There was a positive correlation between ISS and IL-10‡ whilst both IL-23§ Figure 1 (abstract P27). Cytokine mRNA levels in trauma (time 0) and control groups. P27 Cytokine gene expression can predict infectious complications following severe trauma HD Torrance, K Brohi, CJ Hinds, MJ O’Dwyer Bart’s Health NHS Trust, London, UK Critical Care 2013, 17(Suppl 2):P27 (doi: 10.1186/cc11965) S11 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Methods From November 2010 to November 2011 we collected clinical data, drug administration, scores and PCT values of 420 consecutive patients during hospitalization. Statistical analysis was made using SPSS software. We calculated ICU mortality, 1-month mortality and 1-year mortality. Median percentage daily variation was calculated as: (PCT day after – PCT of the date value) / PCT of the date value×100. PCT variation in the last 48 hours of hospitalization was calculated as: (PCT at discharge – PCT at 48 hours before discharge) / PCT 48 hours before discharge×100. We compared peak values in dead patients versus alive patients. A logistic regression was performed in order to assess mortality odds ratio. and RORγT‡ were negatively correlated at time 0. TNFα†, IL-10 and IL-27‡ increased and IFNγ†, IL-12, IL-17A§, RORγT and T bet* mRNA levels decreased over the initial 24 hours. Subsequent bacteraemia (18/121 patients) was associated with a lower TNFα/IL-10 ratio‡ at baseline. Similarly, higher IL-10‡ and lower T bet‡ mRNA at 24 hours also predicted later bacteraemic episodes. Development of pneumonia followed a similar pattern. A multivariate logistical regression model proved highly accurate in predicting infectious complications from mRNA analysis of early blood samples. See Figure 1. y Conclusion Cytokine gene expression patterns indicate an immediate and sustained impairment in Th1, Th17 and innate immunity with concurrent upregulation of the Th2 response following major trauma. The magnitude of this response predicts subsequent infectious complications. y Results Of the 420 patients, 63 (15%) died in the ICU, 12 (2.86%) died 1 month after ICU discharge and 16 (3.80%) died 1 year after ICU discharge. PCT values were higher during the last day of hospitalization in dead patients versus alive patients. PCT percentage variation during the last 48 hours of hospitalization had a slower trend in patients who died than in those who survived; these diff erences are even more marked in patients who had a septic event. A slower descending trend of daily PCT values was found in patients who died than in those who survived. PCT peak levels during the ICU stay were higher in dead patients with respect to alive ones. P27 Cytokine gene expression can predict infectious complications following severe trauma HD Torrance, K Brohi, CJ Hinds, MJ O’Dwyer Bart’s Health NHS Trust, London, UK Critical Care 2013, 17(Suppl 2):P27 (doi: 10.1186/cc11965) At logistic regression analysis PCT decrease in the last 48 hours <–30% (OR 3.71), PCT peak higher than 10 ng/ml (OR 2.38), and PCT last day/PCT peak ratio >50% (OR 2.064) were ICU mortality risk factors. PCT values were a higher predictive ICU mortality risk factor than SOFA and APACHE II scores. Other prognostic factors were age and lactate values. Only age was a risk factor in 1-month and 1-year mortality. References 1. Jensen JU, Heslet L, Jensen TH, et al.: Procalcitonin increase in early identifi cation of critically ill patients at high risk of mortality. Crit Care Med 2006, 34:2596-2602. 2. Fritz HG, Brandes H, Bredle DL, et al.: Post-operative hypoalbuminaemia and procalcitonin elevation for prediction of outcome in cardiopulmonary bypass surgery. Acta Anaesthesiol Scand 2003, 47:1276-1283. Results We analyzed 150 consecutive episodes of severe sepsis (16%) or septic shock (84%) in the ICU. The median age of the patients was 64 (interquartile range, 48.7 to 71) years; the main sources of infection were intra-abdomen (45%) and respiratory (38%); 70.7% had medical diseases. The 28-day mortality was 22.7%. The profi le of death patients were men (64.7%, n =22), with signifi cantly higher average age (63 vs. 57 years; P = 0.049), as well as clinical severity scores, APACHE II (29.8 vs. 24.1; P <0.001) and SOFA (12.1 vs. 8.9; P <0.001) and major dysfunction organ number (4.6 vs. 3.6; P <0.001). Bilirubin was the best predictor of 28-day mortality with the largest AUC (0.71), followed by hemoglobin (0.69) and C3 (0.67). The multivariate logistic regression was adjusted for three risk parameters, hemoglobin (OR: 0.68; 95% CI: 0.51 to 0.94), bilirubin (OR:1.63; 95% CI: 1.08 to 2.45) and white blood cells (OR:1.04; 95% CI: 1.01 to 1.08) and with these parameters a ROC analysis was performed, giving an AUC of 0.77 (0.69 to 0.84). A cohort study of routinely used sepsis biomarkers and 28-day mortality 2 M De La Torre-Prados1, A Garcia-De la Torre2, C Trujillano-Férnández1 1Hospital Virgen de la Victoria, Málaga, Spain; 2Hospital Puerto Real, Cádiz, Spain M De La Torre-Prados1, A Garcia-De la Torre2, C Trujillano-Férnández1 1Hospital Virgen de la Victoria, Málaga, Spain; 2Hospital Puerto Real, Cádiz, Spain p Critical Care 2013, 17(Suppl 2):P28 (doi: 10.1186/cc11966) Introduction The evaluation of sepsis severity is complicated by the highly variable and nonspecifi c nature of clinical signs and symptoms. We studied routinely used biomarkers together with clinical parameters to compare their prognostic value for severe sepsis and evaluate their usefulness. Conclusion PCT is a good prognostic marker and is strongly correlated to the clinical status and gravity of the patients, so PCT seems to be a useful marker in an intensive care scenario. Methods A cohort study of 150 patients >18 years with severe sepsis according to the Surviving Sepsis Campaign, in an ICU of a university hospital. Demographic, clinical parameters and coagulation, infection and infl ammation parameters during the fi rst 24 hours from severe sepsis or septic shock onset were studied. Descriptive and comparative statistical analysis was performed using SPSS version 15.0 (SPSS Inc., Chicago, IL, USA). Changes in circulating procalcitonin versus C-reactive protein in predicting evolution of infectious disease in febrile, critically ill patients Changes in circulating procalcitonin versus C-reactive protein in predicting evolution of infectious disease in febrile, critically ill patients S Hoeboer1,2, J Groeneveld1,2 S Hoeboer1,2, J Groeneveld1,2 1VU University Medical Center, Amsterdam, the Netherlands; 2Erasmus Medical Center, Rotterdam, the Netherlands Critical Care 2013, 17(Suppl 2):P30 (doi: 10.1186/cc11968) S Hoeboer , , J Groeneveld , 1VU University Medical Center, Amsterdam, the Netherlands; 2Erasmus Medical Center, Rotterdam, the Netherlands Critical Care 2013, 17(Suppl 2):P30 (doi: 10.1186/cc11968) Introduction Although absolute values for C-reactive protein (CRP) and procalcitonin (PCT) are well known to predict sepsis in the critically ill, it remains unclear if and how changes in CRP and PCT predict evolution of infectious disease and how they compare in this respect. Introduction Although absolute values for C-reactive protein (CRP) and procalcitonin (PCT) are well known to predict sepsis in the critically ill, it remains unclear if and how changes in CRP and PCT predict evolution of infectious disease and how they compare in this respect. Conclusion The assessment of routine biomarkers (bilirubin, white blood cells and hemoglobin) may be a helpful tool in the decision- making process at the bedside, for the evaluation of early ICU admission of recoverable patients, as indicators of infl ammatory response, organ dysfunction or catabolism level, and their signifi cant predictive value on mortality. Methods In 72 critically ill patients with new-onset fever, CRP and PCT were measured on day 0, 1, 2 and 7 after inclusion, and their clinical course was documented over 1 week with follow-up to day 28. Infection was microbiologically defi ned, as was bloodstream infection; septic shock was defi ned as infection plus shock. p Reference 1. White M, et al.: Chest 2011, 139:626-632. y Reference i Results From peak at day 0 to 2 to day 7, CRP decreases most when (bloodstream) infection and septic shock (day 0 to 2) resolve and increases most when complications such as a new (bloodstream) infection or septic shock (day 3 to 7) supervene (area under the receiver operating characteristic curve 0.70 or higher, P = 0.04 or lower). PCT decreases most when septic shock resolves (AUC 0.72, P = 0.007) and increases most when a new bloodstream infection or septic shock supervenes (AUC 0.82 or higher, P <0.001). The day 7 value of PCT rather than of CRP was predictive for 28-day outcome (AUC 0.70, P = 0.005). i Results From peak at day 0 to 2 to day 7, CRP decreases most when (bloodstream) infection and septic shock (day 0 to 2) resolve and increases most when complications such as a new (bloodstream) infection or septic shock (day 3 to 7) supervene (area under the receiver operating characteristic curve 0.70 or higher, P = 0.04 or lower). PCT decreases most when septic shock resolves (AUC 0.72, P = 0.007) and increases most when a new bloodstream infection or septic shock supervenes (AUC 0.82 or higher, P <0.001). The day 7 value of PCT rather than of CRP was predictive for 28-day outcome (AUC 0.70, P = 0.005). Conclusion The data, obtained during ICU-acquired fever and infections, suggest that CRP and PCT changes predict the course of infectious disease and its complications. CRP may be favoured over PCT courses in decisions on appropriateness and duration of antibiotic treatment, whereas PCT rather than CRP courses may help predicting complications such as bloodstream infection, septic shock and mortality. 1. Glickman SW, Cairns CB, Otero RM, et al.: Disease progression in hemodynamically stable patients presenting to the emergency department with sepsis. Acad Emerg Med 2010, 17:383-390. 1. Glickman SW, Cairns CB, Otero RM, et al.: Disease progression in hemodynamically stable patients presenting to the emergency department with sepsis. Acad Emerg Med 2010, 17:383-390. Procalcitonin as prognostic marker of mortality Introduction Diff erentiation of acute heart failure from infection in patients with respiratory symptoms and a history of congestive heart failure (CHF) is challenging due to overlap of clinical symptoms and X-ray fi ndings. The BACH study found higher mortality rates if patients presenting with dyspnea were treated with antibiotics and their procalcitonin (PCT) levels were low indicating absence of bacterial infection. Yet the BACH study was observational and causal inference cannot be drawn. Herein, we analyzed the eff ects of PCT-guided antibiotic stewardship in CHF patients from a previous trial (ProHOSP). p p p Methods This is a secondary analysis of a previous randomized trial of adult ED patients with respiratory symptoms and a history of CHF. Patients were randomized to administration of antibiotics based on a PCT algorithm (PCT group) or standard guidelines without knowledge of PCT levels (control group). The primary endpoint of this analysis is the risk of adverse outcome defi ned as death or ICU admission within 30 days after ED admission. Figure 1 (abstract P32). Pro-ADM transpulmonary gradient at diff erent time points. Results A total of 233 patients met the inclusion criteria, with 116 in the PCT-guided group and 117 in the control group. In the subgroup of patients with low initial PCT levels <0.25 ng/l (n = 110), PCT-guided patients had a signifi cant reduction in antibiotic exposure (mean 3.7 vs. 6.5 days, diff erence –2.8 (95% CI –4.4, –1.2), P <0.001). Furthermore, PCT-guided patients had a signifi cant lower risk for death and ICU admission (4% vs. 20%, odds ratio 6.0 (1.3, 28.2), P = 0.02). See Figure 1. Conclusion In CHF patients with suspicion of respiratory infection, use of a PCT protocol resulted in a signifi cant decrease of antibiotic exposure and signifi cantly improved outcomes in patients with low PCT levels indicating absence of bacterial infection. Whether inadequate antibiotic therapy in these CHF patients requiring diuretic treatment explains this diff erence in clinical outcomes needs verifi cation. Methods In this prospective cohort study, 39 patients undergoing cardiac surgery using CPB were included. Blood was collected before surgery (T0), after induction of anesthesia (T1), after termination of CPB (T2), at ICU arrival (T3) and 3 hours (T4), 6 hours (T5) and 18 hours (T6) after arrival. Pro-ADM was measured with a sandwich immunoassay. Primary endpoints were length of ICU and hospital stay (ICU-LOS, hospital-LOS). Procalcitonin as prognostic marker of mortality p Results An increase of arterial and venous pro-ADM plasma con- centrations was observed after surgery. Immediately after termination of CPB the venous concentration was signifi cantly lower than arterial pro- ADM concentration, but at T6 the venous concentration was signifi cantly higher, indicating a switch from a negative to positive transpulmonary gradient (Figure 1). The pro-ADM venous–arterial diff erence at T5 was a signifi cant predictor of ICU-LOS (P = 0.032) and the diff erence at T3 was a signifi cant predictor of hospital-LOS (P = 0.001). Figure 1 (abstract P31). Time to adverse outcome according to group allocation. gi Conclusion We found that the transpulmonary gradient of pro-ADM was a predictor for ICU-LOS and hospital-LOS at T3 and T5, respectively. Pro-ADM might be a promising marker for prediction on outcome of patients undergoing cardiac surgery on CPB. The transpulmonary shift of pro-ADM might be caused by an infl ammatory response. References 1. Apostolakis E, et al.: J Card Surg 2010, 25:47-55. 1. Apostolakis E, et al.: J Card Surg 2010, 25:47-55. 2. Kamei M, et al.: Acta Anaesthesiol Scand 2004, 48:980-985. 3. Ueda S, et al.: Am J Respir Crit Care Med 1999, 160:132-136. 1. Apostolakis E, et al.: J Card Surg 2010, 25:47 55. 2. Kamei M, et al.: Acta Anaesthesiol Scand 2004, 48:980-985. 3. Ueda S, et al.: Am J Respir Crit Care Med 1999, 160:132-136. 2. Kamei M, et al.: Acta Anaesthesiol Scand 2004, 48: 2. Kamei M, et al.: Acta Anaesthesiol Scand 2004, 48:980 985. 3. Ueda S, et al.: Am J Respir Crit Care Med 1999, 160:132-136. Procalcitonin as prognostic marker of mortality Procalcitonin as prognostic marker of mortality S Zampieri1, P Bettonte2, M Ortolani1, G Frison1, V Schweiger1, L Gottin3, E Polati1 1Policlinico G.B. Rossi, Verona, Italy; 2Santa Chiara Hospital, Trento, Italy; 3Ospedale Maggiore Civile, Verona, Italy Critical Care 2013, 17(Suppl 2):P29 (doi: 10.1186/cc11967) y Conclusion The data, obtained during ICU-acquired fever and infections, suggest that CRP and PCT changes predict the course of infectious disease and its complications. CRP may be favoured over PCT courses in decisions on appropriateness and duration of antibiotic treatment, whereas PCT rather than CRP courses may help predicting complications such as bloodstream infection, septic shock and mortality. Introduction We analyze procalcitonin (PCT) as a prognostic marker, in order to assess the clinical impact of a daily PCT measure. Introduction We analyze procalcitonin (PCT) as a prognostic marker, in order to assess the clinical impact of a daily PCT measure. S12 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Figure 1 (abstract P32). Pro-ADM transpulmonary gradient at diff erent time points. P31 Procalcitonin-guided antibiotic therapy in patients with congestive heart failure and suspicion of lower respiratory tract infection: results from a randomized trial P Schuetz, E Grolimund, A Kutz, S Haubitz, B Mueller Kantonsspital Aarau, Switzerland Critical Care 2013, 17(Suppl 2):P31 (doi: 10.1186/cc11969) Introduction Diff erentiation of acute heart failure from infection in patients with respiratory symptoms and a history of congestive heart failure (CHF) is challenging due to overlap of clinical symptoms and X-ray fi ndings. The BACH study found higher mortality rates if patients presenting with dyspnea were treated with antibiotics and their procalcitonin (PCT) levels were low indicating absence of bacterial infection. Yet the BACH study was observational and causal inference cannot be drawn. Herein, we analyzed the eff ects of PCT-guided antibiotic stewardship in CHF patients from a previous trial (ProHOSP). Methods This is a secondary analysis of a previous randomized trial of adult ED patients with respiratory symptoms and a history of CHF. Patients were randomized to administration of antibiotics based on a PCT algorithm (PCT group) or standard guidelines without knowledge of PCT levels (control group). The primary endpoint of this analysis is the risk of adverse outcome defi ned as death or ICU admission within 30 days after ED admission. P33 The median age of the patients was S13 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 been standardized in early goal-oriented therapies, which may benefi t from circulating biomarkers for early risk stratifi cation. We aimed to evaluate the prognostic value of presepsin (sCD14-ST), a novel marker of bacterial infection. 64 (interquartile range, 53 to 72) years; the main sources of infection were respiratory tract (46%) and intra-abdomen (21%). The 28-day mortality was 32.5%. The profi le of death patients had a signifi cantly higher average age (64.7 vs. 57.6 years; P = 0.024), as well as clinical severity scores, APACHE II (26.6 vs. 23; P = 0.006) and SOFA (11.6 vs. 89.2; P <0.001). Kaplan–Meier survival analysis was signifi cant. P = 0.0017 for patients with pADM <1.2 nmol/l. Cox regression analysis also showed statistical signifi cance (P = 0.0033) and a likelihood ratio = 1.18 per each 1 nmol/l increase in pADM. Methods We performed a nested case–control study from the randomized controlled Albumin Italian Outcome Sepsis (ALBIOS) trial, enrolling patients with severe sepsis or septic shock from 100 ICUs in Italy. Fifty survivors and 50 nonsurvivors at ICU discharge were selected, matched for age, sex, center and time of enrollment after inclusion criteria were present. EDTA-plasma samples were collected at days 1, 2 and 7 after enrolment for presepsin (immune- chemiluminescence assay PATHFAST Presepsin, URL 320 pg/ml, CV 5%; Mitsubishi Chemicals) and procalcitonin assay (PCT, Elecsys BRAHMS Cobas® PCT, URL 0.046 ng/ml, CV 8.8%; Roche Diagnostics). p Conclusion The protein pADM is an important prognostic biomarker of survival when measured on admission of septic patients to the ICU. References 1. Pezzilli R, Barassi A, Pigna A, et al.: Time course of proadrenomedullin in the early phase of septic shock. A comparative study with other proinfl ammatory proteins. Panminerva Med 2012, 54:211-217. g g Results Clinical characteristics were similar between the two groups, except for a worse SOFA score at day 1 in decedents. Presepsin at day 1 was signifi cantly higher in decedents (2,268 (1,145 to 4,305) pg/ ml, median (Q1 to Q3)) than in survivors (1,184 (855 to 2,158) pg/ml, P = 0.001), while PCT did not diff er (18.5 (3.3 to 45.7) vs. 10.8 (2.6 to 46.4) ng/ml, P = 0.31). P33 P33 Pro-adrenomedullin as prognostic biomarker in the sepsis M De La Torre-Prados, A Garcia-De la Torre, A Enguix, M Mayor, N Zamboschi, C Trujillano-Fernández, A Garcia-Alcantara Hospital Virgen de la Victoria, Málaga, Spain Critical Care 2013, 17(Suppl 2):P33 (doi: 10.1186/cc11971) Pro-adrenomedullin as prognostic biomarker in the sepsis M De La Torre-Prados, A Garcia-De la Torre, A Enguix, M Mayor, N Zamboschi, C Trujillano-Fernández, A Garcia-Alcantara Hospital Virgen de la Victoria, Málaga, Spain Critical Care 2013, 17(Suppl 2):P33 (doi: 10.1186/cc11971) Figure 1 (abstract P31). Time to adverse outcome according to group allocation. p g , g , p Critical Care 2013, 17(Suppl 2):P33 (doi: 10.1186/cc11971) Introduction Measurement of biomarkers is a potential approach to early assessment and prediction of mortality in septic patients. The purpose of this study was to ascertain the prognostic value of pro- adrenomedullin (pADM), measured in all patients admitted to the ICU of our hospital with a diagnosis of severe sepsis or septic shock during 1 year. P32 Pro-adrenomedullin as a clinical predictor after cardiac surgery J Van Fessem, F De Graaf, J Van Paassen, S Arbous LUMC, Leiden, the Netherlands Critical Care 2013, 17(Suppl 2):P32 (doi: 10.1186/cc11970) y Methods A cohort study of 117 patients >18 years with severe sepsis according to the Surviving Sepsis Campaign, in an ICU of a university hospital. Demographic, clinical parameters and pADM, C-reactive protein and procalcitonin were studied during 1 year. Descriptive and comparative statistical analysis was performed using the statistical software packages Statistica Stat Soft Inc 7.1 and MedCalc 9.2.1.0. Results We analyzed 117 consecutive episodes of severe sepsis (15%) or septic shock (85%) in the ICU. The median age of the patients was Introduction Pulmonary complications after cardiac surgery like ARDS are frequent and linked to high mortality [1]. Pro-adrenomedullin (pro-ADM) has a possible role in the development of ARDS [2] and a positive correlation between levels of pro-ADM and infl ammation was found [3]. In this study we investigated whether intraoperative and postoperative pro-ADM transpulmonary gradient could predict postoperative morbidity. g Results We analyzed 117 consecutive episodes of severe sepsis (15%) or septic shock (85%) in the ICU. Early IL-6 response in sepsis is correlated with mortality and severity score P Srisangthong, A Wongsa, P Kittiworawitkul, A Wattanathum Phramongkutklao Hospital, Bangkok, Thailand C iti l C 2013 17(S l 2) P34 (d i 10 1186/ 11972) P Srisangthong, A Wongsa, P Kittiworawitkul, A Wattanathum Phramongkutklao Hospital, Bangkok, Thailand C iti l C 2013 17(S l 2) P34 (d i 10 1186/ 11972) g p g Critical Care 2013, 17(Suppl 2):P34 (doi: 10.1186/cc11972) Introduction IL-6, a proinfl ammatory cytokine, is synthesized from fi bro- blasts, T lymphocytes, endothelial cells and monocytes. It serves as an important mediator during the acute phase response to infl ammation in sepsis. We hypothesized that the plasma IL-6 is correlated with mortality and severity scores in critically ill patients with sepsis. Methods We conducted a prospective study of plasma IL-6 level at the initial phase of sepsis and the risk of mortality. A total of 203 patients with sepsis, who were admitted to the medical ICU at Phramongkutklao Hospital, Bangkok during January to December 2011, were analyzed. Serum IL-6, C-reactive protein (CRP), and lactate were measured within the fi rst 24 hours of ICU admission. Severity scores (APACHE II, SAP II, and SOFA scores) were measured. The primary outcome variable was 28-day all-cause mortality. Conclusion Early presepsin measurements may provide important prognostic information in patients with severe sepsis or septic shock, and may be of crucial importance for early risk stratifi cation. P36 References References 1. Pathan N, et al.: Crit Care Med 2005, 33:1839-1844. 2. Harbarth S, et al.: Am J Respir Crit Care Med 2001, 164:396-402. 1. Pathan N, et al.: Crit Care Med 2005, 33:1839-1844. 2. Harbarth S, et al.: Am J Respir Crit Care Med 2001, 164:396-402. p p y Methods This study was conducted as a single-center retrospective study. Blood samples were collected from patients admitted to the emergency room at Fukuoka University Hospital between June 2010 and October 2012. We enrolled 254 patients with suspected sepsis and other disease patients. We classifi ed the patients into an AKI group according to the RIFLE criteria (Risk n = 52, Injury n = 39, Failure n = 41, Loss of kidney function and End-stage kidney disease n = 7) and a non- AKI group (n = 115). The AKI patient group was further classifi ed into a sepsis group and a nonsepsis group in each AKI stage and we analyzed the diagnostic accuracy of presepsin in patients with sepsis. P33 Presepsin decreased over time in survivors, but remained elevated in decedents (974 (674 to 1,927) vs. 2,551 (1,438 to 5,624) pg/ml at day 7, P = 0.02 for time–survival interaction); PCT decreased similarly in the two groups (P = 0.19). Patients with early elevated presepsin had worse SOFA score, higher number of MOFs, hemodynamic instability (lower mean arterial pressure at baseline and after 6 hours), and mortality rate at 90 days (75% vs. 42%, log- rank P <0.001). The association between presepsin and outcome was more marked in patients with late enrollment (6 to 24 hours), and in septic shock. Early presepsin had better prognostic accuracy than PCT (AUROC 0.69 vs. 0.56, P = 0.07), and improved discrimination over SOFA score, especially in septic shock. pl y p 2. Wang RL, Kang FX: Prediction about severity and outcome of sepsis by proatrial natriuretic peptide and pro-adrenomedullin. Chin J Traumatol 2010, 13:152-157. Usefulness of presepsin in the diagnosis of sepsis in acute kidney injury patients Usefulness of presepsin in the diagnosis of sepsis in acute kidney injury patients Usefulness of presepsin in the diagnosis of sepsis in acute kidney injury patients Y Nakamura, H Ishikura, T Nishida, Y Kawanno, R Yuge, R Ichiki, A Murai Fukuoka University, Fukuoka, Japan Critical Care 2013, 17(Suppl 2):P36 (doi: 10.1186/cc11974) y y Results We found that the overall 28-day mortality was 46% (93 out of 203 patients). There was a signifi cantly positive correlation between mortality rate and plasma IL-6 (survivors vs. nonsurvivors; 74 (4.4 to 1,718) vs. 206 (19 to 5,000) pg/ml, P <0.05), lactate (survivors vs. nonsurvivors; 1.65 (0.7 to 11.61) vs. 2.47 (0.94 to 19.13) mmol/l, P <0.05), but not CRP levels (P = 0.24). Compared with the patients with plasma IL-6 <100 pg/ml, septic patients with IL-6 levels ≥100 were associated with an increased 28-day mortality with the odd ratio of 2.99 (95% CI 1.42 to 6.29, P <0.05). We also found that plasma IL-6 levels were well correlated with APACHE II (P <0.05), SAPS II (P <0.05), and SOFA (P <0.05) scores. Y Nakamura, H Ishikura, T Nishida, Y Kawanno, R Yuge, R Ichiki, A Murai Fukuoka University, Fukuoka, Japan Critical Care 2013, 17(Suppl 2):P36 (doi: 10.1186/cc11974) Introduction The level of presepsin is useful for diff erentiating sepsis from noninfectious systemic infl ammatory response syndrome. It has been reported that the presepsin levels in patients with chronic renal failure are abnormally high. However, there are no studies investigating the usefulness of presepsin for diagnosis of sepsis in patients with acute kidney injury (AKI). Our purpose of this study is to clarify the diagnostic accuracy of presepsin in patients with AKI and the relationship between presepsin level and AKI severity. Conclusion The initial phase plasma IL-6 levels were correlated with severity and mortality in critically ill patients with sepsis. Serum levels of presepsin refl ects the APACHE II and SOFA scores in patients with sepsis Serum levels of presepsin refl ects the APACHE II and SOFA scores in patients with sepsis R Sato, Y Suzuki, M Sato, G Takahashi, M Kojika, Y Inoue, S Endo Iwate Medical University, Morioka, Japan Critical Care 2013, 17(Suppl 2):P37 (doi: 10.1186/cc11975) R Sato, Y Suzuki, M Sato, G Takahashi, M Kojika, Y Inoue, S Endo Iwate Medical University, Morioka, Japan Critical Care 2013, 17(Suppl 2):P37 (doi: 10.1186/cc11975) Methods We analyzed data from a large multinational database of patients with CAP (CAPO) [2] between 2001 and 2012. We performed logistic regression analysis and the area under the receiver operating characteristics (AUC) curve to study the association of these variables with the diagnosis of Legionella. Introduction As a method for the diagnosis of sepsis, we previously reported an ELISA method for measuring the serum levels of presepsin. That method, however, took approximately 2 hours to yield results.i Introduction As a method for the diagnosis of sepsis, we previously reported an ELISA method for measuring the serum levels of presepsin. That method, however, took approximately 2 hours to yield results. Methods To resolve this problem, we later developed a simplifi ed assay kit making use of immunochromatography (Point of Care test; POC test), and are currently evaluating the usefulness of this kit for diagnosis of sepsis and evaluation of its severity. In 21 septic patients with sepsis, we analyzed the serum levels of presepsin in relation to APACHE II and SOFA scores. pp y y Methods To resolve this problem, we later developed a simplifi ed assay kit making use of immunochromatography (Point of Care test; POC test), and are currently evaluating the usefulness of this kit for diagnosis of sepsis and evaluation of its severity. In 21 septic patients with sepsis, we analyzed the serum levels of presepsin in relation to APACHE II and SOFA scores. g g Results Data for 8,278 CAP patients were analysed; the infectious organism was known in 2,321 cases (28%), including a total of 101 patients with urinary antigen-confi rmed Legionnaires’ disease and 983 patients with confi rmed pneumococcal disease. All variables were predictors for Legionella with odds ratios ranging from 1.002 to 5.767. Combining the variables in a joint logistic regression model showed a high predictive accuracy with an AUC of 0.86. Results APACHE II and SOFA scores at baseline were 28.7 ± 7.5 and 10.8 ± 3.9, respectively, in these patients. Host genetic variants associated with community-acquired pneumonia LE Salnikova1, TV Smelaya1, VV Moroz1, A Golubev1, AV Rubanovich2 1V.A. Negovsky Research Institute of General Reanimatology, Moscow, Russia; 2N.I. Vavilov Institute of General Genetics, Russian Academy of Sciences, Moscow, Russia Critical Care 2013, 17(Suppl 2):P40 (doi: 10.1186/cc11978) Introduction Serum nitrite/nitrate (NOx) levels in the early phase of septic acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) have recently been reported to possibly play a key role in pathogenesis of ALI/ARDS. p g Methods NOx levels in the early phase of septic ALI/ARDS were measured by autoanalyzer (TCI-NOX 1000; Tokyo Kasei Kogyo Co., Ltd, Tokyo, Japan). Cytokine was measured by ELISA (Medogenix, Fleurus, Belgium). Introduction It has been shown that polymorphic variants at some host genes can modify risk of community-acquired pneumonia (CAP), including those critical for the host response to CAP – innate immune system, the lung’s defense against inhaled microorganisms, and inhibition of fi brinolysis and the renin–angiotensin system. The aim of the study was to analyze polymorphisms in genes potentially relevant to CAP pathogenesis mechanisms to reveal novel and confi rm reported genetic risk factors in the general Russian population. g Results Both NOx and TNFα levels were signifi cantly higher in the ARDS group than in the ALI group. A negative correlation was found between the PaO2/FIO2 (P/F) ratio and serum NOx levels. In addition, a positive correlation was found between the TNFα and serum NOx levels. The 30-day, 60-day and 90-day mortality rates were 8.7%, 15.2% and 19.6%, respectively, in the patients with ALI/ARDS. There were no diff erences in the P/F ratio, serum NOx levels or TNFα levels in the early phase of ALI/ARDS between the 30-day survival and death groups. On the other hand, the P/F ratio, serum NOx levels and TNFα levels in the early phase of ALI/ARDS were signifi cantly higher in the 60-day and 90-day death groups than in the corresponding survival groups. There were no signifi cant diff erences in the 90-day mortality rates between the ALI and ARDS groups.i Methods Patients with CAP (n = 334), volunteers without a previous history of CAP, control group A (n = 141) and a second control group B (n = 314) were included in the study. P38 P38 Relationship between serum nitrite/nitrate levels in the early phase of septic acute lung injury and prognosis M Sato, Y Suzuki, T Masuda, G Takahashi, M Kojika, Y Inoue, S Endo Iwate Medical University, Morioka, Japan Critical Care 2013, 17(Suppl 2):P38 (doi: 10.1186/cc11976) References 1. Fiumefreddo R, Zaborsky R, Haeuptle J, et al.: Clinical predictors for Legionella in patients presenting with community-acquired pneumonia to the emergency department. BMC Pulm Med 2009, 9:4. 2. Ramirez JA: Fostering international multicenter collaborative research: the CAPO Project. Int J Tuberc Lung Dis 2007, 11:1062-1065. Serum levels of presepsin refl ects the APACHE II and SOFA scores in patients with sepsis These were signifi cant correlations between the serum presepsin levels and the APACHE II score, and also between the serum presepsin levels and the SOFA score. Conclusion Furthermore, there was also a signifi cant correlation between the results of the POC test and the serum presepsin levels. These results indicate that measurement of the serum presepsin might be useful for evaluating the severity of sepsis. Conclusion This analysis validates the Legionella score in an inde- pendent sample and shows high diagnostic accuracy. Interventional trials with adapted antibiotic regimes for non-inferiority in a real live population are warranted. R f Compared values of presepsin (sCD14-ST) and procalcitonin as early markers of outcome in severe sepsis and septic shock: a preliminary report from the Albumin Italian Outcome Sepsis (ALBIOS) study Compared values of presepsin (sCD14-ST) and procalcitonin as early markers of outcome in severe sepsis and septic shock: a preliminary report from the Albumin Italian Outcome Sepsis (ALBIOS) study P Caironi1, S Masson2, E Spanuth3, R Thomae4, R Fumagalli5, A Pesenti5, M Romero6, G Tognoni6, R Latini2, L Gattinoni1 1Fondazione IRCCS Ca’ Granda – Ospedale Maggiore Policlinico, Università degli Studi di Milano, Milan, Italy; 2Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy; 3Diagnostic Engineering & Research GmbH, Heildelberg, Germany; 4Mitsubishi Chemical Europe GmbH, Munich, Germany; 5Ospedale San Gerardo, Monza, Italy; 6Consorzio Mario Negri Sud, Santa Maria Imbaro, Italy Critical Care 2013, 17(Suppl 2):P35 (doi: 10.1186/cc11973) , g , , 1Fondazione IRCCS Ca’ Granda – Ospedale Maggiore Policlinico, Università degli Studi di Milano, Milan, Italy; 2Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy; 3Diagnostic Engineering & Research GmbH, Heildelberg, Germany; 4Mitsubishi Chemical Europe GmbH, Munich, Germany; 5Ospedale San Gerardo, Monza, Italy; 6Consorzio Mario Negri Sud, Santa Maria Imbaro, Italy Results For the non-AKI patients, the median of presepsin in patients with nonsepsis (n = 78) and the sepsis group (n = 37) were 406 pg/ml (range: 86 to 4,374) and 1,065 pg/ml (range 86 to 9,960), respectively (P <0.0001). For the Risk patients, the median of presepsin in patients with nonsepsis (n = 25) and the sepsis group (n = 27) were 299 pg/ml (range: 71.2 to 3,361) and 831 pg/ml (range: 233 to 16,759), respectively (P <0.01). For the Injury patients, the median of presepsin in patients with nonsepsis (n = 12) and the sepsis group (n = 27) were 463 pg/ml (range: 122 to 1,197) and 1,451 pg/ml (range: 237 to 4,200), respectively (P <0.001). For the Failure patients, the median of presepsin in patients y Critical Care 2013, 17(Suppl 2):P35 (doi: 10.1186/cc11973) Introduction Sepsis results from complex interactions between infecting microorganisms and host responses, often leading to multiple organ failures and death. Over the years, its treatment has Introduction Sepsis results from complex interactions between infecting microorganisms and host responses, often leading to multiple organ failures and death. Over the years, its treatment has S14 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 P39 with nonsepsis (n = 14) and the sepsis group (n = 27) were 1,607 pg/ml (range: 454 to 8,516) and 1,523 pg/ml (range: 293 to 16,764), respectively (P = 0.175). The diagnostic accuracy of presepsin in patients with sepsis was determined by ROC analysis, the area under the curve was 0.789 for the non-AKI patient group, 0.735 for the Risk patient group, 0.855 for the Injury patient group and 0.593 for the Failure patient group. Conclusion In Failure and more progressed AKI patients, the diagnostic accuracy of the presepsin level was lower than the other groups. References with nonsepsis (n = 14) and the sepsis group (n = 27) were 1,607 pg/ml (range: 454 to 8,516) and 1,523 pg/ml (range: 293 to 16,764), respectively (P = 0.175). The diagnostic accuracy of presepsin in patients with sepsis was determined by ROC analysis, the area under the curve was 0.789 for the non-AKI patient group, 0.735 for the Risk patient group, 0.855 for the Injury patient group and 0.593 for the Failure patient group. Simple and fast prediction of Legionella sp. in community-acquired pneumonia: validation of a prediction rule S Haubitz1, F Hitz2, L Graedel2, T Wiemken3, P Peyrani Dicastelnuovo3, J Ramirez3, M Batschwaroff 1, C Fux1, B Mueller1, P Schütz1 1Kantonsspital Aarau, Switzerland; 2University of Basel, Switzerland; 3University of Louisville School of Medicine, Louisville, KY, USA Critical Care 2013, 17(Suppl 2):P39 (doi: 10.1186/cc11977) Conclusion In Failure and more progressed AKI patients, the diagnostic accuracy of the presepsin level was lower than the other groups. References 1. Endo S, et al.: J Infect Chemother 2012, 18:891-897. doi:10.1007/s10156-012-0435-2 2. Bellomo R, et al.: Crit Care 2004, 8:R204-R212. doi:10.1186/cc2872 1. Endo S, et al.: J Infect Chemother 2012, 18:891-897. doi:10.1007/s10156-012-0435-2 Introduction Ruling out Legionella sp. in patients presenting with community-acquired pneumonia (CAP) is important due to diff erences in treatment regimens. Yet antigen tests as well as blood cultures have low sensitivity and an important time delay, making empirical broad spectrum coverage necessary particularly in severe cases. Fiumefreddo and colleagues recently proposed a clinical score based on six clinical and laboratory variables (fever, cough, sodium, lactate-dehydrogenase, C-reactive protein, platelet count) which allowed assessing the likelihood of Legionella [1]. Yet these variables need validation in an independent patient cohort before implementation into clinical routine. 2. Bellomo R, et al.: Crit Care 2004, 8:R204-R212. doi:10.1186/cc2872 2. Bellomo R, et al.: Crit Care 2004, 8:R204-R212. doi:10.1186/cc2872 Eff ectiveness of early ureteric stenting for urosepsis associated with urinary tract calculi S Nishiguchi , Y Tokuda 1Shonan Kamakura General Hospital, Kamakura, Japan; 2Mito Kyodo General Hospital, Mito, Japan Critical Care 2013, 17(Suppl 2):P43 (doi: 10.1186/cc11981) S Nishiguchi , Y Tokuda 1Shonan Kamakura General Hospital, Kamakura, Japan; 2Mito Kyodo General Hospital, Mito, Japan Critical Care 2013, 17(Suppl 2):P43 (doi: 10.1186/cc11981) p Results A total of 536 (14%) of the 3,766 enrolled patients had SCAP. They were mostly male (66%) with median age 59 (29 to 82) years, median SAPS II 44 (21 to 80) and total SOFA score 8 (3 to 16). Thirty- seven per cent of the cases were microbiologically documented (St. pneumoniae – 24%; Enterobacteriaceae – 20%; infl uenza A (H1N1) virus – 18%) and 45% had septic shock. Antibiotic combination was used in 76% of the patients and 61% received a macrolide. Median hospital length of stay was 19 (3 to 70) days and hospital mortality was 35%. Comorbidities were present in 70% of the patients. The most frequent were: diabetes mellitus (21%), chronic respiratory failure (18%) and alcoholism (15%). Median Charlson’s comorbidity index (CCI) was 4 (0 to 13). In univariate analysis, the presence of at least one comorbidity (odds ratio (OR) 2.29; 95% CI 1.49 to 3.52), namely cancer (OR 3.80; 95% CI 2.14 to 6.74; P <0.001), chronic renal failure (OR 3.23; 95% CI 1.53 to 6.82; P = 0.001), immunosuppression (OR 2.12; 95% CI 1.15 to 3.92; P = 0.014) and neurologic disease (OR 1.87; 95% CI 1.10 to 3.17; P = 0.02), increased the chances of dying in the hospital. Median CCI was also signifi cantly higher in nonsurvivors (5 vs. 3; P <0.001; OR per point 1.10 (95% CI: 1.05 to 1.15)). The only independent risk factor for hospital mortality was the presence of at least one comorbidity (OR 2.09; 95% CI 1.13 to 3.85). Introduction Urosepsis associated with urinary tract calculi is a critical disease. Patients with this condition occasionally require drainage, mostly ureteric stent placement and these patients need longer hospitalization. However, indications for timely ureteric stenting for urosepsis associated with urinary tract calculi have not been clearly determined. The objective of the study was to evaluate whether earlier stent placement might lead to shorter length of stay (LOS) in hospitals. Outcome of severe community-acquired pneumonia: the impact of comorbidities Outcome of severe community-acquired pneumonia: the impact of comorbidities JM Pereira1, JA Paiva1, F Froes2, JP Baptista3, J Gonçalves-Pereira4 1Centro Hospitalar S. João, Porto, Portugal; 2Hospital Santa Maria-CHLN, Lisboa, Portugal; 3Hospitais da Universidade de Coimbra – CHUC, Coimbra, Portugal; 4Hospital São Francisco Xavier, Lisboa, Portugal Critical Care 2013, 17(Suppl 2):P41 (doi: 10.1186/cc11979) Conclusion In early surgery IE, it appears to be associated with mortality: age >70 years, previous heart disease, emergency surgery, antibiotics within 7 days before surgery, preoperative MOF, and high scores on scales of Pons and Ontario. The causal agent and echocardiography have no relation with worse prognosis. The general syndrome debut and the Streptococcus spp. etiology seem to have lower mortality. IE has pathologies with high .preoperative severity scores but these are not suffi cient to guide therapeutic decisions. Introduction Several comorbidities have been independently asso- ciated with both predisposition to community-acquired pneumonia and a worse outcome. The goal of this study was to evaluate the impact of comorbidities on the outcome of patients with severe community- acquired pneumonia (SCAP). Methods A prospective, multicentre, observational cohort study of all patients with SCAP consecutively admitted to 14 Portuguese ICUs during a 12-month period. Several comorbidities were evaluated: congestive heart failure, cancer, chronic renal failure, chronic respira- tory failure, chronic hepatic disease, alcoholism, diabetes mellitus, neurologic disease, immunosuppression, HIV infection. To evaluate the impact of comorbidities associated with hospital mortality in univariate analysis, a logistic regression analysis adjusted to other variables (clinical relevant or statistically signifi cant in univariate analysis) was performed. P42 P42 Prognostic scores and infective endocarditis P Fernandez Ugidos1, R Gomez Lopez1, P Vidal Cortes1, A Ceniceros Barros1, L Seoane Quiroga1, JM Lopez Perez2 1Complejo Hospitalario Universitario Ourense, Spain; 2Complejo Hospitalario Universitario A Coruña, Spain Critical Care 2013, 17(Suppl 2):P42 (doi: 10.1186/cc11980) Introduction Infective endocarditis (IE) is a high-mortality disease, especially in the early surgery subgroup. The aim of this study was try to identify prognostic factors of IE that will require surgery during the same admission of their diagnosis, including evaluation of surgical severity scores. Conclusion We have provided the fi rst experimental evidence for the associations of genes coding detoxifi cation enzymes with the risk of CAP. Our results also demonstrate that predisposition to CAP is strongly attributed to the eff ects of a number of genes with low penetrance and therefore imply that inter-locus interactions may be regarded as an important component of polygenic and multifactorial factors of susceptibility to CAP. Methods A retrospective study (5 years) of all patients admitted to a tertiary hospital in northern Spain with diagnosis of IE (modifi ed Duke criteria) who required early surgery. Demographic, clinical and microbiology data were collected. Chi-square and Student t tests. Signifi cance: P <0.05. SPSS17. gi Results We had 73 patients, 79.5% men, age 65 years. Eighty-two percent had positive blood cultures. Forty-one percent of cases required previous ICU admission. Surgery was urgent in 35%. Fifty-six per cent of patients had postoperative shock and 58% suff ered post- surgery ARF. Hospital mortality was 31.5%. Regarding prognostic scales: the mean EuroSCORE was 11.38 ± 3.93 points. No patient was placed in the low-risk group. Ninety-four per cent of cases were at high risk. The Parsonnet mean score was 27.7 ± 11.6. The mean Beth Israel Medical Center was 37.6 ± 10 points. The mean Ontario scale was 7.09 ± 2.7. The mean Surgical risk scale of Roques was 6.69. And the mean by Pons Scale was 24.02. Dead patients are older, with previous heart disease, require urgent surgery and have previous ICU stay; usually they have MOF and they had received less than 7 days of antibiotic treatment. The etiology did not worsen the prognosis. Outcome of severe community-acquired pneumonia: the impact of comorbidities JM Pereira1, JA Paiva1, F Froes2, JP Baptista3, J Gonçalves-Pereira4 1Centro Hospitalar S. P42 João, Porto, Portugal; 2Hospital Santa Maria-CHLN, Lisboa, Portugal; 3Hospitais da Universidade de Coimbra – CHUC, Coimbra, Portugal; 4Hospital São Francisco Xavier, Lisboa, Portugal Critical Care 2013, 17(Suppl 2):P41 (doi: 10.1186/cc11979) Host genetic variants associated with community-acquired pneumonia Using allele-specifi c tetraprimer PCR, all subjects were genotyped for 13 polymorphic variants in the genes of xenobiotic detoxifi cation CYP1A1 (rs2606345, rs4646903, rs1048943), GSTM1 (Ins/Del), GSTT1 (Ins/Del), ABCB1 (rs1045642); immune and infl ammation response IL-6 (rs1800795), TNFα (rs1800629), MBL2 (rs7096206), CCR5 (rs333), NOS3 (rs1799983), angiotensin-converting enzyme (ACE; rs4340), and occlusive vascular disease/hyperhomocysteinemia MTHFR (rs1801133). Conclusion Our fi ndings suggested that NOx may be involved in the pathogenesis of ALI/ARDS. S15 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Results Seven genes CYP1A1 rs2606345T/T, GSTM1 Ins/, ABCB1 C/C, IL-6 C/C-G/G, NOS3 T/T-G/G, CCR5 Ins/Ins, and ACE Del/Del were associated with CAP. The highest eff ect was detected for the CYP1A1 rs2606345: in comparison with the control A, P = 3.9×10–5, OR = 2.40, 95% CI: 1.59 to 3.64; and in comparison with the control B, P = 1.4×10–5, OR = 2.0, 95% CI: 1.46 to 2.74. For the two genes CYP1A1 and GSTM1, associations remained signifi cant after correction for multiple comparisons. Multiple analysis by the number of all risk genotypes showed a highly signifi cant association with CAP (P = 2.4×10–7, OR = 3.03, 95% CI 1.98 to 4.64) with the threshold for three risk genotypes. Using the ROC analysis, the AUC value for multi-locus model was estimated as 68.38, which is rather high for genetic markers.i Yamamoto Y, et al.: BMC Urol 2012, 12:4. Yoshimura K, et al.: J Urol 2005, 173:458-462. Yamamoto Y, et al.: BMC Urol 2012, 12:4. Yoshim ra K et al J Urol 2005 173 458 462 2. Yoshimura K, et al.: J Urol 2005, 173:458-462. P46 Abdominal infection plays a role in the incidence of ventilator- associated pneumonia GB Bouroche1, S Ruckly2, B Misset3, JF Timsit4, F Philippart3 1Institut Gustave Roussy, Villejuif, France; 2Institut Albert Bonniot, La Tronche, France; 3Hôpital Saint Joseph, Paris, France; 4CHU Grenoble, France Critical Care 2013, 17(Suppl 2):P46 (doi: 10.1186/cc11984) g References 1. Clec’h C, Schwebel C, Français A: Does catheter-associated urinary tract infection increase mortality in critically ill patients. Infect Control Hosp Epidemiol 2007, 28:1367-1373. 1. Clec’h C, Schwebel C, Français A: Does catheter-associated urinary tract infection increase mortality in critically ill patients. Infect Control Hosp Epidemiol 2007, 28:1367-1373. p Methods To investigate, in humans, the potential involvement of previous intra-abdominal infection (IIA) in preventing or promoting VAP, we realized a prospective observational study using data from a multicenter database (OUTCOMEREA), including all patients admitted 2. Marx G, Reinhart K: Urosepsis: from the intensive care viewpoint. Int J Antimicrob Agents 2008, 31S:S79-S84. 2. Marx G, Reinhart K: Urosepsis: from the intensive care viewpoint. Int J Antimicrob Agents 2008, 31S:S79-S84. 2. Marx G, Reinhart K: Urosepsis: from the intensive care viewpoint. Int J Antimicrob Agents 2008, 31S:S79-S84. Figure 1 (abstract P46). Cumulative incidence of VAP. Eff ectiveness of early ureteric stenting for urosepsis associated with urinary tract calculi Methods For patients who required ureteric stent procedures for urosepsis associated with urinary tract calculi in our hospital from July 2008 to July 2012, we compared the LOS in our hospital between patients with earlier stenting and those without it. A linear regression model was used for multivariable-adjusted comparison. j Results In a total of 30 patients (mean age, 72; 13 males), the mean days from emergency room admission to ureteric stenting was 3.5 days (range, 1 to 14 days) and the overall mean LOS was 36 days (range, 8 to 102 days). The early-stenting group (mean LOS, 21 days) had signifi cantly shorter LOS than the delayed-stenting group (mean LOS, 50 days) with adjusted β coeffi cient of –26 days (95% CI, –46, –6). Conclusion In patients with urosepsis associated with urinary tract calculi, earlier stenting within 2 days of admission may reduce the hospital LOS. References Conclusion In SCAP, the presence of at least one comorbidity doubles the chances of dying in the hospital and is an independent risk factor for hospital mortality. Yamamoto Y, et al.: BMC Urol 2012, 12:4. Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S16 (n = 13, 13.4%) and intraabdominal infections (n = 13, 13.4%). Patients with pneumonia were treated according to knowledge in a specifi c moment, thus diff erent antibiotics were used. The duration of antibiotic therapy was 20 ± 15.5 days. In nine episodes of pneumonia according to the CDC no germ was isolated in the cultures. Six of the episodes were polymicrobial infections. The most frequent microbes isolated were E. coli (n = 7, 22.5% of pneumonia cases), A. fumigatus (n = 7, 22.5%), S. aureus (n = 3, 9.68%), P. aeruginosa (n = 3, 9.68%), P. mirabilis, K. pneumoniae, E. cloacae, E. faecalis, C. glabrata, and S. marcescens (one case each, 3.22%). Pneumonia was suspected but not confi rmed in 75 patients. Despite this, antibiotic treatment was maintained for a media of 17.35 ± 7.01 days: 56 wide-spectrum treatments and 18 targeted therapy after knowing the antibiogram. The length of ICU stay was 38.4 ± 70.8 (3 to 264) days, of hospital stay was 66.2 ± 80.5 (3 to 304) days and of mechanical ventilation was 27.3 ± 50.2 (3 to 264) days. The mortality of patients with pneumonia was 32.3%. Abdominal infection plays a role in the incidence of ventilator- associated pneumonia GB Bouroche1, S Ruckly2, B Misset3, JF Timsit4, F Philippart3 1Institut Gustave Roussy, Villejuif, France; 2Institut Albert Bonniot, La Tronche, France; 3Hôpital Saint Joseph, Paris, France; 4CHU Grenoble, France Critical Care 2013, 17(Suppl 2):P46 (doi: 10.1186/cc11984) Introduction Despite many therapeutic interventions, ventilator- acquired pneumonias (VAP) are frequent in the ICU and are associated with major morbidity and mortality. Sepsis causes a time-dependent modifi cation of the infl ammatory response. This reprogramming could promote the occurrence of a secondary infection and worsen the prognosis. In animals, peritonitis is associated with an alteration of pulmonary immunity and an increasing mortality from secondary pneumonia. y Conclusion Urinary sepsis mortality is associated with late-onset and/ or inappropriate antibiotic use, as well as with immunodepression and advanced age. Introduction Analysis of mortality-related factors in urinary sepsis patients. Methods A retrospective descriptive study of urologic sepsis patients in the ICU from 2008 to 2010. Clinical, epidemiological and outcome variables were analysed. Quantitative variables are expressed as either mean and standard deviation or as median and interquartile range for asymmetric variables. Qualitative variables are expressed as percentages and absolute values. Mann–Whitney’s U test and Fisher’s exact test were applied (α error was 5% in both cases), as well as binary logistic regression for multivariate analysis. Results There was a total number of 44 patients (aged 59.39 ± 17.71; 63.8% females). APACHE II score upon admission was 18 ± 6. Out of these patients, 27.3% showed no underlying disorder and 18.2% (no = 8) showed chronic renal failure; 25% were immunodepressed patients; 31% underwent urinary instrumentation in the previous 15 days, yet only three of them had undergone permanent urine catheterization. Observed mortality was 25%, while sepsis-related mortality was 22.7%. The patients who died were, on average, older that those who survived (67.9 ±7 10.2 vs. 56.8 ± 18.7; P = 0.02). Besides, the former also reported greater delay in turning to the hospital after symptom onset (13.4 ± 6.6 vs. 6.2 ± 4.7 days; P = 0.0001). Immunodepressed patients presented higher mortality rate: OR 8.7 (95% CI 1.7 to 42.3), as well as those who underwent inappropriate initial antibiotic treatment: OR 10.8 (95% CI 2.1 to 54.7). No relation was observed between germ typology or resistance to β-lactam antibiotics and mortality. After adjustment of mortality due to APACHE II score upon admission, delay in the onset of appropriate antibiotic treatment was an independent predictor of mortality in our patients: OR 1.2, 95% CI (1.02 to 1.42). Conclusion Nosocomial pneumonia is the most frequent infection in our series. Despite when infection was not confi rmed, antibiotic therapy was maintained in suspect cases. We found a high incidence of aspergillosis. Limitations because of wide duration of this study should be considered. P46 P44 Outcomes in urinary sepsis Outcomes in urinary sepsis C Joya-Montosa, E Trujillo-Garcia, MJ Delgado-Amaya, E Curiel-Balsera Hospital Regional Carlos Haya, Málaga, Spain Critical Care 2013, 17(Suppl 2):P44 (doi: 10.1186/cc11982) Outcomes in urinary sepsis y p C Joya-Montosa, E Trujillo-Garcia, MJ Delgado-Amaya, E Curiel-Balsera Hospital Regional Carlos Haya, Málaga, Spain Critical Care 2013 17(Suppl 2):P44 (doi: 10 1186/cc11982) Introduction Analysis of mortality-related factors in urinary sepsis patients. Nosocomial pneumonia in the postoperative period after heart transplantation transplantation R Gómez López1, P Fernández Ugidos1, P Vidal Cortes1, M Bouza Vieiro2, J Miñiz2, S Fojon Polanco2, M Paniagua Martin2, R Marzoa Rivas2, E Barge Caballero2, M Crespo Leiro2 1Complexo Hospitalario Universitario de Ourense, Spain; 2Complexo Hospitalaro Universitario de A Coruña, Spain Critical Care 2013, 17(Suppl 2):P45 (doi: 10.1186/cc11983) p R Gómez López1, P Fernández Ugidos1, P Vidal Cortes1, M Bouza Vieiro2, J Miñiz2, S Fojon Polanco2, M Paniagua Martin2, R Marzoa Rivas2, E Barge Caballero2, M Crespo Leiro2 1Complexo Hospitalario Universitario de Ourense, Spain; 2Complexo Hospitalaro Universitario de A Coruña, Spain Critical Care 2013, 17(Suppl 2):P45 (doi: 10.1186/cc11983) Hospitalaro Universitario de A Coruña, Spain p p Critical Care 2013, 17(Suppl 2):P45 (doi: 10.1186/cc11983) Introduction Infections are a major complication during the postoperative period after heart transplantation (HT). In our hospital, nosocomial pneumonia is the most frequent infection in this period. The objective of this study is to determine the epidemiological and microbiological characteristics of this disease in our centre. Methods A descriptive retrospective study of all medical records of HT performed in a single institution from 1991 to 2009 followed until June 2010. Clinical and microbiological variables were considered. Centre for Diseases Control (CDC) criteria were used to defi ne nosocomial infections. Invasive aspergillosis was considered if there were criteria for probable aspergillosis according to IDSA criteria. Results In 594 HTs there were 97 infectious episodes in 75 patients (12.6%). Eighty-fi ve patients (14.3%) died during hospitalization. Infection is the second cause of mortality during the postoperative period (17.9% of dead patients). The most common locations of infections were pneumonia (n = 31, 31.9% of infection episodes), bloodstream (n = 24, 24.7%), urinary tract (n = 14, 14.4%), surgical site Figure 1 (abstract P46). Cumulative incidence of VAP. S17 Critical Care 2013, Volume 17 Suppl 2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Figure 2 (abstract P46). Cumulative occurrence of death. that numbers of CVC, intubation and surgery, the use of muscle relaxant and steroid were independent risk factors for developing VAP. Ventilator days and ICU length of stay were longer in the VAP group (25 vs. 6 and 25 vs. 7 days, respectively). Lastly, the hospital mortality rate was signifi cantly higher in the VAP group (33% vs. 12%, P = 0.008). Nosocomial pneumonia in the postoperative period after heart transplantation Conclusion The incidence of VAP was 9.2% in the SICU of Siriraj Hospital, which was comparable with previous reports. Bundles of care to prevent VAP should include weaning from a ventilator. Muscle relaxant and steroid should be administered according to strong indication. Meticulous care of the airway should be implemented as protocol in order to prevent complications that can result in the development of VAP. Hospital-acquired bloodstream infection: Indian perspective g , FEHI, New Delhi, India FEHI, New Delhi, India Critical Care 2013, 17(Suppl 2):P48 (doi: 10.1186/cc11986) Figure 2 (abstract P46). Cumulative occurrence of death. Introduction This is a 1-year prospective study to determine the incidence, source and etiology of hospital-acquired bloodstream infection (HABSI) in the Indian context. The resistance pattern was also reviewed. Introduction This is a 1-year prospective study to determine the incidence, source and etiology of hospital-acquired bloodstream infection (HABSI) in the Indian context. The resistance pattern was also reviewed. to the ICU for severe sepsis or septic shock who required mechanical ventilation for at least 72 hours. Methods A single-centre prospective study in a 35-bed ICU. HABSI was defi ned according to current CDC guidelines. HCAP, catheter- associated UTI (CAUTI) and skin-related infections causing BSI was also defi ned according to recent guidelines and analysed. Results In total, 2,623 patients were included, of which 290 had an IIA. A total of 862 patients (33%) developed a VAP, 56 (19%) in the IIA group and 806 (34%) in the non-IIA group (P <0.01). VAP, after sepsis, occurred less frequently and later in patients with IIA. The occurrence of IIA, in comparison with another sepsis, is a protective factor against VAP (HR = 0.643 (0.478 to 0.863), P = 0.003). There is, however, no signifi cant diff erence between the groups in terms of ICU mortality (28% vs. 32%, P = 0.16). See Figures 1 and 2. i g g y Results Out of 332 positive samples, 90 samples (n = 45) were HABSI. The microbiological analysis showed 60% were Gram-negative, 6% were candida and 27% were Gram-positive. The commonest isolate was klebsiella and MRSA was commonest in Gram-positive. The source of HABSI showed CRBSI was the commonest cause at 69%, which correlates with international data. Ventilator-associated pneumonia and CAUTI caused 9.5% BSI respectively. The resistance pattern among Gram-negative bacteria showed multidrug-resistant (MDR) and extreme drug-resistant (XDR) isolates were highest. See Tables 1 and 2. g Conclusion In this study, the presence of an abdominal sepsis, in a context of severe sepsis or septic shock, was associated with a lower incidence of later VAP. These results have to be confi rmed in other studies, especially prospective. They open interesting new research directions. Table 1 (abstract P48). Source Source Total (%) CRBSI 69 HCAP 9.5 CAUTI 9.5 Devices 7 Skin 5 Table 2 (abstract P48). Reference 1. Werarak P, Kiratisin P, Thamlikitkul V: Hospital acquired pneumonia and ventilator associated pneumonia in adults at Siriraj hospital, etiology, clinical outcomes and impact of antimicrobial resistance. J Med Assoc Thai 2010, 93(Suppl 1):126-138. Hospital-acquired bloodstream infection: Indian perspective Resistance pattern Resistance Total (%) Non-MDR 15 MDR 44 XDR 33 PDR 8 Conclusion The incidence of HABSI is 27%. Of this, CRBSI cause 70% and Gram-negative bacteria were commonest with high resistance. This is in contrast to western data where Gram-positive infections are common. Our study highlights need for stringent guidelines for CRBSI prevention. References 1. Richard et al.: Crit Care Med 1999, 27:887-892. 2. Valles et al. : J Infect 2008, 56:27-34. P50 Analysis of risk factors for catheter-related bloodstream infection in a parenteral nutrition population P52 Results for introduction of a new hand hygiene program in the ICU P Vos, A Harts-Laurijsen, R Snoeren, R Ramnarain, JA Van Oers St Elisabeth Hospital, Tilburg, the Netherlands Critical Care 2013, 17(Suppl 2):P52 (doi: 10.1186/cc11990) Analysis of risk factors for catheter-related bloodstream infection in a parenteral nutrition population I Conrick-Martin1, M McGovern2, K Boner1, J Bourke1, E Fitzgerald1, R Hone1, M Lynch1, D Phelan1, C Walshe3 I Conrick-Martin1, M McGovern2, K Boner1, J Bourke1, E Fitzgerald1, R Hone1, M Lynch1, D Phelan1, C Walshe3 1Mater Misericordiae University Hospital, Dublin, Ireland; 2Harvard School of Public Health, Cambridge, MA, USA; 3Beaumont Hospital, Dublin, Ireland Critical Care 2013, 17(Suppl 2):P50 (doi: 10.1186/cc11988) 1Mater Misericordiae University Hospital, Dublin, Ireland; 2Harvard School of Public Health, Cambridge, MA, USA; 3Beaumont Hospital, Dublin, Ireland Critical Care 2013, 17(Suppl 2):P50 (doi: 10.1186/cc11988) 1Mater Misericordiae University Hospital, Dublin, Ireland; 2Harvard School of Public Health, Cambridge, MA, USA; 3Beaumont Hospital, Dublin, Ireland Critical Care 2013, 17(Suppl 2):P50 (doi: 10.1186/cc11988) Conclusion A low proinfl ammatory response is a key facilitating factor for the development of infection. Measuring serum TNFα levels before and after operations can thus predict the outcome. Introduction Catheter-related bloodstream infection (CRBSI) is a complication of central venous catheters (CVCs) with an attributable morbidity, mortality and cost [1]. We examined patient risk factors for CRBSI in an adult parenteral nutrition (PN) population. Incidence and risk factors for ventilator-associated pneumonia in Siriraj Hospital O Chaiwat, S Nakaviroj Siriraj Hospital Mahidol University, Bangkok, Thailand Critical Care 2013, 17(Suppl 2):P47 (doi: 10.1186/cc11985) Introduction Ventilator-associated pneumonia (VAP) is a serious illness with substantial morbidity and mortality and increases the cost of hospital care. Even when bundles of care to prevent VAP have been implemented, the incidence of VAP was not dramatically improved. This study aims to determine the incidence and risk factors of VAP in the SICU of Siriraj Hospital. Methods During a 1-year period, 228 patients admitted to the SICU were enrolled. All patients required ventilatory support longer than 48 hours. Data were collected by reviewing patient medical records including demographic data, onset of VAP, type of organisms, medication used, number of central venous catheters (CVC) used and blood transfusion. VAP outcomes were also reported. Results VAP occurred in 21 patients (9.21%) or 8.21 per 1,000 ventilator- days. The onset of VAP was late in the majority of patients. The most common organism was A. baumannii (66%), followed by P. aeuruginosa (19%). Compared with non-VAP groups, patients in the VAP group had higher APACHE II score (18 vs. 13, P <0.001), blood transfusion (95% vs. 75%, P = 0.04), numbers of CVC used (3 vs. 1, P <0.001), muscle relaxant used (43% vs. 3%, P <0.001) and steroid used (33% vs. 4%, P <0.001). The VAP group also had a signifi cantly higher number of intubation, reintubation and self-extubation. Multiple logistic regression showed Conclusion The incidence of HABSI is 27%. Of this, CRBSI cause 70% and Gram-negative bacteria were commonest with high resistance. This is in contrast to western data where Gram-positive infections are common. Our study highlights need for stringent guidelines for CRBSI prevention. S18 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 P49 Risk factors for catheter-related bloodstream infections in a surgical ICU A Kundakci, O Ozkalayci, P Zeyneloglu, H Arslan, A Pirat Baskent University Hospital, Ankara, Turkey Critical Care 2013, 17(Suppl 2):P49 (doi: 10.1186/cc11987) P49 Risk factors for catheter-related bloodstream infections in a surgical ICU A Kundakci, O Ozkalayci, P Zeyneloglu, H Arslan, A Pirat Baskent University Hospital, Ankara, Turkey Critical Care 2013, 17(Suppl 2):P49 (doi: 10.1186/cc11987) Results The study population was 1,961 patients in whom 3,213 CVCs were utilised over 19,511 CVC days. There were 256 CRBSI episodes in 216 patients. Median (IQR) patient age was 62 (23), and 58% were male. The incidence of CRBSI decreased signifi cantly (P <0.001) during the study period from 16% of patients in the period 1997 to 2003 to 7% in 2004 to 2010. The corresponding rate of CRBSI infection (per 1,000 CVC days) decreased from 18 to 10. There was a signifi cant decrease (P <0.001) in numbers of CVCs inserted per patient (from 1.87 to 1.49). Each extra CVC PN day was associated with an increased risk of developing CRBSI of 3.4% (OR 1.034, P <0.05). The number of PN CVCs was associated with developing CRBSI (OR 1.218, P <0.10). Patient factors signifi cantly associated with CRBSI included perioperative PN use (compared with medical patients) (OR 2.414, P <0.01), and male sex (OR 1.952, P <0.01). Introduction Preventing catheter-related bloodstream infections (CR-BSI) can reduce the duration of hospital stay, healthcare costs, and mortality rates. Identifying the risk factors and correction of modifi able factors should be one of the main objectives of infection control measures. The aim of this study is to determine the risk factors of CR-BSI in a cohort of surgical ICU patients admitted to Baskent University Hospital. p Methods Following Institutional Review Board approval, data for 876 patients admitted to the surgical ICU between January 2009 and July 2012 were reviewed retrospectively. After completing the review, 25 patients diagnosed with CR-BSI were compared with 66 appropriate matches who did not have CR-BSI. Demographical features, underlying diseases, APACHE II (Acute Physiology and Chronic Health Evaluation) and SOFA (Sequential Organ Failure Assessment) scores, length of stay, organ dysfunctions, laboratory values, use of vasopressors, mechanical ventilation, nutrition, antibiotics, transfusions, and features related to central venous catheterization were recorded. Patients who did not have a central venous catheter and were discharged within 2 days of ICU stay were excluded. Conclusion This prospective study demonstrated that perioperative PN use was associated with increased risk of CRBSI. The association between CRBSI and CVC PN days is consistent with the theory suggesting benefi t to limiting CVC duration and changing from PN to enteral nutrition as soon as appropriate. Can we predict the postoperative infections? 2 3 2 O’Grady NP: Guidelines for the prevention of intravascular catheter-related infections. Clin Infect Dis 2011, 52:e162-e193. 2. Bouza E: Catheter-related infections: diagnosis and intravascular treatment Clin Microbiol Infect 2002 8 265 274 2. Bouza E: Catheter-related infections: diagnosis and intravascular treatment. Clin Microbiol Infect 2002, 8:265-274. Results From 22 patients, eight deep wound and urine infections were found in 14 days and six urine and wound infections in 30 days after surgery, all survived. All patients were bacterially contaminated, as wound samples taken at the end of operation demonstrated. On day 0 the circulating TNFα values were lower in infected patients. TNF started to increase from day 3 to day 9, never reaching values of the uneventful healing group. Soluble TNF receptor I, IL-6, ACTH, and cortisol concentrations did not demonstrate any diff erence on day 0 but from day 1 started to increase transiently, reaching higher levels in septic patients.l Can we predict the postoperative infections? 2 3 2 y Results Out of 91 patients included in the fi nal analysis, 25 (27%) patients had CR-BSI. When compared with patients who did not have CR-BSI, those who did were older (P = 0.029), required more blood product transfusions during the fi rst 3 days of ICU (P = 0.016), had a longer duration of catheter stay (P = 0.019), and were more frequently catheterized via the internal jugular vein (IJV) (P = 0.022). A logistic regression model revealed that advanced age (OR: 1.037; 95% CI: 1.001 to 1.073; P = 0.042) was an independent risk factor for CR-BSI. Fourteen- day and 28-day mortality rates for CR-BSI were 12% (P = 0.749) and 28% (P = 0.406), respectively. p p p Mózes1, K Gornicsar2, A Grosz3, ZS Domján2, I Buzogány2 p p p T Mózes1, K Gornicsar2, A Grosz3, ZS Domján2, I Buzogány2 1Ministry of Defense Health Centre, Budapest, Hungary; 2Péterfy Hospital, Budapest, Hungary; 3University of Szeged, Hungary Critical Care 2013, 17(Suppl 2):P51 (doi: 10.1186/cc11989) T Mózes1, K Gornicsar2, A Grosz3, ZS Domján2, I Buzogány2 1Ministry of Defense Health Centre, Budapest, Hungary; 2Péterfy Hospital, Budapest, Hungary; 3University of Szeged, Hungary Critical Care 2013, 17(Suppl 2):P51 (doi: 10.1186/cc11989) Introduction Many patients develop infections following operations. Decreased immune competence has been demonstrated in acute neurological conditions. A strong cytokine-mediated anti- infl ammatory response was observed in stroke patients at infection, although infection due to the decreased proinfl ammatory mediators can be expected as well. To investigate this question the following experiment was performed. y Conclusion Although age, blood product transfusion, duration of catheter stay, and use of IJV were diff erent between patients who did and did not have CRBSI, advanced age was the only independent risk factor for CR-BSI. Early suspicion of CR-BSI by the other well-known risk factors has a substantial eff ect on the treatment of CR-BSI. References Methods Twenty-two urinary bladder cancer patients with radical cystectomy and lymphadenectomy were studied. Blood samples were taken on day 0 (before) and days 1, 3, 6, 9 and 14 after operation as well as on days 30, 60, 90 and 270 during follow-up. TNFα, soluble TNFα receptor I and IL-6 levels in sera were determined by HS ELISA and/or ELISA. Plasma ACTH and cortisol values were measured by RIA kits. 1. O’Grady NP: Guidelines for the prevention of intravascular catheter-related infections. Clin Infect Dis 2011, 52:e162-e193. 1. Reference 1. Blot SI, et al.: Clinical and economic outcomes in critically ill patients with nosocomial catheter-related bloodstream infection. Clin Infect Dis 2005; 41:1591-1598. Results for introduction of a new hand hygiene program in the ICU P Vos, A Harts-Laurijsen, R Snoeren, R Ramnarain, JA Van Oers St Elisabeth Hospital, Tilburg, the Netherlands Critical Care 2013, 17(Suppl 2):P52 (doi: 10.1186/cc11990) Methods The study was carried out in a 525-bed tertiary-referral teaching hospital over a 14-year study period (1997 to 2010). All in- patients referred for PN via CVCs were included. Prospectively collected data were recorded in a specifi c PN record. The CRBSI audit group met quarterly to review all sepsis episodes, assigning a diagnostic category (CRBSI or non-CRBSI). Patient risk factors for development of CRBSI were examined using a logistic regression model to take account of the dichotomous nature of the outcome. Odds ratios from a model incorporating demographic and clinical data were tested for statistical signifi cance. Introduction Although hand hygiene is known as a core element in prevention of healthcare-associated infections, compliance to its program is not high. With a new campaign we tried to enlarge awareness on hand hygiene. Introduction Although hand hygiene is known as a core element in prevention of healthcare-associated infections, compliance to its program is not high. With a new campaign we tried to enlarge awareness on hand hygiene. Methods We performed an experimental, before and after, study design on our 30-bed, mixed medical–surgical ICU. We conducted a baseline S19 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 evaluation during 3 days in May 2012 including direct observation of hand hygiene compliance by control nurses and hand cultures of 50 healthcare workers (HCW). Based on the WHO Guidelines on Hand Hygiene in Health Care [1], cleaning of hands with alcohol-based hand rubs (Sterillium) was prescribed before touching a patient and before aseptic procedures, after body fl uid exposure risk and after touching a patient and touching his/her surroundings. Promotion of the hand hygiene program consisted of lectures and web-based self-learning, posters located near points of care and verbal reminders by control nurses. New observations of hand hygiene by control nurses during 3 days and hand cultures of 50 healthcare providers were performed in September 2012. Consumption of alcohol-based hand rub (product volume use per patient-days) was used as a surrogate marker of hand hygiene over time. The diff erence in hand hygiene compliance during the two periods was examined using a chi-squared test. Diff erences in hand cultures were examined using a Student’s t test. Time trends in the consumption of alcohol-based hand rub were examined using linear correlation. P <0.05 was considered statistically signifi cant. Results for introduction of a new hand hygiene program in the ICU P Vos, A Harts-Laurijsen, R Snoeren, R Ramnarain, JA Van Oers St Elisabeth Hospital, Tilburg, the Netherlands Critical Care 2013, 17(Suppl 2):P52 (doi: 10.1186/cc11990) The study was approved by the institutional Ethics Review Board. from March through October 2012, and intervened to change behavior by providing monthly feedback to specifi c provider groups and services. We made use of the Unit Coordinator to measure compliance of all individuals in the ICU. Results Overall compliance by physicians was 82.1%, for nonphysicians was 84.8%. Feedback to physicians, individually and by service, dramatically increased hand hygiene compliance, defi ned as both on entry and exit from the patient room, over the study period. See Figure 1. Conclusion Physician behavior is responsive to monthly feedback that is specifi c to the individual or surgical service. Use of the Unit Coordinator was very eff ective at gathering a very large sample size in a short period of time. P54 Compliance with the implementation of an ICU cluster-randomized trial assessing the benefi ts and potential harms of universal glove and gowning D Kett1, DJ Morgan2, L Pineles2, MJ Zervos3, LS Munoz-Price1, AD Harris2, BUGG Investigators2 1University of Miami/Jackson Memorial Hospital, Miami, FL, USA; 2University of Maryland/VA Maryland Healthcare System, Baltimore, MD, USA; 3Henry Ford Health System, Detroit, MI, USA Critical Care 2013, 17(Suppl 2):P54 (doi: 10.1186/cc11992) Reference 1. Pittet D, et al.: Infect Control Hosp Epidemiol 2009, 30:611-622. Compliance with the implementation of an ICU cluster-randomized trial assessing the benefi ts and potential harms of universal glove and gowning Results During the survey, in May 158 opportunities to observe hand hygiene were presented and 286 in September. Overall compliance improved from 34.2% (54/158) to 51% (146/286), χ2 = 11.7 (P <0.001). In May, 50 HCW had a mean of 63.20 ± 39.37 colony-forming units (CFU) on their hands compared with 43.0 ± 40.19 CFU on the hands of 50 HCW in September (P = 0.024). We also observed an initial increased use of alcohol-based hand rubs from 21 ml per patient-day in May to a maximum 72 ml per patient-day in June, but a decline to 44 ml per patient-day in September, Pearson correlation coeffi cient = 0.31 (P = 0.61). D Kett1, DJ Morgan2, L Pineles2, MJ Zervos3, LS Munoz-Price1, AD Harris2, BUGG Investigators2 g 1University of Miami/Jackson Memorial Hospital, Miami, FL, USA; 2University of Maryland/VA Maryland Healthcare System, Baltimore, MD, USA; 3Henry Ford Health System, Detroit, MI, USA Critical Care 2013, 17(Suppl 2):P54 (doi: 10.1186/cc11992) Introduction The Benefi ts of Universal Glove and Gowning (BUGG) study is a cluster-randomized trial to evaluate the use of wearing gloves and gowns for all patient contact in the ICU. The primary outcome is VRE and MRSA acquisitions; secondary outcomes include frequency of healthcare worker visits, infection rates, hand hygiene compliance and adverse events. Conclusion Implementation of a new hand hygiene program at our ICU resulted in improved hand hygiene compliance and less CFU on the hands of HCW. There was no signifi cant increased use of alcohol-based hand rubs over time. The results indicate that constant awareness is vital for success. Methods We enrolled 20 ICUs in 15 states. ICUs collected nasal and perianal swabs on all patients at admission and discharge/transfer. After a 3-month baseline period, 10 units were randomized to the intervention arm and required to wear gloves and gowns for all patient contact. An intervention toolkit was created based on site feedback and compliance reports. Swab collection compliance was fed back and discussed during site conference calls on a weekly basis. Site coordinators monitored compliance with gloves and gowns, hand hygiene and frequency of HCW visits and reviewed patient charts for adverse events. Sinks as a correctable source of ESBL contamination for patients in the ICU p yf g p Methods Thirty-nine medical wards (MW) and 12 emergency depart- ments (ED) were enrolled. Every unit was asked to fi ll in a pre-agreed HOR Checklist focused on the main requirements suggested by the Guidelines. I Wolf1, P Bergervoet1, W Van der Zwet1, H Van den Oever1, P Savelkoul2, F Sebens1 Results Analysing the human resources available, we see that the bed- to-doctor ratio signifi cantly (P <0.01) increases from the day to the night shift: from 6 to 43 beds per doctor on the MW (median). Otherwise, the ED staff remains roughly the same: from 3.5 to 2.5 doctors on duty (median). The analysis of the organizational tools (Table 1) points out Introduction The incidence of patients carrying ESBL-positive bacteria in our ICU (12 in 780 admissions in 2011) was not considered problematic. However, routine cultures had identifi ed ESBL-negative patients who had become colonized with ESBL strains during their ICU stay. Self-disinfecting siphons, preventing bacterial growth by antibacterial coating and intermittent heating, and biofi lm formation by electromechanical vibration, were placed in all sinks in the ICU. The aim of the present study was to evaluate the eff ect of this intervention. Methods An intervention study in a 12-bed ICU. The intervention involved placement of 19 self-disinfecting siphons (Biorec). All patients with an expected ICU stay of 2 days or more between January 2011 and December 2012 were studied. Samples of throat, sputum and rectum were taken at admission and twice weekly, and cultured for ESBLs. Between June 2011 and October 2011, sinks in patient rooms were cultured regularly for ESBLs. After the intervention in April 2012, multiple repeat cultures were taken. Whenever the species and antibiogram of bacteria cultured from patients and sinks matched, they were typed by AFLP. P55 Fighting hospital sepsis l a low percentage of hospitals having: a Diagnostic and Therapeutic Protocol for sepsis management (8.3%), some Hospital Empirical Antibiotic Therapy Guidelines (0%) and an Infective Source Eradication Protocol (8.3%). Moreover, just 25% of hospitals involve an infectious diseases expert in every case of severe sepsis or septic shock. p y p p Conclusion We guess that the poor availability of HOR showed by the hospitals could have a role in the Guidelines implementation and in the patient’s outcome. Only a comparison between these results and data collected from a Clinical Checklist, focused on sepsis bundle compliance, and from a patient’s outcome summary could confi rm our hypothesis. This is the aim for our next part of the study. Introduction Sepsis accounts for a very high mortality. The Surviving Sepsis Campaign recommends a fi rst 6 hours resuscitative bundle to improve patient outcome. Despite this, the bundle is poorly performed because of several organizational and cultural barriers. In recognition of this, we guess that an Educational and Organizational Intervention out of the ICUs could impact on septic patient outcome. In order to test our hypothesis we carried out, in 12 hospitals, a pre-intervention survey of the human and organizational resources (HOR) available in the management of septic patients. The aim is to seek any barrier potentially aff ecting correct Guidelines implementation. 1. Dellinger RP, et al.: Intensive Care Med 2008, 34:17-60. Improving hand hygiene performance in a surgical ICU: a novel method for data collection to change physician behavior SA Nasraway Tufts Medical Center, Boston, MA, USA Critical Care 2013, 17(Suppl 2):P53 (doi: 10.1186/cc11991) Results During the 12-month study period, 100,210 swabs were collected. After the baseline period, we were able to achieve and maintain swab compliance rates between 85 and 97%. Monthly discharge compliance increased by 21% by the beginning of the intervention period (Figure 1). Observers found 86% compliance with universal glove and gowning over 1,242 30-minute observation periods (Figure 1). Ninety charts at each site were reviewed for adverse events. Conclusion Over a diverse group of US hospitals, we achieved high compliance with surveillance cultures and implementing universal gloving and gowning was achieved quickly with high compliance. Critical Care 2013, 17(Suppl 2):P53 (doi: 10.1186/cc11991) Introduction ICU-acquired infection is directly related to hospital mortality. Hand hygiene is an eff ective, low-cost intervention that can prevent the spread of bacterial pathogens, including multidrug- resistant organisms. Historical compliance with hand hygiene guidelines by physicians, nurses and other care providers is poor. Methods Present expectations by the Infection Control Committee are to ‘pump in, pump out’ of every room, using 63% isopropyl alcohol. We performed 17,622 observations of hand hygiene in the surgical ICU Figure 1 (abstract P54). Swab collection and gown/glove compliance. Figure 1 (abstract P53). Figure 1 (abstract P54). Swab collection and gown/glove compliance. Figure 1 (abstract P53). Figure 1 (abstract P54). Swab collection and gown/glove compliance. Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S20 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 P55 Fighting hospital sepsis AR Calini, S Vesconi, R Fumagalli, S Marchesi, L Ghezzi, G Monti Ospedale Niguarda Ca’ Granda, Milan, Italy Critical Care 2013, 17(Suppl 2):P55 (doi: 10.1186/cc11993) P55 Fighting hospital sepsis AR Calini, S Vesconi, R Fumagalli, S Marchesi, L Ghezzi, G Monti Ospedale Niguarda Ca’ Granda, Milan, Italy Critical Care 2013, 17(Suppl 2):P55 (doi: 10.1186/cc11993) Clinical effi cacy of recombinant human soluble thrombomodulin in patients with septic disseminated intravascular coagulation Y Suzuki, R Sato, M Sato, S Endo Iwate Medical University, Morioka, Japan Critical Care 2013, 17(Suppl 2):P59 (doi: 10.1186/cc11997) Results All enalapril pigs received the maximum NE dose without reaching the target MAP. Enalapril resulted in lower MAP, higher CO, and transiently increased regional blood fl ows (Table 1). Fluid administration and urinary output were similar among groups. Liver mitochondrial respiration was reduced (State 3, State 4) in the enalapril group. One animal died in each ML and enalapril group. y p Critical Care 2013, 17(Suppl 2):P59 (doi: 10.1186/cc11997) Introduction Thrombomodulin is an endothelial cell cofactor and glycoprotein for thrombin-catalyzed activation of protein C. A recom- binant human soluble thrombomodulin (rhsTM) has been recently developed, and this new agent has a unique amino-terminal structure exhibiting anti-infl ammatory activity including sequestraction and cleavage of high-mobility group box 1(HMGB-1). Conclusion Enalapril enhances cardiac output and early regional blood fl ows in sepsis despite lower MAP, and reduces liver mitochondrial respiration, suggesting that RAS is involved in the hemodynamic and metabolic changes in sepsis. Methods In this study, 13 patients with septic disseminated intravascular coagulation (DIC) were treated with rhsTM, which is Recomodulin® Inj. 12800 (Asahi Kasei Pharma Co., Tokyo, Japan). Patients with septic DIC were treated with 130 to 380 U/kg/day.i Eff ects of intraperitoneal immunoglobulin therapy on behavior and cognitive functions in CLP-induced sepsis model in rats F Esen1, E Senturk1, P Ergin Ozcan1, G Orhun1, S Gümrü2, M Kaya1, N Orhan1, N Arıcan1, F Arıcıoglu2 1University of Istanbul, Turkey; 2Marmara University Faculty of Pharmacy, Istanbul, Turkey Critical Care 2013, 17(Suppl 2):P58 (doi: 10.1186/cc11996) Results There were signifi cant results for improvement of APACHE II score and DIC diagnostic criteria score for critically ill patients after treatment using rhsTM (P <0.01). Improvement for platelet count and D-dimer level were also observed in this study (P <0.05). Activation of antithrombin (AT) also was signifi cantly increased after treatment (P <0.05). Hospital mortality was 15.4% in this study. Critical Care 2013, 17(Suppl 2):P58 (doi: 10.1186/cc11996) Introduction The present study investigated the eff ects of a single dose of intraperitoneal (i.p.) IgG and IgGAM administration on various behavioral alterations in a cecal ligation perforation (CLP)-induced sepsis model in rats.i Conclusion The rhsTM might be one of most important endogenous regulators of coagulation, acting as the major inhibitor of thrombin as well as AT III. This new agent may play an important role in treatment for septic DIC. Sinks as a correctable source of ESBL contamination for patients in the ICU Table 1 (abstract P55) Availability Resource (%) Diagnostic and therapeutic protocol for septic patient management 8.3 Use of early warning score for diagnosis and management 8.3 Sepsis team 25 Microbiology laboratory: open 7 days a week 66.6 Lactate dosage: 24 hours a day availability 83.3 Central venous catheter insertion (CVC) available 24 hours a day 91.6 Hospital empirical antibiotic therapy guidelines 0 Infectious diseases team advice in any case of severe sepsis septic shock 25 Infective source eradication Infective source eradication protocols 8.3 Intervention radiology available 24 hours a day 50 Operating room available 24 hours a day 58.3 Specifi c infection management protocol (that is, CVC infection) 58.3 Imaging reporting service available 7 days a week 41.6 yp y Results Before intervention Multiple ESBL-forming strains were found in sinks of all patient rooms. Eighteen patients who were ESBL-negative on ICU admission became colonized with 11 diff erent ESBL strains, that were present in sinks of their admission rooms (Figure 1). Four contaminations were proven by AFLP-tying. One patient died of ESBL- positive E. cloacae pneumonia. After intervention All sinks were negative for ESBL strains. No further patients became ESBL colonized during the ICU stay. y Conclusion Wastewater sinks were the likely source of ESBL colonization for 18 ICU patients. After placing self-disinfecting siphons Figure 1 (abstract P56). Design and results of the intervention. Figure 1 (abstract P56). Design and results of the intervention. Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S21 Table 1 (abstract P57). Hemodynamic variables, among times and groups there were no new ESBL colonizations in patients. This coincided with th di f ESBL t i f ll i k of immunoglobulin therapy on behavioral changes in CLP-induced i i t Table 1 (abstract P57). Clinical effi cacy of recombinant human soluble thrombomodulin in patients with septic disseminated intravascular coagulation Y Suzuki, R Sato, M Sato, S Endo Iwate Medical University, Morioka, Japan Critical Care 2013, 17(Suppl 2):P59 (doi: 10.1186/cc11997) Methods Female Wistar albino rats (200 to 250 g) were divided into fi ve groups (n = 8): a naive Control group, a Sham operated group receiving conventional antibiotic treatment, a CLP group receiving CLP procedure and conventional antibiotic treatment, and IgG and IgGAM groups which were also applied 1 g/kg, i.p. IgG and IGAM therapy 5 minutes after the CLP procedure. Ten, 30 and 60 days after the surgery, animals underwent three behavioral tasks: an open fi eld test to evaluate the locomotor activity, an elevated plus maze test to measure the level of anxiety, and a forced swim test to assess the possible depressive state. The results acquired from these tests were used to estimate the eff ect Sinks as a correctable source of ESBL contamination for patients in the ICU Immunoglobulin therapy prevented the occurrence of these behavioral changes. Especially, animals in the IgGAM group conserved the values quite near to those of the control group in measured parameters. Sinks as a correctable source of ESBL contamination for patients in the ICU Hemodynamic variables, among times and groups P BL R0h R12h R24h R36h R48h (time×group) MAP (ENL/MH/ML) 66 (9)/72 (15)/ 65 (11)/73 (10)/ 57 (4)/82 (6)/ 58 (8)/78 (6)/ 56 (12)/80 (5)/ 56 (11)/79 (9)/ 0.003 65 (9) 81 (16) 72 (13) 67 (12) 61 (6) 56 (13) CO (ENL/MH/ML) 106 (13)/89 (7)/ 92 (17)/67 (11)/ 169 (16)/95 (19)/ 187 (22)/129 (28)/ 161 (25)/139 (32)/ 157 (37)/140 (21)/ <0.0001 105 (13) 88 (22) 104 (13) 143 (31) 145 (13) 150 (18) Carotid fl ow (ENL/MH/ML) 6 (2)/7 (2)/7 (2) 5 (1)/4 (1)/5 (2) 10 (3)/5 (1)/6 (2) 10 (3)/8 (1)/9 (2) 8 (2)/8 (2)/8 (2) 8 (3)/8 (2)/8 (2) <0.0001 Femoral fl ow (ENL/MH/ML) 4 (1)/3 (1)/4 (2) 2 (1)/2 (0)/2 (1) 4 (1)/2 (1)/2 (0) 4 (1)/3 (1)/3 (1) 3 (1)/3 (1)/4 (1) 3 (2)/3 (1)/4 (1) 0.004 of immunoglobulin therapy on behavioral changes in CLP-induced sepsis in rats.ii Results In the open fi eld test, the CLP group showed a signifi cant decrease in total squares passed on days 10 and 30. Similarly, total numbers of rearing and grooming were dramatically decreased in the CLP group in comparison with control and sham groups (P <0.005). In the elevated plus maze test, the number of entries to open arms decreased in the CLP group. In the forced swim test, there was a tendency for increase in immobility time in the CLP group, although the data were statistically insignifi cant. All of these values which were indicating the importance of behavioral alterations were improved on day 60. Immunoglobulin therapy prevented the occurrence of these behavioral changes. Especially, animals in the IgGAM group conserved the values quite near to those of the control group in measured parameters. Results In the open fi eld test, the CLP group showed a signifi cant decrease in total squares passed on days 10 and 30. Similarly, total numbers of rearing and grooming were dramatically decreased in the CLP group in comparison with control and sham groups (P <0.005). In the elevated plus maze test, the number of entries to open arms decreased in the CLP group. In the forced swim test, there was a tendency for increase in immobility time in the CLP group, although the data were statistically insignifi cant. All of these values which were indicating the importance of behavioral alterations were improved on day 60. P57 Hemodynamic and mitochondrial eff ects of enalapril in experimental sepsis AJ Pereira, V Jeger, T Corrêa, M Vuda, S Djafarzadeh, J Takala, S Jakob University Hospital (Inselspital)/University of Bern, Switzerland Critical Care 2013, 17(Suppl 2):P57 (doi: 10.1186/cc11995) Introduction Sepsis may cause mitochondrial dysfunction. The renin–angiotensin system (RAS) activity is increased in sepsis, and can interfere with the mitochondrial function either directly or by modifying hemodynamics. We studied the eff ects of ACE inhibition by enalapril on hemodynamics and hepatic mitochondrial function in sepsis. Two septic groups without enalapril and with diff erent blood pressure targets served as controls. Conclusion Sepsis, even though it has been treated with conventional antibiotics, caused a negative eff ect on behavioral parameters. In this study, IgG and IgGAM treated animals in the presence of CLP did not show these behavioral changes. Therefore our results suggest that a single dose of i.p. IgG and IgGAM treatment, which was applied immediately after the sepsis procedure, prevents behavioral defects observed following sepsis. p g Methods Sepsis (fecal peritonitis) was induced in 24 anesthetized, mechanically ventilated pigs (40.1 ± 2.1 kg), divided into three groups (n = 8 for each): ENL (enalapril pretreatment for 7 days and at 0.005 to 0.02 mg/kg/hour during the study; MAP target 75 to 85 mmHg), ML (target MAP 50 to 60 mmHg), and MH (target MAP 75 to 85 mmHg). Hemodynamic support with fl uids, norepinephrine (NE; maximum dose 5,000 μg/hour), and antibiotics were started after 12 hours of peritonitis and continued for 48 hours. P59 59 Clinical effi cacy of recombinant human soluble thrombomodulin in patients with septic disseminated intravascular coagulation Y Suzuki, R Sato, M Sato, S Endo Iwate Medical University, Morioka, Japan Critical Care 2013, 17(Suppl 2):P59 (doi: 10.1186/cc11997) P61 A clinical randomized trial on the use of atorvastatin in patients with sepsis or septic shock: eff ects on endothelial function K Prado, C Ribeiro, T Furian, R Pinto Ribeiro, D Silvello, L Rohde, N Clausell, L Becker Hospital de Clinicas de Porto Alegre, Brazil Critical Care 2013, 17(Suppl 2):P61 (doi: 10.1186/cc11999) Results Forty-three patients (18 treated, 25 control) completed the study and were further analyzed. Incidence of organ dysfunction at enrollment (treatment vs. control) was: septic shock (94% vs. 100%, P = 0.42), acute respiratory distress syndrome (67% vs. 56%, P = 0.33), and renal failure (39% vs. 24%, P = 0.54). During 115 treatments no serious device-related adverse events occurred. On average, there were no changes in hematology and other blood parameters except for a modest reduction in platelet count (<10%) and albumin (<5%) with treatment. Hemoperfusion decreased IL-6 blood concentration signifi cantly (–49.1%, P = 0.01), with similar reductions of MCP-1 (–49.5%, P = 0.002), IL-1ra (–36.5%, P = 0.001), and IL-8 (–30.2%, P = 0.002). The 28-day mortality (28% vs. 24% control, P = 0.84) and 60- day mortality (39% vs. 32% control, P = 0.75) did not diff er signifi cantly between the two studied groups.i Introduction Sepsis and septic shock are complex infl ammatory syndromes. Multiple cellular activation processes are involved, and many humoral cascades are triggered. Presumably, endothelial cells play a pivotal rule in the pathogenesis of sepsis, not only because they may infl uence the infl ammatory cascade but also because, upon interaction with excessive amounts of infl ammatory mediators, the function of these cells may become impaired. It is likely that a general dysfunction of the endothelium is a key event in the pathogenesis of sepsis [1]. HMG-CoA-reductase inhibitors have been shown to exhibit pronounced immunomodulatory eff ects independent of lipid lowering. Most of these benefi cial eff ects of statins appear to involve restoring or improving endothelial function [2]. We hypothesize that statins can improve endothelial dysfunction in septic patients. Conclusion In this fi rst clinical study of a novel sorbent hemoadsorption device in patients with severe sepsis and ALI, the device appeared to be safe and decreased the blood concentration of several cytokines. Further research is needed to study the eff ect of the device on the clinical outcome of septic patients. y Methods A double-blinded, placebo-controlled, randomized trial was undertaken. A multicenter randomized controlled study of an extracorporeal cytokine hemoadsorption device in septic patients D Schädler1, C Porzelius2, A Jörres3, G Marx4, A Meier-Hellmann5, C Putensen6, M Quintel7, C Spies8, C Engel2, N Weiler1, M Kuhlmann9 1University Medical Center Schleswig-Holstein, Campus Kiel, Germany; 2University of Leipzig, Germany; 3Charité University Hospital Campus Virchow- Klinikum, Berlin, Germany; 4RWTH University Hospital Aachen, Germany; 5HELIOS Klinikum, Erfurt, Germany; 6University of Bonn, Germany; 7University Hospital Göttingen, Germany; 8Campus Charité Mitte and Campus Charité Virchow-Klinikum, Charité-University Medicine Berlin, Germany; 9Vivantes Klinikum im Friedrichshain, Berlin, Germany i Results There were 85 patients (52 men, 33 women; age range 19 to 92 years (mean 69.0 ± 16.9)), and the mortality rate was 27.1% and APACHE II score was 20.1 ± 7.6. WBC, CRP, PT and SOFA score were signifi cantly improved after AT III concentrate therapy (13,176 ± 9,400 vs. 11,693 ± 7,089, P = 0.011, 15.9 vs. 13.3, P = 0.0015, 16.5 ± 4.4 vs. 15.9 ± 5.9, P = 0.0045, and 9.7 ± 3.8 vs. 8.6 ± 4.6, P = 0.01, respectively). Platelet was signifi cantly decreased (11.7 ± 9.7 vs. 10.6 ± 9.0, P = 0.028), while there were no signifi cant diff erences in fi brinogen, FDP and DIC score (387.3 ± 202.6 vs. 381.8 ± 163.2, P = 0.088, 36.9 ± 52.7 vs. 28.8 ± 39.9, P = 0.059, and 2.9 ± 1.6 vs. 2.6 ± 1.8, P = 0.25, respectively) after the therapy. One week after the therapy, platelet and DIC score were signifi cantly improved compared with before the therapy (15.6 ± 10.0, P = 0.0036 and 1.8 ± 1.9, P = 0.0041). , , y Critical Care 2013, 17(Suppl 2):P62 (doi: 10.1186/cc12000) Introduction A novel sorbent hemoadsorption device for cytokine removal (CytoSorbents, USA) was developed and successfully tested in animal models of sepsis. The experience in the clinical setting is still limited to case reports. In this fi rst clinical trial, we tested the hypothesis that treatment with sorbent hemoadsorption could safely and eff ectively reduce cytokines in septic patients with acute lung injury (ALI). Introduction A novel sorbent hemoadsorption device for cytokine removal (CytoSorbents, USA) was developed and successfully tested in animal models of sepsis. The experience in the clinical setting is still limited to case reports. In this fi rst clinical trial, we tested the hypothesis that treatment with sorbent hemoadsorption could safely and eff ectively reduce cytokines in septic patients with acute lung injury (ALI). References 1. Hack EC, et al.: The endothelium in sepsis: source of and a target for infl ammation. Crit Care Med 2001, 29:S21-S27.if 1. Hack EC, et al.: The endothelium in sepsis: source of and a target for infl ammation. Crit Care Med 2001, 29:S21-S27.if l p Methods From January 2009 to December 2011, adult septic patients, whom were given AT III concentrate in our medico-surgical ICU, were included in this study. AT III concentrate was administered 30 to 60 U/kg intravenously every 24 hours for 3 days in the patients with DIC status and AT III defi ciency. Between before and after the AT III concentrate therapy, WBC (/mm3), CRP (mg/dl), platelet (×104/μl), PT (seconds), fi brinogen (mg/dl), FDP (μg/ml), SOFA score and DIC score were evaluated. Values are expressed as mean ± SD. Data were analyzed by Wilcoxon signed-rank test. P <0.05 was considered signifi cant. 2. Davignon J: Benefi cial cardiovascular pleiotropic eff ects of statins. Circulation 2004, 109(Suppl III):III-39-III-43. 2. Davignon J: Benefi cial cardiovascular pleiotropic eff ects of statins. Circulation 2004, 109(Suppl III):III-39-III-43. P60 Antithrombin III concentrate therapy may have an effi cacy in sepsis N Saito, M Takeda, T Harada, R Moroi, M Namiki, A Yaguchi Tokyo Women’s Medical University, Tokyo, Japan Critical Care 2013, 17(Suppl 2):P60 (doi: 10.1186/cc11998) Introduction Antithrombin III (AT III) has been known to contribute to anti-infl ammatory response as well as its anticoagulation. Our previous S22 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Conclusion Our data showed no benefi t with the use of a potent statin acutely in patients with sepsis or septic shock with regards to improvement in endothelial function. study showed AT III defi ciency happened in the early stage of sepsis with no relation to DIC status. However, whether AT III concentrate is a benefi cial therapy or not for septic patients is controversial. Our hypothesis is that AT III concentrate may have effi cacy as anti- infl ammation for sepsis. study showed AT III defi ciency happened in the early stage of sepsis with no relation to DIC status. However, whether AT III concentrate is a benefi cial therapy or not for septic patients is controversial. Our hypothesis is that AT III concentrate may have effi cacy as anti- infl ammation for sepsis. P61 We enrolled adult patients within 24 hours of severe sepsis or septic shock diagnosis and randomized them to placebo or atorvastatin 80 mg/day for a short term. Endothelial dysfunction was assessed measuring plasmatic levels of IL-6, ET-1, VCAM-1 by ELISA and measuring fl ow-mediated vasodilatation of the brachial artery at basal, 24 and 72 hours after randomization. A multicenter randomized controlled study of an extracorporeal cytokine hemoadsorption device in septic patients Conclusion In the patients with septic DIC, WBC, CRP and SOFA score were immediately improved after the AT III concentrate therapy, while platelet and DIC score were improved later. AT III concentrate may also contribute to anti-infl ammatory for septic DIC with anticoagulation. Methods Ventilated patients fulfi lling the criteria for severe sepsis and ALI were enrolled in this multicenter randomized, controlled, open- label study comparing standard of care with or without hemoperfusion treatment. Primary endpoints were safety and IL-6 reduction. Treated patients underwent hemoperfusion at fl ow rates of ~200 to 300 ml/ minute for 6 hours per day for 7 consecutive days. The overall mean reduction in individual plasma cytokines for the control and treatment groups during the treatment period was calculated using a generalized linear model. P64 Methods Adult patients with septic shock who required renal replacement therapy (RRT), with a confi rmed endotoxemia, and suspected Gram-negative infection were consecutively added to the study within the fi rst 24 hours after diagnosis. All patients received full standard treatment for septic shock. Endotoxin elimination was performed using the membrane oXiris (Gambro, Sweden), a medical device for continued RRT with the unique feature of endotoxin adsorbtion. An endotoxin activity assay was used to monitor endotoxin elimination therapy at baseline (T0), 3 hours (T1), 12 hours (T2), 24 hours (T3), 48 hours (T4), and 72 hours (T5). Our key indicators were the improvement in hemodynamics and organ function, and decrease of endotoxin activity (EA) in blood. Continuous variables are presented as mean values with standard deviations. Combined use of pumpless extracorporeal lung assist system and continuous renal replacement therapy with citrate anticoagulation in polytrauma patients y HK Atalan, M Dumantepe, TB Denizalti, IA Tarhan, A Ozler Atasehir Memorial Hospital, Istanbul, Turkey Critical Care 2013, 17(Suppl 2):P64 (doi: 10.1186/cc12002) Introduction The usefulness of a pumpless extracorporeal lung assist system (pECLA) and continuous renal replacement therapy (CRRT) in critically ill patients has been demonstrated in previous studies [1,2]. The aim of this report was to examine combined use of pECLA and CRRT to improve carbon dioxide and infl ammatory mediator removal, which allows for lung protective ventilation strategies. Results High EA level at baseline (0.74 ± 0.14 endotoxin activity units (EAU)) signifi cantly decreased during RRT with oXiris membrane to 0.46 ± 0.02 (T1), 0.34 ± 0.01 (T2), 0.4 ± 0.02 (T3), 0.46 ± 0.04 (T4), 0.35 ± 0.07 (T5) EAU (P <0.05). MAP increased from baseline 72 ± 14 to 81 ± 18, 76 ± 6, 77 ± 7, 83 ± 13, 87 ± 10 mmHg (P <0.05), and the mean norepinephrine use decreased from 0.23 ± 0.04 to 0.19 ± 0.02, 0.11 ± 0.01, 0.09 ± 0.01, 0.04 ± 0.01, 0.0 μg/kg/minute (P <0.05) at T0, T1, T2, T3, T4, T5, respectively. The SOFA score had decreased from 14 ± 4 to 12 ± 2, 9 ± 3, 7 ± 3 points (P <0.05), and the procalcitonin level declined from 107 ± 123 to 45 ± 41, 29 ± 30, 17 ± 157 ± 1 ng/ml (P <0.05) at T0, T3, T4, T5. Methods In our 10 patients with ARDS due to polytrauma and sepsis, pECLA was established by insertion of cannulae to the femoral artery and vein. CRRT cannulae were introduced by venous line of the same vascular access (Figure 1). We preferred regional anticoagulation with trisodium citrate for both CRRT and ILA. Figure 1 (abstract P64). ILA and CRRT connected to the patient. Conclusion RRT with oXiris membrane resulted in the eff ective elimination of endotoxins from the blood. The therapy was associated with an increase in blood pressure, a reduction of vasopressor requirements, and an improvement of organ function. The application of the endotoxin activity assay was useful for bedside monitoring of endotoxemia in ICU patients. P63 Continuous renal replacement therapy with the adsorbent membrane oXiris in septic patients: a clinical experience F Turani, F Candidi, R Barchetta, E Grilli, A Belli, E Papi, A di Marzio, M Falco Aurelia Hospital/European Hospital, Rome, Italy Critical Care 2013, 17(Suppl 2):P63 (doi: 10.1186/cc12001) Results We studied 47 patients, 24 in the placebo group (mean age 52 ± 20 years, 29.1% male; APACHE II risk score 23.5 ± 7.3) and 23 in the statin group (mean age 49.5 ± 18 years, 53.4% male; APACHE II risk score 23 ± 6.9). The baseline characteristics of the placebo group were similar to statin patients as well as the mean length of stay in the ICU (8.6 ± 7.4 and 9.1 ± 8 days, respectively) and the time on vasopressors (49.3 ± 47.1 and 59 ± 91.1 hours, respectively). No signifi cant diff erence was observed on the temporal variation of biomarker levels (IL-6, VCAM-1, ET-1) between treatment and control groups. The intrahospital mortality rate was 26% in the statin group and 45% in the placebo group (P = 0.17). Introduction Renal failure is an important complication of sepsis and CRRT with adsorbing membranes may be useful in this clinical setting [1]. The aims of the study in septic/septic shock patients are to evaluate: the safety of a new hemofi lter membrane oXiris with adsorbing properties and anti-endotoxin activity; the renal and hemodynamic Introduction Renal failure is an important complication of sepsis and CRRT with adsorbing membranes may be useful in this clinical setting [1]. The aims of the study in septic/septic shock patients are to evaluate: the safety of a new hemofi lter membrane oXiris with adsorbing properties and anti-endotoxin activity; the renal and hemodynamic S23 Table 1 (abstract P63) Parameter Units T0 T1 Creatinine mg/dl 1.9 ± 0.1 1.18 ± 0.1 Diuresis ml/24 hours 1,284 ± 78 1,573 ± 98 Norepinephrine μg/kg/minute 0.17 ± 0.2 0.06 ± 0.1* IL-6 pg/ml 572 ± 78 278 ± 57* Procalcitonin ng/ml 35 ± 7 15 ± 2* Endotoxin Level/U 0.64 ± 0.2 0.49 ± 0.1 P <0.05 vs. T0. Results Mean SAPS II and APACHE II scores were 55 and 23 respectively. Mean time on mechanical ventilation was 22 days. Mean ICU stay was 30 days for survivors and 38 days for nonsurvivors. 1. Marshall JC, Foster D, Vincent JL, Cook DJ, Cohen J, Dellinger RP, Opal S, Abraham E, Brett SJ, Smith T, Mehta S, Derzko A: Diagnostic and prognostic implications of endotoxemia in critical illness: results of the MEDIC Study. J Infect Dis 2004, 190:527-534. P66 Use of extracorporeal endotoxin elimination therapy for septic shock gi Results Table 1 presents the main results of this study. y Conclusion In septic/septic shock patients with renal failure, CRRT with a new treated heparin-coated membrane (oXiris; Gambro) is clinically feasible, and has a positive eff ect on renal function and hemodynamics. An adsorbing eff ect on proinfl ammatory mediators may have a role in these results. These data and the trend toward a decrease of endotoxin during the treatment warrant further investigation. Reference Introduction Endotoxin, a component of the outer membrane of Gram- negative bacteria, is considered an important factor in pathogenesis of septic shock [1]. The aim of our study was to determine whether endotoxin elimination treatment added to the standard treatment would improve organ function in patients with septic shock. 1. Rimmelé T, et al.: Nephrol Dial Transplant 2009, 24:421-427. P63 When compared with baseline values most relevant parameters were the improvement in tidal volumes, plateau pressures, PaCO2 levels and pH (Figure 2). Four patients survived while six patients died from sepsis-MOF.f Results Mean SAPS II and APACHE II scores were 55 and 23 respectively. Mean time on mechanical ventilation was 22 days. Mean ICU stay was 30 days for survivors and 38 days for nonsurvivors. When compared with baseline values most relevant parameters were the improvement in tidal volumes, plateau pressures, PaCO2 levels and pH (Figure 2). Four patients survived while six patients died from sepsis-MOF. Conclusion We concluded that pECLA can eff ectively address the impaired gas exchange in ARDS and CRRT is a safe procedure with potential therapeutic value for treating MOF. Citrate anticoagulation was well tolerated and fi lter life was appropriate. The use of the same vascular access for ILA and CRRT may minimize invasive procedures and related side eff ects. Conclusion We concluded that pECLA can eff ectively address the impaired gas exchange in ARDS and CRRT is a safe procedure with potential therapeutic value for treating MOF. Citrate anticoagulation was well tolerated and fi lter life was appropriate. The use of the same vascular access for ILA and CRRT may minimize invasive procedures and related side eff ects. References 1. Bein T, et al.: Int J Emerg Med 2010, 3:177-179. 2. Demetrios J, et al.: Kidney Int 2005, 67:2361-2367. 1. Bein T, et al.: Int J Emerg Med 2010, 3:177-179. response; and the changes of endotoxin and proinfl ammatory molecules. response; and the changes of endotoxin and proinfl ammatory molecules. Methods Forty septic/septic shock patients with renal failure were enrolled in the study. All patients had preoperative endotoxin >0.6 level/units (EAA Spectral D) and were submitted to high-volume hemodiafi ltration (50 ml/kg/hour, Prismafl ex; Gambro) with a new treated heparin-coated membrane (oXiris; Gambro). At T0 (pre- treatment) and T1 (24 hours) the main clinical and biochemical data were evaluated. All data are expressed as mean ± SD. One-way ANOVA test with Bonferroni correction was used to evaluate the data changes. P <0.05 was considered signifi cant. P65 Abstract withdrawn Reference 1. Marshall JC, Foster D, Vincent JL, Cook DJ, Cohen J, Dellinger RP, Opal S, Abraham E, Brett SJ, Smith T, Mehta S, Derzko A: Diagnostic and prognostic implications of endotoxemia in critical illness: results of the MEDIC Study. J Infect Dis 2004, 190:527-534. Figure 1 (abstract P64). ILA and CRRT connected to the patient. S24 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum com/supplements/17/S2 Figure 2 (abstract P64). Changes in tidal volume, plateau pressure, pH and PaCO2. CO Figure 2 (abstract P64). Changes in tidal volume, plateau pressure, pH and PaCO2. P67 (133; 286) to 132 (68; 155) mg/l, P = 0.015 and SOFA score from 7.0 (3,0; 8.0) to 6.0 (3,0; 7.0), P = 0.007. At 24 hours after Alteco LPS, signifi cantly decreased PCT from 8.7 (3.0; 25.9) to 4.8 (2.1; 10.0) ng/ml. The 28-day mortality rate was 14.2% (n = 2) in the PMX-F group and 31.3% (n = 10) in the Alteco LPS group. P67 Selective adsorption of lipopolysaccharide in the complex treatment of patients with severe sepsis S Rei, I Aleksandrova, V Kiselev, M Ilynskiy, G Berdnikov, L Marchenkova, G Bulava, N Borovkova Hospital Research Institute for Emergency Medicine named after N.V. Sklifosovsky, Moscow, Russia Critical Care 2013, 17(Suppl 2):P67 (doi: 10.1186/cc12005) Table 1 (abstract P67). Clinical characteristics of the groups Characteristic PMX-F Alteco LPS Sex M/F 9/5 17/15 Age (years) 41.6 ± 17.1 42.1 ± 13.5 Shock 6 (42.9%) 13 (40.6%) APACHE II 21 (17; 25) 21 (16; 24) SOFA 7 (3.0; 8) 7 (3.5; 11) Table 1 (abstract P67). Clinical characteristics of the groups Introduction Severe sepsis and septic shock remain the most serious problem of critical care medicine with a mortality rate of 30 to 55% [1]. Several studies have demonstrated positive eff ects of selective adsorption of LPS on blood pressure, PaO2/FiO2 ratio, endotoxin removal and mortality [2,3]. The purpose of the study was to evaluate the effi ciency of using the selective adsorption of LPS, Toraymyxin – PMX-F (Toray, Japan) and Alteco® LPS Adsorber (Alteco Medical AB, Sweden), in the complex treatment of patients with severe sepsis. Methods Forty-six patients with Gram-negative sepsis in the postoperative period were enrolled into the study. Toraymyxin – PMX-F was used in the PMX-F group (n = 14), while Alteco LPS adsorption was used in the Alteco LPS group (n = 32). P69 Results Survival is shown in Figure 1. Experiments in each animal were starting at 7:00 am. At 8:00 am, respective mean free cortisol of groups A, C and D was 1.81, 0.55 and 2.05 μg/dl; at 1:00 pm they were 0.92, 0.47 and 1.40 μg/dl. Reference The clinical characteristics are listed in Table 1. The SOFA score, PaO2/FiO2, procalcitonin (PCT), C-reactive protein (CRP), endotoxin activity assay (EAA) was noted before, 24 and 48 hours after the selective adsorption of LPS. Conclusion The use of the LPS-selective adsorption (particularly PMX-F) in patients with severe sepsis leads to improvement of systemic infl ammation and organ dysfunction. References 1. Vincent JL, et al.: Crit Care Med 2006, 34:344-353. Results At 48 hours after PMX-F, signifi cantly decreased PCT from 17.5 (5.0; 40.9) to 7.1 (4.8; 13.0) ng/ml, P = 0.028, decreased CRP from 180 2. Cruz DN, et al.: JAMA 2009, 301:2445-2452. 3. Yaroustovsky M et al.: Blood Purif 2009, 28:227-233. S25 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 or when vasopressor infusion is terminated whilst 11% after a fi xed duration. Withdrawal of CS also diff ered, in that 25% tapered/weaned steroids, 31% stopped it abruptly and 44% of CCPs would base their CS cessation pattern on the clinical context. Only 46% of CCPs believe that CS is benefi cial whereas 44% were unsure of the benefi ts in septic shock. Only 29 (36%) responders indicated that their critical care unit had a written protocol for CS in septic shock. References References 1. Dellinger et al.: Crit Care Med 2008, 36:296-327. 2. Bourne et al.: Anaesthesia 2003, 58:591-596. References 1. Dellinger et al.: Crit Care Med 2008, 36:296-327. 2. Bourne et al.: Anaesthesia 2003, 58:591-596. 1. Dellinger et al.: Crit Care Med 2008, 36:296-327. 2. Bourne et al.: Anaesthesia 2003, 58:591-596. Need for restoration of cortisol serum levels for successful antimicrobial therapy in experimental sepsis T Doulias1, A Pistiki2, P Christopoulos1, V Papaziogas1, E Giamarellos-Bourboulis2, I Koutelidakis1 1University of Thessaloniki, Medical School, Thessaloniki, Greece; 2University of Athens, Medical School, Athens, Greece Critical Care 2013, 17(Suppl 2):P70 (doi: 10.1186/cc12008) T Doulias1, A Pistiki2, P Christopoulos1, V Papaziogas1, E Giamarellos-Bourboulis2, I Koutelidakis1 1University of Thessaloniki, Medical School, Thessaloniki, Greece; 2University of Athens, Medical School, Athens, Greece Critical Care 2013, 17(Suppl 2):P70 (doi: 10.1186/cc12008) yg y y Results This study was terminated early as interim analysis identifi ed no signifi cant clinical benefi t. Baseline characteristics (age/APACHE IV score) were similar between two groups of patients. No signifi cant diff erence was noted between two groups with respect to change in total SOFA score (+1 vs. –5.5 for AT vs. ST, P = 0.382), vasopressor score (–29 vs. –46.6, P = 0.775), PaO2/FiO2 ratio (–26 vs. +163, P = 0.199), total urine output from 0 to 72 hours (3,850 ml vs. 4,570 ml, P = 0.355) and 28-day mortality (14.3% vs. 37.5%, P = 0.569). No signifi cant side eff ect was noted when using this new hemoadsorption device. Introduction It is postulated that clinical benefi t of low-dose hydrocortisone in septic shock is related to reversal of relative adrenal insuffi ciency [1]. This was proved in an animal model of sepsis. fi Methods Sixty-nine Wistar male rats were assigned to the following groups: A, sham-operation; B, sepsis; C, bilateral adrenalectomy and sepsis; D, bilateral adrenalectomy, sepsis and hydrocortisone treatment; E, bilateral adrenalectomy, sepsis and ertapenem treatment; and F, bilateral adrenalectomy, sepsis, hydrocortisone and ertapenem treatment. Sepsis was induced by the i.p. infusion of 1×106 cfu/ml of Escherichia coli after adrenalectomy. Hydrocortisone 10 mg/kg was infused i.v. bid starting 1 hour after bacterial challenge. Ertapenem 5 mg/kg was infused i.v. once daily starting 1 hour after bacterial challenge. Survival was recorded. In a separate set of experiments in 18 rats, animal sacrifi ce was performed to measure the free cortisol concentration. Conclusion This small study cannot identify any extra clinical benefi t on addition of Alteco endotoxin hemoadsorption to standard therapy in patients suff ering from intra-abdominal sepsis with shock due to Gram-negative bacterial infection. g Reference 1. Cruz DN, et al.: JAMA 2009, 301:2445-2452. 1. Cruz DN, et al.: JAMA 2009, 301:2445-2452. P68 Alteco endotoxin hemoadsorption in Gram-negative septic shock patients HP Shum, KC Chan, WW Yan Pamela Youde Nethersole Eastern Hospital, Hong Kong Critical Care 2013, 17(Suppl 2):P68 (doi: 10.1186/cc12006) Introduction Septic shock is a common cause of mortality and morbidity in the ICU. Endotoxin hemoadsorption using a Polymyxin B fi ber column can improve patient outcome [1]. This study investigated the therapeutic eff ect of a new endotoxin hemoadsorption device in Gram-negative septic shock patients. Conclusion The perceptions, usage and cessation of CS in septic shock vary but do appear to have shifted in the last decade. A UK survey in 2003 identifi ed that only 60% of ICUs used CS for septic shock and over 22% perform a short synacthen test [2]. It appears that many intensivists are using CS for septic shock, despite confl icting outcome data. We all strive to practice evidence-based medicine but until we have a robust, reliable and methodical randomised control trial that attempts to resolve the CS debate, practice will remain diverse on this subject, as refl ected by our survey. g p p Methods An open, controlled, prospective, randomized, single-centre trial conducted between February 2010 and June 2012. Patients with septic shock due to intra-abdominal sepsis were recruited and randomized to either standard therapy (ST, n = 8) or standard therapy plus two 2 hourly sessions of Alteco endotoxin hemoperfusion (AT, n = 7). Standard therapy included infective source control, appropriate early antibiotic, low-dose steroid, early continuous renal replacement therapy in the presence of acute kidney injury (RIFLE Injury class or more), hemodynamic optimization and lung-protected ventilatory support. Primary outcome was change in organ dysfunction at 48 hours measured by Sequential Organ Failure Assessment (SOFA) scores. Secondary outcomes were change in vasopressor requirement (measured by vasopressor score), PaO2/FiO2 (fraction of inspired oxygen) ratio, total urine output at 72 hours and 28-day mortality.i Effi cacy and utility of preemptive anti-Gr(+) coverage in selected critically ill patients Effi cacy and utility of preemptive anti-Gr(+) coverage in selected critically ill patients VT Todorova, SM Milanov, G Georgiev, M Milanov Emergency Hospital ‘Pirogov’, Sofi a, Bulgaria Critical Care 2013, 17(Suppl 2):P73 (doi: 10.1186/cc12011) critically ill patients VT Todorova, SM Milanov, G Georgiev, M Milanov Emergency Hospital ‘Pirogov’, Sofi a, Bulgaria Critical Care 2013, 17(Suppl 2):P73 (doi: 10.1186/cc12011) Introduction Based on the results of our previous studies [1] we have identifi ed clinical risk factors for the emergence of Gr(+) infections in our ICU and we have developed a new algorithm for combating them. The choice of the particular antibiotic drug is guided by additional risk factors for severity of illness and data on the infectious focus. The response to therapy and its duration are also stated. The aim of the current study was to evaluate the effi cacy and safety of this preemptive approach. Results In total, 383 patients were admitted with septic shock, of whom 253 (66%) were included; 231(92%) had appropriate antibiotic therapy, of whom 52 (22.5%) died at 7 days. Average time to initiation of appropriate antibiotic therapy was 9 ± 23 hours. By bivariate analysis, body mass index (BMI) <20, SAPS II ≥56, SOFA score ≥11 and bacteremia were signifi cantly associated with 7-day mortality. Urinary tract infection (UTI) was a protective factor. Age, sex, comorbidities (particularly immunosuppression), Knaus score, nosocomial infection and type of germ had no infl uence on 7-day mortality. By multivariate logistic regression, BMI <20 (OR = 4.87, 95% CI = 1.36 to 17.43, P = 0.01) and SOFA score ≥11 (OR = 7.99, 95% CI = 3.11 to 20.5, P <0.001) were the only factors signifi cantly associated with 7-day mortality. UTI was a signifi cant protective factor (OR = 0.30, 95% CI = 0.10 to 0.88, P = 0.03). Time to initiation of appropriate antibiotherapy was not associated with 7-day mortality (OR = 0.99, 95% CI = 0.99 to 1.00, P = 0.48). pp Methods A randomized prospective controlled trial was carried out from September 2010 to September 2012. Patients were submitted to block randomization and stratifi ed on the basis of their initial SAPS II exp score. Antibiotic therapy was started on the day of inclusion in the treatment group and only with proven Gr(+) pathogen in the control group. Steroid therapy in septic shock: survey of practice amongst UK critical care physicians G Rajendran, K Dasari, A Dhrampal j , , p Norfolk & Norwich University Hospital, Norwich, UK Critical Care 2013, 17(Suppl 2):P69 (doi: 10.1186/cc12007) Figure 1 (abstract P70). Survival of rats. Introduction Corticosteroid (CS) therapy in sepsis remains contro- versial and was fi rst introduced in sepsis management for its anti- infl ammatory property. CS has found a role in septic shock amelioration with inconsistent outcomes. The Surviving Sepsis Campaign (SSC) includes CS as a Level 2C recommendation in septic shock [1]. Adapting and practicing SSC guidelines vary between critical care units. Accordingly, a survey was conducted to elucidate the usage of CS for septic shock by UK critical care physicians (CCPs). p y p y Methods Following approval by the UK Intensive Care Society (ICS), the survey was publicised on the ICS website and its newsletter. y Results A total of 81 intensivists responded to this online survey. Seventy-four (92.5%) CCPs prescribed CS only if the septic shock is poorly responsive to fl uid resuscitation and vasopressor therapy. Six (7.5%) initiated CS at the same time as vasopressor therapy. None initiated CS for patients with severe sepsis. No CS other than hydrocortisone is being used. The most commonly used intravenous regimen is 50 mg 6 hourly (65%) followed by 50 mg 8 hourly (11%). Only 10% of CCPs would prescribe it by infusion. Less commonly used regimens were 100 mg 8 hourly (6%) and 100 mg 6 hourly (5%). Only 5% would consider adding fl udrocortisone. Prior to initiating CS, 5% of CCPs would perform a short synacthen test, while 94% would not. The majority (89%) of CCPs would stop CS after resolution of shock state Figure 1 (abstract P70). Survival of rats. S26 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 cases during the outbreak. Completed questionnaires describing 27 patients, 11 nonsurvivors and 16 survivors, were returned.i cases during the outbreak. Completed questionnaires describing 27 patients, 11 nonsurvivors and 16 survivors, were returned.i Conclusion Even when eff ective antimicrobial treatment is adminis- tered, administration of hydrocortisone at a regimen restoring normal secretion is mandatory for survival. Milanov S, et al.: Gram positive nosocomial infections in a general ICU – emerging new clues. Crit Care 2011, 15(Suppl 1):P224. Steroid therapy in septic shock: survey of practice amongst UK critical care physicians p Results In preliminary analysis of categorical data, a signifi cantly (Fisher exact test) greater proportion of patients with compared with without the following fi ndings did not survive; history of alcohol use (P = 0.05); the presence of lethargy (P = 0.01), confusion (P = 0.03), nausea (P = 0.04), abdominal pain (P = 0.02), or the need for vasopressors (P = 0.002), oxygen, mechanical ventilation, or steroids (all P = 0.004) at presentation; and excessive bleeding at surgery (P = 0.01). Initial analysis of continuous data demonstrated that, compared with survivors at presentation, nonsurvivors had signifi cantly (one-way ANOVA) increased respiratory rate, percent neutrophils on complete blood count, hemoglobin, INR, C-reactive protein, and bilirubin and signifi cantly decreased temperature, systolic blood pressure, platelets, sodium, albumin, calcium (corrected for albumin), base excess and bicarbonate (all P ≤0.05).f 1. Annane D, et al.: JAMA 2002, 288:862-871. Effi cacy and utility of preemptive anti-Gr(+) coverage in selected critically ill patients Initial data were gathered on demographics, diagnosis, proven risk factors for sepsis-related mortality, severity of infl ammatory response, ventilator-associated pneumonia and organ dysfunction. Dynamics of SIRS, CPIS and SOFA scores, subsequent infectious isolates, ventilator-free days, length of ICU stay and outcome were followed for each patient. y y Conclusion Prognosis at 7 days of patients with septic shock is largely related to the number of failing organs. The majority received appropriate antibiotic therapy although time to initiation is longer than recommended. Further eff orts are warranted to reduce mortality in patients with septic shock. p Results A total of 170 patients were enrolled. No statistically signifi cant diff erences in their basal characteristics were found. The subsequent score values, length of ICU stay and the number of ventilator-free days were also comparable between groups. The majority of Gr(+) pathogens were isolated between 6 and 10 days of inclusion. No diff erences were found regarding the concomitant Gr(–) fl ora and the related antibiotic therapy. The new organ dysfunction severity was similar in both groups (P = 0.37). The in-hospital mortality was 26.2% in the treatment group versus 18.6% in the control group (P = 0.56). Signifi cant diff erences between the Kaplan–Meier estimates of survival were also not found (log-rank test P = 0.81). No major adverse reactions were observed. Results A total of 170 patients were enrolled. No statistically signifi cant diff erences in their basal characteristics were found. The subsequent score values, length of ICU stay and the number of ventilator-free days were also comparable between groups. The majority of Gr(+) pathogens were isolated between 6 and 10 days of inclusion. No diff erences were found regarding the concomitant Gr(–) fl ora and the related antibiotic therapy. The new organ dysfunction severity was similar in both groups (P = 0.37). The in-hospital mortality was 26.2% in the treatment group versus 18.6% in the control group (P = 0.56). Signifi cant diff erences between the Kaplan–Meier estimates of survival were also not found (log-rank test P = 0.81). No major adverse reactions were observed. Conclusion The implementation of this new policy failed to reduce the degree of organ dysfunction severity and was not associated with signifi cant survival benefi t. Moreover, even though it did not reach statistical signifi cance, a second peak of Gr(+) isolates was observed as a possible complication of the preemptive therapy. P71 Impact of time to initiation of appropriate antibiotic therapy on early mortality of patients with septic shock JP Quenot1, C Binquet2, S Vinault2, A Pavon1 1University Hospital Dijon, France; 2CHU de Dijon, Centre d’Investigation Clinique – Epidémiologie Clinique, Dijon, France Critical Care 2013, 17(Suppl 2):P71 (doi: 10.1186/cc12009) P71 Impact of time to initiation of appropriate antibiotic therapy on early mortality of patients with septic shock JP Quenot1, C Binquet2, S Vinault2, A Pavon1 1University Hospital Dijon, France; 2CHU de Dijon, Centre d’Investigation Clinique – Epidémiologie Clinique, Dijon, France Critical Care 2013, 17(Suppl 2):P71 (doi: 10.1186/cc12009) Impact of time to initiation of appropriate antibiotic therapy on early mortality of patients with septic shock Impact of time to initiation of appropriate antibiotic therapy on early mortality of patients with septic shock JP Quenot1, C Binquet2, S Vinault2, A Pavon1 Introduction The impact of appropriate antibiotic therapy on prognosis of patients with septic shock is well established. However, the prognostic infl uence of time to initiation of antibiotics remains debated. We evaluated the eff ect on 7-day mortality of time to initiation of appropriate antibiotic therapy in patients hospitalised in critical care for septic shock. Conclusion The implications of the apparent diff erences noted between nonsurvivors and survivors in this survey of cases from the fi rst known outbreak of injectional anthrax require further study. However, these diff erences might inform the design of research during future outbreaks or of methods to identify patients most in need of anthrax-specifi c therapies such as toxin-directed antibodies. Methods Secondary analysis from the EPISS cohort. We included only patients admitted to the University Hospital Dijon. Septic shock was defi ned as initiation of vasopressors in a patient with suspected or documented infection with at least one criterion of hypoperfusion (metabolic acidosis and/or renal insuffi ciency and/or hepatic dys func- tion). We excluded patients with no available bacteriological data. Anti- biotherapy was considered appropriate if at least one of the antibiotics prescribed was active against the germ identifi ed. Bivariate and multivariate logistic regression analysis was used to assess the impact of time to initiation of appropriate antibiotherapy on 7-day mortality. Effi cacy and utility of preemptive anti-Gr(+) coverage in selected critically ill patients Whether this approach could lead to vancomycin MIC creep or there could still be a niche for it later in the course of treatment and/or in nontrauma patients remains to be further explored. P72 P74 Gram-negative resistance and need for ICU among urinary tract infections in the United States M Zilberberg1, A Shorr2 1EviMed Research Group, LLC, Goshen, MA, USA; 2Washington Hospital Center Washington, DC, USA Critical Care 2013, 17(Suppl 2):P74 (doi: 10.1186/cc12012) t e p e a e ce o ect o t e t e u t d ug es sta t ( ) o CPE. We defi ned MDR-PA as any PA isolate resistant to ≥3 drug classes. Enterobacteriaceae were classifi ed as CPE if resistant to both a third- generation cephalosporin and a carbapenem. We evaluated specimens from respiratory, bloodstream, urinary tract (UTI) and complicated intra-abdominal infections. Introduction Urinary tract infection (UTI) can lead to both hospitali- za tion and severe sepsis. We theorized that UTI due to Gram- negative (GN) multidrug-resistant P. aeruginosa (MDR-PA), extended- spectrum β-lactamase (ESBL), E. coli (EC) and Klebsiella sp. (KP), and carbapenemase-producing Enterobacteriaceae (CPE) would be frequently isolated in the ICU. Results We identifi ed 327,912 PA (60,695 (18.5%) MDR-PA) and 279,600 Enterobacteriaceae (2,558 (0.9%) CPE) specimens. More than one- quarter (26.1%) of all PA were recovered from ICU patients as compared with 17.9% of all Enterobacteriaceae specimens. Of those specimens originating in the ICU, MDR-PA represented 21.9% of all PA organisms, while CPE represented 1.6% of all Enterobacteriaceae. Pneumonia and UTI accounted for 92.4% of all PA and 84.0% of all Enterobacteriaceae specimens. The proportion of both MDR-PA and CPE was highest in pneumonia (22.0% and 1.6%, respectively) and lowest in UTI (13.7% and 0.6%, respectively). Over the time frame of the study, CPEs emerged and stabilized at approximately 2.8% of all Enterobacteriaceae, while MDR-PA increased slightly from 16.0% of all PA in 2000 to 17.8% in 2009. Conclusion Although CPE organisms have emerged as an important pathogen, MDR-PA remains an order of magnitude more prevalent in the United States. Pneumonia patients and those in the ICU are at an increased risk for both MDR-PA and CPE infections compared with those outside the ICU. y Methods We analyzed a large US-based microbiology database, Eurofi ns TSN, between the years 2000 and 2009. We determined the proportion of isolates caused by MDR-PA, ESBL-EC, ESBL-KP, and CPE relative to their susceptible counterparts. We defi ned MDR-PA as any isolate resistant to ≥3 drug classes. ESBL organisms were defi ned as E. coli and K. pneumoniae resistant to a third-generation cephalosporin. Prevalence of multidrug resistance, extensive drug resistance and pandrug resistance among multiple Gram-negative isolates: experience in a tertiary-care hospital ICU in North India S Dewan, T Sahoo, N Chandra, A Varma Fortis Escorts Heart Institute, New Delhi, India Critical Care 2013, 17(Suppl 2):P76 (doi: 10.1186/cc12014) Prevalence of multidrug resistance, extensive drug resistance and pandrug resistance among multiple Gram-negative isolates: experience in a tertiary-care hospital ICU in North India S Dewan, T Sahoo, N Chandra, A Varma Fortis Escorts Heart Institute, New Delhi, India Critical Care 2013, 17(Suppl 2):P76 (doi: 10.1186/cc12014) Prevalence of multidrug resistance, extensive drug resistance and pandrug resistance among multiple Gram-negative isolates: experience in a tertiary-care hospital ICU in North India g y g Conclusion The microbiology of GN UTI hospitalizations has shifted over the last decade and greater antimicrobial resistance is evident. The prevalence of MDR-PA, ESBL-EC, ESBL-KP, and CPE is higher in the ICU than in other hospital locations. Introduction Antibiotic resistance is a major worldwide problem in the ICU [1]. The situation in developing countries like India is particularly serious. Since the presence of drug-resistant bacteria in the environment is a threat for public health, up-to-date information on local pathogens and the drug sensitivity pattern is very crucial to treat patients. This study was carried out to evaluate the prevalence of multidrug resistance (MDR), extensive drug resistance (XDR) and pandrug resistance (PDR) among multiple Gram-negative isolates in a medical–surgical ICU in a tertiary care hospital in North India. P75 Multidrug resistance among P. aeruginosa and Enterobacteriaceae in US hospitals, 2000 to 2009 M Zilberberg1, A Shorr2 1EviMed Research Group, LLC, Goshen, MA, USA; 2Washington Hospital Center, Washington, DC, USA Critical Care 2013, 17(Suppl 2):P75 (doi: 10.1186/cc12013) Introduction Gram-negative resistance remains a major challenge in the care of critically ill patients. Traditionally, P. aeruginosa (PA) has represented the most concerning pathogen. However, carbapenemase- producing Enterobacteriaceae (CPE) have emerged as a challenge, but h id i l f hi h i l d d Figure 1 (abstract P74). Figure 1 (abstract P74). Figure 1 (abstract P74). Methods We conducted a prospective observational study. All data were analysed using descriptive statistics. All Gram-negative culture isolates over a period of 13 months (October 2011 to October 2012) were included in this study. Isolation and identifi cation were performed using the bact alert system and VITEK2, respectively. Sensitivities were determined by Kirby Bauer disc diff usion and broth dilution using VITEK2-AST cards and interpreted according to Clinical and Laboratory Standards Institute criteria. P72 Enterobacteriaceae were considered CPE if resistant to both a third- generation cephalosporin and a carbapenem. We further examined the evolution of the frequency of resistance among GN UTIs over time. Results We identifi ed 115,201 PA (13.7% MDR-PA), 359,090 EC (5.6% ESBL), 97,419 KP (12.9% ESBL), and 176,110 Enterobacteriaceae (0.6% CPE) UTI specimens. The prevalence of resistance rose for each organism of interest from 2000 through 2009: MDR-PA, 11.6 to 12.3%; ESBL-EC, 3.3 to 8.0%; ESBL-KP, 9.1 to 18.6%; CPE 0 to 2.3%. For each organism the proportion of resistant pathogens was consistently higher among ICU specimens than among specimens from other hospital locations, reaching nearly 20% for MDR-PA (Figure 1). P72 Comparison of survivors and nonsurvivors in 27 confi rmed injectional anthrax cases from the 2009 outbreak in Scotland M Booth1, L Donaldson1, C Xizhong2, S Junfeng2, P Eichacker2 1Glasgow Royal Infi rmary, Glasgow, UK; 2National Institutes for Health, Bethesda, MD, USA Critical Care 2013, 17(Suppl 2):P72 (doi: 10.1186/cc12010) Critical Care 2013, 17(Suppl 2):P72 (doi: 10.1186/cc12010) ( g ) j Conclusion The implementation of this new policy failed to reduce the degree of organ dysfunction severity and was not associated with signifi cant survival benefi t. Moreover, even though it did not reach statistical signifi cance, a second peak of Gr(+) isolates was observed as a possible complication of the preemptive therapy. Whether this approach could lead to vancomycin MIC creep or there could still be a niche for it later in the course of treatment and/or in nontrauma patients remains to be further explored. Introduction From December 2009 to December 2010, 47 patients in Scotland presented with confi rmed anthrax infection manifested by soft tissue disease related to heroin injection. These cases represent the fi rst known outbreak of a recently recognized form of anthrax, termed injectional anthrax, which appears to be associated with a high mortality rate (28% in confi rmed cases from the UK outbreak). While epidemiologic data from this outbreak have been published, no report has systematically described fi ndings in patients at presentation or compared these fi ndings in nonsurvivors and survivors. p Reference Methods To better describe injectional anthrax, we developed a questionnaire and sent it to clinicians who had cared for confi rmed Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S27 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 P74 Methods We analyzed a large US-based microbiology database, Eurofi ns TSN, between the years 2000 and 2009. We aimed to describe the prevalence of infection with either multidrug-resistant (MDR) PA or CPE. We defi ned MDR-PA as any PA isolate resistant to ≥3 drug classes. Enterobacteriaceae were classifi ed as CPE if resistant to both a third- generation cephalosporin and a carbapenem. We evaluated specimens from respiratory, bloodstream, urinary tract (UTI) and complicated intra-abdominal infections. Risk factors associated with Acinetobacter baumannii septicemia and its mortality rates in critically ill patients Risk factors associated with Acinetobacter baumannii septicemia and its mortality rates in critically ill patients K Kontopoulou, K Tsepanis, I Sgouropoulos, A Triantafyllidou, D Socratous, P Tassioudis, E Chasou, E Antypa, F Renta, E Antoniadou, K Mandraveli General Hospital G. Gennimatas, Thessaloniki, Greece Critical Care 2013, 17(Suppl 2):P77 (doi: 10.1186/cc12015) Results Sixty-three patients were admitted. The mean length of stay (MLS) was 3 ± 3 days, and the mean age was 25 ± 14 (13 patients <15 years). Patients were hospitalized for combat-related trauma (74%), noncombat-related trauma, medical pathologies (10%), and postoperative care (8%). They were Afghans (92%) or westerners (8%). Swabs were not realized for eight patients. Forty-three percent revealed an ESBLB colonization: Escherichia coli (22 patients), Klebsiella pneumoniae (one patient), Acinetobacter baumanii (one patient). No patients were colonized with MRSA. Ten patients (16%) were directly admitted to the ICU, 12 (19%) had been hospitalized before admission, 39 (62%) were transferred after resuscitative and stabilization care in a level 2 unit. For the two last categories, the MLS (for previous hospitalization) was respectively 14 ± 28 days and 8 ± 6 hours. Among patients transferred after care in a level 2 unit, MLS was no diff erent between colonized and noncolonized patients: 8 ± 7 versus 9 ± 6 hours (P = 0.5, Mann–Whitney test). y y p K Kontopoulou, K Tsepanis, I Sgouropoulos, A Triantafyllidou, D Socratous, P Tassioudis, E Chasou, E Antypa, F Renta, E Antoniadou, K Mandraveli General Hospital G. Gennimatas, Thessaloniki, Greece Critical Care 2013, 17(Suppl 2):P77 (doi: 10.1186/cc12015) K Kontopoulou, K Tsepanis, I Sgouropoulos, A Triantafyllidou, D Socratous, P Tassioudis, E Chasou, E Antypa, F Renta, E Antoniadou, K Mandraveli General Hospital G. Gennimatas, Thessaloniki, Greece Critical Care 2013, 17(Suppl 2):P77 (doi: 10.1186/cc12015) Introduction Acinetobacter baumannii (A. baum) is a leading cause of septicemia of patients hospitalized in the ICU with high mortality rates. The aim of our study is to investigate the risk factors associated with A. baum bacteremia and its mortality rates. y Methods A total of 937 patients (457 women and 480 men, median age 59) admitted to the ICU during the period 1 January 2009 to 30 September 2012 were enrolled in our retrospective study. Blood cultures were obtained from all patients. The identifi cation and the antimicrobial susceptibility testing were performed by the automated system VITEK2 (Bio Merieux, France). Prevalence of multidrug resistance, extensive drug resistance and pandrug resistance among multiple Gram-negative isolates: experience in a tertiary-care hospital ICU in North India S Dewan, T Sahoo, N Chandra, A Varma Fortis Escorts Heart Institute, New Delhi, India Critical Care 2013, 17(Suppl 2):P76 (doi: 10.1186/cc12014) For the purpose of this study, we used MDR to denote isolates resistant to representatives three or more classes of antimicrobial agents, XDR as those resistant to all but one or two classes and PDR as those resistant to all classes of antimicrobial agents available [2,3]. Results Out of a total 2,796 culture specimens sent over 13 months, 250 isolates were Gram-negative (8.9%). Among these 250 (n) Gram- negative isolates, 195 (78%) were extended-spectrum β-lactamase (ESBL) producers and the remaining 55 (22%) were non-ESBL producers. Among the ESBL producers, PDR, XDR and MDR isolates were 14 (5.6%), 113 (45.2%) and 68 (27.2%), respectively (Figure 1). Among the XDR-positive organisms, seven (6.1%) organisms were New Delhi metallo-β-lactamase-1 (NDM-1) producers and fi ve (4.4%) organisms were NDM-2 producers. Among ESBL-positive isolates, the most predominant isolate was Klebsiella pneumoniae (29.7%) followed by Acinetobacter aeruginosa (22.5%) and Escherichia coli (20.5%) (Figure 2). Among non-ESBL-positive isolates, the most predominant isolate was Escherichia coli (34.5%) followed by Klebsiella pneumoniae (21.8%) and Pseudomonas aeruginosa (14.5%) (Figure 2). P75 Multidrug resistance among P. aeruginosa and Enterobacteriaceae in US hospitals, 2000 to 2009 M Zilberberg1, A Shorr2 1EviMed Research Group, LLC, Goshen, MA, USA; 2Washington Hospital Center, Washington, DC, USA Critical Care 2013, 17(Suppl 2):P75 (doi: 10.1186/cc12013) Multidrug resistance among P. aeruginosa and Enterobacteriaceae in US hospitals, 2000 to 2009 Introduction Gram-negative resistance remains a major challenge in the care of critically ill patients. Traditionally, P. aeruginosa (PA) has represented the most concerning pathogen. However, carbapenemase- producing Enterobacteriaceae (CPE) have emerged as a challenge, but the epidemiology of this pathogen is poorly understood. Conclusion ESBL producers were the most frequently isolated Gram- negative bacterial isolates in this tertiary-care hospital in north India. Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S28 pp http://ccforum.com/supplements/17/S2 Figure 1 (abstract P76). Prevalence of drug-resistant Gram-negative isolates. Figure 2 (abstract P76). Prevalence of Gram-negative isolates. P <0.05 from the previous model and those mentioned at recent medical literature as signifi cantly related with A. baum septicemia and its mortality. Figure 1 (abstract P76). Prevalence of drug-resistant Gram-negative isolates. y Results A total of 101 patients (10.78%) developed bloodstream infection caused by A. baum and the mortality rate due to A. baum septicemia was estimated as 49.5% (50/101). Whitman TJ: Infection control challenges related to war wound infections in the ICU setting. J Trauma 2007, 62:S53. Prevalence of multidrug resistance, extensive drug resistance and pandrug resistance among multiple Gram-negative isolates: experience in a tertiary-care hospital ICU in North India S Dewan, T Sahoo, N Chandra, A Varma Fortis Escorts Heart Institute, New Delhi, India Critical Care 2013, 17(Suppl 2):P76 (doi: 10.1186/cc12014) Multiple regression analysis revealed adjusted mortality >55% (Exp(B) = 2.01, P = 0.013), MIS (Exp(B) = 1.97, P = 0.017), ABL (Exp(B) = 2.34, P = 0.009), LS >14 days (Exp(B) = 1.34, P = 0.034), MV (Exp(B) = 2.67, P = 0.005) and INV.PR (Exp(B) = 3.27, P = 0.001) as independent risk factors associated with A. baum septicemia. p Conclusion The fact that the probability of A. baum bacteremia increases in immunocompromised patients refl ects the opportunistic characteristic of these infections. MV accelerates respiratory A. baum colonization, which is a risk factor for AB bacteremia. Recent INV. PR increased the incidence of A. baum bacteremia and this result is probably related to the severe status in patients with central venous catheter. The administration of carbapenems inhibits the growth of other more susceptible bacteria, allowing the growth of multidrug- resistant A. baum. Figure 1 (abstract P76). Prevalence of drug-resistant Gram-negative isolates. Figure 2 (abstract P76). Prevalence of Gram-negative isolates. P78 Prevalence of colonization by multiresistant bacteria on admission to the ICU in the French military hospital in Kabul, Afghanistan JV Schaal, P Pasquier, H Delacour, A Salvadori, A Jarrassier, JR Renner, SM Mérat Military Teaching Hospital Bégin, Saint-Mandé, France Critical Care 2013, 17(Suppl 2):P78 (doi: 10.1186/cc12016) Prevalence of colonization by multiresistant bacteria on admission to the ICU in the French military hospital in Kabul, Afghanistan Prevalence of colonization by multiresistant bacteria on admission to the ICU in the French military hospital in Kabul, Afghanistan JV Schaal, P Pasquier, H Delacour, A Salvadori, A Jarrassier, JR Renner, SM Mérat Military Teaching Hospital Bégin, Saint-Mandé, France Critical Care 2013, 17(Suppl 2):P78 (doi: 10.1186/cc12016) Military Teaching Hospital Bégin, Saint-Mandé, France Military Teaching Hospital Bégin, Saint-Mandé, France Critical Care 2013, 17(Suppl 2):P78 (doi: 10.1186/cc12016) Figure 2 (abstract P76). Prevalence of Gram-negative isolates. Introduction The French military hospital at the Kaboul International Airport (KaIA) base provides surgical care for International Force and Afghan National Army soldiers, and also local patients. The development of multiresistant bacteria (MRB) nosocomial infections has raised a major problem complicating the care of combat casualties [1]. The aim of this study is to assess the prevalence of MRB carriage on admission to the ICU in this combat support hospital. Introduction The French military hospital at the Kaboul International Airport (KaIA) base provides surgical care for International Force and Afghan National Army soldiers, and also local patients. The development of multiresistant bacteria (MRB) nosocomial infections has raised a major problem complicating the care of combat casualties [1]. The aim of this study is to assess the prevalence of MRB carriage on admission to the ICU in this combat support hospital. Among ESBL producers, XDR organisms were most frequent, followed by MDR and PDR organisms. Few of the XDR isolates were NDM producers, which have propensity to spread to other bacteria. In view of signifi cant prevalence of multidrug resistance amongst Gram-negative organisms in the ICU, regular surveillance of antibiotic susceptibility patterns plays a crucial role for setting orders to guide the clinician in choosing empirical or directed therapy of infected patients. References Methods We used a prospective observation study on patients admitted to the French military ICU in KaIA over 3 months (July to September 2012). All hospitalized patients were assessed for the presence of colonization with MRB: nasal and rectal swabs were performed to identify, respectively, methicillin-resistant Staphylococcus aureus (MRSA) and extended-spectrum β-lactamases bacteria (ESBLB). The following data were recorded for each patient on admission: demographic characteristics, bacteriological results, length of stay, type of previous hospitalization. References 1. Radji M, et al.:Asian Pac J Trop Biomed 2011, 1:39-42. 2. Falagas ME, et al.: Clin Infect Dis 2008, 46:1121-1122. 3. Souli M, et al.: Euro Surveill 2008, 13:30-40. 1. Radji M, et al.:Asian Pac J Trop Biomed 2011, 1:39-42. 2. Falagas ME, et al.: Clin Infect Dis 2008, 46:1121-1122. 3. Souli M, et al.: Euro Surveill 2008, 13:30-40. References 1. Wu HS, et al.: Clin Microbiol Infect 2012, 18:E373-E376 2. Huang ST, et al.: J Microbiol Immunol Infect 2012, 45:356-362. Risk factors associated with Acinetobacter baumannii septicemia and its mortality rates in critically ill patients Data collected included underlying diseases, malignancies and immune suppression (MIS), prognostic factors (APACHE score, adjusted mortality), age, sex, length of ICU stay (LS), recent administration of broad-spectrum β-lactam antibiotics (especially carbapenems; ABL), mechanical ventila- tion (MV), implementation of invasive procedures (central venous catheter and urine catheter; INV.PR) and outcome. At fi rst a univariate statistical model was used with signifi cance level set at P = 0.05. For the multivariate statistical analysis model we used all variables with Conclusion In this study, prevalence of colonization with ESBLB at admission is very high, suggesting a high prevalence of MDR colonization in the local population in Afghanistan. It remains important to intensify the prevention policy against MRB cross-transmission in the deployed ICU. p y Reference p y Reference Whitman TJ: Infection control challenges related to war wound infections in the ICU setting. J Trauma 2007, 62:S53. Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S29 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 (63 patients: 30 received SD and 33 HD), the clinical cure rate and microbiological eradication percentage were higher when TGC was used at higher doses (57.6% vs. 33.3%; P = 0.08 and 57.1% vs. 30.4%; P = 0.1). Table 1 shows multivariate analysis of clinical cure predictors in the VAP subgroup. (63 patients: 30 received SD and 33 HD), the clinical cure rate and microbiological eradication percentage were higher when TGC was used at higher doses (57.6% vs. 33.3%; P = 0.08 and 57.1% vs. 30.4%; P = 0.1). Table 1 shows multivariate analysis of clinical cure predictors in the VAP subgroup. P79 Extended-spectrum β-lactamase Providencia stuartii in a general ICU P Myrianthefs1, E Evodia2, G Fildissis1, G Baltopoulos1 1Athens University, Athens, Greece; 2Iatriko Kentro Athinon, Athens, Greece Critical Care 2013, 17(Suppl 2):P79 (doi: 10.1186/cc12017) Table 1 (abstract P80). Predictors of clinical cure in patients with VAP (multivariate analysis) OR (95% CI) P value HD TGC 5.93 (1.38 to 25.6) 0.02 IIAT 0.23 (0.06 to 0.85) 0.03 SOFA score 0.62 (0.45 to 0.86) <0.01 Table 1 (abstract P80). Predictors of clinical cure in patients with VAP (multivariate analysis) Introduction Providencia stuartii, a member of the family of Entero- bacteriacea, is a Gram-negative pathogen causing colonization and opportunistic infections in ICU patients. pp p Methods We retrospectively recorded the characteristics of P. stuartii infections in our ICU in a total period of 1 year (six-bed general ICU). Methods We retrospectively recorded the characteristics of P. stuartii infections in our ICU in a total period of 1 year (six-bed general ICU). Results A total of 116 patients (80 males, 68.9%) were hospitalized in our ICU of mean age 58.5 ± 1.8, mean ICU stay: 23.2 ± 3.3 days, APACHE II: 19.3 ± 0.7, SAPS II: 45.7 ± 1.6, SOFA: 7.9 ± 0.4 and mortality: 18.9%. Admission diagnosis was multiple trauma (29.3%), emergency surgery (37.1%), and medical (33.6%). Of them 21 (18.1%) developed P. Reference 1. Tasina E, et al.: Effi cacy and safety of tigecycline for the treatment of infectious diseases: a meta-analysis. Lancet Infect Dis 2011, 11:834-844. P81 p y Reference stuartii infection that was related to multiple trauma (P = 0.0289), length of ICU stay 69.8 ± 12.6 (median 51.0) versus 12.6 ± 1.7 (median 6.0) (P <0.0001) and illness severity APACHE II (21.7 ± 1.3 vs. 17.5 ± 0.8; P = 0.0056), SAPS II (54.9 ± 2.9 vs. 43.6 ± 1.8; P = 0.0296) and SOFA (10.6 ± 0.5 vs. 7.2 ± 0.4; P <0.0001). There was no statistically signifi cant diff erence regarding sex, age or mortality (P = 0.3789). Mean day of fi rst isolation was 31.1 ± 2.1 (median 28.0). The number of isolations per site were – blood: 11 (median day: 25.0), tracheal aspirates: 9 (median: 28.5), catheter tip: 15 (median: 31.0), urine: 6 (median: 32.0), wound: 12 (median: 25.0), biological fl uids: 4 (median: 29.5), other catheters: 1 (median: 28.0), middle ear: 2 (median: 32.5), and nose: 1 (median: 25.0). Six patients had only one site isolation and the remaining 15 had multiple sites of P. stuartii isolation. We totally recorded 63 isolates of which 82.5% were second-generation and third-generation cephalosporin-resistant, 80.3% aztreonam-resistant and 81% carbapenem-resistant strains expressing an extended-spectrum β-lactamase phenotype. All patients had previously received colistin or meropenem or tygecycline for a median period of 18.5, 15 and 10.5 days, respectively. Conclusion In critically ill patients, HD TGC use seems to be safe and, combined with other active antibiotics, may increase the rate of MDR germ VAP clinical success. IIAT and the severity degree of patients’ clinical condition still remain major determinants of VAP treatment failure. In vitro effi ciency of Amikacin Inhale, a novel drug-device delivery system y N Kadrichu1, S Boc1, K Corkery1, P Challoner2 1Novartis Pharmaceuticals, San Carlos, CA, USA; 2Nektar Therapeutics, San Francisco, CA, USA Critical Care 2013, 17(Suppl 2):P81 (doi: 10.1186/cc12019) Introduction Amikacin Inhale (NKTR-061, BAY41-6551) is a drug- device combination in clinical development for adjunctive treatment of intubated and mechanically ventilated patients with Gram-negative pneumonia. The product uses a proprietary vibrating mesh nebulizer system (PDDS Clinical) with amikacin sulfate formulated for inhalation (3.2 ml of 125 mg/ml amikacin solution) for a 10-day twice-daily course of therapy. It is designed for use with two delivery systems: one system for intubated patients (On-vent; Figure 1), and a second Handheld (HH) system for patients who are extubated before completing the course of therapy (Figure 2). We investigated in vitro the amikacin lung dose delivered by PDDS Clinical. Conclusion P. stuartii infection/colonization may develop in critically ill patients, especially those with multiple trauma and prolonged ICU stay, and maybe isolated in blood after the 25th day of ICU stay. Also, infections due to extended-spectrum β-lactamase-producing multidrug-resistant P. stuartii are an emerging problem. P80 High-dose tigecycline use in severe infections G De Pascale1, L Montini1, T Spanu2, V Bernini1, A Occhionero1, DL Grieco1, M Biancone1, P De Santis1, ES Tanzarella1, SL Cutuli1, MA Pennisi1, MA Antonelli1 1Sacro Cuore Catholic University, Rome, Italy; 2Institute of Microbiology, Sacro Cuore Catholic University, Rome, Italy Critical Care 2013, 17(Suppl 2):P80 (doi: 10.1186/cc12018) P80 High-dose tigecycline use in severe infections G D P l 1 L M i i1 T S 2 V B i i1 A O Methods An estimated lung dose (ELD) for On-vent setting was measured in vitro after collecting aerosolized amikacin from a fi lter at the end of an endotracheal tube during ventilation. The ELD for the HH device was calculated from the fi ne particle fraction (FPF <5 μm) post- mouthpiece, multiplied by the in vitro delivered dose post-mouthpiece. FPF <5 μm refl ects lung deposition observed during phase 2 clinical trials [1]. Eighty-one nebulizers with volume median diameter (VMD) Introduction The aim of this study is to describe the clinical and epidemiological profi le of ICU patients receiving tigecycline (TGC) and to evaluate the potential benefi ts of TGC higher doses. Figure 1 (abstract P81). On-vent system with brown blinder shell for clinical trials. i Methods All patients admitted to our ICU between 1 June 2009 and 31 May 2012 who received TGC were evaluated. Cases were excluded when infections were not microbiologically confi rmed.i i Results Over the study period, 100 patients fulfi lled the inclusion criteria: 54 in the SD group (50 mg every 12 hours) and 46 in the HD group (100 mg every 12 hours). The SD group and the HD group were not signifi cantly diff erent in terms of age, severity of disease, duration of TGC therapy, rate of concomitant other active antibiotic use and of inadequate empirical antimicrobial therapy (IIAT) (P = NS). MDR A. baumannii and K. pneumoniae were the main pathogens isolated. The percentage of germs other than A. baumannii and K. pneumoniae was higher in the SD TGC group (P <0.01). Otherwise infections due to less susceptible germs (TGC MIC value ≥1 μg/ml) were mainly treated with TGC higher doses (P <0.01). No signifi cant diff erences were found in terms of ICU mortality (P = 0.8). The rate of abnormal laboratory measures during TGC treatment was similar between the two groups (P = NS). No patients required TGC discontinuation or dose reduction because of suspected adverse events. In the VAP subpopulation Figure 1 (abstract P81). On-vent system with brown blinder shell for clinical trials. Critical Care 2013, Volume 17 Suppl 2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S30 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 parameters infl uencing the relation between amikacin dose and peak concentration. Figure 2 (abstract P81). Handheld system with brown blinder shell for clinical trials. Cost implication of antibiotic-associated diarrhoea and the fi nancial impact of probiotic use in its prevention p p p C Ebm, M Cecconi, C Moran, A Rhodes, T Rahman C Ebm, M Cecconi, C Moran, A Rhodes, St Georges Healthcare Trust, London, UK St Georges Healthcare Trust, London, UK Reference g Critical Care 2013, 17(Suppl 2):P83 (doi: 10.1186/cc12021) 1. Fink J, et al.: High in vivo amikacin lung deposition after NKTR-061 dosing correlates with in vitro aerosol characterization. Am J Respir Crit Care Med 2008, 177:A530. Introduction Antibiotic-associated diarrhoea (AAD) occurs in as many as 30% of patients receiving antibiotics, often leading to increased morbidity, prolonged in-hospital stay and additional healthcare resource utilisation. Age, antibiotics and prolonged postoperative ward and ICU stay have been suggested to be independent risk factors. In such patient populations, probiotics may be used to prevent antibiotic- associated diarrhoea, yet they are not routinely recommended as a component of perioperative care. The aim of this study was to model the long-term costs associated with AAD and to assess the eff ectiveness of probiotics as a preventive strategy. P80 High-dose tigecycline use in severe infections G D P l 1 L M i i1 T S 2 V B i i1 A O Methods In this retrospective study we looked at 47 patients (128 peak levels) between 2003 and 2012. Multivariate linear regression analysis was done for several parameters: administered dose calculated with total body weight, ideal body weight, adjusted body weight, type of intensive care patient, BMI, daily fl uid balance, SOFA score and APACHE score, and patient characteristics were analyzed. Results A linear correlation between dose and amikacin peak level was confi rmed (Figure 1). A total 54.69% of all amikacin administrations did not result in a therapeutic peak level. The multivariate linear regression analysis showed the best linear correlation with adjusted body weight and SOFA score. The comparison of variables between four patient groups, based on the deviation between measured peak level and predicted peak level (according the linear correlation), showed new variables that may infl uence peak level. yl Conclusion This confi rms that low doses (<18 mg/kg) of amikacin in intensive care patients seldom result in a therapeutic peak level. The proposed loading dose of 25 mg/kg is good for reaching a therapeutic level, although 29.6% remains subtherapeutic. Due to the linear correlation, more therapeutic levels may be reached with higher doses (25 to 30 mg/kg). New variables need further investigation to explain the high variability in achieved peak level. Figure 2 (abstract P81). Handheld system with brown blinder shell for clinical trials. of 4.4 ± 0.5 μm and output rates of 0.23 ± 0.10 ml/minute were tested for each system. Delivered dose data were fi t to the independent variables (that is, VMD and output rates) using a least-squares fi t with 95% confi dence limits. P83 y y Conclusion The results support the use of the PDDS Clinical with either system to administer aerosolized amikacin with high effi ciency and no dose adjustment is required when switching from the On-vent to the HH system for extubated patients. References References 1. Delattre IK, et al.: Ther Drug Monit 2010, 32:749-756. 2. Taccone FS, et al.: Crit Care 2010, 14:R53. 3. Gálvez R, et al.: Int J Antimicrobial Agents 2011, 38:146-151. 4. Avent ML, et al.: Internal Med J 2011, 41:441-449. 5. Fernández de Gatta MM, et al.: J Clin Pharm Ther 1996, 21:417-421. 1. Delattre IK, et al.: Ther Drug Monit 2010, 32:749-756. 2. Taccone FS, et al.: Crit Care 2010, 14:R53. 3. Gálvez R, et al.: Int J Antimicrobial Agents 2011, 38:146-151. 4. Avent ML, et al.: Internal Med J 2011, 41:441-449. 5. Fernández de Gatta MM, et al.: J Clin Pharm Ther 1996, 21:417-421. 1. Delattre IK, et al.: Ther Drug Monit 2010, 32:749-756. 2. Taccone FS, et al.: Crit Care 2010, 14:R53. 2. Taccone FS, et al.: Crit Care 2010, 14:R53. 3. Gálvez R, et al.: Int J Antimicrobial Agents 2011, 38:146-151. i Results Total percentage recoveries for On-vent and HH test runs were between 85% and 115% of the nominal dose. The mean ELDs were 50 ± 9% (On-vent) and 49 ± 11% (HH) of the nominal dose. Nebulizers with longer dosing times and lower VMDs had higher ELD values for both delivery systems. 4. Avent ML, et al.: Internal Med J 2011, 41:441-449. 4. Avent ML, et al.: Internal Med J 2011, 41:441-449. 5 Fernández de Gatta MM et al : J Clin Pharm Ther 1996 21:417 42 5. Fernández de Gatta MM, et al.: J Clin Pharm Ther 1996, 21:417-42 Empirical versus preemptive antimycotic therapy in terms of outcome benefi t l d l Figure 1 (abstract P85). Cologne antifungal strategy. Figure 1 (abstract P85). Cologne antifungal strategy. Introduction Novel treatment strategies for invasive candidiasis (IC) are constantly emerging. Nevertheless, diffi culties in diagnosis pose a challenge on their reliability, effi cacy and safety. We have previously developed and approbated in our ICU an algorithm for empirical antimycotic therapy, combining the most signifi cant risk factors for IC with three major clinical criteria for persistent nonbacterial sepsis [1]. On the other hand, preemptive therapy, based on identifi cation of mycotic antigens and/or anti-mycotic antibodies in serum, is regarded as more reliable, even though it is known for its low sensitivity. The aim of the current study was to compare and evaluate the possible outcome benefi t of our protocol implementation versus detection of galactomanan in patient’s serum as a trigger for antimycotic treatment initiation. Results Mean age was 67.4 years with 64% male patients. Most patients had combined CABG and valve procedure (n = 20), other groups were HTX and LTX (n = 4), assist therapy (n = 4), TAVI (n = 3) and other procedures. Mean predicted mortality using the logarithmic CASUS score at the onset of IFI was 59%. C. albicans was isolated in 73%, C. glabrata in 21%. Length of antifungal treatment using micafungin in 30 cases was 14 ± 5 days. Eradication of yeast was successful in 79% but mortality of all patients remains high at 36.8% but was lower than predicted in the CASUS score. Mortality was not yeast related. Methods A randomized prospective controlled trial was carried out from September 2010 to September 2012. After the implication of the inclusion and exclusion criteria, patients were submitted to block randomization and stratifi ed on the basis of their initial SAPS II exp score. Antimycotic therapy was started on the day of inclusion in the control group and only with positive galactomanan serum test in the preemptive therapy group. Initial data were gathered on demographics, proven risk factors for IC-related mortality, severity of infl ammatory response and organ dysfunction. Dynamics of SIRS and SOFA values, Candida colonization index, ventilator-free days, length of ICU stay and outcome were followed for each patient.i p y y Conclusion Our described treatment approach shows encouraging results for the treatment of IFI especially in high-risk cardiothoracic patients. Results A total of 106 patients were enrolled. Empirical versus preemptive antimycotic therapy in terms of outcome benefi t l d l No statistically signifi cant diff erences in their basal characteristics were found. The subsequent SIRS and SOFA scores showed fi rm dynamics in the control group, although the new organ dysfunction severity was insignifi cantly lower. The length of ICU stay and the number of ventilator-free days were comparable. The in-hospital mortality was 47.1% in the preemptive therapy group versus 31.3% in the control group (P = 0.94). A total of seven adverse reactions were observed among treated patients, yet not associated with higher mortality risk. Analysis of parameters in critically ill patients infl uencing amikacin peak levels p M Hollevoet1, SJ Toye2, J De Waele1 p IM Hollevoet1, SJ Toye2, J De Waele1 Introduction Recent studies demonstrate that a loading dose of 25 mg/kg (total body weight) of amikacin in septic patients is required to reach a suffi cient peak concentration. This study examines Methods We developed a simulation model to determine clinical costs and outcomes attributable to AAD. To assess the cost-eff ectiveness of probiotics, as part of a perioperative regime, we constructed a decision Figure 1 (abstract P82). Correlation between dose of amikacin and peak concentration. Figure 1 (abstract P82). Correlation between dose of amikacin and peak concentration. S31 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 tree. The model observes long-term costs and outcomes of probiotics as compared with conventional therapy, from a societal perspective. Input parameters, extracted from meta-analysis, clinical trials and national databases, include incidence numbers, costs and quality- adjusted health states for the remaining life (QALYs). Outcomes assessed were overall costs attributable to ADD and the cost-eff ectiveness of probiotics, described as costs/QALY. References . Eggiman et al.: Invasive Candida infections in the ICU. Mycoses 20 1. Eggiman et al.: Invasive Candida infections in the ICU. Mycoses 2012, 55(Suppl):65-72. 55(Suppl):65-72. 2. Andes et al.: Impact of treatment strategy on outcomes in patients with candidemia and other forms of invasive candidiasis: a patient-level quantitative review of randomized trials. Clin Infect Dis 2012, 54:1110-1122. P86 Conclusion The choice of empirical versus preemptive therapy led to earlier and more stable reduction in the degree of organ dysfunction severity. It showed to be at least not inferior if not equal; in terms of survival benefi t and expediency of treatment. Moreover, galactomanan detection fails to guide the choice of the individual antimycotic, based on the expected Candida spp. Impact of echinocandins as fi rst-choice therapy in cardiothoracic high-risk patients with candidemia Impact of echinocandins as fi rst-choice therapy in cardiothoracic high-risk patients with candidemia g p G Langebartels, Y Choi, J Catteleans, T Wahlers University of Cologne, Germany Critical Care 2013, 17(Suppl 2):P85 (doi: 10.1186/cc12023) g p G Langebartels, Y Choi, J Catteleans, T Wahlers University of Cologne, Germany Critical Care 2013, 17(Suppl 2):P85 (doi: 10.1186/cc12023) Introduction Invasive candidemia is a major cause of increased mortality among ICU patients. Antifungal agents like liposomale amphotericin B and azoles could not accomplish the claim to be fi rst choice in the treatment of invasive fungal infection (IFI) because of side eff ects and eff ectiveness. Especially, cardiothoracic surgery patients as a group of high-risk patients are in a focus for new strategies and agents. A new class of antimycotic agents, the echinocandins, with a low profi le of side eff ects, low interactive potential and high eff ectiveness in the treatment of candidemia, is a powerful option in the treatment of IFI. We report our single-center experience with a modifi ed clinical treatment approach based on clinical score of Leon and using echinocandins as fi rst-line therapy for proven and suspected fungal infection. Results Our results indicate an estimated incremental lifetime cost of £13,272.53 per ADD patient, largely driven by increased ICU length of stay and readmission rates. The addition of probiotics to the standard perioperative regime is associated with a small survival benefi t of 1.2 months, yet a cost reduction of £917.3/ADD patient. The main cost was increased duration of ICU stay and readmissions, which contribute to 85% of total expenses. p Conclusion AAD is associated with a signifi cant increase in costs from a societal perspective. The provision of probiotics can achieve substantial cost savings and can be recommended as a cost-eff ective regime in the perioperative setting. Preventing ADD off ers a potentially signifi cant reduction of in-hospital costs and resource expenditures. g Methods From May 2011 to October 2012, 2,844 patients were treated on our cardiothoracic ICU. We evaluated 37 cardiothoracic postoperative patients with proven or suspected IFI or prophylaxis (Figure 1). The records were evaluated for cardiothoracic procedures, microbiological and yeast date, cardiothoracic surgery score (CASUS), ICU and clinical data. P84 Empirical versus preemptive antimycotic therapy in terms of outcome benefi t S Milanov, VT Todorova, G Georgiev, M Milanov Emergency Hospital ‘Pirogov’, Sofi a, Bulgaria Critical Care 2013, 17(Suppl 2):P84 (doi: 10.1186/cc12022) Cost-eff ectiveness analysis of anidulafungin in the treatment of candidaemia g Results A total of 2,426 patients were admitted to Heart of England NHS Foundation Trusts ICUs during the study period. Of these, 31 patients were identifi ed as having an IFI (1.3%). Baseline demographic data were similar between groups. Patients with IFI had signifi cantly longer ICU length of stay (19 days vs. 5 days, P <0.0001) and required more days of advanced organ support (12 days vs. 3 days, P <0.0001). A trend towards higher hospital mortality (41% vs. 27%, P = 0.08) was observed. Candida albicans was the predominant organism cultured (64%), followed by Candida glabrata (23%), with other Candida species accounting for the remaining 13% of IFIs. Sites of IFIs were blood (45%), intra-abdominal (39%), and pleural (16%). Most patients (52%) who acquired an IFI had had intra-abdominal surgery prior to ICU admission. The majority of patients (71%) were treated with echinocandins, whilst of the nine patients who were initially treated with fl ucanazole, six (67%) required therapy escalation to an echinocandin. G Auzinger1, G Playford2, C Graham3, H Narula3, C Charbonneau4, D Weinstein4, M Kantecki4, H Schlamm5, M Ruhnke6 1King’s College Hospital, London, UK; 2Princess Alexandra Hospital, Brisbane, Australia; 3RTI Health Solutions, Durham, NC, USA; 4Pfi zer International Operations, Paris, France; 5Pfi zer Inc., New York, NY, USA; 6Charité University Medicine, Berlin, Germany Critical Care 2013, 17(Suppl 2):P87 (doi: 10.1186/cc12025) G Auzinger1, G Playford2, C Graham3, H Narula3, C Charbonnea D Weinstein4, M Kantecki4, H Schlamm5, M Ruhnke6 1King’s College Hospital, London, UK; 2Princess Alexandra Hospital, Brisbane, Australia; 3RTI Health Solutions, Durham, NC, USA; 4Pfi zer International Operations, Paris, France; 5Pfi zer Inc., New York, NY, USA; 6Charité University Medicine, Berlin, Germany y Critical Care 2013, 17(Suppl 2):P87 (doi: 10.1186/cc12025) Critical Care 2013, 17(Suppl 2):P87 (doi: 10.1186/cc12025) Introduction Echinocandins are recommended fi rst-line treatment for candidaemia [1]. A cost-eff ectiveness model developed from a UK perspective examined costs and outcomes of antifungal treatment for candidaemia and other forms of invasive candidiasis based on European clinical guidelines [1]. Methods Costs and treatment outcomes with the echinocandin anidula fungin were compared with caspofungin, micafungin, fl uconazole, voriconazole and amphotericin B. The model included non-neutropenic patients aged ≥16 years with confi rmed candidaemia/ another form of invasive candidiasis receiving intravenous fi rst-line treatment [2]. Fungal ventricular assist device infections occur in colonized patients and are associated with high mortality rate Fungal colonization (FC), infection and outcome during VAD support n FC Mortality No IFI 24 12 9 IFI 10 9 8 P value 0.029 0.024 Conclusion In our center, we observed a high incidence of IFI in IC patients with VAD that was associated with a mortality rate of 80% Screening of fungal colonization appears to be very important durin the ICU stay for VAD patients. Trials are needed for investigating th use, the drug choice and the timing of antifungal prophylaxis for suc high-risk patients. Reference 1. Aslam S, et al.: Clin Infect Dis 2010, 50:664-671. P87 were all-cause 6-week mortality, cost of treatment-related adverse events (AEs) and other medical resource use costs. Life-years were calculated using a published model [4]. Antifungal agent-related AEs were taken from the product label/literature. Resource use was derived from the literature and discussion with clinical experts. Drug acquisition/ administration costs were taken from standard UK costing sources. with fungi [1]. The relationship between colonization and invasive fungal infection (IFI) in severely ill ICU patients with a VAD support is not described. This study analyzes the incidence and outcome of fungal infection and colonization in VAD patients in bridge to transplantation or in destination therapy. py Methods We conducted a retrospective review of all VAD implantations in our surgical ICU between 2007 and 2012. The incidence of fungal colonization, antifungal prophylaxis, bacterial sepsis and the mortality of IFI versus no IFI patients were compared. Results First-line anidulafungin for treatment of candidaemia was cost-eff ective per life-year gained versus fl uconazole (incremental cost-eff ectiveness ratio £813). Anidulafungin was cost saving versus caspofungin and micafungin in terms of life-years gained due to lower ICU costs and a higher rate of survival combined with a higher probability of clinical success.f Results In the study period, 34 patients with severe heart failure or cardiogenic shock were selected for a VAD implantation (nine in destination therapy). The overall mortality rate was 50% during mechanical assistance. Confi rmed (n = 8) and highly suspected (n = 2) IFI occurred during the ICU stay in 29% of patients who were treated with echinocandins, voriconazole and/or liposomal amphotericin B. The isolated fungi were: six Candida albicans, two parapsilosis, one glabrata and one invasive pulmonary aspergillosis. Antifungal prophylaxis with fl uconazole was administered to 18% of patients at mean for 5 days mainly in the more recent implantations. Allou N, et al.: Curr Infect Dis Rep 2011, 13:426-432. Zaragosa R, et al.: Adv Sepsis 2008, 6:90-98. Fungal ventricular assist device infections occur in colonized patients and are associated with high mortality rate In the no IFI population, 54% (n = 13) had a systemic or VAD bacterial sepsis with a mortality rate about 54%. The mortality without any sepsis was reduced to 18%. Fungal colonization was signifi cantly more present (90% vs. 50%) before IFI in VAD patients. The mortality rate was dramatically higher with IFI (80% vs. 38%) in accordance with the literature [1]. See Table 1. y Conclusion Anidulafungin was cost-eff ective compared with fl uconazole for treatment of candidaemia and was cost saving versus other echinocandins in the UK. European guidelines recommend echinocandins as fi rst-line treatments for candidaemia [1]; this model indicates that anidulafungin marries clinical eff ectiveness and cost-eff ectiveness. f References 1. Cornely OA: Clin Microbiol Infect 2012, 18:19-37. 2. Reboli AC, et al.: N Engl J Med 2007, 356:2472-2482. 3. Mills EJ, et al.: Ann Clin Microbiol Antimicrob 2009, 8:23. 4. Sidhu MK, et al.: Curr Med Res Opin 2009, 25:2049-2059 2. Reboli AC, et al.: N Engl J Med 2007, 356:2472 2482. 3. Mills EJ, et al.: Ann Clin Microbiol Antimicrob 2009, 8:23. 4. Sidhu MK, et al.: Curr Med Res Opin 2009, 25:2049-2059 Table 1 (abstract P86). Fungal colonization (FC), infection and outcome during VAD support Table 1 (abstract P86). Fungal colonization (FC), infection and outcome during VAD support n FC Mortality No IFI 24 12 9 IFI 10 9 8 P value 0.029 0.024 Reference 1. Aslam S, et al.: Clin Infect Dis 2010, 50:664-671. Cost-eff ectiveness analysis of anidulafungin in the treatment of candidaemia Patients were categorised as a clinical success or failure (patients with persistent/breakthrough infection); frequency data for each outcome were taken from a mixed-treatment comparison [3]. Successfully treated patients switched to oral therapy. Clinical failures switched to a diff erent antifungal class. It was assumed that second-line treatment duration was equivalent to that of fi rst-line treatment and only two lines of therapy were required to treat infection. Other inputs Conclusion The results of our study are consistent with other published data, in that whilst IFI prevalence is low, they are associated with increased morbidity in critically ill patients. This study has led to a change in hospital policy regarding antifungal use in the ICU, with echinocandins being fi rst-line in the pre-emptive treatment of IFI. We keenly await the results of the FIRE study, which will provide important insights to identifi cation of patients at risk of IFIs and optimal drug therapy. Prevalence and impact of invasive fungal infections in intensive care JM Patel K Couper T Melody R O’Brien D Parekh Introduction Invasive fungal infections (IFI) aff ect 1% of ICU patients and are increasing in incidence. IFIs are associated with a poor prognosis, which is further complicated by diffi culties in identifi cation of fungal organisms by traditional culture methods and the emergence of Candida species resistant to triazole therapy [1,2]. This study aimed to assess the prevalence of IFIs, the organisms responsible and outcomes of patients aff ected. Conclusion In our center, we observed a high incidence of IFI in ICU patients with VAD that was associated with a mortality rate of 80%. Screening of fungal colonization appears to be very important during the ICU stay for VAD patients. Trials are needed for investigating the use, the drug choice and the timing of antifungal prophylaxis for such high-risk patients. R f f Methods Patients admitted to the Heart of England NHS Foundation Trust ICUs who acquired an IFI were identifi ed through the hospital Fungal Infection Risk Evaluation (FIRE) study database. All ICU patients admitted between October 2009 and March 2011 were used as a comparative cohort. Data collected included: baseline demographics, length of stay, ICU and hospital mortality, and nature of IFI. Data were analysed using Student’s t test for continuous data and Fischer’s exact test for categorical data. Fungal ventricular assist device infections occur in colonized patients and are associated with high mortality rate P Gaudard, J Eliet, N Zeroual, G Culas, P Colson CHRU Montpellier, France Critical Care 2013, 17(Suppl 2):P86 (doi: 10.1186/cc12024) Referencefi Introduction Infection is a common complication of the ventricular assist device (VAD) and is associated with poor outcome especially 1. S Milanov, et al.: Effi cacy and utility of a protocol for pre-emptive antimycotic therapy. Crit Care 2010, 14(Suppl 1):P70. 1. S Milanov, et al.: Effi cacy and utility of a protocol for pre-emptive antimycotic therapy. Crit Care 2010, 14(Suppl 1):P70. Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S32 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 with fungi [1]. The relationship between colonization and invasiv fungal infection (IFI) in severely ill ICU patients with a VAD support not described. This study analyzes the incidence and outcome of fung infection and colonization in VAD patients in bridge to transplantatio or in destination therapy. Methods We conducted a retrospective review of all VAD implantation in our surgical ICU between 2007 and 2012. The incidence of fung colonization, antifungal prophylaxis, bacterial sepsis and the mortali of IFI versus no IFI patients were compared. Results In the study period, 34 patients with severe heart failu or cardiogenic shock were selected for a VAD implantation (nin in destination therapy). The overall mortality rate was 50% durin mechanical assistance. Confi rmed (n = 8) and highly suspected (n = IFI occurred during the ICU stay in 29% of patients who were treate with echinocandins, voriconazole and/or liposomal amphotericin B. Th isolated fungi were: six Candida albicans, two parapsilosis, one glabra and one invasive pulmonary aspergillosis. Antifungal prophylaxis wi fl uconazole was administered to 18% of patients at mean for 5 da mainly in the more recent implantations. In the no IFI populatio 54% (n = 13) had a systemic or VAD bacterial sepsis with a mortali rate about 54%. The mortality without any sepsis was reduced to 18% Fungal colonization was signifi cantly more present (90% vs. 50% before IFI in VAD patients. The mortality rate was dramatically high with IFI (80% vs. 38%) in accordance with the literature [1]. See Table Table 1 (abstract P86). References Allou N, et al.: Curr Infect Dis Rep 2011, 13:426-432. Zaragosa R, et al.: Adv Sepsis 2008, 6:90-98. S33 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 P89 Beliefs and actual practice of oxygen therapy in the ICU HJ Helmerhorst1, MJ Schultz2, PH Van der Voort3, E De Jonge1, DJ Van Westerloo1 1Leiden University Medical Center, Leiden, the Netherlands; 2Academic Medical Center, Amsterdam, the Netherlands; 3Onze Lieve Vrouwe Gasthuis, Amsterdam, the Netherlands Critical Care 2013, 17(Suppl 2):P89 (doi: 10.1186/cc12027) P90 FiO2/PEEP index: a simple tool for opitimizing ventilator settings D Trasy, M Nemeth, K Kiss, Z Till, Z Molnar University of Szeged, Hungary Critical Care 2013, 17(Suppl 2):P90 (doi: 10.1186/cc12028) Introduction During mechanical ventilation, oxygenation can be infl uenced by adjusting FiO2 and positive end-expiratory pressure (PEEP). There have been recommendations for how the FiO2 and PEEP should be set [1]. However, in a recent audit we found that the compliance of doctors of these recommendations is very low [2]. Therefore we invented a simple parameter called the FiO2/PEEP index (FPi) of which the physiologic value is ≤7 (that is, FiO2 = 21%/PEEP = 3 cmH2O), which corresponds to the ARDSNet trial’s minimum FiO2/ PEEP settings: 35%/5 cmH2O [2]. The aim of this case–control study was to investigate the impact of an FPi ≤7 targeted protocol on clinical practice. Introduction The aim of this study was to compare self-reported beliefs with actual clinical practice of oxygen therapy in the ICU. Hyperoxia is frequently encountered in ventilated patients and prolonged exposure has repeatedly been shown to induce lung injury and (systemic) toxicity. y Methods An online questionnaire for ICU clinicians was conducted to investigate beliefs and motives regarding oxygen therapy for critically ill patients. Furthermore, arterial blood gas (ABG) samples and corresponding ventilator settings were retrieved to retrospectively assess objective oxygenation between 1 April 2011 and 31 March 2012 in the ICUs of three teaching hospitals in the Netherlands. p Methods A prospective observational study in 2010 and 2012. Every mechanically ventilated patient was recruited. Demographics, outcome data and Lung Injury Score (LIS) were recorded after admission. Respiratory settings, oxygenation and ventilation parameters were recorded after mechanical ventilation was commenced and the fi rst arterial blood gas sample was taken (T0). Measurements were repeated in 24 hours (T1). Data are presented as mean ± SD, paired-sample and independent-sample t test and chi-square tests were used for statistics. References Results In 2010, 75 patients, and in 2012, 130 patients were included. There was no diff erence in demographics, disease severity, but LIS was higher in 2012: 1.34 ± 1.13 versus 0.84 ± 0.98, P = 0.001. There was no signifi cant diff erence in FPi between the two groups at T0: 10.91 ± 4.25 versus 10.26 ± 5.01 (2012 vs. 2010, respectively). At T24 the FPi was signifi cantly lower in 2012 as compared with 2010: 7.28 ± 2.58 versus 8.17 ± 3.3, P = 0.001; which was due to the higher PEEP applied: 7.08 ± 2.87 versus 6.63 ± 2.91, P = 0.014. Although in 2012 signifi cantly more patients, 112 (86%), were ventilated with FiO2 ≥50% at T0 as compared with 2010, 44 (58%) (P = 0.001), by T24 signifi cantly less patients received FiO2 ≥50%, 46 (35%) vs. 34 (45%) (P = 0.011). There was no signifi cant diff erence between the two groups regarding FiO2, PaO2 and PaCO2 at T0 and T24.i Results Analyzable questionnaire responses were received from 200 ICU physicians and nurses. The majority of respondents believed that oxygen-induced lung injury is a concern, although barotrauma and volutrauma are generally considered to impose a greater risk in mechanical ventilation. Frequently allowed minimal saturation ranges in the questionnaire were 85 to 95% and 7 to 10 kPa (Figure 1). Self- reported FiO2 adjustment in hypothetical patient cases with variable saturation levels was moderately impacted by the underlying clinical condition. To study actual clinical practice, a total of 107,888 ABG samples with corresponding ventilator settings, covering 5,565 patient admissions, were retrieved. Analysis showed a median (IQR) PaO2 of 11.7 kPa (9.9 to 14.3), median FiO2 was 0.4 (0.4 to 0.5), median PEEP was 5 (5 to 8). A total 63.5% of all PaO2 registries were higher than previously suggested oxygenation goals (7.3 to 10.7 kPa) [1]. In 56.8% of cases with PaO2 higher than the target range, neither FiO2 nor PEEP levels had been lowered when the next ABG sample was taken. Figure 1 (abstract P89). Conclusion Most clinicians acknowledge the detrimental eff ects of prolonged exposure to hyperoxia in the ICU and report a low tolerance for high saturation levels. 1. Brower RG, et al.: N Engl J Med 2004, 351:327-336. References However, the self-reported intention for conservative oxygen therapy is not consistently expressed in our objective data of actual clinical practice and a large proportion of patients was exposed to high and potentially toxic oxygen levels. Reference 1. Brower RG, et al.: N Engl J Med 2004, 351:327-336. Figure 1 (abstract P89). 2 2 Conclusion Implementing an FPi ≤7-based algorithm signifi cantly reduced the FiO2 and increased the PEEP applied in mechanically ventilated within the fi rst 24 hours. Whether this has any impact on earlier weaning due to reaching the weaning criteria of FiO2 sooner, and as a result shortening the duration of mechanical ventilation, has to be investigated in the future. g References References 1. ARDSNet: N Engl J Med 2000, 342:1301. 2. Kiss K, et al.: Intensive Care Med 2011, 37(Suppl 1):S195. 1. ARDSNet: N Engl J Med 2000, 342:1301. 1. ARDSNet: N Engl J Med 2000, 342:1301. 2. Kiss K, et al.: Intensive Care Med 2011, 37(Suppl 1):S195. g , 2. Kiss K, et al.: Intensive Care Med 2011, 37(Suppl 1):S195. 2. Kiss K, et al.: Intensive Care Med 2011, 37(Suppl 1):S195. P92 9 Relation between PaO2/FiO2 ratio, SpO2/FiO2 ratio, oxygenation index and ventilation ratio in critically ill patients T Aslanidis, A Myrou, E Chytas, E Anastasiou, E Geka, E Efthimiou, V Ourailoglou, I Soultati, S Primikiri, M Giannakou-Peftoulidou A.H.E.P.A. University Hospital, Thessaloniki, Greece Critical Care 2013, 17(Suppl 2):P92 (doi: 10.1186/cc12030) Relation between PaO2/FiO2 ratio, SpO2/FiO2 ratio, oxygenation index and ventilation ratio in critically ill patients y A Batchinsky1, C Necsoiu1, T Langer2, V Vecchi2, W Baker1, J Salinas1, L Cancio1 1US Army Institute of Surgical Research, Fort Sam Houston, TX, USA; 2University of Milan, Italy Critical Care 2013, 17(Suppl 2):P93 (doi: 10.1186/cc12031) A Batchinsky1, C Necsoiu1, T Langer2, V Vecchi2, W Baker1, J Salinas1, L Cancio1 1US Army Institute of Surgical Research, Fort Sam Houston, TX, USA; 2University of Milan, Italy Critical Care 2013, 17(Suppl 2):P93 (doi: 10.1186/cc12031) Introduction Respiratory complexity (RC) as assessed by sample entropy (SampEn) is lower in patients failing spontaneous breathing trials. We evaluated the role of RC in monitoring subjects with severe acute respiratory distress syndrome (ARDS) treated with the Cardiohelp (Maquet, Rastatt, Germany) extracorporeal life-support system (ECLS). We hypothesized that RC is reduced during apnea. Introduction Many authors have proposed less invasive ways to measure oxygenation in patients with ALI. The aim of the present study is to compare the already popular oxygenation index (OI) and PaO2/ FiO2 ratio (PFr) with other recently proposed indices such as SpO2/FiO2 ratio (SFr) and ventilation ratio (VR) in a mixed ICU population. Methods During a 6-month prospective observational study carried out in a polyvalent 10-bed adult ICU, ABGs were obtained from 145 patients. Two independent measurements were taken from each patient under the same mode of ventilation (SIMVPSV). PFr, SFr, VR and OI were calculated. Demographic data (APACHE II score, age, sex, yp g p Methods Six sheep sedated with midazolam were connected to the Cardiohelp via a 23F Avalon catheter. Blood fl ow was ~2 l/minute and FiO2 was 0.5. Sheep were on CPAP of 8 cmH2O with FiO2 of 1 via tracheostomy. P91 Use of high-fl ow nasal canulae: eff ect on alveolar pressure and its limitation limitation H Hayami, K Mizutani, M Shioda, S Takaki, H Maejima, K Ueno, Y Yamaguchi, T Kariya, T Gotoh Yokohama City University Hospital, Yokohama, Japan Critical Care 2013, 17(Suppl 2):P91 (doi: 10.1186/cc12029) Figure 1 (abstract P89). Introduction High-fl ow nasal canulae (HFNC) deliver high-fl ow humidifi ed gas at up to 60 l/minute. There are two types of respiratory circuit to generate mix gas fl ow, Blender type (typeB) and Venturi type (typeV). The therapy is well established in the pediatric population and HFNC use has been described in the adult population. It has been reported that HFNC provide higher FIO2 compared with low-fl ow canulae, and also create mild positive pharyngeal airway pressure, but the eff ect on alveolar pressure is unknown. We aimed to investigate the eff ect of HFNC on alveolar pressure, by measuring intratracheal pressure in patients with a cricothyrotomy catheter (CTC). At the same time, we measured the actual gas fl ow rate (AGFR) by fl owmeter and compared it with assumed fl ow. Conclusion Most clinicians acknowledge the detrimental eff ects of prolonged exposure to hyperoxia in the ICU and report a low tolerance for high saturation levels. However, the self-reported intention for conservative oxygen therapy is not consistently expressed in our objective data of actual clinical practice and a large proportion of patients was exposed to high and potentially toxic oxygen levels. Reference l Methods Seven patients with a CTC were participated. A tube was connected to the CTC and the tube was then connected to a pressure transducer to measure intratracheal pressure. The HFNC (Optifl ow S34 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 diagnosis, body weight) were also recorded. Kolmogorov–Smirnov test for normality was calculated followed by bivariate nonparametric analysis (statistical signifi cance: P <0.05). system) were applied with the humidifi er to optimize humidication. TypeB was used in three patients and typeV in four patients. The fl ow was started at 10 l/minute. This fl ow rate was titrated upwards to a maximum of 60 l/minute (10, 25, 30, 40, 50, 60 l/minute) and the AGFR was measured. Intratracheal pressure tracing was done over 1 minute. Airway pressure measurement was repeated and the maximal expiratory pressure was measured in mmHg. y gi Results A total of 290 measurements were included for further analysis. References References 1. Rice TW, et al.: Chest 2007, 132:410-417. 2. Sinha P, et al.: Br J Anaesth 2009, 102:692-697. 1. Rice TW, et al.: Chest 2007, 132:410-417. 1. Rice TW, et al.: Chest 2007, 132:410-417. 1. Rice TW, et al.: Chest 2007, 132:410-417. 2. Sinha P, et al.: Br J Anaesth 2009, 102:692-697. Conclusion HFNC are eff ective in providing higher expiratory pressure. It is important to know the fl ow rate is lower than expected when the Venturi type is used. 1. Rice TW, et al.: Chest 2007, 132:410-417. 2. Sinha P, et al.: Br J Anaesth 2009, 102:692-697. Signifi cant diff erence versus Baseline. #Signifi cant diff erence between Preinjury and Postinjury apnea. P91 Use of high-fl ow nasal canulae: eff ect on alveolar pressure and its limitation Mean ± SD values for age and APACHE II score were 61.2 ± 16 years and 15.4 ± 1.8, respectively, while median ± SD values for PFr are 257 ± 135, for SFr 211 ± 58, for OI 4.8 ± 4.46 and for VR 1.19 ± 0.31. Relations between PFr, SFr and OI are displayed in Table 1. p y p g Results The AGFR in the respiratory circuit was almost same in typeB, but there was obvious decrease in the AGFR in typeV (7.1 ± 1.0, 17.7 ± 0.8, 21.9 ± 0.9, 29.9 ± 3.6, 36.9 ± 2.7, 45.0 ± 5.5 l/minute at assumed fl ow, 10, 25, 30, 40, 50, 60 l/minute, respectively). HFNC signifi cantly increased maximal expiratory pressure in both groups, 1.5 ± 2.1, 2.0 ± 1.0, 3.0 ± 2.8, 4.5 ± 3.5 mmHg for typeV and 2.5 ± 0.7, 5.8 ± 2.4, 6.0 ± 2.8, 8.0 ± 2.8 mmHg (maximum 10 mmHg) for typeB, when AGFR was set at 30, 40, 50, 60 l/minute. Higher AGFRs were found to result in larger increase in maximum expiratory pressure. The data indicate that HFNC are associated with an increase in intratracheal expiratory pressure. Because it was diffi cult to determine end-expiratory pressure, we chose maximal expiratory pressure for a substitute. The reason why AGFR in typeV was lower than assumed fl ow may be the resistance generated by NC. The larger increase in expiratory pressure in our study than previously reported may be due to the eff ect of high respiratory resistance of Japanese who have relatively small airway structure compared with western people.f Table 1 (abstract P92). Correlations (Spearman ρ) between VR, PFr, SFr and OI Table 1 (abstract P92). Correlations (Spearman ρ) between VR, PFr, SFr and OI PFr SFr OI Table 1 (abstract P92). Correlations (Spearman ρ) between VR, PFr, SFr and OI PFr SFr OI VR –0.326* –0.325* 0.373* PFr – 0.759* –0.939* SFr – – –0.773* P <0.01. Conclusion Our study identifi ed a strong relation between PFr, SFr and OI but not VR. Thus, these markers may be used interchangeably as bedside indices of oxygenation in critically ill patients. Yet larger studies are needed to come to a safer conclusion. R f Acute respiratory distress syndrome – the Berlin defi nition: impact on an ICU of a university hospital Acute respiratory distress syndrome – the Berlin defi nition: impact on an ICU of a university hospital A Agrifoglio, J Lopez, J Figueira, M Hernández, L Fernández, M Irazabal, S Yus, M Jiménez Hospital Universitario La Paz, Madrid, Spain Critical Care 2013, 17(Suppl 2):P94 (doi: 10.1186/cc12032) y A Agrifoglio, J Lopez, J Figueira, M Hernández, L Fernández, M Irazabal, S Yus, M Jiménez Hospital Universitario La Paz, Madrid, Spain Critical Care 2013, 17(Suppl 2):P94 (doi: 10.1186/cc12032) g Results A weaning-induced pulmonary edema was diagnosed in 12 instances (PAOP signifi cantly increased from 15.6 ± 0.6 to 25.8 ± 0.9 in these cases). EVLWI, BNP, plasma protein and hemoglobin concentrations signifi cantly increased in these instances (28.3 ± 5.7%, 20.2 ± 7.8%, 9.6 ± 0.8% and 9.3 ± 1.3%, respectively) while they did not signifi cantly changed in cases without weaning-induced pulmonary edema. The increase of EVLWI ≥8.5% (+1.5 ml/kg), an increase in BNP ≥6.7% (+23 pg/ml), an increase in plasma protein concentration ≥5% and in hemoglobin concentration ≥5% exhibited good areas under the ROC curves to predict weaning-induced pulmonary edema (0.97 ± 0.03, 0.80 ± 0.11, 1.0 ± 0.00 and 0.92 ± 0.05, respectively). These areas under the ROC curves were not statistically diff erent. The baseline values of EVLWI, BNP, plasma protein and hemoglobin concentrations did not predict weaning-induced pulmonary edema. Introduction The current defi nition of ARDS was established in 1994 by the AECC. In 2011 emerged the Berlin defi nition. The aim of this study was to fi nd a new approach from the impact of ARDS in an ICU and to analyze APACHE II score, days of mechanical ventilation (MV), duration of ARDS and mortality. y Methods We analyzed the database of our ICU collected prospectively from October 2008 to January 2011 and regarded all patients undergoing MV for 48 hours or more and who met the AECC criteria. All patients were ventilated according to the ARDSNet protocol. p g p Results During this period 1,900 patients were admitted, 607 were under MV for at least 48 hours and 104 had ARDS criteria. Moderate ARDS 60.6%: 60% male, median age 53 years, APACHE II mean 22.49 (95% CI = 20.71 to 24.27), mean duration MV 23.84 (95% CI = 18.08 to 29.60), ARDS duration 12.87 (95% CI = 9.83 to 15.90), average stay 29.22, mortality 52.4%. P92 Reference ICU admission have a longer duration of ARDS, which means more days on MV and therefore increased ICU stay. Reference of 0.1 ml/kg oleic acid. Apneas (defi ned as >60% reduction in minute ventilation) developed both before (Preinjury) and after ARDS (Postinjury). Heart rate (HR), mean arterial pressure (MAP), respiratory rate (RR) and tidal volume (TV) were recorded, and the PaO2:FiO2 ratio (PFR) calculated corresponding to sections of respiratory pressure waveforms containing 200 breaths during Baseline, during Preinjury and Postinjury apnea periods. Similar periods were selected before and after apneas. Inter-breath interval (IBI) and its deviation, SampEn, percentage of forbidden words (FW), bit per-word entropy (BPWEn), symbol distribution entropy (DisNEn), and stationarity (StatAv) were calculated using software. Statistics by one-way ANOVA with adjustment for multiple comparisons. 1. Ranieri M, et al.: JAMA 2012, 307:2526-2533. P95 Extravascular lung water, B-type natriuretic peptide and blood volume contraction for diagnosing weaning-induced pulmonary edema M Dres, JL Teboul, N Anguel, C Richard, X Monnet Service de réanimation médicale, Hôpital de Bicêtre, Le Kremlin Bicêtre, France Critical Care 2013, 17(Suppl 2):P95 (doi: 10.1186/cc12033) Extravascular lung water, B-type natriuretic peptide and blood volume contraction for diagnosing weaning-induced pulmonary edema M Dres, JL Teboul, N Anguel, C Richard, X Monnet Service de réanimation médicale, Hôpital de Bicêtre, Le Kremlin Bicêtre, France Critical Care 2013, 17(Suppl 2):P95 (doi: 10.1186/cc12033) M Dres, JL Teboul, N Anguel, C Richard, X Monnet Service de réanimation médicale, Hôpital de Bicêtre, Le Kremlin Bicêtre, France Critical Care 2013, 17(Suppl 2):P95 (doi: 10.1186/cc12033) Results See Table 1. SampEn was associated with PFR (r2 = 0.31, P <0.001). Introduction We compared three diff erent methods for diagnosing pulmonary edema induced by weaning from mechanical ventilation: the increase in extravascular lung water index (EVLWI), the increase in B-type natriuretic peptide (BNP) and blood volume contraction, refl ected by increases in plasma protein and in hemoglobin concen- trations, all observed during a spontaneous breathing trial (SBT).fi Conclusion Respiratory complexity during apnea and ECLS remains unchanged in a healthy state but decreases after ARDS. It has a signifi cant association with PFR and may serve as an index of injury severity. P92 After ~6 hours of ECLS, ARDS was induced by injection Table 1 (abstract P93) Preinjury Preinjury Preinjury Postinjury Postinjury Postinjury BL pre-apnea apnea post-apnea pre-apnea apnea post-apnea RR (breaths/minute) 22 ± 2 22 ± 3 17 ± 2 23 ± 4 24 ± 7 5 ± 1 25 ± 7 TV (ml) 388 ± 31 217 ± 32 145 ± 14 205 ± 21 211 ± 51 116 ± 30* 229 ± 40 HR (beats/minute) 156 ± 15 153 ± 21 152 ± 21 166 ± 21 162 ± 22 165 ± 21 151 ± 16 MAP (mmHg) 102 ± 4.12 102 ± 6.13 103 ± 6.91 104 ± 6.08 106 ± 3.76 108 ± 3.86 111 ± 5.69 PFR (unitless) 435 ± 24.80 417 ± 48.06 354 ± 49.52 429 ± 37.87 139 ± 50.90* 92 ± 30.43# 91 ± 27.25 IBI (ms) 2,765.1 ± 230.5 3,149.8 ± 378.0 5,151.7 ± 467.7 3,404.7 ± 663.0 2,253.2 ± 427.0 10,334.0 ± 1,739.2# 2,162.0 ± 387.6 IBI Dev (ms) 771.8 ± 117.49 1,630.0 ± 230.64 4,505.7 ± 785.34 1,598.3 ± 292.45 1,404.8 ± 445.04 25,065.0 ± 11,480.37# 844.6 ± 236.65 SampEn (unitless) 1.7 ± 0.12 1.6 ± 0.16 1.3 ± 0.13 1.6 ± 0.11 1.2 ± 0.14 0.5 ± 0.25# 1.3 ± 0.20 FW (%) 29.9 ± 4.60 41.5 ± 7.43 41.8 ± 10.15 34.5 ± 6.10 44.0 ± 8.80 62.2 ± 4.16 33.6 ± 5.26 BPwEn (unitless) 4.9 ± 0.18 4.3 ± 0.27 4.1 ± 0.37 4.6 ± 0.22 4.1 ± 0.42 2.7 ± 0.38# 4.4 ± 0.24 DisNEn (unitless) 0.8 ± 0.03 0.7 ± 0.04 0.7 ± 0.06 0.8 ± 0.05 0.7 ± 0.07 0.4 ± 0.06# 0.7 ± 0.04 StatAv (unitless) 0.42 ± 0.06 0.37 ± 0.09 0.37 ± 0.04 0.41 ± 0.10 0.31 ± 0.05 0.51 ± 0.07 0.42 ± 0.08 Signifi cant diff erence versus Baseline. #Signifi cant diff erence between Preinjury and Postinjury apnea. S35 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 ICU admission have a longer duration of ARDS, which means more days on MV and therefore increased ICU stay. Reference 1. Ranieri M, et al.: JAMA 2012, 307:2526-2533. ICU admission have a longer duration of ARDS, which means more days on MV and therefore increased ICU stay. P94 Methods We included 12 diffi cult-to-wean patients (22 recordings). Before and at the end of a SBT (T tube), we measured pulmonary occlusion arterial pressure (PAOP), EVLWI (PiCCO device), BNP, plasma protein and hemoglobin concentrations. Weaning-induced pulmonary edema was confi rmed if a clinical intolerance to SBT was associated with an increase of PAOP >18 mmHg at the end of SBT. Acute respiratory distress syndrome – the Berlin defi nition: impact on an ICU of a university hospital Severe ARDS 39.4%: 63% male, median age 45 years, APACHE II mean score 23.19 (95% CI = 20.62 to 25.75), mean duration MV 18.52 (95% CI = 13.01 to 24.03), ARDS duration 12.15 (95% CI = 8.72 to 15.58), average stay 22.65, mortality 48.8% with no statistically signifi cant diff erence with moderate ARDS (P = 0.72). Leading cause of death in both groups: multiorgan dysfunction (60%). See Tables 1 and 2. Conclusion In this new approach of our ARDS patients we found no statistically signifi cant diff erences in relation to the variables of interest to analyze in the two groups. Patients with severe ARDS who survive Conclusion The increases in EVLWI, in plasma protein and hemoglobin concentration and in BNP are valuable alternatives to the pulmonary artery catheter for diagnosing weaning-induced pulmonary edema. P96 P96 Airway pressures and S36 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 the other hand, auto open lung management off ers a distinctive result of ventilation. A signifi cant improvement of oxygenation and lung compliance was observed within a few breaths after the recruitment maneuvers. Both subjects were ventilated at the same tidal volume of 6 ml/kg and the comparative results of automatic ventilation settings and BGA are provided in Table 1 for every 2 hours. Conclusion The auto open lung management concept gave much better gas exchange than the auto ARDSNet protocol. These preliminary results showed a necessity to evaluate the two diff erent ventilation strategies. Therefore, further experiments with pig models will be implemented in the near future to obtain results with statistical signifi cance and to ensure the safety of automation in a mechanical ventilation system. the other hand, auto open lung management off ers a distinctive result of ventilation. A signifi cant improvement of oxygenation and lung compliance was observed within a few breaths after the recruitment maneuvers. Both subjects were ventilated at the same tidal volume of 6 ml/kg and the comparative results of automatic ventilation settings and BGA are provided in Table 1 for every 2 hours. ventilatory mechanics were measured twice daily. Data are presented as mean ± 95% CI. P > 0.05. the other hand, auto open lung management off ers a distinctive result of ventilation. A signifi cant improvement of oxygenation and lung compliance was observed within a few breaths after the recruitment maneuvers. Both subjects were ventilated at the same tidal volume of 6 ml/kg and the comparative results of automatic ventilation settings and BGA are provided in Table 1 for every 2 hours. Results A total of 166 patients with mean age 55 (52 to 58) years, LIS of 2.27 (2.16 to 2.37) and MODS of 3.13 (2.9 to 3.3) were enrolled and stratifi ed by LISL (65.8%) and LISH (34.2%). Mortality, and incidence of pneumothorax and atelectasis were 21% (14.9 to 28.2), 1.6% (0.8 to 3.1) and 6.4 (3.2 to 11.2). Duration of MV was 6 days (4.83 to 7.16). Pneumothorax in LISL and LISH was 1% (0 to 5.2) versus 2.4% (0.7 to 6.1), and mortality 11.4% (3.8 to 24.6) versus 25% (9.8 to 46.7). P97 Comparison of a fully automatic ARDSNet protocol and a feedback-controlled open lung management concept A Pomprapa1, D Schwaiberger2, B Lachmann2, S Leonhardt1 1RWTH Aachen University, Aachen, Germany; 2Charite Berlin, Germany Critical Care 2013, 17(Suppl 2):P97 (doi: 10.1186/cc12035) Introduction Intellivent-ASV has been developed to provide fully closed loop mechanical ventilation using a ventilation controller keep- ing EtCO2 and SpO2 within expert-based ranges. Ventilation of ARDS patients focuses on delivering adequate oxygenation and allowing elimination of CO2 while protecting the lung. The objectives were to compare Intellivent-ASV with conventional ventilation on safety and effi cacy, and to compare the number of manual adjustments between the two ventilatory modalities. Introduction The aim of this study is to compare two ventilation strategies, the ARDSNet protocol and open lung management, using computer control for 6 hours. The standard therapy for patients with ARDS does typically apply a mechanical ventilator to support breathing. The cost of therapy is high and it requires much attention from physicians to adjust the proper ventilation settings in a timely manner. A closed-loop ventilation concept has therefore been developed and tested with two induced ARDS pigs. y Methods A randomized, controlled study including all consecutive patients receiving mechanical ventilation for at least 48 hours. Patients were randomly ventilated either with Intellivent-ASV or conventional ventilation, with a S1 (Hamilton, Bonaduz, Switzerland). Parameters were adjusted by the clinician in charge of the patient. Ventilatory and oxygenation parameters were recorded cycle by cycle during 48 hours and blood gases were performed every 6 hours. g Methods The hardware system is composed of a ventilator (Servo 300), a spectrophotometry (CEVOX), a capnography device (CO2SMO+), an electrical impedance tomography device (GOE MF II) and a patient monitor (Sirecust). The software is developed with Labview 7.1. With approval from the ethical committee, two 27 kg pigs were exposed to surfactant depletion with a warm saline washout to induce ARDS (PaO2/ FiO2 <200 mmHg). One pig model was ventilated with an automatic ARDSNet protocol and another was automatically ventilated with open lung management. Blood gas analysis (BGA) was carried out every half an hour.i Results Twenty-four patients with ARDS were included, 10 female, 14 male, median age 58 (46 to 63) years, APACHE II score 22 (17 to 29), PaO2/FiO2 at inclusion 136 (107 to 154). Eleven were ventilated in the conventional group and 13 in the Intellivent-ASV group. P96 Pressure support ventilation with minimal sedation as the main ventilatory mode in critically ill patients with lung injury: eff ect on mortality and incidence of complications A Gomez1, A Leon1, G Fernandez1, G Montenegro1, H Gomez2 1Clinica Palermo, Bogota, Colombia; 2University of Pittsburgh, PA, USA Critical Care 2013, 17(Suppl 2):P96 (doi: 10.1186/cc12034) Pressure support ventilation with minimal sedation as the main ventilatory mode in critically ill patients with lung injury: eff ect on mortality and incidence of complications A Gomez1, A Leon1, G Fernandez1, G Montenegro1, H Gomez2 1Clinica Palermo, Bogota, Colombia; 2University of Pittsburgh, PA, USA Critical Care 2013, 17(Suppl 2):P96 (doi: 10.1186/cc12034) Table 1 (abstract P94). Subgroup analysis: moderate ARDS Variable Survivors Nonsurvivors P value APACHE II 20.06 ± 6.96 24.83 ± 6.17 0.006 MV (days) 25.63 ± 29.13 22.21 ± 15.43 0.557 ARDS (days) 10.29 ± 9.07 15.20 ± 12.92 0.107 ICU stay 34.33 ± 33.26 24.57 ± 16.91 0.142 Table 2 (abstract P94). Subgroup analysis: severe ARDS Variable Survivors Nonsurvivors P value APACHE II 20.82 ± 8.89 25.31 ± 5.63 0.761 MV (days) 26.95 ± 18.03 10.10 ± 11.64 0.001 ARDS (days) 15.8 ± 11.60 8.5 ± 8.6 0.031 ICU stay 33.52 ± 18.25 11.25 ± 11.80 0.001 Table 1 (abstract P94). Subgroup analysis: moderate ARDS Introduction The primary aim of this study is to assess the impact of pressure support ventilation (PSV) on the rate of pneumothorax and mortality in critically ill patients with lung injury. The secondary aim is to evaluate pressure–volume (P–V) relationships. Spontaneous modes of ventilation have been associated with lower rates of atelectasis, less muscle atrophy, better airfl ow distribution and importantly lower sedation requirements, which relates to lower mortality. Accordingly, we hypothesized that the use of PSV in patients with moderate/severe lung injury would have rates of pneumothorax and mortality within the standard of care. We further hypothesized that given its spontaneous nature, set pressures (PEEP and PS) but not tidal volume (Vt) would be related to airway pressures. Methods All adult patients admitted to two surgical/medical ICUs subjected to invasive mechanical ventilation (MV) were enrolled. Patients were stratifi ed by Lung Injury Score (LIS) in two groups: <2.5 (LISL); ≥2.5 (LISH). Exclusion criteria included pneumothorax on admission, use of other ventilatory strategies, and inability to trigger ventilation. Patients were ventilated with PSV, and treated only with pro re nata haldol, morphine and clozapine. P96 Mean parameters were: Vt 10.3 ml/kg (10.1 to 10.6); PEEP 10.6 (9.9 to 11); PS 16.6 (15.9 to 17.1); plateau pressure (Ppl) 25.7 (25.1 to 26.2). Ppl was >26.2 only in 2.5%. PEEP and PS (P <0.0001), and MODS were associated with Pplt, but not Vt* or LIS. Only lower Vt was associated with barotrauma (OR = 0.996, P = 0.02). p y Conclusion The auto open lung management concept gave much better gas exchange than the auto ARDSNet protocol. These preliminary results showed a necessity to evaluate the two diff erent ventilation strategies. Therefore, further experiments with pig models will be implemented in the near future to obtain results with statistical signifi cance and to ensure the safety of automation in a mechanical ventilation system. Conclusion We demonstrate that PSV in minimally sedated patients with severe lung injury is safe as it is associated with low incidence of barotrauma, atelectasis and mortality, and with Ppl and duration of MV within standard of care. We also demonstrate in PSV that P–V relationships may diff er and that in this setting higher Vt may not be deleterious. P98 Comparison of an entirely automated ventilation mode, Intellivent-ASV, with conventional ventilation in ARDS patients: a 48-hour study E Bialais1, L Vignaux2, X Wittebole1, D Novotni2, J Meyer2, M Wysocki2, T Sottiaux3, G Reychler1, J Roeseler1, P Laterre1, P Hantson1 1Cliniques Universitaires Saint Luc, Brusells, Belgium; 2Hamilton Medical AG, Bonaduz, Switzerland; 3Clinique Notre-Dame de Grâce, Gosselies, Belgium Critical Care 2013, 17(Suppl 2):P98 (doi: 10.1186/cc120236) P97 The study was stopped for one patient from the Intellivent-ASV group because of a pneumothorax not caused by ventilation. The delivered Vt was slightly higher during Intellivent-ASV (7.9 (7.5 to 8.5) vs. 7.2 (6.8 to 7.8) ml/kg, P = 0.013). The time spent by the various parameters in the suboptimal zone (safety) is the same for the two ventilation modes. The time spent in the optimal zone (effi cacy) is the same for the two ventilation modes, Results Artifi cial ventilation using the auto ARDSNet protocol success- fully stabilized oxygenation, minimized plateau pressure (<30 cmH2O), and controlled the pH value for acidosis and alkalosis management. On Table 1 (abstract P97). Comparative results between auto ARDSNet protocol and auto open lung management Before auto ventilation After 2 hours After 4 hours After 6 hours Auto ARDSNet Auto OLM Auto ARDSNet Auto OLM Auto ARDSNet Auto OLM Auto ARDSNet Auto OLM FiO2 1.0 1.0 0.40 0.25 0.40 0.25 0.50 0.25 RR (bpm) 22 40 23 38 23 39 23 35 I:E ratio 1:2 1:1 1:2 1:1 1:2 1:1 1:2 1:1 VT (ml/kg) 6 6 6 6 6 6 6 6 Pplat /PIP (mmHg) 22 22 18 20.6 15.8 17.7 14.6 15.4 PEEP (cmH2O) 5 10 8 15 2 13 10 11 pH 7.34 7.30 7.32 7.52 7.37 7.60 7.38 7.51 pO2/FiO2 (mmHg) 59 59 180 380.8 155 387.2 122 404 pCO2 (mmHg) 52 64 56 39.6 58 31.5 54 41 OLM, open lung management. Table 1 (abstract P97). Comparative results between auto ARDSNet protocol and auto open lung management . Comparative results between auto ARDSNet protocol and auto open lung management S37 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 expect for SpO2 (78% (75 to 89) with Intellivent-ASV vs. 44% (41 to 58), P = 0.001). Intellivent-ASV required less manual adjustments during 48 hours (2 (0 to 7) vs. 17 (10 to 27), P <0.001). The total number of adjustments during 48 hours, including automatic regulation, was higher in the Intellivent-ASV group (379 (274 to 493) vs. 17 (10 to 27), P <0.001). Methods This observational study in ICU mechanically ventilated septic patients with intra-abdominal surgical infections was carried out at the V.A. Negovsky Research Institute of General Reanimatology in 2010 to 2012. ARDS was diagnosed and staged according to the research institute criteria [1] and the American–European criteria. Diff erence in pulmonary permeability between indirect and direct acute respiratory distress syndrome assessed by the transpulmonary thermodilution technique K Morisawa1, Y Taira1, M Yanai1, Y Takamatu1, S Kushimoto2, S Fujitani3 1St Marianna University, Kawasaki-Shi, Japan; 2Tohoku University Graduate School of Medicine, Sendai-shi, Japan; 3Tokyo Bay Urayasu/Ichikawa Medical Center, Urayasu-shi, Japan Critical Care 2013, 17(Suppl 2):P99 (doi: 10.1186/cc12037) Introduction ARDS is characterized by increased pulmonary capillary permeability secondary to diff use alveolar infl ammation and injury. Common risk factors can be classifi ed into two groups: extrapulmonary causes (indirect etiologies: ARDSexp) or pulmonary causes (direct etiologies: ARDSp). There are few quantitative methods to distinguish between the diff erences in these two ARDS categories. Conclusion Plasma SPD level ≤253.0 ng/ml is a sensitive and specifi c biomarker of the early stage of ARDS in septic patients. Reference f g Methods A subanalysis of the trial by the PiCCO Pulmonary Edema Study (prospective, observational, multi-institutional study) in 23 ICUs of academic tertiary referral hospitals in Japan. All consecutive adult patients requiring mechanical ventilation with the diagnosis of ARDS were monitored by the transpulmonary thermodilution technique system (PiCCO; Pulsion Medical Systems, Munich, Germany) for 3 days: day 0, day 1 and day 2. The pulmonary vascular permeability index (PVPI), extravascular lung water index and intrathoracic blood volume index were measured concurrently. Three experts retrospectively determined the pathophysiological mechanism causing ARDS by patient history, clinical presentation, chest computed tomography and radiography. Patients were classifi ed into two groups: patients with ARDS triggered by ARDSexp and ARDSp. 1. Kuzovlev A, et al.: Semin Cardiothorac Vasc Anesth 2010, 14:231-24 P101 Intravenous perfl uorocarbons for prevention of ventilator-associated ARDS A Scultetus1, A Haque1, F Arnaud1, G McNamee2, C Auker1, R McCarron1, P McKay2, R Mahon1 1Naval Medical Research Center, Silver Spring, MD, USA; 2Uniformed Services University of the Health Sciences, Bethesda, MD, USA Critical Care 2013, 17(Suppl 2):P101 (doi: 10.1186/cc12039) A Scultetus1, A Haque1, F Arnaud1, G McNamee2, C Auker1, R McCarron1, P McKay2, R Mahon1 Results During the study period from March 2009 to August 2011, a total of 173 patients were assessed including 56 ARDSexp patients and 117 ARDSp patients, with the most common cause of ARDSexp secondary to sepsis (71%) and of ARDSp pneumonia (80%). The measure ment of PVPI was signifi cantly elevated in the ARDSp group on all days. There were no signifi cant diff erences in mortality at 28 days, mechanical ventilation days, and hospital length-of-stay between the two groups, while the ARDSexp group seemed to be associated with prolonged mechanical ventilation days and hospital length-of-stay.f Introduction Emulsifi ed perfl uorocarbons (PFC) are synthetic hydro- carbons that can carry 50 times more oxygen than human plasma. Their properties may be advantageous in applications requiring preservation of tissue viability in oxygen-deprived states [1,2], making them a potential candidate for combat and civilian prehospital resuscitation. Our hypothesis is that an intravenous dose of PFC increases vital organ tissue oxygenation, improves survival after hemorrhagic shock (HS) and may reduce or prevent the development of ventilator-associated ARDS. Here we report data from the fi rst part (HS only) of a multiphase swine study to study the benefi ts of PFC in treating HS and preventing ARDS. This initial study was designed to ensure safe use of PFC in traumatized animals. Conclusion This study suggests the existence of several diff erences in pathogenetic pathways and the degree of pulmonary permeability between patients with ARDSexp and ARDSp. We therefore believe that using the PVPI may provide us with timely quantitative information to make clinical decisions. Methods Anesthetized Yorkshire swine were hemorrhaged 55% of their estimated blood volume (Time 0 (T0)) over 15 minutes. At T15 minutes, pigs received a bolus of the PFC Oxycyte or 10% hydroxyethyl starch (HES). At T60 animals underwent continuous hemorrhage (0.5 cm3/kg/ minute) until death. Time to death and physiological parameters were primary endpoints. P97 Plasma concentrations of SPD and CCP were measured on ARDS diagnosis (day 0) and days 3, 5, and 7 by immunoenzyme essay (Bio Vendor, USA). Data were statistically analyzed by STATISTICA 7.0, ANOVA method, and presented as mean ± σ, ng/ml. P <0.05 was considered statistically signifi cant. Areas under the receiver operating curves (ROC) were calculated. Conclusion Over 48 hours, Intellivent-ASV allowed safe ventilation for ARDS patients, with better oxygenation and an identical ventilation effi cacy when compared with conventional ventilation, with less manual adjustments. Results Fifty-fi ve patients (out of 250 screened) were enrolled according to the inclusion/exclusion criteria. Patients were assigned into groups: ARDS (n = 30; subgroups – ARDS stage 1 (n = 15), stage 2 (n = 15)) and noARDS (n = 25). In the ARDS group SPD was higher at all points than in the noARDS group (day 0 – 352.5 ± 287.8 vs. 141.0 ± 103.4; day 3 – 278.6 ± 204.8 vs. 151.8 ± 125.9; day 5 – 339.9 ± 331.4 vs. 162.3 ± 138.8; day 7 – 339.9 ± 300.1 vs. 169.8 ± 154.5; P <0.05). SPD levels were lower at ARDS stage 1 in comparison with stage 2 (day 0 – 154.6 ± 125.2 vs. 451.8 ± 299.2; day 3 – 129.3 ± 74.9 vs. 362.9 ± 202.0; day 5 – 167.8 ± 120.4 vs. 428.5 ± 372.4; day 7 – 186.2 ± 127.1 vs. 415.5 ± 337.0; P <0.05). Plasma SPD had a good diagnostic capacity for stage 1 ARDS: SPD on day 0 ≤253.0 ng/ml yielded a sensitivity of 90% and specifi city of 74% (AUC = 0.83; 95% CI = 0.631 to 0.951; P = 0.0001). No such diff erences for CCP were detected.i P103 End-expiratory esophageal pressure versus lower infl ection point in acute lung injury Results In the multivariable model, age, lung compliance, serum lactate and serum ACE levels were signifi cantly associated with both 28-day and 90-day mortality. No signifi cant correlation was found between serum and BALF ACE levels (Spearman’s ρ = 0.054; P = 0.66). Serum ACE concentrations were signifi cantly higher (P = 0.046) in patients with D/D genotype versus the two other groups combined (I/D and I/I genotypes). A meta-analysis of six studies (including ours) provided evidence that the D allele is signifi cantly associated with increased mortality in ALI/ARDS patients, yielding a per-allele odds ratio of 1.76 (95% CI: 1.19 to 2.59). See Figure 1 and Table 1.f Introduction No recommendations available concerning protocols of static PV loop and esophageal pressure measurements use set positive end-expiratory pressure (PEEP). The aim of the study was evaluation of the signifi cance of the lower infl ection point (LIP) and esophageal pressure monitoring for PEEP adjustment in ALI and ARDS. Conclusion Serum ACE levels appear to be aff ected by the I/D poly- morphism and are correlated with prognosis in patients with ALI/ARDS, indicating that further investigation of the clinical signifi cance of the ACE in ARDS might be of value. Methods A prospective study performed in one general ICU. We include 72 patients who received mechanical ventilation before evaluation and met ARDS criteria by AECC (1994) – acute onset, PaO2/FiO2 lower than 250 Torr, bilateral infi ltrates on chest X-ray. Exclusion criteria were age <15 years and pregnancy. We drew a static pressure–volume loop with sustained infl ation 40×30 (PV loop) for all patients using a low- fl ow technique (Hamilton G5) and measured the esophageal pressure (Avea) in 36 of 72 patients. After that PEEP was set according to zero end-expiratory transpulmonary pressure. We compare PV loop data with esophageal pressure measurements.l Figure 1 (abstract P102). Figure 1 (abstract P102). Results The low infl ection point median was 8 (95% CI = 5 to 10.5) mbar, which does not correspond to the empirically set optimal PEEP of 13 (95% CI = 12 to 15) mbar (P <0.001, Wilcoxon test). End-expiratory esophageal pressure (EEEP) median was 14 (95% CI = 12 to 18) mbar, the correlation between LIP and EEEP was poor (ρ = 0.279, P = 0.049). We fi nd signifi cant correlation between static compliance and EEEP (ρ = –0.421, P = 0.005). P100 P102 Eff ect of angiotensin-converting enzyme gene I/D polymorphism and its expression on clinical outcome in acute respiratory distress syndrome I Tsangaris, A Tsantes, P Kopterides, G Tsaknis, S Kokkori, I Karampela, D Konstantonis, S Karabi, E Vrigkou, A Pappas, S Orfanos, A Armaganidis University Hospital ‘Attikon’, University of Athens Medical School, Athens, Greece Critical Care 2013, 17(Suppl 2):P102 (doi: 10.1186/cc12040) Introduction The role of the D allele of the angiotensin-converting enzyme (ACE) gene I/D polymorphism in the clinical outcomes of patients with acute lung injury and acute respiratory distress syndrome (ALI/ARDS) remains controversial. We assessed simultaneously the eff ect of the ACE I/D polymorphisms as well as the serum and BALF ACE levels on prognosis of ARDS patients. Methods We recruited 69 mechanically ventilated ALI/ARDS patients. ACE activity levels in serum and BALF were assessed by chemical th d P ti t t d f ACE I/D l hi Ti Table 1 (abstract P102). Characteristics of the study population (n = 69) Age (years) 64.4 ± 17.9; 69 (28 to 89) Sex (males, %) 43 (62.3%) APACHE II score 22.1 ± 6.2; 21 (14 to 35) SOFA score 8.4 ± 3.2; 8 (4 to 16) Lung compliance (ml/cmH2O) 29.4 ± 7.2; 30 (17 to 40) PaO2/FiO2 139, 1 ± 47.1; 140 (72 to 223) PEEP 8.1 ± 2.9; 8 (5 to 15) Lung Injury Score 2.5 ± 0.6; 2.5 (1.75 to 3.50) Blood lactate (mmol/l) 1.7 ± 1.7; 1.3 (0.5 to 5.2) Septic status 39 (56.5%) Serum ACE (U/l) 16.5 ± 10.8; 13.6 (4.8 to 39.8) BALF (U/l) 2.3 ± 1.4; 2.0 (0.3 to 4.7) ACE I/D polymorphism: D/D 27 (39.1%) I/D 28 (40.6%) I/I 14 (20.3%) 28-day mortality 34/69 (49.3%) 90-day mortality 43/69 (62.3%) Ventilator-free days 5.1 ± 7.8; (0 to 23) Days w/o cardiovascular failure 14.0 ± 10.2; 16 (0 to 27) Days w/o renal failure 15.4 ± 10.6; 16 (0 to 28) Table 1 (abstract P102). 0 Eff ect of angiotensin-converting enzyme gene I/D polymorphism and its expression on clinical outcome in acute respiratory distress syndrome Eff ect of angiotensin-converting enzyme gene I/D polymorphism and its expression on clinical outcome in acute respiratory distress syndrome y I Tsangaris, A Tsantes, P Kopterides, G Tsaknis, S Kokkori, I Karampela, D Konstantonis, S Karabi, E Vrigkou, A Pappas, S Orfanos, A Armaganidis University Hospital ‘Attikon’, University of Athens Medical School, Athens, Greece Critical Care 2013, 17(Suppl 2):P102 (doi: 10.1186/cc12040) Introduction The role of the D allele of the angiotensin-converting enzyme (ACE) gene I/D polymorphism in the clinical outcomes of patients with acute lung injury and acute respiratory distress syndrome (ALI/ARDS) remains controversial. We assessed simultaneously the eff ect of the ACE I/D polymorphisms as well as the serum and BALF ACE levels on prognosis of ARDS patients. p g p Methods We recruited 69 mechanically ventilated ALI/ARDS patients. ACE activity levels in serum and BALF were assessed by chemical methods. Patients were genotyped for ACE I/D polymorphisms. Time- to-event analysis evaluated the variables associated with the 28-day and 90-day mortality. P100 Characteristics of the study population (n = 69) Age (years) 64.4 ± 17.9; 69 (28 to 89) Sex (males, %) 43 (62.3%) APACHE II score 22.1 ± 6.2; 21 (14 to 35) SOFA score 8.4 ± 3.2; 8 (4 to 16) Lung compliance (ml/cmH2O) 29.4 ± 7.2; 30 (17 to 40) PaO2/FiO2 139, 1 ± 47.1; 140 (72 to 223) PEEP 8.1 ± 2.9; 8 (5 to 15) Lung Injury Score 2.5 ± 0.6; 2.5 (1.75 to 3.50) Blood lactate (mmol/l) 1.7 ± 1.7; 1.3 (0.5 to 5.2) Septic status 39 (56.5%) Serum ACE (U/l) 16.5 ± 10.8; 13.6 (4.8 to 39.8) BALF (U/l) 2.3 ± 1.4; 2.0 (0.3 to 4.7) ACE I/D polymorphism: D/D 27 (39.1%) I/D 28 (40.6%) I/I 14 (20.3%) 28-day mortality 34/69 (49.3%) 90-day mortality 43/69 (62.3%) Ventilator-free days 5.1 ± 7.8; (0 to 23) Days w/o cardiovascular failure 14.0 ± 10.2; 16 (0 to 27) Days w/o renal failure 15.4 ± 10.6; 16 (0 to 28) Table 1 (abstract P102). Characteristics of the study population (n = 69) on these data we will continue to the next phase of this project and test PFC in the prevention of ARDS alone, and in combination with HS. References on these data we will continue to the next phase of this project and test PFC in the prevention of ARDS alone, and in combination with HS. References 1. Keipert PE, et al.: Enhanced oxygen delivery by perfl ubron emulsion during acute hemodilution. Artif Cells Blood Substit Immobil Biotechnol 1994, 22:1161-1167.l 2. Habler O, et al.: IV perfl ubron emulsion versus autologous transfusion in severe normovolemic anemia: eff ects on left ventricular perfusion and function. Res Exp Med (Berl) 1998, 197:301-318. 1. Keipert PE, et al.: Enhanced oxygen delivery by perfl ubron emulsion during acute hemodilution. Artif Cells Blood Substit Immobil Biotechnol 1994, 22:1161-1167. P100 Biomarkers for early stage of acute respiratory distress syndrome in septic patients: surfactant protein D and Clara cell protein A Kuzovlev1, V Moroz1, A Goloubev1, S Polovnikov2 1V.A. Negovsky Scientifi c Research Insitute of General Reanimatology RAMS, Moscow, Russia; 2N.N. Burdenko Main Military Hospital, Moscow, Russia Critical Care 2013, 17(Suppl 2):P100 (doi: 10.1186/cc12038) Results Average survival time after onset of second hemorrhage was 60 minutes in PFC-treated animals versus 65 minutes in the HES group. Mean arterial pressure (MAP) was similar between T0 and T60, thereafter PFC-treated animals had lower MAP, mean pulmonary artery pressure (MPAP), heart rate and cardiac output (P >0.05).if Conclusion There was no signifi cant diff erence in survival time, MAP and MPAP in the PFC group compared with HES control. These data suggest that it is safe to administer in this HS model. Regulatory approval of hemoglobin-based oxygen carriers has been halted due to possible side eff ects related to vasoconstriction. In this model, and with this class of oxygen-carrying drugs (PFCs), we did not observe evidence of vasoconstriction. Using PFCs did not result in a survival advantage here; however, there was also no observation of adverse events. Based Introduction The injury of alveolar epithelium and endothelium is the basis of pathogenesis for the early stage of acute respiratory distress syndrome (ARDS). A prompt detection of this injury will provide us with a possibility for early ARDS diagnosis and treatment. Currently no biomarkers of alveolar epithelial injury are clinically available. The purpose of our study was to investigate the role of surfactant protein D (SPD) and Clara cell protein (CCP) as biomarkers of early ARDS. S38 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 on these data we will continue to the next phase of this project and test PFC in the prevention of ARDS alone, and in combination with HS. References 1. Keipert PE, et al.: Enhanced oxygen delivery by perfl ubron emulsion during acute hemodilution. Artif Cells Blood Substit Immobil Biotechnol 1994, 22:1161-1167. 2. Habler O, et al.: IV perfl ubron emulsion versus autologous transfusion in severe normovolemic anemia: eff ects on left ventricular perfusion and function. Res Exp Med (Berl) 1998, 197:301-318. P103 P103 End-expiratory esophageal pressure versus lower infl ection point in acute lung injury A Yaroshetskiy1, D Protsenko1, E Larin2, O Ignatenko1, B Gelfand1 1Russian National Research Medical University, Moscow, Russia; 2City Hospital #7, Moscow, Russia Critical Care 2013, 17(Suppl 2):P103 (doi: 10.1186/cc12041) 2. Habler O, et al.: IV perfl ubron emulsion versus autologous transfusion in severe normovolemic anemia: eff ects on left ventricular perfusion and function. Res Exp Med (Berl) 1998, 197:301-318. Quantifi cation of stress raisers in ARDS Quantifi cation of stress raisers in ARDS M Cressoni1, M Amini1, P Cadringher2, C Chiurazzi1, D Febres1, E Gallazzi1, A Marino1, M Brioni1, F Menga1, I Cigada1, E Carlesso1, D Chiumello2, L Gattinoni2 1Università degli Studi di Milano, Milan, Italy; 2Fondazione IRCCS Ca’ Granda- Ospedale Maggiore Policlinico, Milan, Italy Critical Care 2013, 17(Suppl 2):P105 (doi: 10.1186/cc12043) Results We included in our study the CT scans of 89 patients (24 mild, 55 moderate and 10 severe ARDS, age 58 ± 17 years, BMI 25.6 ± 5.4, PaO2/FiO2 165 ± 66, PaCO2 42 ± 9, 60 discharged alive from ICU (67%)). At PEEP 5 cmH2O 39 ± 16% of the lung parenchyma was not infl ated, 33 ± 13% poorly infl ated, 28 ± 15% well infl ated and 0 ± 1% overinfl ated. The median lung recruitability between 5 and 15 cmH2O PEEP was 6% (3 to 10%) of lung parenchyma (71 g (26 to 182 g)). Mean EELV at PEEP 5 cmH2O was 1,233 ± 709 while it increased to 1,784 ± 848 at PEEP 15 cmH2O (ΔEELV = 551 ± 407 ml). More than one-half (52% (31 to 75%)) of the increase in end-expiratory lung volume (EELV) was due to an increase in infl ation of the already well-aerated tissue; 38% (23 to 43%) was due to the infl ation of poorly aerated tissue, while only 4% (1 to 8%) to the recruitment of lung tissue. The CLsp of the well, poorly and recruited tissue was 0.07 (0.031 to 0.13), 0.03 (0.01 to 0.06) and 0.02 (0.01 to 0.04) ml/cmH2O/g, respectively (P <0.0001). Introduction Ventilator-induced lung injury (VILI) is a well-known side eff ect of mechanical ventilation. The pressures and volumes needed to induce VILI in healthy animals are far greater than pressure and volumes applied in clinical practice [1]. A possible explanation may be the presence of local pressure multipliers (stress raisers). Methods We retrospectively analyzed CT scans of 147 patients with ARDS and CT scans of 100 healthy subjects. A homogeneous lung would have the same gas/tissue ratio in all its regions. If a lung region expands less than the neighbour regions these will be more strained to vicariate the non/less expanding region. Recruited lung tissue does not resume normal mechanical properties g Results Patients were 65 ± 13 years old, ventilation days were 9 ± 10 and PaO2/FiO2 was 194 ± 49 mmHg at PEEP 8 ± 2 cmH2O. Introduction of Sigh improved PaO2/FiO2 (P <0.001) and ΔEELVgl (P <0.001). The increase in EELV was diff use across all lung regions as both ΔEELVnondep and ΔEELVdep changed (P <0.01 and P = 0.06). minute ventilation, arterial blood pH and hemodynamics were not signifi cantly aff ected by introduction of Sigh, while mean airway pressure increased (P <0.001), albeit only at the highest Sigh rate and by a limited extent (~2 cmH2O). Patients with mean ΔEELVgl over the three Sigh phases >200 ml (that is, Sigh-responders) had signifi cantly lower PaO2/ FiO2 values at baseline (that is, at Sigh rate = 0/minute) than Sigh- nonresponders (163 ± 25 mmHg vs. 222 ± 39 mmHg, P <0.01). p p M Cressoni1, C Chiurazzi1, M Amini1, D Febres1, E Gallazzi1, E Carlesso1, P Cadringher1, T Langer1, D Chiumello1, L Gattinoni2 1Università degli Studi di Milano, Milan, Italy; 2Fondazione IRCCS Ca’ Granda – Ospedale Maggiore Policlinico, Milan, Italy Critical Care 2013, 17(Suppl 2):P106 (doi: 10.1186/cc12044) Introduction Positive end-expiratory pressure (PEEP) is commonly used in patients with ARDS to prevent end-expiratory collapse. The increase in gas volume and/or change in mechanical properties of the lung are used at the bedside to estimate lung recruitment and individualize PEEP selection. Methods Retrospective analysis of patients who underwent whole- lung CT scan at 5 and 15 cmH2O PEEP. Every chest CT was divided both into 10 apex-base levels of equal height and into 10 sterno-vertebral levels, yielding 100 units per scanned lung (200 units per patient); each unit was classifi ed according to its average CT number (<–100 not infl ated, –100 to –500 poorly infl ated, –500 to –900 well infl ated and –900 overinfl ated). Lung regions defi ned as not infl ated at PEEP 5 cmH2O and infl ated (poorly, well or over) at PEEP 15 cmH2O were classifi ed as recruited. Moreover, a surrogate specifi c lung compliance (CLsp) was determined on the expiratory limb of the pressure–volume curve (15 to 5 cmH2O PEEP at CT) and was defi ned as Δgas/cmH2O PEEP/gram of tissue. Gattinoni L, et al.: Int Care Med 2005. 31:776-784. Lung recruitment induced by Sigh in hypoxemic intubated critically ill patients p TM Mauri1, G Bellani1, A Coppadoro1, P Tagliabue2, A Barletta1, V Meroni2, M Teggia Droghi1, N Patroniti1, A Pesenti1 1University of Milan-Bicocca, Monza, Italy; 2San Gerardo Hospital, Monza, Italy Critical Care 2013, 17(Suppl 2):P104 (doi: 10.1186/cc12042) of the stress raisers the fraction of lung volume above this threshold. Results The extent of stress raisers increased with the severity of ARDS (14 ± 5, 18 ± 8, 23 ± 1% of lung parenchyma in mild, moderate and severe ARDS, P <0.0001). The extent of stress raisers correlated with the dead space fraction (r2 = 0.34, P <0.001), with the fraction of poorly aerated tissue (r2 = 0.36, P <0.0001) and also has a negative correlation with the fraction of well infl ated tissue (r2 = 0.47, P <0.0001). The response to PEEP, passing from 5 to 45 cmH2O is minimal (average decrease of stress raiser extent 6 ± 5%) and inter-individual variability is great (in 11 patients, stress raisers increased passing from PEEP 5 to PEEP 45). Stress raisers turn out to be greater in nonsurvivor patients than in survivor patients (17 ± 7 vs. 20 ± 9% of lung volume, P = 0.03). Introduction In intubated critically ill patients, cyclic short recruitment manoeuvres (Sigh) introduced during pressure support ventilation (PSV) improve oxygenation, probably by increasing end-expiratory lung volume (EELV). We assessed Sigh eff ects on regional distribution of EELV by electrical impedance tomography (EIT): a bedside, radiation- free lung imaging technique. Moreover, we investigated baseline characteristics correlated with response to Sigh. Methods We enrolled 20 intubated critically ill patients undergoing PSV with PaO2/FiO2 ≤300 mmHg and PEEP ≥5 cmH2O. We applied on each patient’s thorax a 16-electrode belt connected to an EIT monitor (PulmoVista 500®; Dräger Medical GmbH, Lübeck, Germany). Sigh was introduced as 35 cmH2O continuous positive airway pressure phase lasting 3 to 4 seconds at diff erent rates (0, 0.5, 1, 2 per minute, random order) for 20 minutes. From raw EIT data, we calculated global changes in EELV (ΔEELVgl), calculated as changes in end-expiratory lung impedance calibrated versus tidal volume to estimate changes in ΔEELV and considering 0 Sigh/minute as baseline; and ΔEELV of nondependent and dependent lung regions (ΔEELVnondep and ΔEELVdep). Together, we collected ventilation parameters, hemodynamics and arterial blood gases. Reference 1. Protti A: Am J Respir Crit Care Med 2011, 183:1354-1362. 1. Protti A: Am J Respir Crit Care Med 2011, 183:1354-1362. Recruited lung tissue does not resume normal mechanical properties p g g Conclusion Introduction of Sigh in intubated critically ill patients undergoing PSV improves gas exchange by inducing alveolar recruitment across all lung regions with minimal increase of airway pressure. Patients with more severe gas exchange impairment present the highest degree of potentially recruitable lung. Quantifi cation of stress raisers in ARDS We measured the stress raisers by computing the ratio between the gas fraction of the region of interest and the neighbouring regions: if the infl ation would be the same (homogeneity), the ratio will be equal to one; if the infl ation of the surrounding regions would be greater than the region of interest (that is, more strained), the ratio between the two will be greater than one 2 Conclusion Most of the PEEP-related EELV increase is due to an increased infl ation of already well aerated lung tissue. In agreement with the baby lung theory [1], the well-aerated tissue showed near to normal mechanical properties. Lung recruitment induced by Sigh in hypoxemic intubated critically ill patients P106 Recruited lung tissue does not resume normal mechanical properties M Cressoni1, C Chiurazzi1, M Amini1, D Febres1, E Gallazzi1, E Carlesso1, P Cadringher1, T Langer1, D Chiumello1, L Gattinoni2 1Università degli Studi di Milano, Milan, Italy; 2Fondazione IRCCS Ca’ Granda – Ospedale Maggiore Policlinico, Milan, Italy Critical Care 2013, 17(Suppl 2):P106 (doi: 10.1186/cc12044) P103 End-expiratory esophageal pressure versus lower infl ection point in acute lung injury Sustained infl ation did not lead to improved oxygenation (P >0.05). PEEP adjustment by EEEP led to an increase in PaO2/FiO2 – median 107 mmHg (95% CI = 18 to 147, P <0.001). EEEP was similar to empirically set PEEP (P >0.05). Conclusion LIP has poor correlation with EEEP. PEEP adjustment by esophageal pressure was close to empirically set PEEP and can improve oxygenation. S39 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 P104 and was taken as a measure of stress raiser. We considered pathological stress raisers as the regions showing infl ation ratio greater than the 95th percentile of the control group (1.61) and defi ned as the extent P104 Lung recruitment induced by Sigh in hypoxemic intubated critically ill patients TM Mauri1, G Bellani1, A Coppadoro1, P Tagliabue2, A Barletta1, V Meroni2, M Teggia Droghi1, N Patroniti1, A Pesenti1 1University of Milan-Bicocca, Monza, Italy; 2San Gerardo Hospital, Monza, Italy Critical Care 2013, 17(Suppl 2):P104 (doi: 10.1186/cc12042) Reference Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S40 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 P107 Intraoperative management of hypoxemia with recruitment maneuvers: are the benefi ts worth the costs? V Grosomanidis, B Fyntanidou, K Karakoulas, K Kotzampasi, E Oloktsidou, C Nouris, C Skourtis Aristotle Medical School, Thessaloniki, Greece Critical Care 2013, 17(Suppl 2):P107 (doi: 10.1186/cc12045) P107 and without inotropic support were enrolled. A transthoracic echo- cardiography was performed to assess diastolic function: E/A, isovolumic relaxation time (IVRT), deceleration time (DT), E/e’ were assessed. Eight patients (fi ve with poor relaxation pattern, three with decreased left ventricle compliance) were identifi ed in diastolic dysfunction group (Group 1) and eight were included in the normal diastolic function group (Group 2). A recruitment manoeuvre was performed by applying a PEEP of 40 cmH2O for 30 seconds. Haemodynamic parameters of cardiac output (CO), stroke volume (SV), heart rate (HR), pulse pressure variation (PPV), and cardiac cycle effi ciency (CCE) were continuously recorded during the manoeuvre using the MostCare pulse contour method.ii and without inotropic support were enrolled. A transthoracic echo- cardiography was performed to assess diastolic function: E/A, isovolumic relaxation time (IVRT), deceleration time (DT), E/e’ were assessed. Eight patients (fi ve with poor relaxation pattern, three with decreased left ventricle compliance) were identifi ed in diastolic dysfunction group (Group 1) and eight were included in the normal diastolic function group (Group 2). A recruitment manoeuvre was performed by applying a PEEP of 40 cmH2O for 30 seconds. Haemodynamic parameters of cardiac output (CO), stroke volume (SV), heart rate (HR), pulse pressure variation (PPV), and cardiac cycle effi ciency (CCE) were continuously recorded during the manoeuvre using the MostCare pulse contour method. Introduction Several strategies can be applied for the prevention and management of anesthesia-related hypoxemia. The aim of our study was to investigate the cardiovascular eff ects of a specifi c recruitment maneuver (RM) used for the management of intraoperative hypoxemia. Methods Forty-nine patients (29 male/20 female) of mean age 57.4 ± 9.9 years, mean weight 80.2 ± 13.6 kg and ASA-PS classifi cation I to IV (I: 2, II: 27, III: 16, IV: 6), undergoing general surgery procedures, who developed intraoperative hypoxemia (PaO2/FiO2 <200), were enrolled in our study. For the management of hypoxemia a total of 67 RMs have been applied, which consisted of a manual increase of the airway pressure to 40 cmH2O. Reference This rise has been maintained for 10 seconds and was followed by an increase of positive end-expiratory pressure from 5 to 10 cmH2O. Before RM application an oesophageal Doppler monitor probe was inserted into the patients for measuring stroke volume (SV), cardiac output (CO), peak velocity (PV) and fl ow corrected time (FTc). Standard monitoring also included ECG, IBP, ETCO2 and SpO2. Heart rate (HR), systemic arterial pressure (SAP), SV, CO, PV and FTc were recorded directly before, during, right after and 5 minutes after RM application (T1 to T4). Kolmogorov–Smirnoff was used to test normal distribution of data and ANOVA was used for the statistical analysis. P <0.05 was considered statistically signifi cant.i Results No signifi cant haemodynamic changes happened in the fi rst part of the recruitment (t0 to t15 seconds). A signifi cant decrease in CO (P <0.05), SV (P <0.01), systolic pressure (P <0.04) and PPV (P <0.04) occurred in Group 1 during the remaining part of the manoeuvre. No signifi cant decrease in the same haemodynamic parameters were noted when only E/A was used to discriminate the diastolic dysfunction. See Table 1. Table 1 (abstract P108). CO diff erence associated with diastolic dysfunction parameters E/A <1 E/A >1 P value CO 15 to 30 3.9 4.8 0.20 E/e’ <1 E/e’ >1 CO 15 to 30 3.92 5.6 0.02 Table 1 (abstract P108). CO diff erence associated with diastolic dysfunction parameters y gi Results HR, SAP, SV, CO, PV and FTc showed a statistically signifi cant decrease during and right after RM compared with the baseline values but they were gradually restored to control values after RM discontinuation (Table 1). Conclusion The recruitment manoeuvre can compromise left diastolic fi lling by increasing the transpulmonary pressure. In patients with diastolic dysfunction a signifi cant decrease in SV can be observed during the recruitment manoeuvre with no compensatory mechanisms evoked determining a signifi cant decrease in CO. The E/A ratio is not able to discriminate between the two groups, confi rming the power of tissue Doppler imaging to recognize the correct diastolic pattern. Reference Table 1 (abstract P107) T1 T2 T3 T3 HR (beats/minute) 69.8±7.1 60.2±10.6* 67.1±7.4^ 68.4±6.9^ SAP mean (mmHg) 89.8±7.7 53.4±5.7 81.5±7.7^ 87.6±6.7^ SV (ml) 82.2±21.2 22.5±15.5 75.4±21.7^ 79.6±6.4^ CO (l/minute) 5.75±1.7 1.4±0.9 5.1±1.7^ 5.5±1.5^ PV (cm/second) 72.6±17.1 26±15.9 68±16.8^ 70.7±15.9^ FTc (seconds) 0.4±0.06 0.19±0.08 0.36±0.06^ 0.36±0.06^ P <0.05 versus T1. ^P <0.05 versus T2. 1. Reference Gernoth C, et al.: Respiratory and haemodynamic changes during decremental open lung positive end-expiratory pressure titration in patients with acute respiratory distress syndrome. Crit Care 2009, 13:R59. P109 Alveolar Recruitment for ARDS Trial: preliminary results AB Cavalcanti1, EA Suzumura1, M Abreu1, GF Ribeiro1, A Kodama1, F Moreira1, HP Guimarães1, E Romano1, MB Amato2, O Berwanger1, CR Carvalho2, ART Investigators1 1Hospital do Coração – HCor, São Paulo, Brazil; 2Universidade de São Paulo, Brazil Critical Care 2013, 17(Suppl 2):P109 (doi: 10.1186/cc12047) Conclusion According to our results, RM application causes a profound impairment of the cardiovascular system. This can be attributed to the increase of the airway pressures, which results in preload decrease and afterload increase of the right ventricle and cardiac contractility attenuation. Nevertheless, these eff ects are transient and reversible and RM application is a safe technique for the management of intraoperative hypoxemia, provided that adequate cardiac preload is ensured and the anesthesiologist is alert to discontinue the RM if it exceeds cardiovascular reserves. Critical Care 2013, 17(Suppl 2):P109 (doi: 10.1186/cc12047 Introduction The aim of the ongoing Alveolar Recruitment for ARDS Trial (ART) is to evaluate whether a maximum stepwise alveolar recruit- ment maneuver (MSARM) followed by ventilation at optimal PEEP may decrease 28-day mortality in patients with moderate to severe ARDS compared with ventilation with the ARDSNet strategy (NCT01374022). Here we report the results of a planned a priori preliminary analysis involving 100 patients to assess feasibility, physiologic variables and safety outcomes. P108 P108 Haemodynamic changes during alveolar recruitment manoeuvre in patients with diastolic dysfunction A Rubino, E Marini, B Ferro, M Collareta, F Forfori, F Guarracino, F Giunta Azienda Ospedaliero Universitaria Pisana, Pisa, Italy Critical Care 2013, 17(Suppl 2):P108 (doi: 10.1186/cc12046) y Methods ART is an event-driven multicenter randomized controlled trial planned to last until 520 deaths within 28 days are observed. Patients assigned to the experimental group receive a MSARM achieving PEEP of 45 cmH2O and plateau pressure of 60 cmH2O plus PEEP titrated according to the static compliance of the respiratory system (ART strategy). The target tidal volume is 4 to 6 ml/kg predicted body weight (PBW) and plateau pressure ≤30 cmH2O in both groups. Results We randomized 101 patients between November 2011 and October 2012 in 51 centers. Considering 124 active sites in July 2013 and the current recruitment rate, we will fi nish enrollment by November 2014. MSARM was complete in 34/47 (72%) of patients allocated to the P112f Eff ect of protective ventilation on organ-specifi c cytokine production in an experimental postoperative sepsis model J Sperber1, M Lipcsey2, A Larsson3, A Larsson2, J Sjölin3, M Castegren1 1Centre for Clinical Research Sörmland, Uppsala University, Uppsala, Sweden; 2Surgical Sciences, Uppsala University, Uppsala, Sweden; 3Medical Sciences, Uppsala University, Uppsala, Sweden Critical Care 2013, 17(Suppl 2):P112 (doi: 10.1186/cc12050) Methods We prospectively evaluated 270 patients immediately after cardiac surgery, presenting hypoxemia and PaO2/FiO2 <250. Patients were randomized to an intensive alveolar recruitment maneuver (ARM) or a moderate ARM strategy. Intensive ARM group: recruitment with an inspiratory pressure amplitude of 15 cmH2O and PEEP of 30 cmH2O, followed by ventilation with PEEP = 13 cmH2O, during 4 hours of protective mechanical ventilation with VT = 6 ml/kg/pbw. Moderate ARM group: recruitment with opening pressures of 20 cmH2O in the airways, followed by ventilation with PEEP = 8 cmH2O, during 4 hours of protective mechanical ventilation with VT = 6 ml/kg/pbw. The primary outcome was a composite endpoint of severe pulmonary complications in the postoperative period defi ned as intra-hospital death, need for mechanical ventilation for more than 48 hours after surgery, pulmonary infection or after reintubation within 28 days after randomization. The secondary outcome was the incidence of nonpulmonary complications as postoperative myocardial ischemia, acute renal failure (RIFLE-R), respiratory mechanics and blood gas analysis after ARM, ICU length of stay, hospital length of stay and 30-day mortality. Introduction Low tidal volume (VT) ventilation in intensive care patients without lung injury attenuates the systemic infl ammatory response [1]. The contribution of the specifi c organ infl ammatory responses to the systemic picture remains to be elucidated. We investigated the eff ect of low VT ventilation compared with medium high VT on hepatic, splanchnic and cerebral cytokine responses in an experimental large animal postoperative sepsis model. Methods Twenty pigs, group Protective Ventilation (PV), were ventilated with low VT (6 ml/kg) and PEEP 10 cmH2O while 10 pigs, group Control (C), were ventilated with a VT of 10 ml/kg and PEEP 5 cmH2O. Catheters were introduced into an artery, the jugular bulb, the hepatic vein and the portal vein. Laparotomy for 2 hours simulated a surgical procedure after which baseline ensued and a continuous endotoxin infusion was started at 0.25 μg/kg/hour for 5 hours. Diff erences were analyzed with ANOVA for repeated measures. 1. Amato MB, et al.: N Engl J Med 1998, 338:347-354. P110 0 Intensive alveolar recruitment protocol reduces pulmonary complications and intensive care permanence after cardiac surgery A Leme1, L Hajjar1, E Nozawa1, C Hashizume1, J Almeida1, J Fukushima1, J Auler Jr1, R Santiago1, R Ianotti1, M Amato2, E Osawa1, M Feltrim1, F Galas1 1Heart Institute, São Paulo, Brazil; 2Faculdade de Medicina da Universidade de São Paulo, Brazil Critical Care 2013, 17(Suppl 2):P110 (doi: 10.1186/cc12048) Conclusion The reliability of pressure measurements and also of compliance estimation via the tested catheters is high. Only in two catheters was the fi lling volume a critical point for a precise measurement of pressure or for estimation of compliance. Immediately after unpacking, adhesion of the balloon material might prevent reliable pressure measurement, therefore before the fi rst measurement overfi lling of the balloon and retention of the excess gas seems strongly recommended. Introduction Cardiac surgical procedures are associated with a high incidence of postoperative complications, increasing costs and mortality. The purpose of this study is to evaluate prospectively the impact of two protective mechanical ventilation strategies, both using low-tidal volume ventilation (6 ml/kg/ibw) after cardiac surgery. Haemodynamic changes during alveolar recruitment manoeuvre in patients with diastolic dysfunction Introduction To investigate the haemodynamic eff ects of an alveolar recruitment manoeuvre in intubated patients with diastolic dysfunction compared with patients with normal diastolic function in a mixed ICU. Methods Between October 2011 and February 2012, 16 mechanically ventilated patients admitted to the ICU with normal systolic function 2 Results We randomized 101 patients between November 2011 and October 2012 in 51 centers. Considering 124 active sites in July 2013 and the current recruitment rate, we will fi nish enrollment by November 2014. MSARM was complete in 34/47 (72%) of patients allocated to the S41 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 ART group. The main reason for MSARM interruption was hypotension (8/9 (90%)). Mean titrated PEEP in the ART group was 16.1 ± 3.5 versus 12.9 ± 3.4 cmH2O in ARDSNet (P <0.001). One hour after randomization, tidal volume was similar between the ART and ARDSNet groups (5.1 ± 0.8 vs. 5.3 ± 0.7 ml/kg PBW, respectively; P = 0.08). Mean values remained below 6 ml/kg PBW up to day 3. Few patients had plateau pressure >30 cmH2O 1 hour after randomization in the ART and ARDSNet groups (3/43 (7.0%) vs. 4/47 (8.5%), respectively; P = 1.00) and on subsequent days. PaO2/FiO2 was signifi cant higher in the ART group (179.5 ± 84.5 vs. 143.3 ± 46.8, P = 0.01) and increased over time up to 7 days after randomization (272.3 ± 136.5 vs. 192.6 ± 72.3, P = 0.003). The ART strategy did not increase the risk of barotrauma (relative risk (RR) = 0.78, 95% CI = 0.19 to 3.30) in the fi rst 7 days after randomization or the need to initiate or increase vasopressors or mean arterial pressure <65 mmHg (RR = 1.14, 95% CI = 0.65 to 2.02, P = 0.67) 1 hour after randomization. However, the ART strategy increased the risk for severe acidosis (pH <7.10) 1 hour after randomization (RR = 3.20, 95% CI = 1.12 to 9.20, P = 0.03). Reliability of pressure measurements via balloon catheters is high: an evaluation of six esophageal catheters Reliability of pressure measurements via balloon catheters is high: an evaluation of six esophageal catheters p g S Walterspacher, L Isaak, HJ Kabitz, J Guttmann, S Schumann University Medical Center Freiburg, Germany Critical Care 2013, 17(Suppl 2):P111 (doi: 10.1186/cc12049) y g y Critical Care 2013, 17(Suppl 2):P111 (doi: 10.1186/cc12049) Introduction Reliable measurement of esophageal pressure is a prerequisite for analysis of respiratory system mechanics in spontaneous breathing patients. For that purpose, various types of balloon catheters exist that diff er in material, size and shape. In physical models we studied the quality of pressure measurement via six diff erent balloon catheters, three of them containing a second balloon for measurement of gastric pressure. g p Methods Nine balloons of six esophageal catheters were investigated in three conditions: measurement of balloon pressure during initial infl ations immediately after unpacking; measurement of static pressures at diff erent fi lling volumes; and compliance estimation in physical models (27, 54, 90 ml/cmH2O) at diff erent levels of superimposed pressure. Conclusion ART is feasible. The incidence of adverse events was similar between groups except for severe acidosis 1 hour after randomization. Hence we adjusted the study protocol, increasing the respiratory rate (from 10 to 15/minute) during MSARM. Results During the initial infl ation most catheters showed pressure artifacts resulting from material adhesion. Those artifacts disappeared during following infl ations. Static pressure measurements could be performed with an error below 1 cmH2O if the balloon was fi lled appropriately. Overfi lling of the balloon resulted in larger errors only in two catheters. Compliance estimations resulted in errors below 1 ml/ cmH2O. Superimposed pressure had no relevant eff ect on compliance estimation. P112f y y y y Results The intensive ARM group compared with the moderate ARM group had lower incidence of the primary outcome, mainly due to the reduced rate of pulmonary infection (2.3% vs. 10.1%, P = 0.009). Moreover, the intensive ARM group presented higher lung compliance (68 ± 19 vs. 51 ± 17 ml/cmH2O, P <0.001) and PaO2/FiO2 ratio (360 ± 68 vs. 240 ± 74, P <0.001) after intervention when compared with the moderate group. Also, the intensive ARM presented a lower length of ICU stay (3 days vs. 4 days, P = 0.027) than the moderate ARM. There are no diff erences regarding severe nonpulmonary complications and 30- day mortality between groups. Results TNFα levels were higher in the hepatic vein than in the artery, the jugular bulb and the portal vein. IL-6 levels were higher in the artery and the jugular bulb compared with the portal and hepatic veins. IL-10 levels were higher in the portal vein compared with the jugular bulb and hepatic vein. The organ-specifi c IL-10 concentrations were all higher than the arterial concentration. Comparison between the ventilation groups showed that TNFα, IL-6 and IL-10 in the hepatic vein were higher in group C compared with group PV at the end of the experiment. Peak concentrations of TNFα and IL-6 in the portal vein were higher in group C compared with group PV. Conclusion An intensive ARM strategy reduces postoperative pulmo- nary complications, reduces hypoxemia, increases lung compliances and decreases the length of ICU stay after cardiac surgery. Reference S42 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Methods Mechanically ventilated patients (PEEP 10 cmH2O and TV 7 ml/kg – Baseline) were randomized to two groups: in the PEEP 15 group, PEEP was increased from 10 to 15 cmH2O; while in the PEEP 5 group, PEEP was decreased from 10 to 5 cmH2O. Arterial gas analyses were performed in both groups after 5, 15, 30 and 60 minutes from the change of PEEP. Conclusion In this experiment TNFα was mainly generated in the liver while the results point to signifi cant nonhepatic IL-6 and IL-10 production. Ventilation with low VT and medium-high PEEP attenuated hepatic and splanchnic cytokine production compared with medium- high VT and lower PEEP. Reference 1. N Engl J Med 2006, 354:1775-1786. 1. N Engl J Med 2006, 354:1775-1786. 1. N Engl J Med 2006, 354:1775-1786. P112f Reference g Results We enrolled 44 ARDS patients: 23 in the PEEP 15 group and 21 in the PEEP 5 group. At Baseline, PaO2/FiO2 (P/F) and PaCO2 were similar in both groups (P/F 169.5 ± 78.8 vs. 165.4 ± 80.6; PaCO2 45.6 ± 8.5 vs. 41.7 ± 5.0 mmHg, PEEP 15 vs. PEEP 5, respectively). In the PEEP 15 group, P/F signifi cantly continuously increased over time. In PEEP 5, P/F signifi cantly decreased after 5 minutes and remained stable over time. In the PEEP 15 group, PaCO2 did not change within 60 minutes after PEEP increase. When PEEP was reduced (PEEP 5) PaCO2 remained stable for the fi rst two steps, while at 30 and 60 minutes PaCO2 was signifi cantly higher than at Baseline (Figures 1 and 2). 1. Determann et al.: Crit Care 2010, 14:R1. doi:10.1186/cc8230 P114 Comparison between the standard and low-dose chest CT scans on the lung quantitative analysis in critically ill patients. S Froio1, D Chiumello1, I Cigada2, M Brioni2, S Coppola1, F Menga2, L Gattinoni2 1Fondazione IRCCS Ca’ Granda – Ospedale Maggiore Policlinico, Milan, Italy; 2Università degli Studi di Milano, Milan, Italy Critical Care 2013, 17(Suppl 2):P114 (doi: 10.1186/cc12052) Comparison between the standard and low-dose chest CT scans on the lung quantitative analysis in critically ill patients. S Froio1, D Chiumello1, I Cigada2, M Brioni2, S Coppola1, F Menga2, L Gattinoni2 Comparison between the standard and low-dose chest CT scans on the lung quantitative analysis in critically ill patients. S Froio1, D Chiumello1, I Cigada2, M Brioni2, S Coppola1, F Menga2, L Gattinoni2 Figure 2 (abstract P113). Two-way ANOVA RM. P <0.05, #versus 5 minutes. Figure 1 (abstract P113). Two-way ANOVA RM. P <0.05, versus Time 0, #versus 5 minutes, °versus 15 minutes. Comparison between the standard and low do the lung quantitative analysis in critically ill pat S Froio1, D Chiumello1, I Cigada2, M Brioni2, S Coppo L Gattinoni2 1Fondazione IRCCS Ca’ Granda – Ospedale Maggiore P 2Università degli Studi di Milano, Milan, Italy Critical Care 2013, 17(Suppl 2):P114 (doi: 10.1186/cc Introduction Quantitative analysis of a lung C the reference method to study lung recruitab ventilatory strategy in ARDS patients. However radiation exposure, especially when CT analysis is the evolution of ARDS. The aim of this study was t of a lower radiation dose on lung CT quantitati with analysis computed by standard CT. Methods Sedated and paralyzed patients und scans: a Standard CT (120 kV, 110 mAs, pitch 1.2 Care Dose Technology) and a CT performed by a (Lowdose: 120 kV, 60 mAs). CTs were perform inspiratory or expiratory hold, for diff erent valu Each CT image was manually delineated exclud and mediastinal structures. We analyzed Stand images with dedicated software to quantify lung regions with diff erent degrees of infl atio data computed by Standard and by Lowdose s according to Bland–Altmann analysis. Figure 2 (abstract P113). Two-way ANOVA RM. P <0.05, #versus 5 minutes. Figure 1 (abstract P113). Two-way ANOVA RM. P <0.05, versus Time 0, #versus 5 minutes, °versus 15 minutes. Figure 1 (abstract P114). Total tissue weight. Figure 1 (abstract P113). Two-way ANOVA RM. P <0.05, versus Time 0, #versus 5 minutes, °versus 15 minutes. Time required for gas exchange equilibration after a change of positive end-expiratory pressure in acute respiratory distress syndrome y S Coppola1, D Chiumello1, F Menga2, M Brioni2, I Cigada2, S Froio1 1Fondazione IRCCS Ca’ Granda – Ospedale Maggiore Policlinico, Milan, Italy; 2Università degli Studi di Milano, Milan, Italy Critical Care 2013, 17(Suppl 2):P113 (doi: 10.1186/cc12051) i y Conclusion Our data indicate that it is important in critically ill patients to allow suffi cient time for the full eff ect of PEEP increase on oxygenation and to prevent excessive delay when P/F decrease occurs following the application of a lower level of PEEP. Introduction Positive end-expiratory pressure (PEEP) is fundamental to prevent lung collapse in ARDS patients. A common method to titrate PEEP is to perform a PEEP test, recording the variation of cardio- respiratory parameters after a PEEP change [1]. The aim of this study was to evaluate the time-course changes of gas exchanges following a PEEP test. P116 P116 Feasibility and eff ectiveness of prone position in morbidly obese ARDS patients: a case–control clinical study A De Jong, N Molinari, M Sebbane, A Prades, S Jaber Montpellier University Hospital, Montpellier, France Critical Care 2013, 17(Suppl 2):P116 (doi: 10.1186/cc12054) Introduction Obese patients are at risk of developing atelectasis and acute respiratory distress syndrome (ARDS) [1]. The prone position (PP) may reduce atelectasis, and improves oxygenation and outcome in severe hypoxemic patients in ARDS [2], but little is known about its eff ect in obese ARDS patients. Results We enrolled 13 patients admitted to our ICU. In the Bland– Altman analysis of the lung total tissue and not infl ated tissue, the bias and agreement bands for Standard CT scan and Lowdose CT quantitative analysis were –16.97 g (–77.43 to +43.49 g) and –9.65 g (–116.81 to 77.51 g), respectively (Figures 1 and 2). Results We enrolled 13 patients admitted to our ICU. In the Bland– Altman analysis of the lung total tissue and not infl ated tissue, the bias and agreement bands for Standard CT scan and Lowdose CT quantitative analysis were –16.97 g (–77.43 to +43.49 g) and –9.65 g (–116.81 to 77.51 g), respectively (Figures 1 and 2). f Methods Morbidly obese patients (body mass index (BMI) ≥35 kg/m2) in PP with ARDS (PaO2/FiO2 ratio ≤200 mmHg) were matched to nonobese (BMI <30 kg/m2) ARDS patients in a case–control clinical study. The primary endpoints were safety and complications of PP; the second endpoints were the eff ect on oxygenation (PaO2/FiO2 ratio at the end of PP), length of mechanical ventilation and ICU stay, nosocomial infections and mortality. g p y g Conclusion Lung CT quantitative analysis computed by Lowdose CT scans could be a useful tool for monitoring and ventilatory management of ARDS patients. y Results Between January 2005 and December 2009, 149 patients were admitted for ARDS. Thirty-three obese patients were matched with 33 nonobese patients. Median PP duration was 9 (6 to 11) hours in obese patients and 8 (7 to 12) hours in nonobese patients (P = 0.28). We collected 51 complications, of which 25 in obese patients and 26 in nonobese patients. The number of patients with at least one complication was similar across groups (n = 10, 30%). P114 1Fondazione IRCCS Ca’ Granda – Ospedale Maggiore Policlinico, Milan, Italy; 2Università degli Studi di Milano, Milan, Italy Critical Care 2013, 17(Suppl 2):P114 (doi: 10.1186/cc12052) Introduction Quantitative analysis of a lung CT scan is considered the reference method to study lung recruitability to optimize the ventilatory strategy in ARDS patients. However, CT implies a risk of radiation exposure, especially when CT analysis is necessary to monitor the evolution of ARDS. The aim of this study was to evaluate the impact of a lower radiation dose on lung CT quantitative analysis compared with analysis computed by standard CT. y p y Methods Sedated and paralyzed patients underwent two chest CT scans: a Standard CT (120 kV, 110 mAs, pitch 1.2, collimation 0.6 mm; Care Dose Technology) and a CT performed by a lower radiation dose (Lowdose: 120 kV, 60 mAs). CTs were performed during the same inspiratory or expiratory hold, for diff erent values of airway pressure. Each CT image was manually delineated excluding pleural eff usions and mediastinal structures. We analyzed Standard and Lowdose CT images with dedicated software to quantify the tissue weight of lung regions with diff erent degrees of infl ation. Lung quantitative data computed by Standard and by Lowdose scans were compared according to Bland–Altmann analysis. Figure 1 (abstract P113). Two-way ANOVA RM. P <0.05, versus Time 0, #versus 5 minutes, °versus 15 minutes. Figure 1 (abstract P113). Two-way ANOVA RM. P <0.05, versus Time 0, #versus 5 minutes, °versus 15 minutes. Figure 1 (abstract P113). Two-way ANOVA RM. P <0.05, versus Time 0, #versus 5 minutes, °versus 15 minutes. Figure 2 (abstract P113). Two-way ANOVA RM. P <0.05, #versus 5 minutes according to Bland Altmann analysis. Figure 2 (abstract P113). Two-way ANOVA RM. P <0.05, #versus 5 minutes. Figure 1 (abstract P114). Total tissue weight. Figure 1 (abstract P114). Total tissue weight. Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S43 Critical Care 2013, Volume 17 Suppl 2 , pp http://ccforum.com/supplements/17/S2 therapy was higher in APRV than the CMV group (72% vs. 49%, respectively, P <0.0001). The mortality rate was signifi cantly higher in CMV (n = 46, 51%) as compared with APRV (n = 11, 29%) (P = 0.022). Conclusion The use of APRV in septic shock patients restores hemodynamic stability earlier than the CMV mode. There was a signifi cant improvement in ICU survival using APRV over CMV. P114 Early initiation of APRV in ventilated septic shock patients was associated with a decrease in ICU mortality. Figure 2 (abstract P114). Not infl ated tissue weight. Figure 2 (abstract P114). Not infl ated tissue weight. P116 The PaO2/FiO2 ratio (Figure 1) increased signifi cantly more in obese patients (from 118 ± 43 to 222 ± 84 mmHg) than in nonobese patients (from 113 ± 43 mmHg to 174 ± 80 mmHg, P = 0.03). Length of mechanical ventilation, ICU stay and nosocomial infections did not diff er signifi cantly, but mortality at 90 days was signifi cantly lower in obese patients (27 vs. 48%, P <0.05). 1. N Engl J Med 2006, 354:1775-1786. Benefi cial eff ects of intrapulmonary percussive ventilation in patients with respiratory insuffi ciency in the ICU I Blum, R Janssen-Dean, A Overdijk van, B Speelberg St Anna Hospital Geldrop, Geldrop, the Netherlands Critical Care 2013, 17(Suppl 2):P119 (doi: 10.1186/cc12057) I Blum, R Janssen-Dean, A Overdijk van, B Speelberg St Anna Hospital Geldrop, Geldrop, the Netherlands Critical Care 2013, 17(Suppl 2):P119 (doi: 10.1186/cc12057) l Methods A case–control study was conducted following ethics approval. A case was defi ned as a ventilated pregnant/postpartum woman reported to the Australian and New Zealand INFINITE H1N1 09 study from 1 June 2009 to 31 August 2009. Controls were ventilated nonpregnant women (15 to 49 years) reported to the INFINITE H1N1 09 study during the same time frame. Data were entered into SPSS and analysed using nonparametric statistics; two-tailed P <0.05 was considered signifi cant. Introduction Intrapulmonary percussive ventilation (IPV) is a therapy that is used to clear endobronchial secretions. The IPV ventilator was designed and developed by FM Bird in 1979. The ventilator consists of a phasitron that delivers rapid, high-fl ow, mini bursts of oxygen, mixed with air. The potential aims of this mechanism are pulmonary recruitment, improved mucus clearance with a direct high-frequency oscillatory eff ect. We investigated whether IPV has a positive eff ect on ventilatory values in adult patients on the ICU. i Results We studied 36 index cases and 38 controls. Index cases were more likely to have a single arterial blood gas (ABG) taken prior to intubation (P <0.05). Similar reasons were given for the trigger to intubate (high respiratory rate, low PaO2, increased work of breathing) apart from a high PaCO2, which was a trigger in the control group only (P <0.05). There were no diff erences in the pre-intubation and post- intubation ABGs apart from a lower PaCO2 (P <0.05) and lower HCO3 (P <0.05) in cases, and cases presented with a lower haemoglobin (P <0.05). There were six diffi cult intubations documented with no diff erences between groups. Initial ventilator settings including mode, tidal volume, minute volume and respiratory rate demonstrated no diff erences. Both groups showed increases in PaO2 and PaCO2, and a decrease in pH from the pre-intubation to post-intubation ABGs (P <0.05).f y p Methods All patients presenting during a 4-month period in 2011 with respiratory insuffi ciency on our mixed adult ICU were included in this study. Patients were monitored before, directly after and 15 minutes after therapy with IPV using a Bird Intrapulmonary Ventilator Model IPV-2C. Preliminary experience with airway pressure release ventilation on hemodynamics in patients with septic shock in a medical/surgical ICU A Hussain, H Lababidi, A Mir, A AlHamoud, A Al Oheli, A Al Enezi King Fahd Medical City, Riyadh, Saudi Arabia Critical Care 2013, 17(Suppl 2):P115 (doi: 10.1186/cc12053) Introduction Airway pressure release ventilation (APRV) allows spontaneous breathing throughout the ventilation cycle. It increases venous return and cardiac index, which will signifi cantly improve organ perfusion. This is important in septic shock patients to prevent extrathoracic organ system failure secondary to poor perfusion. Benefi ts of APRV with cardiovascular changes are noticed in patients with acute lung injury and acute respiratory distress syndrome. It is not well established whether applying APRV will improve the survival outcome for septic shock patients. The primary outcome is whether the use of APRV in septic shock patients restores hemodynamic stability earlier than the CMV mode. The secondary hypothesis is whether the use of APRV in septic shock patients improves their survival in the ICU. Methods After Institutional Review Board approval, we retrospectively analyzed the clinical data of 129 septic shock patients who received ventilator support between January and December 2011 at a tertiary care hospital. The Cox proportional hazards model was used in adjusting potential confounding factors. The nonparametric Wilcoxon rank sum test was used to assess signifi cant outcome diff erences between groups. Time to event/survival data will be analyzed using Kaplan–Meier methods. These analyses were accomplished using SAS, version 9.3. Figure 1 (abstract P116). Individual variations of PaO2/FiO2 ratio between supine and prone positions in obese and nonobese patients. Results Among the 187 patients, 58 were excluded as per the exclusion criteria: incomplete data (n = 28), do not resuscitate (n = 16), ICU readmission (n = 12) and head injury (n = 4). Finally, 129 patients were included, from these 91 received CMV and 38 received APRV. At the beginning of the study, there were no diff erences between the groups in relation to hemodynamic parameters. Reversal of shock achieved in less than 72 hours was statistically signifi cant between the groups (APRV, n = 16 (42%) and CMV, n = 8 (9%), P = 0.0101). The proportion of patients recovering from septic shock after initiation of ventilator Figure 1 (abstract P116). Individual variations of PaO2/FiO2 ratio between supine and prone positions in obese and nonobese patients. S44 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 years. Provision of mechanical ventilation to pregnant/postpartum women with H1N1 infl uenza: a case–control study l WE Pollock1, R Bellomo2, S Webb3, I Seppelt4, A Davies5, E Sullivan6, S Morrison7, B Howe7 1Mercy Hospital for Women, Heidelburg, Australia; 2Austin Health, Heidelberg, Australia; 3Royal Perth Hospital, Perth, Australia; 4Nepean Hospital, Penrith, Australia; 5Alfred Hospital, Prahran, Australia; 6University of New South Wales, Randwick, Australia; 7Monash University, Prahran, Australia Critical Care 2013, 17(Suppl 2):P117 (doi: 10.1186/cc12055) Conclusion In this study, inhalation injury is common in burn patients. They had higher severity scores (APACHE II, ABSI) and higher mortality. These patients had a higher need for mechanical ventilation and lower PaO2/FiO2 ratio, but there was no signifi cant increase in ARDS or respiratory sepsis. Introduction During the infl uenza pandemic of 2009, clinicians delivered mechanical ventilation to pregnant women with little evidence to guide practice. The objective of this study was to compare the provision of mechanical ventilation to pregnant/postpartum women and a nonpregnant matched control group admitted to the ICU with H1N1 infl uenza. P120 P120 High-frequency ventilation for acute traumatic and nontraumatic lung injury R Varutti1, R Bigai1, M Fiorillo1, D Tomasello1, W Mercante1, G Trillò2 1Azienda Ospedaliera Santa Maria degli Angeli, Pordenone, Italy; 2Udine University Hospital, Udine, Italy Critical Care 2013, 17(Suppl 2):P120 (doi: 10.1186/cc12058) Preliminary experience with airway pressure release ventilation on hemodynamics in patients with septic shock in a medical/surgical ICU The mean total burn surface area (TBSA) was 28% (± 25). Forty- fi ve patients (53.5%) had TBSA >20%. These patients had higher severity scores: ABSI 8 (± 2.8) versus 6 (± 2.2) (P = 0.0001), APACHE II 13 (± 6.9) versus 7 (± 6.7) (P = 0.0001). They also had a higher need for mechanical ventilation (88.1% vs. 34.9%, P = 0.001) and shock (59.5% vs. 27.0%, P = 0.001). The PaO2/FiO2 ratio at admission with and without inhalation was 245 versus 359 (P = 0.003), and at 72 hours was 207 versus 306 (P = 0.002). There were no signifi cant diff erences in the appearance of sepsis, ARDS and renal failure. The length of stay with and without inhalation was 25.2 (1 to 95) days versus 22.5 (1 to 92) days (P = 0.4). The inhalation injury group showed a signifi cantly higher mortality (9.0% vs. 28.9%, P = 0.001). Conclusion PP seems safe in obese patients and may improve oxygenation more than in nonobese patients. Obese patients could be a subgroup of ARDS patients who may benefi t most from PP. References 1. Gong MN, et al.: Thorax 2010, 65:44-50. 2. Charron C, et al.: Intensive Care Med 2011, 37:785-790. References 1. Gong MN, et al.: Thorax 2010, 65:44-50. 2. Charron C, et al.: Intensive Care Med 2011, 37:785-790. 1. Gong MN, et al.: Thorax 2010, 65:44-50. 2. Charron C, et al.: Intensive Care Med 2011, 37:785-790. Benefi cial eff ects of intrapulmonary percussive ventilation in patients with respiratory insuffi ciency in the ICU All patients received IPV for a period of 20 minutes consisting of two cycles of 10 minutes. Peripheral oxygen saturation (SpO2), tidal volume (Vt), respiratory rate, end-tidal CO2 (ET-CO2), dynamic lung compliance (C-dyn) and work of breathing (WOB) were monitored at the diff erent time points. Paired Student t tests were performed in order to compare the values immediately before IPV, with directly after therapy and 15 minutes later. P <0.05 was considered signifi cant.i yi Results Eighty-three patients were examined. SpO2 improved signifi - cantly from 93.7 ± 3.7 before IPV to 95.7 ± 2.8 after IPV (P <0.001) and 15 minutes later to 95.2 ± 2.8 (P <0.001). Vt improved from 418 ± 111 before IPV to 476 ± 102 directly after (P <0.001) and to 480 ± 131 15 minutes later (P <0.01). Respiratory rate improved from 24 ± 6 to 23 ± 6 only after 15 minutes signifi cantly (P <0.01). WOB and C-dyn did not change. ET-CO2 decreased from 34.9 ± 14.8 to 33.3 ± 13.3 (P <0.05) directly after IPV and to 32.1 ± 12.8 (P <0.01) 15 minutes later.if Conclusion There were physiological diff erences between the two groups with pregnant/postpartum women showing lower PaCO2 and HCO3. However, initial ventilator support was not signifi cantly diff erent for pregnant/postpartum women compared with controls. Reference p p Critical Care 2013, 17(Suppl 2):P118 (doi: 10.1186/cc12056) 1. Salim A, Martin M: High-frequency percussive ventilation. Crit Care Med 2005, 33:S241-S245. Introduction The objective is to analyze the epidemiology and mortality of critical burn patients with inhalation injury. Introduction The objective is to analyze the epidemiology and mortality of critical burn patients with inhalation injury. Methods A prospective, observational and descriptive study was carried out in all patients admitted to an ICU from October 2008 to June 2011. Inhalation injury was defi ned with two or more of the following criteria: history of injury in an enclosed space, facial burns with singed nasal hair, carbonaceus sputum or stridor. If they were intubated it was diagnosed by bronchoscopy. Demographic data, length of stay, ABSI, APACHE II, duration of mechanical ventilation, hospital course and mortality data were collected. Data are presented as number and percentage or as median and interquartile range and were analyzed with the Fisher exact test and Mann–Whitney test. P119i Benefi cial eff ects of intrapulmonary percussive ventilation in patients with respiratory insuffi ciency in the ICU I Blum, R Janssen-Dean, A Overdijk van, B Speelberg St Anna Hospital Geldrop, Geldrop, the Netherlands Critical Care 2013, 17(Suppl 2):P119 (doi: 10.1186/cc12057) Inhalation injury in critical burn patients L C h f M S h E H L F á d Conclusion In this study we demonstrated a benefi cial eff ect of IPV on oxygen saturation, tidal volume and end-tidal CO2. IPV has this eff ect in addition to the mobilization of bronchial secretions. Reference P120 High-frequency ventilation for acute traumatic and nontraumatic lung injury high-frequency oscillatory ventilation (HFOV) was considered mostly a rescue therapy. py Methods Fifteen adult patients admitted to our ICU for acute traumatic and nontraumatic lung injury were submitted to HFOV when conventional mechanical ventilation failed. Acknowledgement This research has been cofi nanced by the European Union and Greek national funds through the operating programme ‘Education and Lifelong Learning’ – Research Funding Programme Heracleitus II. Results Clinical and demographic data are shown in Table 1. Figure 1 shows the trend of gas parameters during the recovery. At baseline PaO2 was 94 ± 28 mmHg; after 6 hours of HFOV: 135 ± 41 mmHg, P <0.01. At baseline PaO2/FiO2 was 182 ± 97 mmHg; after 6 hours of HFOV: 264 ± 101 mmHg, P <0.01. The benefi ts are maintained when returned to conventional ventilation. Reference 1. Zeravik J, et al.: Acta Anaesthesiol Scand 1989, 33(Suppl 90):149-152. Figure 1 (abstract P121). Scatter plot and fi t line of ELWI versus dPaO2/FiO2. Conclusion HFOV may therefore be anticipated to improve end- organ perfusion and gas exchange; it should be considered in severe traumatic and nontraumatic respiratory failure [1]. 1. BMJ 2010, 340:c2327. 1. BMJ 2010, 340:c2327. P120 High-frequency ventilation for acute traumatic and nontraumatic lung injury R Varutti1, R Bigai1, M Fiorillo1, D Tomasello1, W Mercante1, G Trillò2 1Azienda Ospedaliera Santa Maria degli Angeli, Pordenone, Italy; 2Udine University Hospital, Udine, Italy C i i l C 2013 17(S l 2) P120 (d i 10 1186/ 12058) y p y Critical Care 2013, 17(Suppl 2):P120 (doi: 10.1186/cc12058) Introduction ARDS is commonly observed in trauma patients. In some instances the severity of the clinical presentation is such that all conventional ventilatory support mode fails. In this setting, Results Of the 362 patients admitted, 84 (23.2%) had inhalation injury. Seventy-six percent were male and the average age was 52 (± 17.5) S45 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Figure 1 (abstract P120). Mean PaO2, PaCO2, PaO2/FiO2 at baseline and during HFOV (P, percussionator; C, conventional). P <0.05 versus baseline, †P <0.01 versus baseline. Figure 1 (abstract P120). Mean PaO2, PaCO2, PaO2/FiO2 at baseline and during HFOV (P, percussionator; C, conventional). P <0.05 versus baseline, †P <0.01 versus baseline. Figure 1 (abstract P120). Mean PaO2, PaCO2, PaO2/FiO2 at baseline and during HFOV (P, percussionator; C, conventional). P <0.05 v versus baseline. Table 1 (abstract P120). Clinical and demographic data Mean Age (years) 64 Male/female 11/4 Dead 4 Trauma 3 SAPS II 45/40 APACHE 23/8 Table 1 (abstract P120). Clinical and demographic data fl ow were 3.5 Hz, 85 to 95 cmH2O, and 40 l/minute, respectively; a 2.5 to 3.5 cmH2O tracheal tube cuff leak was used. HFO mean airway pressure (mPaw) exceeded preceding conventional ventilation (CV)-mPaw by 7 to 13 cmH2O. PaO2/FiO2, lung mechanics, and hemodynamics were documented during lung-protective CV (baseline) and 1 hour following the initiation of HFO-TGI ventilation. PULSION PICCOplus v7.0 was used for hemodynamic measurements including the ELWI.i y g Results Oxygenation (PaO2/FiO2) improved signifi cantly with HFO- TGI compared with CMV (125.5 ± 54.7 vs. 195.6 ± 108.7, P <0.001). Changes in PaO2/FiO2 were positively correlated with ELWI at baseline (Spearman’s ρ = 0.56, P = 0.016). See Figure 1. There were no signifi cant changes in patients’ fl uid balance and hemodynamics including the ELWI. Conclusion Estimation of the ELWI can help to predict the oxygenation response of ARDS patients considered for HFO-TGI ventilation. The possibility that HFO-TGI exerts an eff ect on pulmonary oedema needs further investigation.i high-frequency oscillatory ventilation (HFOV) was considered mostly a rescue therapy. P121 Improvement in oxygenation with high-frequency oscillatory ventilation combined with tracheal gas insuffl ation is correlated to the extravascular lung water index CS Vrettou, S Malachias, SG Zakynthinos, SD Mentzelopoulos Evaggelismos General Hospital, Athens, Greece Critical Care 2013, 17(Suppl 2):P121 (doi: 10.1186/cc12059) Physiologic eff ects and regional ventilation of high-frequency positive-pressure ventilation using a conventional ventilator in a severe ARDS animal model associated with an inspiratory pause or recruitment maneuvers RL Cordioli1, M Park2, M Amato3, S Gomes3, E Leite2, L Azevedo2 1Hopitaux Universitaire de Geneve, Geneva, Switzerland; 2Hospital Sirio Libanes IEP, São Paulo, Brazil; 3Hospital das Clinicas LIM 09, São Paulo, Brazil Critical Care 2013, 17(Suppl 2):P122 (doi: 10.1186/cc12060) RL Cordioli1, M Park2, M Amato3, S Gomes3, E Leite2, L Azevedo2 1Hopitaux Universitaire de Geneve, Geneva, Switzerland; 2Hospital Sirio Libanes IEP, São Paulo, Brazil; 3Hospital das Clinicas LIM 09, São Paulo, Brazil Critical Care 2013, 17(Suppl 2):P122 (doi: 10.1186/cc12060) i g Results Data displayed by VDR4® overestimated pulmonary pressures by more than 10%, but were reliable for other parameters. During ascent, an off set appeared for all respiratory parameters: Vt increased by 59% and PmEI by 53% between 0 and 8,000 ft. During descent, the off set was reversely directed with a 39% decrease in Vt and a 28% decrease in PmEE between 8,000 and 0 ft. Modifying working pressure adequately corrected PmEI and PmEE, but not Vt. In all cases, manually correcting VDR4® parameters to their 0 ft level also corrected these off sets. Multivariate analysis further established that, adjusting for other parameters, Vt, PmEI and PmEE did practically not depend on altitude. Oxygen consumption of the respirator was high, 25 l/minute at 0 ft, and stable with altitude. It was reduced with percussive rate and with FiO2. Introduction Protective mechanical ventilation (MV) in ARDS is based on reduced stretch of pulmonary tissue, sometimes resulting in severe hypoventilation that can be avoided when using high respiratory rate. High-frequency positive-pressure ventilation (HFPPV) has not been fully explored, especially when associated with other strategies aiming to avoid hypercapnia. y Methods We induced ARDS in eight pigs by lung lavage with saline plus 3 hours of injurious MV with low PEEP and high driving pressure (DP). We then performed a recruitment maneuver (RM) followed by PEEP titration using the amount of alveolar collapse in electrical impedance tomography (EIT). Then stabilization during 1 hours with tidal volume (VT) at 6 ml/kg, respiratory rate (RR) 35 breaths/minute and PEEP selected with the PEEP-FiO2 table (ARMA study), which was kept constant during two steps of HFPPV with a RR 60: one without an inspiratory pause (HFPPV-60), and one with a pause of 30% of inspiratory time (HFPPV-60 w/P30%). In another HFPPV step, we used PEEP titrated with EIT after RM (HFPPV-60 w/RM). Recruitable volume is comparable in acute respiratory distress syndrome and in healthy lungs CA Stahl1, K Moeller2, D Steinmann1, D Henzler3, S Lundin4, O Stenqvist4 1University Medical Center Freiburg, Germany; 2Biomedical Engineering, Villingen-Schwenningen, Germany; 3Klinikum Herford, Germany; 4Sahlgrenska University Hospital, Gothenburg, Sweden Critical Care 2013, 17(Suppl 2):P124 (doi: 10.1186/cc12062) Table 1 (abstract P122). Physiological variables Variable VT = 6, stabilization HFPPV-60 HFPPV-60 w/RM DP (cmH2O) 16 ± 2 16 ± 3 12 ± 2 P/F ratio 95 ± 13 149 ± 60 246 ± 99* Tukey’s post-hoc analysis, P < 0.05 versus others. Table 1 (abstract P122). Physiological variables Introduction The application of PEEP is commonly used in acute respiratory distress syndrome (ARDS) and has been shown to improve oxygenation. To identify patients that most benefi t from the application of PEEP, the discrimination of recruiters and nonrecruiters has been postulated by Gattinoni and colleagues [1]. Recently, Dellamonica and colleagues [2] presented a method to predict alveolar recruitment. We hypothesised that the amount of recruitable volume allows the discrimination between ARDS patients and patients with healthy lungs (HL). Conclusion HFPPV with a conventional mechanical ventilator is able to maintain stable PaCO2 in clinically acceptable values, allowing reductions in VT. HFPPV-60 w/RM and PEEP titration using EIT allowed further physiologic benefi ts in a severe ARDS model. ( ) Methods We recalculated the recruited volume (RV) in 25 patients with ARDS [3] according to the method proposed by Dellamonica and colleagues during an incremental PEEP manoeuvre (PEEP increased until the plateau pressure reached 45 cmH2O). RV was calculated as the change in end-expiratory lung volume minus total respiratory system compliance times the PEEP change (RV = ΔEELV – CTOT×ΔPEEP). For comparison, 12 patients with HL undergoing elective surgery in general anaesthesia were measured using the same protocol. Results Both ARDS and HL patients exhibited typical P–V curves and stepwise recruitment (Figure 1). By raising PEEP from 0 to 12 cmH2O, ARDS patients recruited 331 ± 195 ml (mean ± SD) and HL patients 435 ± 43 ml. There was a strong correlation (R2 = 0.88) of the total RV with the end-inspiratory volume at a plateau pressure of 45 cmH2O in both groups; that is, recruitment was found to the same extent in both groups (Figure 2). Methods We recalculated the recruited volume (RV) in 25 patients with ARDS [3] according to the method proposed by Dellamonica and colleagues during an incremental PEEP manoeuvre (PEEP increased until the plateau pressure reached 45 cmH2O). g References g References 1. Velmahos GC, et al.: Chest 1999, 116:440-446. 2. Chung KK, et al.: Crit Care Med 2010, 38:1970-1977. References 1. Velmahos GC, et al.: Chest 1999, 116:440-446. 2. Chung KK, et al.: Crit Care Med 2010, 38:1970-1977. 2 Results HFPPV allowed reduction in PaCO2 levels: 81 (77, 94) versus 60 (58, 61), 59 (58, 60), 60 (58, 61) mmHg, besides using lower VT: 5.2 (5.0, 5.9), 5.1 (4.5, 6.0), 4.7 (4.2, 5.7) and 4.8 (4.5, 5.6) ml/kg during stabilization, HFPPV-60, HFPPV-60 w/P30% and HFPPV-60 w/RM, respectively. It had no signifi cant diff erent results comparing HFPPV-60 with and without an inspiratory pause. HFPPV-60 w/RM allowed a better alveolar homogenization and improvement in oxygenation, shunt, dead space and DP compared with the other steps. See Table 1. P124 Recruitable volume is comparable in acute respiratory distress syndrome and in healthy lungs CA Stahl1, K Moeller2, D Steinmann1, D Henzler3, S Lundin4, O Stenqvist4 1University Medical Center Freiburg, Germany; 2Biomedical Engineering, Villingen-Schwenningen, Germany; 3Klinikum Herford, Germany; 4Sahlgrenska University Hospital, Gothenburg, Sweden Critical Care 2013, 17(Suppl 2):P124 (doi: 10.1186/cc12062) Recruitable volume is comparable in acute respiratory distress syndrome and in healthy lungs RV was calculated as the change in end-expiratory lung volume minus total respiratory system compliance times the PEEP change (RV = ΔEELV – CTOT×ΔPEEP). For comparison, 12 patients with HL undergoing elective surgery in general anaesthesia were measured using the same protocol. Physiologic eff ects and regional ventilation of high-frequency positive-pressure ventilation using a conventional ventilator in a severe ARDS animal model associated with an inspiratory pause or recruitment maneuvers During each HFPPV step, VT was set to reach a PaCO2 of 60 ± 3 mmHg. Distribution of regional ventilation was analyzed using EIT. Equilibrium was considered if PaCO2 was stable (<5% of variation) for >30 minutes. 2 Conclusion HFPV can be safely used at altitude, provided that VDR4®- displayed parameters are used to manually adjust settings in order to avoid exposing patients to volutrauma or barotrauma during ascent, and to major hypoventilation and alveolar collapse during descent. The high oxygen consumption is currently the main limit to its use for long- range aeromedical evacuations. Improvement in oxygenation with high-frequency oscillatory ventilation combined with tracheal gas insuffl ation is correlated to the extravascular lung water index HFPPV better alveolar homogenization and improvem shunt, dead space and DP compared with the ot Table 1 (abstract P122). Physiological variables Variable VT = 6, stabilization HFPPV-6 DP (cmH2O) 16 ± 2 16 ± 3 P/F ratio 95 ± 13 149 ± 60 Tukey’s post-hoc analysis, P < 0.05 versus others. Conclusion HFPPV with a conventional mechan to maintain stable PaCO2 in clinically accept reductions in VT. HFPPV-60 w/RM and PEEP titra further physiologic benefi ts in a severe ARDS mo P123 High-frequency percussive ventilation at altitu hypobaric chamber with a mechanical test lung AC Cirodde1, S Montmerle2, ND Donat1, CB Bourhill L Bargues1, T Leclerc1 1Military Hospital Percy, Clamart, France; 2IRBA, Brétig Critical Care 2013, 17(Suppl 2):P123 (doi: 10.1186/c Introduction High-frequency percussive ventilat technique for most severe acute lung injury/acu syndrome (ARDS) patients [1], especially with respiratory burns [2]. This study aimed at ch delivered by Percussionnaire VDR4® and at evalua interferes with HFPV, in order to assess its usabili Methods Using a mechanical test lung mimicki 17 ml/cmH2O) with two resistance levels (5 and and ventilated with VDR4® in a hypobaric cham between 0 and 5,000 and then 0 and 8,000 ft were P122 VDR4® parameters were modifi ed one at a time at each altitude. Besides these parameters (cross-measured with standalone hardware), oxygen consumption of the respirator and three calculated parameters were studied: low-frequency tidal volume (Vt, integrated from instantaneous fl ows measured with a Fleisch pneumotachograph), end-inspiratory (PmEI) and end-expiratory (PmEE) mean pressures. PmEI and PmEE in HFPV refl ect plateau pressure and positive end-expiratory pressure in conventional ventilation. The correction of altitude-induced off set with the modifi cation of working pressure was also tested. Physiologic eff ects and regional ventilation of high-frequency positive-pressure ventilation using a conventional ventilator in a severe ARDS animal model associated with an inspiratory pause or recruitment maneuvers RL Cordioli1, M Park2, M Amato3, S Gomes3, E Leite2, L Azevedo2 1Hopitaux Universitaire de Geneve, Geneva, Switzerland; 2Hospital Sirio Libanes IEP, São Paulo, Brazil; 3Hospital das Clinicas LIM 09, São Paulo, Brazil Critical Care 2013, 17(Suppl 2):P122 (doi: 10.1186/cc12060) Physiologic eff ects and regional ventilation of high-frequency positive-pressure ventilation using a conventional ventilator in a severe ARDS animal model associated with an inspiratory pause or recruitment maneuvers Improvement in oxygenation with high-frequency oscillatory ventilation combined with tracheal gas insuffl ation is correlated to the extravascular lung water index Introduction High-frequency oscillatory ventilation combined with tracheal gas insuffl ation (HFO-TGI) can signifi cantly improve oxygenation in patients with ARDS. It has been demonstrated that oxygenation in patients with ARDS has a better response to HFO when extravascular lung water is >15 ml/kg body weight (BW). Our aim is to examine whether the extravascular lung water index (ELWI) correlates with changes in PaO2/FiO2 in patients ventilated with HFO-TGI. Figure 1 (abstract P121). Scatter plot and fi t line of ELWI versus dPaO2/FiO2. 2 2 Methods Data from 18 sessions of HFO-TGI in six patients were included in the analysis. HFO frequency, oscillatory pressure amplitude, and bias Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S46 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 P122 Physiologic eff ects and regional ventilation of h positive-pressure ventilation using a conventio severe ARDS animal model associated with an recruitment maneuvers RL Cordioli1, M Park2, M Amato3, S Gomes3, E Leite2, 1Hopitaux Universitaire de Geneve, Geneva, Switzerlan Libanes IEP, São Paulo, Brazil; 3Hospital das Clinicas LIM Critical Care 2013, 17(Suppl 2):P122 (doi: 10.1186/c Introduction Protective mechanical ventilation on reduced stretch of pulmonary tissue, sometim hypoventilation that can be avoided when using High-frequency positive-pressure ventilation (H fully explored, especially when associated with o to avoid hypercapnia. Methods We induced ARDS in eight pigs by lu plus 3 hours of injurious MV with low PEEP and (DP). We then performed a recruitment man by PEEP titration using the amount of alveolar impedance tomography (EIT). Then stabilization tidal volume (VT) at 6 ml/kg, respiratory rate (R and PEEP selected with the PEEP-FiO2 table (AR kept constant during two steps of HFPPV with an inspiratory pause (HFPPV-60), and one wit inspiratory time (HFPPV-60 w/P30%). In another PEEP titrated with EIT after RM (HFPPV-60 w/RM step, VT was set to reach a PaCO2 of 60 ± 3 m regional ventilation was analyzed using EIT. Equil if PaCO2 was stable (<5% of variation) for >30 mi Results HFPPV allowed reduction in PaCO2 lev 60 (58, 61), 59 (58, 60), 60 (58, 61) mmHg, be 5.2 (5.0, 5.9), 5.1 (4.5, 6.0), 4.7 (4.2, 5.7) and 4.8 ( stabilization, HFPPV-60, HFPPV-60 w/P30% a respectively. It had no signifi cant diff erent results with and without an inspiratory pause. P123 We compared incidence rates of nosocomial bacteremia using the Hospitals in Europe Link for Infection Control through Surveillance (HELICS) between general ICU and VV-ECMO patients and used multiple logistic regression analysis to control for possible confounders. Figure 2 (abstract P124). p Results During the study period 1,146 patients were admitted and 16 received VV-ECMO. The incidence of bloodstream infection in patients with ECMO was 19/1,000 exposure-days versus 4,9/1,000 exposure- days in general ICU patients (incidence rate ratio of 3.82; 95% CI: 2.0 to 7.3; P <0.001). Bacteremia was mostly due to Gram-negative agents (65%). The patients with bloodstream infections under ECMO (n = 10) had a nonsignifi cant younger age (P = 0.08) and a lower SAPS II score (P = 0.03) compared with non-ECMO patients (n = 25). VV-ECMO patients had a signifi cantly higher risk of primary bacteremia than non- ECMO patients (P = 0.04). Patients with bloodstream infections in the VV-ECMO group had a lower crude mortality rate (OR: 0.1, P = 0.04), not confi rmed in the adjusted analysis. There were no crude or adjusted diff erences in the time to bacteremia or infections due to multiple drug-resistant microorganisms (OR: 0.26; P = 0.085) between groups. g g g p Conclusion This study suggests that VV-ECMO patients have a signifi cantly higher risk for primary nosocomial bloodstream infection. A larger study is needed to confi rm such fi ndings and to assess the need for specifi c intervention, namely routine antibiotic prophylaxis. Referencesfi Figure 2 (abstract P124). 1. Peek GJ, et al.: Effi cacy and economic assessment of conventional ventilatory support versus extracorporeal membrane oxygenation for severe adult respiratory failure (CESAR): a multicentre randomised controlled trial. Lancet 2009, 374:1351-1363. 1. Peek GJ, et al.: Effi cacy and economic assessment of conventional ventilatory support versus extracorporeal membrane oxygenation for severe adult respiratory failure (CESAR): a multicentre randomised controlled trial. Lancet 2009, 374:1351-1363. P123 P123 High-frequency percussive ventilation at altitude: study in a hypobaric chamber with a mechanical test lung AC Cirodde1, S Montmerle2, ND Donat1, CB Bourhillon2, P Jault1, L Bargues1, T Leclerc1 1Military Hospital Percy, Clamart, France; 2IRBA, Brétigny, France Critical Care 2013, 17(Suppl 2):P123 (doi: 10.1186/cc12061) Results Both ARDS and HL patients exhibited typical P–V curves and stepwise recruitment (Figure 1). By raising PEEP from 0 to 12 cmH2O, ARDS patients recruited 331 ± 195 ml (mean ± SD) and HL patients 435 ± 43 ml. There was a strong correlation (R2 = 0.88) of the total RV with the end-inspiratory volume at a plateau pressure of 45 cmH2O in both groups; that is, recruitment was found to the same extent in both groups (Figure 2). Introduction High-frequency percussive ventilation (HFPV) is a rescue technique for most severe acute lung injury/acute respiratory distress syndrome (ARDS) patients [1], especially with smoke inhalation or respiratory burns [2]. This study aimed at characterizing HFPV as delivered by Percussionnaire VDR4® and at evaluating how hypobarism interferes with HFPV, in order to assess its usability at altitude. Conclusion The relative contribution of RV to lung volume gain is similar in ARDS and in patients with healthy lungs. Our results question the relevance of recruitability as defi ned by Dellamonica and colleagues as a typical phenomenon of ARDS, but support the baby lung concept, as the recruited volume was closely related to the size of the lung. Methods Using a mechanical test lung mimicking ARDS (compliance 17 ml/cmH2O) with two resistance levels (5 and 15 cmH2O/l/second) and ventilated with VDR4® in a hypobaric chamber, ascents/descents between 0 and 5,000 and then 0 and 8,000 ft were performed. Adjustable Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum com/supplements/17/S2 S47 http://ccforum.com/supplements/17/S2 References 1. Gattinoni et al.: N Engl J Med 2006, 354:1775. 2. Dellamonica et al.: Intensive Care Med 2011, 37:1595. 3. Stahl et al.: Crit Care Med 2006, 34:2090. P125 Nosocomial bloodstream infection and venovenous extracorporeal membrane oxygenation: a retrospective cohort study B Serra de Oliveira, S Mendes Fernandes, C França Hospital de Santa Maria CHLN, Lisboa, Portugal Critical Care 2013, 17(Suppl 2):P125 (doi: 10.1186/cc12063) Methods A retrospective cohort study from patients admitted to our ICU between April 2009 and June 2012. P123 We compared incidence rates of nosocomial bacteremia using the Hospitals in Europe Link for Infection Control through Surveillance (HELICS) between general ICU and VV-ECMO patients and used multiple logistic regression analysis to control for possible confounders. Results During the study period 1,146 patients were admitted and 16 received VV-ECMO. The incidence of bloodstream infection in patients with ECMO was 19/1,000 exposure-days versus 4,9/1,000 exposure- days in general ICU patients (incidence rate ratio of 3.82; 95% CI: 2.0 to 7.3; P <0.001). Bacteremia was mostly due to Gram-negative agents (65%). The patients with bloodstream infections under ECMO (n = 10) had a nonsignifi cant younger age (P = 0.08) and a lower SAPS II score (P = 0.03) compared with non-ECMO patients (n = 25). VV-ECMO patients had a signifi cantly higher risk of primary bacteremia than non- ECMO patients (P = 0.04). Patients with bloodstream infections in the VV-ECMO group had a lower crude mortality rate (OR: 0.1, P = 0.04), not confi rmed in the adjusted analysis. There were no crude or adjusted diff erences in the time to bacteremia or infections due to multiple drug-resistant microorganisms (OR: 0.26; P = 0.085) between groups. Conclusion This study suggests that VV-ECMO patients have a signifi cantly higher risk for primary nosocomial bloodstream infection. A larger study is needed to confi rm such fi ndings and to assess the need for specifi c intervention, namely routine antibiotic prophylaxis. References 1. Peek GJ, et al.: Effi cacy and economic assessment of conventional ventilatory support versus extracorporeal membrane oxygenation for severe adult respiratory failure (CESAR): a multicentre randomised controlled trial. Lancet 2009, 374:1351-1363. 2. Conrick-Martin I, et al.: Nosocomial infections in a cohort of extracorporeal life support patients. Crit Care Resusc 2012, 14:198-201. P126 Monitoring anticoagulation during extracorporeal membrane oxygenation in patients with acute respiratory failure M Panigada1, C Mietto2, F Pagan2, L Bogno2, V Berto2, L Gattinoni2 1F d i IRCCS C ’ G d O d l M i P li li i Mil It l Figure 1 (abstract P124). Figure 2 (abstract P124). Figure 1 (abstract P124). Figure 1 (abstract P124). Figure 2 (abstract P124). Methods A retrospective cohort study from patients admitted to our ICU between April 2009 and June 2012. References 1. Gattinoni et al.: N Engl J Med 2006, 354:1775. 2. Dellamonica et al.: Intensive Care Med 2011, 37:1595. 3. Stahl et al.: Crit Care Med 2006, 34:2090. 2. Conrick-Martin I, et al.: Nosocomial infections in a cohort of extracorporeal life support patients. Crit Care Resusc 2012, 14:198-201. 2. Conrick-Martin I, et al.: Nosocomial infections in a cohort of extracorporeal life support patients. Crit Care Resusc 2012, 14:198-201. P125 Nosocomial bloodstream infection and venovenous extracorporeal membrane oxygenation: a retrospective cohort study B Serra de Oliveira, S Mendes Fernandes, C França Hospital de Santa Maria CHLN, Lisboa, Portugal Critical Care 2013, 17(Suppl 2):P125 (doi: 10.1186/cc12063) P127 Extracorporeal membrane oxygenation: the MOTOR of cytokine production? p Y Hara1, O Nishida1, T Nakamura1, S Uchiyama1, J Shibata1, C Yamashita1, M Yumoto1, Y Shimomura1, N Kuriyama1, N Yasuoka1, M Ito1, K Kawata1, S Hayakawa1, S Yamada2, T Miyasho3, K Moriyama1 1Fujita Health University School of Medicine, Toyoake, Japan; 2Shino Corporation, Tokyo, Japan; 3Rakuno Gakuen University, Ebetsu, Japan Critical Care 2013, 17(Suppl 2):P127 (doi: 10.1186/cc12065) g Results In all patients RR decreased with the increase of extracorporeal CO2 removal and a negative correlation was found between RR and ECCO2 Cl/total VCO2 (r2 = 0.42, P <0.01). In all patients we were able to obtain a reduction of RR below 15 (28 ± 4 vs. 8 ± 4, RR at low gas fl ow vs. RR at maximal gas fl ow, P <0.001). The selected maximal gas fl ow was variable between diff erent patients (6.7 ± 2 l/minute), corresponding to diff erent levels of ECCO2 Cl/total VCO2 (83 ± 17%, range 53 to 100%) and RR response (8 ± 4, range 5 to 14). Introduction The usefulness of extracorporeal membrane oxygenation (ECMO) is being rediscovered in the wake of the pandemic of H1N1 infl uenza. However, it has been reported that patients who received ECMO often developed virus-associated hemophagocytic syndrome (VAHS), compared with those without ECMO support. Although there is ample evidence that extensive cytokine activation is a key factor in VAHS, ECMO itself could be a potential trigger to exacerbate the pathology by amplifying cytokine activation. In this study, we investigated whether mediators such as cytokines may be produced by ECMO. Conclusion In patients with COPD exacerbation, who failed non- invasive ventilation, VV-ECMO allows one to maintain spontaneous breathing. Titration of extracorporeal CO2 removal leads to control RR. This approach could interrupt the vicious circle of dynamic hyperinfl ation and allow the defl ation of lung parenchyma. Figure 1 (abstract P128). y Methods Patients with severe respiratory failure who were placed on ECMO were enrolled between June and July 2012. This study was approved by the ethics committee. Blood specimens were drawn from the blood circuit at the inlet of the centrifugal pump (before) and outlet of the hollow fi ber oxygenator (after) at a frequency of three to four times per day. Blood IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12(p70), IL-13, IL-17, G-CSF, GM-CSF, IFNγ, MCP-1, MIP-1β, and TNFα were measured globally using a multiplex cytokine bead array system (Bio-Plex; Bio-Rad, Tokyo, Japan). Eff ect of extracorporeal CO2 removal on respiratory rate in spontaneously breathing patients with chronic obstructive pulmonary disease exacerbation Table 1 (abstract P126). Comparison between aPTT and K-TEG R Table 1 (abstract P126). Comparison between aPTT and K-TEG R aPTT range (ratio) K-TEG R range Low Therapeutic High (minutes) (<1.5) (1.5 to 2) (>2) Low (<20) 13 (9%) 8 (5%) 0 21 (14%) Therapeutic (20 to 90) 10 (7%) 25 (17%) 11 (7%) 46 (30%) High (>90) 15 (10%) 55 (36%) 15 (10%) 85 (56%) 38 (25%) 88 (58%) 26 (17%) 152 total samples Conclusion Anticoagulation was excessive in more than one-half of the samples according to TEG monitoring, while negligible based on aPTT. Reference 1. Oliver WC: Semin Cardiothorac Vasc Anesth 2009, 13:154-175. P127 Extracorporeal membrane oxygenation: the MOTOR of cytokine production? Y Hara1, O Nishida1, T Nakamura1, S Uchiyama1, J Shibata1, C Yamashita1, M Yumoto1, Y Shimomura1, N Kuriyama1, N Yasuoka1, M Ito1, K Kawata1, S Hayakawa1, S Yamada2, T Miyasho3, K Moriyama1 1Fujita Health University School of Medicine, Toyoake, Japan; 2Shino Corporation, Tokyo, Japan; 3Rakuno Gakuen University, Ebetsu, Japan Critical Care 2013 17(Suppl 2):P127 (doi: 10 1186/cc12065) Table 1 (abstract P126). Comparison between aPTT and K-TEG R aPTT range (ratio) Table 1 (abstract P126). Comparison between aPTT and K-TEG R PTT ( ti ) Table 1 (abstract P126). Comparison between aPTT and K-TEG R aPTT range (ratio) K-TEG R range Low Therapeutic High (minutes) (<1.5) (1.5 to 2) (>2) Low (<20) 13 (9%) 8 (5%) 0 21 (14%) Therapeutic (20 to 90) 10 (7%) 25 (17%) 11 (7%) 46 (30%) High (>90) 15 (10%) 55 (36%) 15 (10%) 85 (56%) 38 (25%) 88 (58%) 26 (17%) 152 total samples Introduction During severe exacerbation of chronic obstructive pulmonary disease (COPD) tachypnea, as a consequence of respiratory acidosis, and airfl ow limitation, due to small airway obstruction, lead to lung hyperinfl ation, respiratory distress and gas exchange impairment. Invasive mechanical ventilation could worsen lung hyperinfl ation and produce a vicious circle. We investigated whether increasing extracorporeal carbon dioxide removal (ECCO2 Cl) could reduce the respiratory rate (RR), so prolonging time for lung emptying and allowing resolution of hyperinfl ation. g yl Methods Six patients with COPD exacerbation with respiratory acidosis (PaCO2 83 ± 27 mmHg, pH 7.19 ± 0.1) and tachypnea (RR 39 ± 5) despite maximal non-invasive ventilation underwent venovenous extracorporeal membrane oxygenation (VV-ECMO). All patients were awake and spontaneously breathing an adequate air–oxygen mixture to correct hypoxemia (PaO2 72 ± 27 mmHg). Monitoring anticoagulation during extracorporeal membrane oxygenation in patients with acute respiratory failure yg p B Serra de Oliveira, S Mendes Fernandes, C França Hospital de Santa Maria CHLN, Lisboa, Portugal p , , g Critical Care 2013, 17(Suppl 2):P125 (doi: 10.1186/cc12063) M Panigada1, C Mietto2, F Pagan2, L Bogno2, V Berto2, L Gattinoni2 1Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy; 2IRCCS Ca’ Granda Maggiore Policlinico Hospital Foundation, Milan, Italy Critical Care 2013, 17(Suppl 2):P126 (doi: 10.1186/cc12064) Introduction Venovenous extracorporeal membrane oxygenation (VV-ECMO) for respiratory failure in the ICU is used in a variety of clinical situations and has been demonstrated to signifi cantly improve survival without disability in adult respiratory distress syndrome [1]. ECMO has been presented as a risk factor for bloodstream infection although recently published data do not support this view or the use of antibiotic prophylaxis [2]. We aimed to examine VV-ECMO as a risk factor for nosocomial bloodstream infection. Introduction aPTT is a common tool for anticoagulation monitor ing during extracorporeal membrane oxygenation (ECMO). Thrombo- elasto graphy (TEG) is another available option in this setting. Methods A prospective observational study on 12 consecutive patients during venovenous ECMO. Anticoagulation was provided g p y ( ) p g Methods A prospective observational study on 12 consecutive patients during venovenous ECMO. Anticoagulation was provided S48 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 0.22; IL-8, P = 0.43; IL-10, P = 0.84; MCP-1, P = 0.10; and MIP-1β, P = 0.65; Wilcoxon signed-rank test). with unfractioned heparin titrated to an aPTT ratio target of 1.5 to 2. Kaolin-activated TEG (K-TEG) was contemporarily measured but did not guide heparin infusion. Baseline K-TEG reaction time (R) >20 minutes is accepted for anticoagulation but when it exceeds 90 minutes anticoagulation may be too great [1]. with unfractioned heparin titrated to an aPTT ratio target of 1.5 to 2. Kaolin-activated TEG (K-TEG) was contemporarily measured but did not guide heparin infusion. Baseline K-TEG reaction time (R) >20 minutes is accepted for anticoagulation but when it exceeds 90 minutes anticoagulation may be too great [1]. g Conclusion The use of ECMO in patients with severe respiratory failure did not induce systemic infl ammatory changes. These observations are preliminary, but may nevertheless have important implications for the future management of patients with severe infections. g y g Results Mean ECMO duration was 9 ± 4 days. Eff ect of extracorporeal CO2 removal on respiratory rate in spontaneously breathing patients with chronic obstructive pulmonary disease exacerbation While keeping the blood fl ow stable (2.9 ± 0.5 l/minute), we changed the gas fl ow of the artifi cial lung to modify the extracorporeal CO2 clearance as a percentage of total patient CO2 production (% ECCO2 Cl/total VCO2) and we observed the variations of RR. We recorded RR at three levels of gas fl ow in each patient (Figure 1). Conclusion Anticoagulation was excessive in more than one-half of the samples according to TEG monitoring, while negligible based on aPTT. Reference Reference 1. Oliver WC: Semin Cardiothorac Vasc Anesth 2009, 13:154-175. Monitoring anticoagulation during extracorporeal membrane oxygenation in patients with acute respiratory failure A total of 152 K-TEGs were collected. Comparison between aPTT and K-TEG R is reported in Table 1. Four patients (33%) had hemorrhagic complications. Neither aPTT nor K-TEG R were signifi cantly diff erent in patients with hemorrhagic events compared with patients without hemorrhagic events but the latter received a signifi cantly lower total heparin dose (P = 0.0097). P128f P128f Eff ect of extracorporeal CO2 removal on respiratory rate in spontaneously breathing patients with chronic obstructive pulmonary disease exacerbation E Spinelli, S Crotti, L Zacchetti, N Bottino, V Berto, R Russo, M Chierichetti, A Protti, L Gattinoni Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy Critical Care 2013, 17(Suppl 2):P128 (doi: 10.1186/cc12066) P131 Feasibility of minimally invasive stimulation of the phrenic nerves for supporting ventilation in fully anesthetized swine J Bijwadia1, M Karamanoglu2 1Regions Hospital, St Paul, MN, USA; 2Respithera, Bloomington, MN, USA Critical Care 2013, 17(Suppl 2):P131 (doi: 10.1186/cc12069) ECCO2 removal with a phosphorylcholine-coated membrane oxygenator in diffi cult respiratory weaning patients Table 1 (abstract P129) p g Results There was a close correlation between the measured build-up of end-expiratory lung volume during a PEEP-step manoeuvre and ΔPEEP/EL where EL was conventionally determined using oesophageal pressure measurements (see Figure 1).fi Conclusion Esophageal pressure measurements are diffi cult to perform [3] and rarely used in routine clinical practice. Our fi ndings indicate that a change in PEEP together with measurements of the resulting change in end-expiratory volume by spirometry in the ventilator could be used to determine lung elastance separately, the relation between lung and chest wall elastance as well as the transpulmonary pressure. References Conclusion ECCO2 removal with a phosphorylcholine-coated mem- brane oxygenator is clinically safe, avoids clotting of the oxygenator circuit and allows adequate CO2 removal. Reference 1. Stenqvist O, et al.: Acta Anaesthesiol Scand 2012, 56:738-747. 2. Baydur A: Chest 2002, 121:324-326. 3. Hedenstierna G: Minerva Anestesiol 2012, 78:959-966. 1. Stenqvist O, et al.: Acta Anaesthesiol Scand 2012, 56:738-747. 2. Baydur A: Chest 2002, 121:324-326. 2. Baydur A: Chest 2002, 121:324 326. 3. Hedenstierna G: Minerva Anestesiol 2012, 78:959-966. 1. Livigni S, et al.: Crit Care 2006, 10:R151. P127 Extracorporeal membrane oxygenation: the MOTOR of cytokine production? One-way ANOVA was used to compare the changes of parameters. P <0.05 was considered signifi cant. Results Table 1 presents the main results. The CO2 removal by mem- brane oxygenator ranged from 56 to 37 ml/minute. All patients survived to the treatment and 7/10 were weaned from the ventilator at the end of ECCO2 removal. Only one oxygenator was used for every patient without clotting of the circuit or any major bleeding problem. Table 1 (abstract P129) Day 0 Day 2 Day 4 pH 7.24 ± 0.06 7.38 ± 0.1 7.41 ± 0.07* PaCO2 (mmHg) 70 ± 5 57 ± 8 52 ± 3* PP (cmH2O) 48 ± 4 28 ± 4* 25 ± 4 VCO2 (ml/minute) 220 ± 15 201 ± 14 180 ± 13* P <0.05 versus T0. Conclusion ECCO2 removal with a phosphorylcholine-coated mem- brane oxygenator is clinically safe, avoids clotting of the oxygenator circuit and allows adequate CO2 removal. Reference 1. Livigni S, et al.: Crit Care 2006, 10:R151. P130 Lung elastance can be determined without esophageal pressure measurements C Grivans, S Lundin, O Stenqvist Sahlgrenska University Hospital, Gothenburg, Sweden Critical Care 2013, 17(Suppl 2):P130 (doi: 10.1186/cc12068) Introduction We have previously shown, in an ex vivo porcine model, that lung elastance calculated as the PEEP change divided by lung volume increase (ΔPEEP/ΔEELV) is closely correlated to conventionally measured lung elastance using oesophageal pressure [1]. In this study we hypothesize that the successive change in lung volume during a PEEP-step manoeuvre could be predicted from ΔPEEP and lung l h b f h d l d h P129 ECCO2 removal with a phosphorylcholine-coated membrane oxygenator in diffi cult respiratory weaning patients F Turani, S Martini, A Marinelli, M Falco, R Barchetta, F Candidi, F Gargano 1Aurelia Hospital/European Hospital, Rome, Italy Critical Care 2013, 17(Suppl 2):P129 (doi: 10.1186/cc12067) Figure 1 (abstract P130). Correlation between spirometrically measured and calculated increase in EELV. ECCO2 removal with a phosphorylcholine-coated membrane oxygenator in diffi cult respiratory weaning patients Introduction ECCO2 removal may be a useful support in patients with diffi cult respiratory weaning. The aim of this study is to evaluate a new phosphorylcholine-coated EECO2 removal system with no thrombogenic activity to assess the clinical safety of the system, the changes of main cardiorespiratory indices and CO2 removal by the system. y Methods Ten patients were enrolled. Before starting with ECCO2 removal all patients were ventilated with TV <6 ml/kg, peak pressure >35 cmH2O and pH <7.25. ECCO2 removal was initiated using a modifi ed continuous venovenous hemofi ltration system with a membrane oxygenator (ABYLCAP; Bellco, Mirandola, Italy; membrane surface area: 0.67 m2, blood fl ow 280 to 350 ml/minute, phosphorylcholine coated). Femoral vein cannulation with a double-lumen catheter was used to connect the patients to the extracorporeal system. Heparin was infused to maintain ACT <190 to 240 seconds. All patients had ECCO2 for 4 days. Every 12 hours the pH, PaCO2, peak pressure and PaO2 were evaluated. VCO2 was determined by indirect calorimetry using a gas analyser equipped on an Engstrom Carestation Ventilator. During the ECCO2 removal the patients were ventilated with TV ≤6 ml/kg and peak pressure <30 cmH2O. All data are expressed as mean ± SD. One-way ANOVA was used to compare the changes of parameters. P <0.05 was considered signifi cant. Figure 1 (abstract P130). Correlation between spirometrically measured and calculated increase in EELV. i Results Table 1 presents the main results. The CO2 removal by mem- brane oxygenator ranged from 56 to 37 ml/minute. All patients survived to the treatment and 7/10 were weaned from the ventilator at the end of ECCO2 removal. Only one oxygenator was used for every patient without clotting of the circuit or any major bleeding problem. pressure minus tidal variation in oesophageal pressure divided by tidal volume. Position of the oesophageal catheter was verifi ed according to Baydur [2]. The measured change in end-expiratory lung volume during the PEEP-step manoeuvre using spirometry was compared with the end-expiratory lung volume change calculated from EL and stepwise changes in PEEP as ΔPEEP/EL. Table 1 (abstract P129) Day 0 Day 2 Day 4 pH 7.24 ± 0.06 7.38 ± 0.1 7.41 ± 0.07 PaCO2 (mmHg) 70 ± 5 57 ± 8 52 ± 3* PP (cmH2O) 48 ± 4 28 ± 4* 25 ± 4 VCO2 (ml/minute) 220 ± 15 201 ± 14 180 ± 13* P <0.05 versus T0. P127 Extracorporeal membrane oxygenation: the MOTOR of cytokine production? HMGB1 was measured using an ELISA kit (Shino-Test, Tokyo, Japan). Results Two patients with interstitial pneumonia were studied. The ECMO system consisted of a Rotafl ow Centrifugal Pump (Maquet Japan, Tokyo, Japan), a Biocube TNC coating 6000 (NIPRO, Osaka, Japan), and a percutaneous cardiopulmonary support system (Capiox EBS; Terumo, Tokyo, Japan). The blood fl ow rate was 2.0 ± 4.0 l/minute. A total of 34 blood sets were collected. In most cases, blood levels of IL-1β, IL-2, IL- 4, IL-5, IL-12(p70), IL-13, IL-17, GM-CSF, IFNγ, and TNFα were below the detection limit and did not increase during ECMO. The other mediators were detected at the inlet (before), but no signifi cant increase was observed at the outlet (after) (HMGB1, P = 0.33; IL-6, P = 0.12; IL-7, P = Figure 1 (abstract P128). S49 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 P129 ECCO2 removal with a phosphorylcholine-coated membrane oxygenator in diffi cult respiratory weaning patients F Turani, S Martini, A Marinelli, M Falco, R Barchetta, F Candidi, F Gargano 1Aurelia Hospital/European Hospital, Rome, Italy Critical Care 2013, 17(Suppl 2):P129 (doi: 10.1186/cc12067) Introduction ECCO2 removal may be a useful support in patients with diffi cult respiratory weaning. The aim of this study is to evaluate a new phosphorylcholine-coated EECO2 removal system with no thrombogenic activity to assess the clinical safety of the system, the changes of main cardiorespiratory indices and CO2 removal by the system. Methods Ten patients were enrolled. Before starting with ECCO2 removal all patients were ventilated with TV <6 ml/kg, peak pressure >35 cmH2O and pH <7.25. ECCO2 removal was initiated using a modifi ed continuous venovenous hemofi ltration system with a membrane oxygenator (ABYLCAP; Bellco, Mirandola, Italy; membrane surface area: 0.67 m2, blood fl ow 280 to 350 ml/minute, phosphorylcholine coated). Femoral vein cannulation with a double-lumen catheter was used to connect the patients to the extracorporeal system. Heparin was infused to maintain ACT <190 to 240 seconds. All patients had ECCO2 for 4 days. Every 12 hours the pH, PaCO2, peak pressure and PaO2 were evaluated. VCO2 was determined by indirect calorimetry using a gas analyser equipped on an Engstrom Carestation Ventilator. During the ECCO2 removal the patients were ventilated with TV ≤6 ml/kg and peak pressure <30 cmH2O. All data are expressed as mean ± SD. P134 Novel biomarkers for prediction of mortality after acute exacerbation of chronic obstructive pulmonary disease H Michalopoulou, H Michalopoulou, P Stamatis, F Kattis, D Stamatis Metaxa Hospital, Athens, Greece Critical Care 2013, 17(Suppl 2):P134 (doi: 10.1186/cc12072) Results Ninety patients were included (77% male, 23% female). Mean age 69 years (± 11.88). Fifty-fi ve percent were smokers, 52% had arterial hypertension, 39% received long-term oxygenotherapy, 18% received antibiotics in the 7 days before, 18% corticosteroids, and 14% were on long-term NIV support at home. An emergency medical ambulance was immediately sent for 86% of patients. Ninety-two percent had normal consciousness (Glasgow Coma Scale 15), 78% had bronchospasm, 71% had signs of respiratory struggle, and 12% were unable to speak. The mean respiratory rate was 31.4 cycles/ minute (± 8.18), the average cardiac pulse was 103.6 beats/minute (± 23.14). Nasal EtCO2 44.92 mmHg (± 16.38), pulse oximetry with air was 83.48% (± 12.09), and the average fl ow rate of oxygen delivered was 5.69 l/minute (± 2.93). None of the patients had fever. Eighty-fi ve percent were supported on spontaneous ventilation, 22% received prehospital non-invasive ventilation, they all showed signs of severity and 3% need tracheal intubation. Seventy-fi ve percent of patients received β2-agonist and anticholinergic nebulization, 45% intravenous corticosteroids. Seventy-one percent were admitted to the emergency room, 29% to the ICU. Introduction Retrospective studies suggest that cardiac troponin levels are often elevated in patients with acute exacerbation of chronic obstructive pulmonary disease (AECOPD) indicating a poor survival. Novel high-sensitivity cardiac troponin (hs-cTnT) assays have better analytical precision than standard troponin (cTnT) assays. We elaborated a prospective cohort study to investigate the prognostic value of this novel biomarker in patients with AECOPD. Methods Fifty-six patients (mean age 64 years, 68% male) with the fi nal diagnosis of AECOPD were enrolled. Those who were diagnosed with acute coronary syndromes were excluded. We measured cardiac troponin T with a standard fourth-generation assay and a high- sensitivity assay. Clinical, electrocardiographic and echocardiographic data were collected at admission and the primary prognostic endpoint was death during 30 days of follow-up. Results Mean hs-cTnT levels at admission were 34 ng/l. During the follow-up period seven patients (12%) died. Thirty-eight percent of patients had hs-cTnT above the range of 14 ng/l. Lung elastance can be determined without esophageal pressure measurements C Grivans, S Lundin, O Stenqvist Sahlgrenska University Hospital, Gothenburg, Sweden Sahlgrenska University Hospital, Gothenburg, Sweden g y g Critical Care 2013, 17(Suppl 2):P130 (doi: 10.1186/cc12068) Critical Care 2013, 17(Suppl 2):P130 (doi: 10.1186/cc12068) Introduction Long-term use of mechanical ventilators may lead to ventilator-induced diaphragmatic dysfunction (VIDD) and increase the duration of weaning from MV [1]. It was hypothesized that stimulating the diaphragm during MV may prevent VIDD and may lead to early weaning [2]. In this study, the feasibility of generating coordinated contraction of both diaphragms was investigated using a novel transvenous diaphragmatic pacing system. Introduction We have previously shown, in an ex vivo porcine model, that lung elastance calculated as the PEEP change divided by lung volume increase (ΔPEEP/ΔEELV) is closely correlated to conventionally measured lung elastance using oesophageal pressure [1]. In this study we hypothesize that the successive change in lung volume during a PEEP-step manoeuvre could be predicted from ΔPEEP and lung elastance as ΔPEEP/EL. The objective of the study was to validate this hypothesis in patients with acute respiratory failure (ARF). Methods Two juvenile pigs were anesthetized with propofol (150 to 250 μg/kg/minute) and ventilated (VENT) with an assist control mode MV (Nellcor Puritan Bennett 840). Using fl uoroscopy, a novel multipolar neurostimulation catheter (Inspirx RL PICC53; Respithera, Bloomington, MN, USA) was threaded into the left internal jugular vein and advanced to the junction of right atrium and the superior vena cava using a Methods Thirteen patients with ARF were studied during an incremental PEEP trial, 0–4–8–12–16 cmH2O. ΔEELV was determined as the change in expiratory tidal volume following each PEEP step. Conventional calculation of lung elastance was obtained from tidal variation in airway S50 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 modifi ed Seldinger technique. The successful capture of the right and left phrenic nerves was confi rmed by fl uoroscopic visualization. Peak airway pressures (PAWP) and blood gases were determined after 10 minutes MV (MV), MV and stimulation applied together (MV+STIM) and stimulation only (STIM). modifi ed Seldinger technique. The successful capture of the right and left phrenic nerves was confi rmed by fl uoroscopic visualization. Peak airway pressures (PAWP) and blood gases were determined after 10 minutes MV (MV), MV and stimulation applied together (MV+STIM) and stimulation only (STIM). P133 N-terminal pro-brain natriuretic peptide as a prognostic marker in patients with exacerbation of chronic obstructive pulmonary disease Table 1 (abstract P131). Ventilation parameters MV MV+STIM STIM PAWP (cmH2O) 20 16 9 PO2 (mmHg) 89 101 95 PCO2 (mmHg) 32 26 26 Table 1 (abstract P131). Ventilation parameters Table 1 (abstract P131). Ventilation parameters P Stamatis, H Michalopoulou, D Stamatis Metaxa Hospital, Athens, Greece Critical Care 2013, 17(Suppl 2):P133 (doi: 10.1186/cc12071) P Stamatis, H Michalopoulou, D Stamatis Metaxa Hospital, Athens, Greece Critical Care 2013, 17(Suppl 2):P133 (doi: 10.1186/cc12071) Introduction Plasma N-terminal pro-brain natriuretic peptide (NT-pro- BNP) levels are elevated in patients with pulmonary disease especially in those with concomitant right ventricular dysfunction. The aim of the present study was to investigate the use of plasma NT-pro-BNP levels as a prognostic tool in patients with exacerbation of chronic obstructive pulmonary disease (COPD). Conclusion It was possible to capture and stimulate both phrenic nerves using a minimally invasive approach to support respiration and sustain blood gases at physiological levels. This development could help wean MV-dependent ICU patients earlier. Further long-term studies are needed to assess the full potential of this novel system. References Methods A retrospective medical records analysis of all patients hospitalized between June 2009 and June 2012 with the fi nal diagnosis of acute exacerbation of COPD who had undergone NT-pro-BNP measurements at admission followed by echocardiogram. 1. Vassilakopoulos T, Petrof BJ: Am J Respir Crit Care Med 2004, 169:336-341. 2. Sassoon CS, Zhu E, Caiozzo VJ: Am J Respir Crit Care Med 2004, 170:626-632. 1. Vassilakopoulos T, Petrof BJ: Am J Respir Crit Care Med 2004, 169:336-341. 2. Sassoon CS, Zhu E, Caiozzo VJ: Am J Respir Crit Care Med 2004, 170:626-632. 1. Vassilakopoulos T, Petrof BJ: Am J Respir Crit Care Med 2004, 169:336-341. y Results Seventy-two patients (mean age 69 years, 58% male) with COPD exacerbation but without clinical or echocardiography-oriented evidence of acute cardiac disease were enrolled. The mean forced expiratory volume in 1 second (FEV 1)/forced vital capacity ratio was 44.2% and the mean FEV 1 was 0.82 l. Median NT-pro-BNP levels at admission were 424.4 pg/ml and the tertile limits were 262.1 and 930 pg/ml. NT-pro-BNP levels signifi cantly predicted 30-day mortality (OR: 7.2, 95% CI: 3.9 to 15.2; P <0.001), (OR: 25, 95% CI: 16.3 to 37.3; P <0.001) and (OR: 44, 95% CI: 44.5 to 76.2) for each tertile. These associations persisted after adjusting for arterial CO2 pressure, body mass index, age, gender and systolic pulmonary artery pressure. References References 1. Antoine G: The epidemiology and outcome of medical emergency team call patients treated with non invasive ventilation. Resuscitation 2011, 82:1218-1223. 2. Schmidbauer W: Early prehospital use of non invasive ventilation improves acute respiratory failure in acute exacerbation of chronic obstructive pulmonary disease. Emerg Med J 2011, 28:626-627. 1. Antoine G: The epidemiology and outcome of medical emergency team call patients treated with non invasive ventilation. Resuscitation 2011, 82:1218-1223. 2. Schmidbauer W: Early prehospital use of non invasive ventilation improves acute respiratory failure in acute exacerbation of chronic obstructive pulmonary disease. Emerg Med J 2011, 28:626-627. y Results No animal–ventilator dyssynchrony during stimulation (MV+STIM) was noted while peak airway pressures were reduced. During STIM there was no discernible paradoxical movement of the diaphragm. In addition, PCO2 and PO2 confi rmed that adequate ventilation and oxygenation can be provided by the system, while PAWP could be reduced (Table 1). Prehospital management of COPD patients in respiratory failure and short-term outcome G Campagne, J Cuny, P Gosselin, P Goldstein, N Assez, E Wiel Lille University Hospital Center, Lille Cedex, France y p Critical Care 2013, 17(Suppl 2):P132 (doi: 10.1186/cc12070) Introduction Respiratory failure in COPD patients is a frequent call in French emergency dispatching centers. We have evaluated the prehospital management of COPD patients and severity signs, and analyse outcome in the emergency department or ICU. Conclusion Among patients with acute exacerbation of chronic pulmonary disease, NT-pro-BNP could be a useful marker for severity and poor prognosis. Methods We conducted an observational, descriptive, retrospective, single-center study during a 4-month period. All COPD patients with respiratory failure and prehospital care were included. Diff erent data were recorded. P134 Prognostic accuracy of hs-cTnT for death was signifi cantly higher, with area under the ROC curve (AUC) of 0.83 (95% CI: 0.72 to 0.86), than that of cTnT (AUC: 0.63, 95% CI: 0.56 to 0.74; P <0.001). After adjustment for age, gender, creatinine levels, heart rate, left ventricular ejection fraction, arterial O2 pressure and systolic pulmonary artery pressure, hs-cTnT above the Conclusion Most of the patients had signs of severity and bronchospasm. The absence of fever and antibiotics allows us to think that the cause of decompensation is not pneumonia. Although most of them were hypoxic and hypercapnic, they seem to be good candidates for NIV support in prehospital care. Very few studies report the use of NIV in cases of COPD respiratory failure in the fi rst care delivered at home. Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S51 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 equal groups: group I (cases, 25 patients) were subjected to additional intervention, early FOB during the fi rst 6 to 12 hours from admission; while group II (control, 25 patients) received the conventional treatment and NIPPV only. Outcome parameters measured were changes in ABG data, duration of NIPPV, rate of its success, ICU stay and mortality as well as the safety of FOB and possible complications.if 99th percentile was associated with a hazard ratio for death of 3.2 (95% CI: 1.5 to 6.7). Conclusion In AECOPD, novel biomarkers such as hs-cTnT appear to be positively associated with COPD severity and could be a determinant of mortality. 99th percentile was associated with a hazard ratio for death of 3.2 (95% CI: 1.5 to 6.7). Conclusion In AECOPD, novel biomarkers such as hs-cTnT appear to be positively associated with COPD severity and could be a determinant of mortality. y Results No signifi cant diff erence was detected between the two groups regarding the baseline characteristics. No serious complications happened from FOB, oxygen desaturation occurred in 4/25 patients (16%), tachycardia in 2/25 patients (8%). In group I, 23 patients (92%) were successfully weaned from NIPPV versus 16 patients (64%) in group II (P = 0.037). Total duration of NIPPV was 28.52 hours in group I versus 56.25 hours in group II (P = 0.001). Length of ICU stay was 4.84 days in group I versus 8.68 days in group II (P = 0.001). SMART-COP score for patients admitted with community-acquired pneumonia (CAP) to an ICU in a district general hospital: a smarter way of identifying patients with severe CAP? SMART-COP score for patients admitted with community-acquired pneumonia (CAP) to an ICU in a district general hospital: a smarter way of identifying patients with severe CAP? y y g p M Pachucki1, F Kelly2, A Padkin2 M Pachucki1, F Kelly2, A Padkin2 y 1Frenchay Hospital, Bristol, UK; 2RUH, Bath, UK Results Thirty-four patients were enrolled. Median age was 66 (54 to 71) years; 65% of the patients were male; median APACHE IV score on admission was 76 (61 to 96). Median ICU stay was 8 (5 to 17) days, with an overall hospital mortality of 12%. Thirteen patients (38%) tested positive for a respiratory virus. The most frequently found virus was infl uenza (37%), followed by RSV (15%), rhinovirus (15%), MPV (15%), corona virus (9%) and parainfl uenza virus (9%). Two of the patients (6%) had a bacterial–viral co-infection (blood culture and PCR positive for Streptococcus pneumonia and rhinovirus in one patient and for Haemophilus infl uenza and infl uenza virus in the second patient). Length of stay in the ICU was signifi cantly longer in PCR-negative patients, in comparison with PCR-positive patients (11 (6 to 20) vs. 7 (2 to 8) days, respectively, P <0.05).l y p Critical Care 2013, 17(Suppl 2):P137 (doi: 10.1186/cc12075) Introduction British Thoracic Society guidelines on community- acquired pneumonia (CAP) advocate ICU referral for patients with CURB65 score of 4 and 5. A recently developed scoring system, SMART- COP, designed to identify patients at need of intensive respiratory or vasopressor support (IRVS), has been validated in a variety of settings. It predicts the need for ICU admission (defi ned as need for IRVS) with greater accuracy than CURB65, but is not used routinely in our UK institution. Methods We retrospectively analysed critical care admissions of patients with a diagnosis of CAP in a UK district general hospital – ICNARC-coded diagnoses of pneumonia (bacterial, viral, no organisms isolated) over a 7-month period (August 2011 to January 2012). We ascertained the CURB65 and SMART-COP scores on referral to the ICU and matched them in relation to the need for IRVS, length of inotropic and ventilatory support and ICU length of stay. Conclusion Respiratory viruses, and particularly infl uenza virus, are frequently found in adult patients with respiratory failure admitted to the ICU. P136 Role of bronchoscopy during non-invasive ventilation in hypercapnic respiratory failure W Rady, A Abouelela, A Aly, W Youssef Alexandria University, Alexandria, Egypt Critical Care 2013, 17(Suppl 2):P136 (doi: 10.1186/cc12074) References 1. Carrat F, et al.: Intensive Care Med 2006, 2:156-159. 2. Choi SH, et al.: Am J Respir Crit Care Med 2012, 186:325-332. 3. Miggins M, et al.: PLoS One 2011, 6:18890. 1. Carrat F, et al.: Intensive Care Med 2006, 2:156-159. 2. Choi SH, et al.: Am J Respir Crit Care Med 2012, 186:325-332. 3. Miggins M, et al.: PLoS One 2011, 6:18890. Results Our search revealed 28 potential matches. Five patients were excluded (not CAP) and the notes for seven patients were not available for analysis. We analysed the notes of 16 patients matching our criteria. In this small sample, there was a strong association between increasing SMART-COP score and the need for IRVS (correlation coeffi cient r = 0.96). There was also a strong correlation with longer inotropic support (r = 0.85) and longer ventilatory support (r = 0.96) with increasing SMART-COP scores but a weaker correlation with length of ICU stay (r = 0.49). Moreover, none of the patients admitted to the ICU had CURB65 score higher than 3 at the time of ICU referral. P134 Only one patient died in group I versus three patients in group II (P = 0.609). High incidence of respiratory viruses in critically ill adult patients with respiratory failure Introduction Laboratory testing for viral infections is not routinely performed in adult patients admitted to the ICU. However, reports indicate that viruses may contribute to signifi cant morbidity and mortality and that viral–bacterial co-infection is associated with poor outcome in this particular patient population. The aim of the study was to document the incidence of respiratory virus infections in critically ill adult patients admitted to the ICU for acute respiratory failure. g p p g p Conclusion The early application of FOB during NIPPV in patients with ARF due to COPD exacerbation was shown to be safe. Signifi cant improvement in the outcome of patients who underwent FOB was noticed in terms of improved ABG data, shorter duration of NIPPV, higher percentage of success and shorter ICU stay while no signifi cant diff erence was detected in mortality. References 1. Scala R, et al.: Crit Care 2010, 14:R80. y Methods The prospective, observational study took place during two consecutive winter seasons in a mixed 16-bed ICU at the Medical Center Leeuwarden, the Netherlands. Adult patients admitted to the ICU, suspected of respiratory failure due to community-acquired pneumonia, were included. After informed consent brushed nasopharyngeal swab (Copan®) samples were taken within 24 hours after admission and analyzed by PCR assays for the presence of respiratory syncytial virus (RSV), infl uenza virus, metapneumovirus (MPV), parainfl uenza virus, adenovirus, rhinovirus and coronavirus. Data are presented as median (IQR). 1. Scala R, et al.: Crit Care 2010, 14:R80. 2. Ambrosino N, et al.: Eur Respir J 2008, 31:874-886. 2. Ambrosino N, et al.: Eur Respir J 2008, 31:874-886. 2. Ambrosino N, et al.: Eur Respir J 2008, 31:874-886. 3. Heunks LM, et al.: Intensive Care Med 2010, 36:143-147. p 3. Heunks LM, et al.: Intensive Care Med 2010, 36:143-147. 3. Heunks LM, et al.: Intensive Care Med 2010, 36:143-147. Role of bronchoscopy during non-invasive ventilation in hypercapnic respiratory failure W Rady, A Abouelela, A Aly, W Youssef W Rady, A Abouelela, A Aly, W Youssef Alexandria University, Alexandria, Egypt Critical Care 2013, 17(Suppl 2):P136 (doi: 10.1186/cc12074) Conclusion In our small sample, higher SMART-COP score was associated with increased likelihood of IRVS. This suggests that a further study with a larger sample size should be performed to investigate whether SMART-COP is an improvement on CURB65 in predicting the need for IRVS in UK intensive care patients. Introduction Non-invasive positive-pressure ventilation (NIPPV) is the fi rst-line treatment for hypercapnic acute respiratory failure (ARF) secondary to COPD exacerbation in selected patients. Limited data exist supporting the use of fi beroptic bronchoscopy (FOB) during this clinical setting. The aim of this study is to assess the role of FOB during NIPPV in patients with decompensated COPD acute exacerbation. P135 High incidence of respiratory viruses in critically ill adult patients with respiratory failure M Sietses1, TE Faber1, L Bont2, H Buter1, EC Boerma1 1Medical Center Leeuwarden, the Netherlands; 2University Medical Center Utrecht, the Netherlands Critical Care 2013, 17(Suppl 2):P135 (doi: 10.1186/cc12073) Ventilation distribution measured with electrical impedance tomography at varying levels of assist during pressure support versus neurally adjusted ventilatory assist in ICU patients MS Van Mourik1, P Blankman1, D Hasan2, D Gommers1 1Erasmus Medical Center, Rotterdam, the Netherlands; 2Maasstad, Rotterdam, the Netherlands Critical Care 2013, 17(Suppl 2):P140 (doi: 10.1186/cc12078) 1Erasmus Medical Center, Rotterdam, the Netherlands; 2Maasstad, Rotterdam, the Netherlands y y Results The APACHE scores in the S group and NS group were 27 ± 10 and 23 ± 4, Sequential Organ Failure Assessment scores were 8 ± 4 and 7 ± 3, respectively. These scores showed no signifi cant diff erence between the groups. Procalcitonin (PCT) in the S and NS groups was 20.7 ± 21.7 and 45.0 ± 47.7 ng/ml, respectively, and there was no signifi cant diff erence between the groups. PCT declined signifi cantly in both groups. PaO2/FiO2 of the NS group was signifi cantly higher than the S group on ICU admission and 4 days after admission, but no signifi cant diff erence on 7 days after ICU admission. IL-6 of the NS group declined signifi cantly after ICU admission, and the S group also tended to decline. Introduction Electrical impedance tomography (EIT) is a non-invasive and nonradiating imaging technique, which can be used to visualize ventilation distribution of the lungs and could distinguish between the dependent (dorsal) and nondependent (ventral) parts. Introduction Electrical impedance tomography (EIT) is a non-invasive and nonradiating imaging technique, which can be used to visualize ventilation distribution of the lungs and could distinguish between the dependent (dorsal) and nondependent (ventral) parts. Methods The aim of this study was to observe ventilation distribution between dependent and nondependent lung regions, for the individual patient, during three diff erent levels of support during pressure support (PS) and neurally adjusted ventilatory assist (NAVA) ventilation. Ten mechanically ventilated patients in the ICU were included. y p Results The ratio for dependent/nondependent distribution of ventilation is signifi cantly higher at lower support levels compared with higher support levels in both PS and NAVA. However, during NAVA there was signifi cantly less impedance loss between the diff erent levels of assist compared with PS. Tidal volumes decreased when decreasing assist levels during PS whereas not during NAVA ventilation. The electrical activity of the diaphragm decreased in both PS and NAVA with higher levels of assist. Three patients showed an increase in dependent tidal impedance variation (TIV) after lowering the assist level from 15 to 10 cmH2O. This increase in TIV did not occur during NAVA ventilation. 3. Robins-Browne K, et al.: The SMART-COP score performs well for pneumonia risk stratifi cation in Australia’s Tropical Northern Territory: a prospective cohort study. Trop Med Int Health 2012, 17:914-919. 4. Fang et al.: Application and comparison of scoring indices to predict outcomes in patients with healthcare associated pneumonia. Crit Care 2011, 15:R32. P138f P138 Eff ects of steroid therapies to severe streptococcal pneumoniae that required mechanical ventilation T Nagura, T Ikeda, K Ikeda, T Ueno, S Suda Tokyo Medical University, Hachioji Medical Center, Tokyo, Japan Critical Care 2013, 17(Suppl 2):P138 (doi: 10.1186/cc12076) Introduction Streptococcal pneumonia remains the most common cause of community-acquired pneumonia (CAP), bacterial meningitis and bacteremia. Severe pneumonia caused by streptococcal pneu- monia frequently exists in the emergency room or ICU. We performed this study to evaluate the eff ect of steroid therapy for severe strepto- coccal pneumonia patients with mechanical ventilation retrospectively. Methods We enrolled 13 adults of streptococcal pneumonia patients who required mechanical ventilation. Seven of 13 patients (S group) were administered with steroid (hydrocortisone 200 to 300 mg/day), and the remaining six patients received no steroid therapy (NS group). As the conventional therapies, mechanical ventilation was commenced when a patient’s PaO2/FiO2 showed less than 200 or they clinically complained of being short of breath. All patients received appropriate fl uid therapies, vasoactive agents and blood transfusion according to the protocol of early goal-directed therapy in the Surviving Sepsis Campaign Guidelines 2008, and also were treated with antibiotics, immunoglobulins (5 g/day for 3 days) and sivelestat sodium hydrate (4.8 mg/kg/day for 7 days). Conclusion The presence of diabetes mellitus and immunosuppression, higher APACHE II scores, longer mechanical ventilation duration, and presence of open wounds were predictors of VAP. Reference 1. Guidelines for the management of adults with hospital-acquired, ventilator-associated, and healthcare-associated pneumonia. Am J Respir Crit Care Med 2005, 171:388-416. Ventilation distribution measured with electrical impedance tomography at varying levels of assist during pressure support versus neurally adjusted ventilatory assist in ICU patients MS Van Mourik1, P Blankman1, D Hasan2, D Gommers1 1Erasmus Medical Center, Rotterdam, the Netherlands; 2Maasstad, Rotterdam, the Netherlands Critical Care 2013, 17(Suppl 2):P140 (doi: 10.1186/cc12078) Conclusion Steroid therapy for severe streptococcal pneumonia patients with mechanical ventilation may have a potential to maintain oxygenation of the lung, but no signifi cant eff ects on changes of infl ammatory markers (IL-6, CRP). P139 Risk factors for ventilator-associated pneumonia in a surgical ICU A Kundakci, O Ozkalaycı, P Zeyneloglu, H Arslan, A Pirat Baskent University Hospital, Ankara, Turkey Critical Care 2013, 17(Suppl 2):P139 (doi: 10.1186/cc12077) 2 Conclusion There is more ventilation in the dependent part of the lung, compared with the nondependent part, at lower levels of assist. This could indicate that at higher support levels the contribution of the diaphragm is reduced. During NAVA ventilation, there is an autoregulation in which the patient is adjusting his tidal ventilation to maintain homogeneous ventilation distribution. Introduction Ventilator-associated pneumonia (VAP) is the most common nosocomial infection in ICU patients who require mechanical ventilation support. The aim of this study was to determine predictors for the development of VAP in surgical ICU patients admitted to Baskent University Hospital. y Methods Following Institutional Review Board approval we performed this retrospective study, including 876 patients admitted to the surgical ICU between January 2009 and July 2012. After completing a review of patient data, 45 patients diagnosed with VAP were compared with 26 appropriate matches who were not. Patients’ demographical features (age, sex, body weight), underlying diseases, etiology for ICU admission, APACHE II and Sequential Organ Failure Assessment scores, duration of hospitalization, organ dysfunctions, fl uid balances, laboratory values, use of vasopressors, mechanical ventilation, nutrition, antibiotics, transfusions, features related to central venous catheterization, urinary catheterization, and intubation were the recorded parameters. Patients P140 Ventilation distribution measured with electrical impedance tomography at varying levels of assist during pressure support versus neurally adjusted ventilatory assist in ICU patients MS Van Mourik1, P Blankman1, D Hasan2, D Gommers1 1Erasmus Medical Center, Rotterdam, the Netherlands; 2Maasstad, Rotterdam, the Netherlands Critical Care 2013, 17(Suppl 2):P140 (doi: 10.1186/cc12078) References 1. Lim WS, et al.: BTS guidelines for the management of community acquired pneumonia in adults: update 2009. Thorax 2009, 64(Suppl 3):iii1-iii55. doi:10.1136/thx.2009.121434 Methods This study is a randomized prospective case–control pilot study carried out on 50 patients admitted to critical care units at Alexandria University Hospital, Egypt suff ering from hypercapnic ARF secondary to COPD exacerbation with Kelly Matthay Score from 2 to 4. All patients received NIPPV. Patients were divided randomly into two doi:10.1136/thx.2009.121434 2. Charles PG, et al.: SMART-COP: a tool for predicting the need for intensive respiratory or vasopressor support in community-acquired pneumonia. J Clin Infect Dis 2008, 47:375-384. 2. Charles PG, et al.: SMART-COP: a tool for predicting the need for intensive respiratory or vasopressor support in community-acquired pneumonia. J Clin Infect Dis 2008, 47:375-384. Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S52 who were not intubated and were discharged or died within 2 days of ICU admission were excluded. Results Out of 71 patients who were included in the fi nal analysis, 45 patients (63%) had VAP. Comparing with the control group, patients who developed VAP were more likely to have diabetes mellitus and immunosuppression (P = 0.020 and P = 0.014, respectively). These patients also had higher APACHE II scores (P = 0.020) and a longer duration of mechanical ventilation (P <0.001), and more frequently had an open wound (P = 0.001). Following regression analysis, presence of diabetes mellitus (OR: 12.048; 95% CI: 1.157 to 125.000; P = 0.037), immunosuppression (OR: 16.949, 95% CI: 2.463 to 111.111; P = 0.004), and open wound (OR: 5.714; 95% CI: 1.017 to 37.258; P = 0.048), higher APACHE II scores (OR: 1.132; 95% CI: 1.022 to 1.254; P = 0.018), and prolonged duration of mechanical ventilation (OR: 1.084; 95% CI: 1.002 to 1.171; P = 0.043) were determined as risk factors for VAP. Fourteen- day and 28-day mortality rates for VAP were 19% and 29%, respectively (P = 0.760 and P = 1.000, respectively). References 1. Marik PE, et al.: J Intensive Care Med 2012. [Epub ahead of print] 2. Piper AJ, et al.: Am J Respir Crit Care Med 2011, 183:292-298. 1. Marik PE, et al.: J Intensive Care Med 2012. [Epub ahead of print] 2. Piper AJ, et al.: Am J Respir Crit Care Med 2011, 183:292-298. 1. Marik PE, et al.: J Intensive Care Med 2012. [Epub ahead of print] 2. Piper AJ, et al.: Am J Respir Crit Care Med 2011, 183:292-298. Non-invasive ventilation in patients with a clinical diagnosis of pneumonia: a clinical audit Results Three hundred and fi fty-nine subjects were analyzed. Table 1 shows the time on BiPAP per visit. Children whose weight was >97 percentile revealed trends towards longer treatment times on BiPAP compared with the other two groups. The moderate subjects who weighed >97 percentile had statistically signifi cant longer treatment periods (P <0.006) when compared with the <90 percentile moderate group. Initial BiPAP settings are listed in Table 2. When controlling for age, higher BiPAP settings correlated with increasing weight. There were no weight-based trends for admissions to the PICU or overall hospital lengths of stay. No serious complications were noted.if Introduction The benefi ts of non-invasive ventilation (NIV) in patients with type 2 respiratory failure secondary to exacerbations of COPD are widely acknowledged [1], but its effi cacy in respiratory failure of other aetiologies is less clear. We describe use of NIV (bilevel and/ or continuous positive airway pressure modes) in the subgroup of patients with a clinical diagnosis of pneumonia in a 35-bed adult critical care unit (ACCU). Methods A retrospective review of data recorded prospectively on an electronic clinical information management system at University College Hospital (UCH), London, UK. Patients requiring NIV acutely at UCH are managed on the ACCU. Electronic records reviewed for patients admitted from 1 November 2010 to 2 December 2011 with clinical diagnosis of pneumonia, and in whom NIV was recorded on ≥6 occasions on the electronic chart. We concede some patients on NIV <6 hours may have been missed. We excluded patients in whom NIV was the ceiling of therapy and those using domiciliary NIV. The following data were collected: baseline patient demographics, comorbidities, therapy received in the ACCU, respiratory physiological variables, length of stay/unit outcome. Descriptive statistics of the cohort and variables associated on univariate analysis with requirement for intubation were generated using MedCalc Version 12.3.0.0 (MedCalc Software, Mariakerke, Belgium).i Conclusion This is the fi rst study to analyze the weight eff ect on BiPAP application in children with status asthmaticus. Subjects who weighed more trended greater mean time on BiPAP and initial BiPAP settings. Weight did aff ect PICU admissions or overall length of hospital stay. Analysis of bilevel positive airway pressure therapy in children based on weight Subjects were divided into moderate and severe exacerbations and then further subdivided into the following growth curve-based weight subgroups: <90 percentile, 90 to 97 percentile and >97 percentile. Subjects received standard asthma therapies in addition to BiPAP. Data were obtained at the bedside by the respiratory therapist or collected retrospectively by study investigators. Data were stored and analyzed using a RedCap database.i in status asthmaticus. Our purpose was to analyze BiPAP use and outcomes for children with status asthmaticus and obesity in our PED. Methods Patients placed on BiPAP in the PED for status asthmaticus from 1 January 2010 to 31 August 2012 were included in the analysis. Subjects were divided into moderate and severe exacerbations and then further subdivided into the following growth curve-based weight subgroups: <90 percentile, 90 to 97 percentile and >97 percentile. Subjects received standard asthma therapies in addition to BiPAP. Data were obtained at the bedside by the respiratory therapist or collected retrospectively by study investigators. Data were stored and analyzed using a RedCap database.i Analysis of bilevel positive airway pressure therapy in children based on weight Analysis of bilevel positive airway pressure therapy in children based on weight A Williams, T Abramo Vanderbilt University Medical Center, Nashville, TN, USA Critical Care 2013, 17(Suppl 2):P141 (doi: 10.1186/cc12079) A Williams, T Abramo Vanderbilt University Medical Center, Nashville, TN, USA Critical Care 2013, 17(Suppl 2):P141 (doi: 10.1186/cc12079) Introduction Obesity as a comorbidity adds a challenge when treating children presenting to the PED in status asthmaticus. Bilevel positive airway pressure (BiPAP) is an accepted treatment modality for children S53 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Table 1 (abstract P141). Time on BiPAP by weight percentile and severity of asthma exacerbation (hours) Moderate exacerbation Severe exacerbation Table 1 (abstract P141). Time on BiPAP by weight percentile and severity of asthma exacerbation (hours) Methods A monocentric prospective observational study including 48 patients with MOHS and treated by NIV for ARF over a 2-year period. Results NIV failed to reverse ARF in only seven patients. NIV failure was associated with a sixfold increase in in-hospital mortality (85.7% vs. 14.6%; P <0.001). Factors associated with NIV failure and need for endotracheal intubation included pneumonia (n = 5, 71.4% vs. n = 8, 19.5%; P = 0.01), higher SOFA (11 vs. 5; P <0.001) and SAPS 2 (69 vs. 39; P = 0.001) score at admission. The only factor associated with successful response to NIV was idiopathic decompensated MOHS (n = 19, 100% vs. n = 0, 0%; P = 0.03). In these patients, pH (7.23 vs. 7.28 vs. 7.44, respectively, at H0, H2, H24; P <0.05) and PCO2 (74.5 vs. 68.5 vs. 57 mmHg, respectively, at H0, H2, H24; P <0.05) dramatically improved with NIV.fi Conclusion NIV provides a very effi cient therapy to reverse hypercapnic ARF in morbidly obese patients. Multiple organ failure and pneumonia are the main factors associated with NIV failure in patients with MOHS. Conclusion NIV provides a very effi cient therapy to reverse hypercapnic ARF in morbidly obese patients. Multiple organ failure and pneumonia are the main factors associated with NIV failure in patients with MOHS. References in status asthmaticus. Our purpose was to analyze BiPAP use and outcomes for children with status asthmaticus and obesity in our PED. Methods Patients placed on BiPAP in the PED for status asthmaticus from 1 January 2010 to 31 August 2012 were included in the analysis. Non-invasive ventilation in patients with a clinical diagnosis of pneumonia: a clinical audit CJ Wright1, J Hornsby2, DA Barr3, SR Moonesinghe2 1Glasgow Royal Infi rmary, Glasgow, UK; 2University College Hospital, London, UK; 3Brownlee Institute, Glasgow, UK Critical Care 2013, 17(Suppl 2):P143 (doi: 10.1186/cc12081) Non-invasive ventilation in patients with a clinical diagnosis of pneumonia: a clinical audit CJ Wright1, J Hornsby2, DA Barr3, SR Moonesinghe2 1Glasgow Royal Infi rmary, Glasgow, UK; 2University College Hospital, London, UK; 3Brownlee Institute, Glasgow, UK Critical Care 2013, 17(Suppl 2):P143 (doi: 10.1186/cc12081) P142 Determinants of non-invasive ventilation success or failure in morbidly obese patients in acute respiratory failure M Lemyze, P Taufour, J Mallat, O Nigeon, D Thevenin Schaff ner Hospital, Lens, France Critical Care 2013, 17(Suppl 2):P142 (doi: 10.1186/cc12080) Introduction Malignant obesity hypoventilation syndrome (MOHS) refers to the association between morbid obesity (body mass index >40 kg/m2), daytime hypercapnia (PCO2 >45 mmHg) after other respiratory or neuromuscular causes of alveolar hypoventilation have been excluded, and multiple organ disorders (diabetes mellitus, arterial hypertension, metabolic syndrome, obstructive sleep apnea syndrome) [1]. Acute respiratory failure (ARF) is a common life-threatening complication of MHOS. Non-invasive ventilation (NIV) provides the cornerstone of the therapeutic management of decompensated MOHS [2]. We aimed to identify the determinants of NIV success or failure in this indication. g Results Fifty-two patients fulfi lled the inclusion criteria, 29 (55.8%) were male, median age was 62.4 (IQR 48.8 to 71.6), median unit stay was 8 days (IQR 4.5 to 16.5), mean duration of NIV use was 2 days (IQR 1 to 3), 27 (51.9%) required intubation of whom 48% died. Total mortality was 13 (25%) – all intubated. Patients with recorded comorbidities were more likely to be intubated than those without (OR = 4.3, P = 0.0362); pH was signifi cantly lower in those requiring intubation at all recorded time points; and mean FiO2 at 4 to 6 hours was signifi cantly higher in those requiring intubation(0.72 vs. 0.56, P = 0.032). There was a trend S54 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 toward patients with COPD requiring intubation more often (OR = 8.4, P = 0.0711). are specialized and relatively invasive. Visualizing diaphragm thickening during inspiration by ultrasound may permit non-invasive monitoring. We explored the feasibility, reliability and physiological signifi cance of diaphragm thickening on ultrasound. Conclusion NIV was successful in 48.1% of patients with pneumonia, the remainder requiring intubation. Given the high mortality in those patients who failed NIV we believe its use in a ward setting is hazardous. We conclude that NIV may be useful in some individuals with pneumonia, but they should be managed in the ACCU setting. Further work is required to identify those patients at risk of deterioration on NIV given the possible excess mortality. Methods Five healthy subjects participated. Analysis of management of non-invasive ventilation support in prehospital care for COPD patients and short-term outcome J Cuny, G Campagne, P Gosselin, P Goldstein, N Assez, E Wiel Lille University Hospital Center, Lille Cedex, France Critical Care 2013, 17(Suppl 2):P144 (doi: 10.1186/cc12082) Analysis of management of non-invasive ventilation support in prehospital care for COPD patients and short-term outcome J Cuny, G Campagne, P Gosselin, P Goldstein, N Assez, E Wiel Lille University Hospital Center, Lille Cedex, France Critical Care 2013, 17(Suppl 2):P144 (doi: 10.1186/cc12082) y Results Diaphragm thickening in the zone of apposition was readily visualized by ultrasound in all fi ve subjects. Mean end-expiratory diaphragm thickness was 2.1 mm (SD = 0.3 mm). During tidal breathing, the diaphragm thickened by a mean of 35% (SD = 31%). The Bland–Altman coeffi cient of reproducibility was 0.5 mm; approximately 50% of measurement variability arose from caliper positioning on the ultrasound machine; diaphragm thickness measurements changed as the probe was placed in diff erent intercostal interspaces. Diaphragm inspiratory thickening increased signifi cantly with increasing inspiratory eff ort but also varied with lung volume independent of eff ort. At inspiratory volumes below 40% of inspiratory capacity, lung volume change contributed minimally to diaphragm thickening. Introduction Benefi ts of the use of NIV in emergency departments are well established. Training and available staff , and choice of respiratory machines are essential criteria for success. Methods We conducted an observational, descriptive, retrospective, single-center study in a 4-month period. COPD patients with respiratory failure who received prehospital NIV were included. We compared two groups: COPD patients with NIV, and COPD patients without NIV. g p p p Results Forty-two patients were included, mean age 68.86 years (± 11.98), 57.14% smokers, 64.28% arterial hypertension, 100% long- term oxygenotherapy, 23.80% antibiotics in the 7 days before, 28.57% corticosteroids. A total of 88.09% had bronchospasm, 78.26% had struggle signs, 28.57% were unable to speak, 14.28% of patients were sweating. The mean respiratory rate was 30.5 cycles/minute (± 7.17), mean pulse rate was 105.76 (± 25.34). Nasal EtCO2 was 47.75 mmHg (± 16.53), pulse oxymetry in air was 85% (± 10.94), oxygen fl ow rate was 5.45 l/minute (± 2.42), temperature was 37.14°C (± 8.15). Twenty patients received NIV. A total of 61.90% were admitted to the emergency department, 35.71% to the ICU, and one patient was left at home. One patient received tracheal intubation in the hosting service. Mortality in the fi rst month was 13.04%. Analysis of management of non-invasive ventilation support in prehospital care for COPD patients and short-term outcome J Cuny, G Campagne, P Gosselin, P Goldstein, N Assez, E Wiel Lille University Hospital Center, Lille Cedex, France Critical Care 2013, 17(Suppl 2):P144 (doi: 10.1186/cc12082) A signifi cant diff erence (P <0.05) was found for: sweats (30%/0), respiratory rate (34 ± 8.23/27 ± 6.11), nasal EtCO2 (55.0 ± 24.4/40.50 ± 9.03), pulse oxymetry in air (80% ± 8.63/90% ± 13.25), pulse oxymetry with oxygen (89.4% ± 4.24/87.90% ± 2.55), β2-mimetic and anti-cholinergic nebulization (60% ± 0.5/90% ± 0.29), emergency room admission (35% ± 0.35/86% ± 0.48), ICU admission (60% ± 0.5/13% ± 0.35), arterial blood gases on arrival in the host service (PaCO2 76.6 ± 18.66/43.93 ± 11.78). No diff erence in mortality at 1 month (2/3). Conclusion Non-invasive ventilation has improved the management and prognosis of COPD patients admitted to the emergency room. Very few studies concern the prehospital management. NIV seems to show an eff ect on prehospital care, especially in patients with signs of severity, hypercapnia, and without fever. Oxygenation and hypercapnia seem to be improved. Also fewer patients are admitted to the ICU. Bronchospasm does not seem to be an obstacle. R f y Conclusion Visualizing diaphragm thickening in the zone of appo- sition by ultrasound provides a feasible non-invasive technique for monitoring diaphragm activation in healthy subjects. Diaphragm thickening primarily refl ects muscular eff ort rather than altered muscle conformation induced by changes in lung volume, especially at lower inspiratory volumes. Reference 1. Brochard L, et al.: Noninvasive ventilation for acute exacerbations of chronic obstructive pulmonary disease. N Engl J Med 1995, 333:817-822. Retrospective review of utilisation and outcomes of diaphragmatic EMG monitoring and neurally adjusted ventilatory assist in a central London teaching hospital over a 3-year periodi Retrospective review of utilisation and outcomes of diaphragmatic EMG monitoring and neurally adjusted ventilatory assist in a central London teaching hospital over a 3-year period A Skorko, D Hadfi eld, A Vercueil, C Bell, A Feehan, K Peters, P Hopkins King’s Health Partners AHSC, London, UK Critical Care 2013, 17(Suppl 2):P146 (doi: 10.1186/cc12084) Introduction The theoretical advantages of monitoring the electrical activity of the diaphragm (EAdi) and neural triggering of support breaths (NAVA-Maquet) have not yet been shown to translate into signifi cant clinical benefi t [1]. Here we assess the eff ect of EAdi monitoring, in patients at risk of prolonged weaning, on outcomes. Figure 1 (abstract P146). Figure 1 (abstract P146). 1. Schmidbauer W, et al.: Early pre hospital use of NIV. Emerg Med J 2011, 28:626-627. P145 P145 A window into diaphragm kinetics: feasibility, precision, and physiological meaning of ultrasound measurements of diaphragm thickness EC Goligher1, F Laghi2, ND Ferguson1 1University of Toronto, Canada; 2Loyola University, Chicago, IL, USA Critical Care 2013, 17(Suppl 2):P145 (doi: 10.1186/cc12083) P142 We monitored inspiratory fl ow, volume, esophageal and gastric pressures, and diaphragm electrical activity (by esophageal and surface electromyography) while subjects performed a series of inspiratory maneuvers: tidal breathing, threshold-loaded breathing, a Muller maneuver, and inspiration to various lung volumes above functional residual capacity. At the end of each inspiratory eff ort, subjects were instructed to close the glottis and relax the respiratory muscles (so as to maintain lung volume while eliminating diaphragm activation). Sonographic images of diaphragm thickening during these maneuvers were obtained using M-mode with a 13 MHz linear array probe placed in the right ninth, 10th, or 11th intercostal space between the middle and anterior axillary lines. Risk of weaning failure with PAOP ≥15 versus PAOP ≥18 Y Nassar, A Abdelbary Risk of weaning failure with PAOP ≥15 versus PAOP ≥18 Y Nassar, A Abdelbary Cairo University, Giza, Egypt Critical Care 2013, 17(Suppl 2):P148 (doi: 10.1186/cc12086) Cairo University, Giza, Egypt Critical Care 2013, 17(Suppl 2):P148 (doi: 10.1186/cc12086) Introduction Despite 18 mmHg being recognized as a classical cutoff value, there is no defi nite value of PAOP above which cardiogenic oedema develops. We aimed to assess the risks of weaning failure with PAOP ≥15 and with PAOP ≥18. Results In total, 493/2,684 (18.3%) patients had heart–lung risk factors for prolonged weaning. One hundred and four patients received EAdi monitoring. Ventilated days were signifi cantly reduced (Figure 1) in the EAdi monitored group (median 9) versus the nonmonitored group (median 12); P = 0.024 (Mann–Whitney U test). ICU mortality was not signifi cantly diff erent and there was no correlation between time spent in NAVA and days ventilated.i Methods A prospective enrollment of 30 adult medical ICU patients on invasive mechanical ventilation who were fulfi lling the criteria for initiation of weaning from mechanical ventilation according to the Sixth International Conference Statement of Intensive Care Medicine. We followed the two-step weaning strategy, which involves assessment regarding readiness for weaning followed by a spontaneous breathing trial (SBT) as a diagnostic test to determine the likelihood of successful extubation. Invasive right heart catheterization to measure PAOP, and non-invasive transthoraxic echo and tissue Doppler parameters E/E’, E/A, DT, IVRT were measured before initiation and after termination of the SBT. Conclusion EAdi catheter insertion was associated with a signifi cant reduction in time spent on MV. A prospective RCT will be needed to confi rm benefi t and explore mechanisms. Reference 1. Brander L, et al.: Chest 2009, 135:695-703. Results E/E’ was signifi cantly correlated with PAOP (r = 503, P <0.001). A cutoff value of septal E/E’ >11.3 predicted PAOP elevation ≥18 mmHg with a sensitivity of 90.9% and specifi city of 87.8%, while a value of septal E/E’ >10.9 predicted PAOP elevation ≥15 mmHg with a sensitivity of 59.1% and specifi city of 87.8%. The incidence of weaning failure was 23%, and 72% of patients who failed to be weaned exhibited signifi cantly higher PAOP during the trial. Patients with PAOP ≥18 at the end of the SBT mostly (72.4%) failed weaning while a minority (27.6%) successfully weaned. 9 Impact of continuous positive airway pressure on the treatment of acute exacerbation of asthma: a randomised controlled trial Y Sutherasan, S Kiatboonsri, P Theerawit, C Kiatboonsri, S Trakulsrichai Ramathibothi Hospital, Bangkok, Thailand Critical Care 2013, 17(Suppl 2):P149 (doi: 10.1186/cc12087) Introduction Despite the extensive use of continuous positive airway pressure (CPAP) in various respiratory failure conditions, its role in acute asthmatic attack is uncertain [1-3]. This study aims at exploring the effi cacy of CPAP when use in addition to the conventional treatment of acute asthma exacerbation in the emergency department (ED). Results Forty-two patients were evaluated (42% male), mean age was 73 years (SD ± 15) and APACHE II score was 18.77 (median 20). Based upon evaluation of the fi ndings of BLUE protocol, a positive correlation was found in 83.78% of the sample. For the diagnosis of pulmonary edema (n = 15), sensitivity was 85% and specifi city was 86%, while for the diagnosis of pneumonia (n = 17), sensitivity was 88% and specifi city was 90%. In this sample there were only four cases of reversible airway obstruction and one case of pulmonary embolism. Five patients were excluded of the analysis because they had rare diagnoses. g y Methods During May to December 2009, acute asthma patients attending ED were randomly assigned to CPAP and control groups. In addition to the conventional treatment, CPAP ventilation of 8 cmH2O was applied to patients in the CPAP group. Data collections in both groups included peak expiratory fl ow rates (PEFR, %predicted) at each 15-minute interval (T0, T15, …), the number of short-acting bronchodilators (SABA) used, ED length of stay, relapse rate and admission rate. The primary outcome is the improvement in peak expiratory fl ow rate during a short period stay in the ED. Results There were 86 patients enrolled, with 43 patients in each study group. PEFR in the CPAP group showed no diff erence from the control group at T0 (52.7 ± 20.33 vs. 47.2 ± 16.64, P = NS) but was signifi cantly better at T15 (65.58 ± 21.64 vs. 56.49 ± 18.53%, P <0.05). Such improvement was consistently observed for all subsequent PEFR measurement, along with the fewer SABA used and relapse rate, but the fi gures did not meet statistical diff erence. Methods During May to December 2009, acute asthma patients attending ED were randomly assigned to CPAP and control groups. In addition to the conventional treatment, CPAP ventilation of 8 cmH2O was applied to patients in the CPAP group. 1. Lichtenstein DA, Mezière GA: Relevance of lung ultrasound in the diagnosis of acute respiratory failure: the BLUE protocol. Chest 2008, 134:117-125. 1. Brander L, et al.: Chest 2009, 135:695-703. 1. Lichtenstein DA, Mezière GA: Relevance of lung ultrasound in the diagnosis of acute respiratory failure: the BLUE protocol Chest 2008 134:117 125 Risk of weaning failure with PAOP ≥15 versus PAOP ≥18 Y Nassar, A Abdelbary The majority (91.3%) of the patients with PAOP <18 had a successful weaning outcome and 8.7% failed weaning outcome. PAOP ≥18 signifi cantly negatively correlated with weaning success (P = 0.001, r = –0.627). On the other hand, PAOP ≥15 did not show a signifi cant correlation (P >0.05, r = –0.274). The PAOP ≥18 weaning failure risk estimate was 8.2, with 95% CI 0.004 to 0.3. The PAOP ≥15 weaning failure risk estimate was 1.83, with a signifi cant 95% CI of 0.913 to 3.648.i P147 Validation of a lung ultrasound protocol in acute respiratory failure: preliminary results RV Cremonese1, AC Tabajara Raupp1, JM Stormovski de Andrade1, RS Townsend1, FS Neres1, AS Machado1, MC Prestes1, JG Maccari2, FL Dexheimer Neto1 1Ernesto Dornelles, Porto Alegre, Brazil; 2Hospital Moinhos de Vento, Porto Alegre, Brazil Critical Care 2013, 17(Suppl 2):P147 (doi: 10.1186/cc12085) P147 Validation of a lung ultrasound protocol in acute respiratory failure: preliminary results RV Cremonese1, AC Tabajara Raupp1, JM Stormovski de Andrade1, RS Townsend1, FS Neres1, AS Machado1, MC Prestes1, JG Maccari2, FL Dexheimer Neto1 1Ernesto Dornelles, Porto Alegre, Brazil; 2Hospital Moinhos de Vento, Porto Alegre, Brazil Critical Care 2013, 17(Suppl 2):P147 (doi: 10.1186/cc12085) Critical Care 2013, 17(Suppl 2):P147 (doi: 10.1186/cc12085) Introduction Patients with acute respiratory failure demand dynamic evaluation and interventions. Lung ultrasound is a bedside technique, very promising in this setting. Previously, Lichtenstein and colleagues proposed an algorithm approach with a diagnostic accuracy of 90.5%, named Bedside Lung Ultrasound in Emergency (BLUE protocol). However, this approach has never been validated in other populations or ultrasound operators. The aim of this study was to evaluate the diagnostic accuracy of the lung ultrasound algorithm (BLUE protocol) in ICU patients admitted with acute respiratory failure. Conclusion E/E’ was signifi cantly correlated with PAOP (r = 503, P <0.001). E/E’ > 11.3 predicted PAOP elevation ≥18 mmHg (sensitivity 90.9% and specifi city 87.8%), while E/E’ >10.9 predicted PAOP elevation ≥15 mmHg (sensitivity 59.1% and specifi city 87.8%). Weaning failure risk is greater (8.2 vs. 1.8) with PAOP ≥18 versus PAOP ≥15, respectively. P149 y Methods A prospective study, single-center consecutive case series evaluating critically ill adult patients in acute respiratory failure, admitted to a 23-bed general ICU between October 2011 and November 2012. The ultrasound examination was performed by an ICU physician until 20 minutes after admission, without interfering with usual care, and the ultrasound operator was blinded to the medical history of the patient. Three items were assessed: artifacts (horizontal A lines or vertical B lines indicating interstitial syndrome), lung sliding, alveolar consolidation and/or pleural eff usion. BLUE protocol results were compared with clinical diagnosis by the medical assistant team at the end of the ICU stay. P149 Impact of continuous positive airway pressure on the treatment of acute exacerbation of asthma: a randomised controlled trial Y Sutherasan, S Kiatboonsri, P Theerawit, C Kiatboonsri, S Trakulsrichai Ramathibothi Hospital, Bangkok, Thailand Critical Care 2013, 17(Suppl 2):P149 (doi: 10.1186/cc12087) A window into diaphragm kinetics: feasibility, precision, and physiological meaning of ultrasound measurements of diaphragm thickness EC Goligher1, F Laghi2, ND Ferguson1 g , g , g 1University of Toronto, Canada; 2Loyola University, Chicago, IL, USA Critical Care 2013, 17(Suppl 2):P145 (doi: 10.1186/cc12083) Introduction Inadequate respiratory muscle activity has been linked to ventilator-induced diaphragm dysfunction. Current techniques for monitoring respiratory muscle activity during mechanical ventilation S55 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 P148 Methods Institutional ethics approval was obtained. The Medtrack clinical information system was searched to identify patients who received MV for >48 hours and who had signifi cant chronic pulmonary disease or left/right ventricular impairment between April 2009 and March 2012. Age, APACHE II score, ventilated days, time in NAVA and outcome were compared between groups who had or had not received EAdi monitoring. P150 Novel method to confi rm tracheal intubation based on airway pressures: validation in patients with lung disease A Stemerdink1, KG Monsieurs2, F Luijmes1, W Dieperink1, JG Zijlstra1, AF Kalmar1 1University Medical Center Groningen, the Netherlands; 2Antwerp University Hospital, Antwerp, Belgium Critical Care 2013, 17(Suppl 2):P150 (doi: 10.1186/cc12088) P150 Novel method to confi rm tracheal intubation based on airway pressures: validation in patients with lung disease A Stemerdink1, KG Monsieurs2, F Luijmes1, W Dieperink1, JG Zijlstra1, AF Kalmar1 1University Medical Center Groningen, the Netherlands; 2Antwerp University Hospital, Antwerp, Belgium Critical Care 2013, 17(Suppl 2):P150 (doi: 10.1186/cc12088) P150 Novel method to confi rm tracheal intubation based on airway pressures: validation in patients with lung disease A Stemerdink1, KG Monsieurs2, F Luijmes1, W Dieperink1, JG Zijlstra1, AF Kalmar1 1University Medical Center Groningen, the Netherlands; 2Antwerp University Hospital, Antwerp, Belgium Critical Care 2013, 17(Suppl 2):P150 (doi: 10.1186/cc12088) Results A total of 470 BC were carried out in 240 patients admitted to the ICU (68% were male and 32% were female). All of the procedures were performed in mechanically ventilated patients. The mean patient age was 61 ± 14 years, with an APACHE II score at admission of 20 ± 7. The main cause for ICU admission was respiratory failure (27%). The most common comorbidities were smoking, chronic obstructive pulmonary disease, cardiovascular risk factors and atrial fi brillation. The main indications for performing the BC were percutaneous tracheostomy endoscopically guided (28%), resolution of atelectasis (27%), aspiration of secretions (24%) and hemoptysis (10%). Bronchial brushing was performed in 435 cases (93%) obtaining microbiological growth in 64% of them, mainly Pseudomonas aeruginosa and Acinetobacter baumanii. Bronchoalveolar lavage was performed in 62 cases (13%) obtaining microbiological growth in 61% of them, mainly cytomegalovirus and Stenotrophomona maltophilia. Citology was performed in 60 cases (13%); 7% showed the presence of microorganism and 13% revealed precancerous/cancerous tissue changes. Introduction Emergency endotracheal intubation results in accidental oesophageal intubation in up to 17% of patients often with disastrous consequences. We have previously published a highly specifi c and sensitive novel method to detect endotracheal intubation based on diff erences in ventilation pressure waveforms in the oesophagus and in the trachea in patients with healthy lungs [1]. A detection algorithm, based on diff erences in compliance/elasticity between the lung and the oesophagus, generated a D-value indicating tracheal intubation if D >0.5 and oesophageal intubation if D <0.5. The aim of the current study was to validate the algorithm in patients with lung disease. p g Conclusion BC can be performed at the bedside, avoiding transfers outside the ICU that may cause clinical worsening. Most frequent indications were bronchoscopy-guided percutaneous tracheostomy, resolution of atelectasis, aspiration of secretions and hemoptysis. P150 Novel method to confi rm tracheal intubation based on airway pressures: validation in patients with lung disease A Stemerdink1, KG Monsieurs2, F Luijmes1, W Dieperink1, JG Zijlstra1, AF Kalmar1 1University Medical Center Groningen, the Netherlands; 2Antwerp University Hospital, Antwerp, Belgium Critical Care 2013, 17(Suppl 2):P150 (doi: 10.1186/cc12088) Microbiological results obtained revealed the presence of aggressive pathogens. BC in ICU provides valuable diagnostic information and has therapeutic utility. y g p g Methods After obtaining institutional approval, 20 intubated and ventilated ICU patients were included. Inclusion criteria were controlled mechanical ventilation and at least mild to moderate lung injury according to a Murray lung injury score >0.1. A connecting piece was placed between the endotracheal tube and the ventilation bag. This piece comprised a thin air-fi lled catheter inserted through the tube lumen at 1 cm from the distal end, and a second catheter located at the proximal end of the tube. We performed three consecutive manual bag ventilations while recording the pressure curves through both catheters. For each ventilation, a D-value was calculated. y References References 1. Shivaram U, et al.: Eff ects of continuous positive airway pressure in acute asthma. Respiration 1987, 52:157-162. 2. Meduri GU, et al.: Noninvasive positive pressure ventilation in status asthmaticus. Chest 1996, 110:767-774. 3. Lin HC, et al.: Eff ect of nasal continuous positive airway pressure on methacholine-induced bronchoconstriction. Respir Med 1995, 89:121-128. 1. Shivaram U, et al.: Eff ects of continuous positive airway pressure in acute asthma. Respiration 1987, 52:157-162. 2. Meduri GU, et al.: Noninvasive positive pressure ventilation in status asthmaticus. Chest 1996, 110:767-774. 3. Lin HC, et al.: Eff ect of nasal continuous positive airway pressure on methacholine-induced bronchoconstriction. Respir Med 1995, 89:121-128. 1. Shivaram U, et al.: Eff ects of continuous positive airway pressure in acute asthma. Respiration 1987, 52:157-162. 2. Meduri GU, et al.: Noninvasive positive pressure ventilation in status asthmaticus. Chest 1996, 110:767-774. 3. Lin HC, et al.: Eff ect of nasal continuous positive airway pressure on methacholine-induced bronchoconstriction. Respir Med 1995, 89:121-128. 1. Shivaram U, et al.: Eff ects of continuous positive airway pressure in acute asthma. Respiration 1987, 52:157-162. Introduction Bronchoscopy (BC) is a frequently used diagnostic and therapeutic tool in our ICU. We planned to review the patients and conditions in which the procedure was performed. The objective was to determine indications, diagnostic and therapeutic yield. 2. Meduri GU, et al.: Noninvasive positive pressure ventilation in status asthmaticus. Chest 1996, 110:767-774. 2. Meduri GU, et al.: Noninvasive positive pressure ventilation in status asthmaticus. Chest 1996, 110:767-774. 3. Lin HC, et al.: Eff ect of nasal continuous positive airway pressure on methacholine-induced bronchoconstriction. Respir Med 1995, 89:121-128. 3. Lin HC, et al.: Eff ect of nasal continuous positive airway pressure on methacholine-induced bronchoconstriction. Respir Med 1995, 89:121-128. 3. Lin HC, et al.: Eff ect of nasal continuous positive airway pressure on methacholine-induced bronchoconstriction. Respir Med 1995, 89:121-128. Methods A retrospective, single-center observational study was carried out. All BC performed in a 20-bed polyvalent ICU of a tertiary university hospital over a period of 30 months (from January 2010 to June 2012) were included. The main variables collected were demographic data and clinical data including comorbidities, endobronchial techniques and results. References 1. Lucena CM, et al.: Med Intensiva 2012, 36:389-395. 2. Estella A, et al.: Med Intensiva 2012, 36:396-401. 3. Shennib H, et al.: Chest Surg Clin N Am 1996, 6:349-361. 4. Turner JS, et al.: Crit Care Med 1994, 22:259-264. 1. Lucena CM, et al.: Med Intensiva 2012, 36:389-395. Results Mean age (SD) of the patients was 60 (16) years, 60% were male. The mean (SD) Murray score was 1.4 (0.6). Pathologies included pulmonary oedema, pneumonia, atelectasis and traumatic lung injury. All 60 D-values are represented in Figure 1. The median (IQR, range) D-value was 38 (16 to 74, 0.8 to 1,272). Our algorithm therefore confi rmed a high sensitivity to detect correct endotracheal intubation also in patients with lung disease. Under the hypothesis that oesophageal compliance does not increase signifi cantly in patients with lung disease, the specifi city of our algorithm will not be aff ected. 9 Impact of continuous positive airway pressure on the treatment of acute exacerbation of asthma: a randomised controlled trial Y Sutherasan, S Kiatboonsri, P Theerawit, C Kiatboonsri, S Trakulsrichai Ramathibothi Hospital, Bangkok, Thailand Critical Care 2013, 17(Suppl 2):P149 (doi: 10.1186/cc12087) Data collections in both groups included peak expiratory fl ow rates (PEFR, %predicted) at each 15-minute interval (T0, T15, …), the number of short-acting bronchodilators (SABA) used, ED length of stay, relapse rate and admission rate. The primary outcome is the improvement in peak expiratory fl ow rate during a short period stay in the ED. Conclusion Preliminary results demonstrate diagnostic accuracy of lung ultrasound examination performed on the admission of patients with acute respiratory failure similar to the original publication. Lung ultrasound, as proposed in the BLUE protocol, has good accuracy, and seems reproducible and useful in this group of patients. f p yl g p y Results There were 86 patients enrolled, with 43 patients in each study group. PEFR in the CPAP group showed no diff erence from the control group at T0 (52.7 ± 20.33 vs. 47.2 ± 16.64, P = NS) but was signifi cantly better at T15 (65.58 ± 21.64 vs. 56.49 ± 18.53%, P <0.05). Such improvement was consistently observed for all subsequent PEFR measurement, along with the fewer SABA used and relapse rate, but the fi gures did not meet statistical diff erence. 1. Lichtenstein DA, Mezière GA: Relevance of lung ultrasound in the diagnosis of acute respiratory failure: the BLUE protocol. Chest 2008, 134:117-125. Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S56 http://ccforum.com/supplements/17/S2 Conclusion The addition of CPAP to conventional acute asthma treatment in the ED could accelerate PEFR improvement with a trend showing fewer SABA needed, lower relapse rate and shorter ED length of stay. P151 Main indications, diagnostic and therapeutic yield of bronchoscopy in the ICU J Iglesias, B Gonzalez, O Rajas La Princesa Hospital, Madrid, Spain Critical Care 2013, 17(Suppl 2):P151 (doi: 10.1186/cc12089) P152 P152 Benchtop study comparing leakages across cuff s of three endotracheal tubes SM Lam, CW Lau, WW Yan Pamela Youde Nethersole Eastern Hospital, Hong Kong Critical Care 2013, 17(Suppl 2):P152 (doi: 10.1186/cc12090) Conclusion The algorithm to detect correct endotracheal intubation performed excellent in patients with lung disease. Reference Introduction The aim was to compare two novel endotracheal tubes (ETT), Mallinckrodt TaperGuard (TG, tapered polyvinyl chloride (PVC) cuff ) and KimVent Microcuff (MC, cylindrical polyurethrane cuff ), 1. Kalmar AF, et al.: Resuscitation 2012, 83:177-182. Figure 1 (abstract P150). D-value of individual test ventilations. Figure 1 (abstract P150). D-value of individual test ventilations. Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 S57 http://ccforum.com/supplements/17/S2 Figure 1 (abstract P152). Downward leak in PEEP 0 cmH2O without suction. Each point represents the mean and standard deviation of eight repeated measures. with SSD. However, the shape most suitable for preventing dripping onto the subglottis has not been determined. The purpose of this study was to determine whether an ET with tapered-type cuff can reduce the incidence of VAP. Methods After approval from the appropriate ethics committee, we conducted a single-institutional prospective randomized clinical trial on the eff ectiveness of using an ET with a diff erent cuff type. Adult patients (n = 399; ≥18 years old) were screened between April 2010 and March 2012, and 289 patients expected to require mechanical ventilation (MV) for at least 48 hours were randomized. Patients were assigned to intubation with one of the following two HVLP ETs with SSD on the experimental tube: tapered type (Taperguard™ Evac®; Covidien, Dublin, Ireland) or spindle type (Hi-Lo Evac®; Covidien). Cuff pressure measurement was carried out every 6 hours, and cuff pressure was maintained between 20 and 30 cmH2O. The primary outcome was VAP incidence based on semiquantitative bronchoalveolar lavage fl uid culture of 3+ or phagocytosis on Gram staining in patients intubated for at least 48 hours. The other outcome was the rate of achieving an appropriate cuff pressure (20 to 30 cmH2O), time to VAP onset, duration of MV, duration of ICU stay, mortality, and adverse events. Figure 1 (abstract P152). Downward leak in PEEP 0 cmH2O without suction. Each point represents the mean and standard deviation of eight repeated measures. Eff ect of body position changes on endotracheal tube cuff pressure in ventilated patients Fernandez JF, et al.: Chest 2012, 142:231-238. 1. Fernandez JF, et al.: Chest 2012, 142:231-238. P152 y y Results The rate of microbiologically confi rmed VAP was 21.7% (23/106) for the tapered-type ET patients and 21.7% (23/106) for the spindle-type ET patients (P = 1.00). The rate of achieving appropriate cuff pressure was 83.2% (332/1,974) for the tapered type and 82.4% (328/1,867) for the spindle type (P = 0.549). No signifi cant diff erences between groups were observed for time to VAP onset, duration of MV, ICU stay. The incidence of reintubation due to laryngeal edema after extubation was slightly higher in patients with the tapered-type ET (11.5%, 6/52) than in patients with the spindle-type ET (2.0%, 1/49), but the diff erence was not signifi cant (P = 0.113).ff with conventional Portex (PT, globular PVC cuff ) in leakages across cuff s (microaspiration) under simulated clinical situations. It has been shown that globular PVC cuff s protect poorly against leakages due to microchannels formed from infolding of redundant cuff material [1]. We hypothesized that TG and MC better prevent microaspiration, which is a major mechanism of ventilator-associated pneumonia (VAP). Methods Each ETT was inserted into a silicone cylinder of 2 cm wide inclined at 35°. Then 20 ml water was added above the cuff and leakage measured every minute under fi ve diff erent simulated clinical conditions: mechanical ventilation for acute severe asthma (positive end-expiratory pressure (PEEP) 0 cmH2O), normal lungs (PEEP 5 cmH2O) and acute respiratory distress syndrome (PEEP 10 cmH2O), and disconnection from the ventilator with and without spontaneous breathing eff ort. Spontaneous breathing was simulated with a respiratory gas exchange simulator. Suction was applied at 200 cmH2O sustained for 3 minutes at the Murphy eye. Each scenario was repeated with cuff pressures (Pcuff ) 10, 20 and 30 cmH2O maintained by a Pcuff maintenance device. Conclusion Diff erences in cuff type and shape under identical conditions of cuff pressure control have no infl uence on the incidence of VAP. Acknowledgement UMIN-CTR UMIN000003371. Acknowledgement UMIN-CTR UMIN000003371. P154f Eff ect of body position changes on endotracheal tube cuff pressure in ventilated patients Eff ect of body position changes on endotracheal tube cuff pressu in ventilated patients C Lizy1, W Swinnen2, S Labeau3, S Blot1 1Gent University, Ghent, Belgium; 2General Hospital Sint Blasius, Dendermonde, Belgium; 3University College Ghent, Belgium Critical Care 2013, 17(Suppl 2):P154 (doi: 10.1186/cc12092) C Lizy1, W Swinnen2, S Labeau3, S Blot1 1Gent University, Ghent, Belgium; 2General Hospital Sint Blasius, Dendermonde, Belgium; 3University College Ghent, Belgium Critical Care 2013, 17(Suppl 2):P154 (doi: 10.1186/cc12092) Results PT leaked grossly in all scenarios without PEEP and at PEEP 5 cmH2O in the presence of suction irrespective of Pcuff (Pcuff 30 cmH2O: PT 19.7 ± 0.4, TG 0.0 ± 0.1, MC 0.0 ± 0.0, P <0.001; Pcuff 20 cmH2O: PT 19.9 ± 0.4, TG 7.4 ± 6.2, MC 0.0 ± 0.0, P <0.001; Pcuff 10 cmH2O: PT 20.0 ± 0.0, TG 12.7 ± 5.1, MC 0.9 ± 0.8, P <0.001). Leakage under these scenarios can be reduced in TG and prevented in MC by Pcuff ≥20 cmH2O (Figure 1).f Introduction In order to avoid either microaspiration (<20 cmH2O) or tracheal injury (>30 cmH2O), the target for endotracheal tube (ETT) cuff pressure (CP) is between 20 and 30 cmH2O. Our objective was to assess the eff ect of patient positioning changes on CP in adult patients. f 2 Conclusion Microcuff outperformed the others in preventing microaspiration, while Portex leaked grossly even at a recommended Pcuff of 20 to 30 cmH2O whenever PEEP was lost. The eff ect of ETT type on the incidence of VAP warrants further investigation. Reference Methods A sample of 12 orally intubated and sedated patients was selected. Patients received neuromuscular blockade and were positioned in a neutral start position (backrest, head of bed elevation (HoB) 30°, head in neutral position) and CP set at 25 cmH2O. Subsequently 16 diff erent position changes were performed: antefl exion head, hyperextension head, left and right lateral fl exion of head, left and right rotation of the head, semirecumbent position (HoB 45°), recumbent position (HoB 10°), horizontal position, trendelenburg (–10°), left and right lateral positioning over 30°, 45° and 90°. Once the patient was correctly positioned, CP was recorded during an end-expiratory ventilatory hold. CP observed was compared with CP at start position (25 cmH2O). Also the number of values outside the target range (20 to 30 cmH2O) was reported as considered clinically relevant. 1. Fernandez JF, et al.: Chest 2012, 142:231-238. 1. P153 Endotracheal tube with tapered-type cuff for preventing ventilator-associated pneumonia: a randomized clinical trial N Saito, T Yagi, Y Hara, H Matsumoto, K Mashiko Nippon Medical School Chiba Hokusoh Hospital, Inzai, Chiba, Japan Critical Care 2013, 17(Suppl 2):P153 (doi: 10.1186/cc12091) P153 Endotracheal tube with tapered-type cuff for preventing ventilator-associated pneumonia: a randomized clinical trial N Saito, T Yagi, Y Hara, H Matsumoto, K Mashiko Nippon Medical School Chiba Hokusoh Hospital, Inzai, Chiba, Japan Critical Care 2013, 17(Suppl 2):P153 (doi: 10.1186/cc12091) 2 y Results A total of 192 measurements were performed (12 subjects×16 positions). Results are shown in Table 1 (CP reported as median, IQR). In every position a signifi cant deviation in CP was observed. In total, 40.6% of values exceeded the upper limit of 30 cmH2O. No values beneath the lower target limit of 20 cmH2O were observed. In each position the upper target limit was exceeded at least once. Conclusion Simple changes in patient positioning may result in potentially harmful CP (>30 cmH2O). These observations call for strict CP monitoring. 2 Results A total of 192 measurements were performed (12 subjects×16 positions). Results are shown in Table 1 (CP reported as median, IQR). In every position a signifi cant deviation in CP was observed. In total, 40.6% of values exceeded the upper limit of 30 cmH2O. No values beneath the lower target limit of 20 cmH2O were observed. In each position the upper target limit was exceeded at least once. Introduction The most common cause of ventilator-associated pneumonia (VAP) is aspiration of oral secretion through the endotracheal tube (ET). Subglottic suction drainage (SSD) has been recommended as a safety measure against aspiration due to its high eff ectiveness. Currently, two types of cuff shape – spindle and tapered – are predominant in high-volume, low-pressure (HVLP) ETs Conclusion Simple changes in patient positioning may result in potentially harmful CP (>30 cmH2O). These observations call for strict CP monitoring. Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S58 Table 1 (abstract P154). P155 Towards safer airway management in the critically ill: lessons from National Audit Project 4 Introduction National Audit Project 4 (NAP4) highlighted the need to improve airway management in ICUs and key recommendations were the continuous use of end-tidal carbon dioxide (ETCO2) monitoring, pre-intubation checklists and diffi cult airway trolleys [1]. This complete cycle audit aimed to quantify the current state of airway management on our ICU and the eff ectiveness of implementing the NAP4 recommendations. Results The studied ETTs remained in place for a median of 7 days (IQR range 4 to 15). The amount of secretions assessed by CT scan was 0.293 ± 0.290 ml (range 0.032 to 0.777 ml). Secretion volumes were not related to patient severity at admission (SAPS 2, P/F ratio) or days of intubation; an inverse correlation with patient’s age was present (P = 0.032, R2 = 0.46). Bacterial growth was present in 9/11 (82%) ETT fl uids cultures and Candida spp. showed an elevated prevalence (6/11, 55%). In the bench tests, the cleaning device reduced resistance to airfl ow (diff erence before and after cleaning 5.5 (95% CI = 8.9 to 1.6) cmH2O/l/second, P = 0.006). After cleaning, resistance resulted higher than intact ETTs, although with a clinically negligible diff erence (diff erence 0.3 (95% CI = 0.2 to 0.6 cmH2O/l/second), P = 0.032). Methods Data collection was carried out prospectively for both phases and included documentation of intubation, use of ETCO2 and the incidence of serious adverse events (SAEs). The contents of the intubation boxes were compared against the Diffi cult Airway Society (DAS) guidelines [2]. The re-audit was carried out 6 months following the introduction of a pre-intubation checklist, a documentation sticker, a diffi cult airway trolley and standardization of the basic bedside airway boxes with a checklist of contents. A training program in airway management for all ICU staff was also introduced. f 2 Conclusion Micro-CT scan is a feasible and promising technique to assess secretions volume in ETTs after extubation. The use of an ETT cleaning device decreases resistance to airfl ow in bench tests; the eff ectiveness of such a device in the clinical setting could be properly assessed by post-extubation CT scan. gf Results The baseline characteristics of both groups were similar. The initial audit included 45 patients and the re-audit 58 patients. In the initial audit 19% of patients had accurate documentation of intubation with the use of ETCO2. The continuous use of ETCO2 was 80%. P153 Endotracheal tube with tapered-type cuff for preventing ventilator-associated pneumonia: a randomized clinical trial N Saito, T Yagi, Y Hara, H Matsumoto, K Mashiko Nippon Medical School Chiba Hokusoh Hospital, Inzai, Chiba, Japan Critical Care 2013, 17(Suppl 2):P153 (doi: 10.1186/cc12091) Endotracheal tube cuff pressure values following patient position changes Cuff P for Δ from Measurements pressure start position >30 cmH2O (cmH2O)a (25 cmH2O) (n (%)) Antefl exion head 33.5 (27.5 to 42.0) 0.005 9 (75) Hyperextension head 30.5 (27.5 to 42.0) 0.004 6 (50) Lateral fl exion head, left 26.5 (25.3 to 28.8) 0.025 1 (8) Lateral fl exion head, right 27.0 (25.3 to 29.8) 0.049 2 (17) Rotation head, left 27.0 (26.3 to 33.8) 0.003 3 (25) Rotation head, right 28.0 (26.3 to 38.8) 0.020 4 (33) Semirecumbent, 45° HoB 28.0 (25.3 to 31.5) 0.009 2 (17) Recumbent, 10° HoB 28.0 (25.3 to 31.5) 0.016 3 (25) Horizontal back rest 28.5 (25.3 to 31.5) 0.011 3 (25) Trendelenburg, 10° 29.0 (25.3 to 32.0) 0.008 5 (42) Left lateral position, 30° 30.5 (25.3 to 32.0) 0.010 6 (50) Left lateral position, 45° 30.5 (27.3 to 32.8) 0.005 6 (50) Left lateral position, 90° 34.0 (27.0 to 36.8) 0.006 7 (58) Right lateral position, 30° 31.0 (27.0 to 34.5) 0.003 6 (50) Right lateral position, 45° 32.5 (28.3 to 35.8) 0.002 8 (50) Right lateral position, 90° 31.0 (29.3 to 35.0) 0.003 7 (58) Total (192 measurements) – – 78 (40.6) HoB, head of bed elevation. aCuff pressure values reported as median (fi rst to third quartile). Table 1 (abstract P154). Endotracheal tube cuff pressure values following patient position changes This audit demonstrated that simple measures in conjunction with a structured training program can help to improve the safety of airway management in critically ill patients. Further work is required to improve compliance with the pre-intubation checklist alongside a continued education program. References 1. The Royal College of Anaesthetists [www.rcoa.ac.uk/nap4] 2. Diffi cult Airway Society Equipment List [http://www.das.uk.com/equipmentlistjuly2005.htm] P156 Quantitative assessment of endotracheal tube (ETT) biofi lm by micro-CT scan: evaluation of the eff ectiveness of ETT cleaning devices A Coppadoro1, G Bellani1, T Mauri1, R Borsa1, V Meroni1, A Barletta1, M Teggia Droghi1, A Lucchini2, R Marcolin2, N Patroniti1, S Bramati2, A Pesenti1 1University of Milan-Bicocca, Monza, Italy; 2San Gerardo Hospital, Monza, Italy Critical Care 2013, 17(Suppl 2):P156 (doi: 10.1186/cc12094) Introduction The goal of this study was to assess the amount of biofi lm within the endotracheal tube (ETT) by CT scan and to evaluate in a bench test a device aimed at ETT biofi lm removal. P157 P157 Early identifi cation of patients at risk of diffi cult intubation in the ICU: development and validation of the MACOCHA score in a multicenter cohort study A De Jong1, N Molinari1, N Terzi2, N Mongardon3, B Jung1, S Jaber1 1Montpellier University Hospital, Montpellier, France; 2Caen University Hospital, Caen, France; 3Cochin University Hospital, Paris, France Critical Care 2013, 17(Suppl 2):P157 (doi: 10.1186/cc12095) P153 Endotracheal tube with tapered-type cuff for preventing ventilator-associated pneumonia: a randomized clinical trial N Saito, T Yagi, Y Hara, H Matsumoto, K Mashiko Nippon Medical School Chiba Hokusoh Hospital, Inzai, Chiba, Japan Critical Care 2013, 17(Suppl 2):P153 (doi: 10.1186/cc12091) Biofi lm grows within the ETT soon after intubation, resulting in increased resistance to airfl ow. Biofi lm detachment from the ETT can result in bacterial colonization of the distal airways. The use of an ETT cleaning device could eff ectively reduce the patient’s airfl ow resistance burden. The quantifi cation and distribution of biofi lm within the ETT might help to evaluate ETT cleaning device eff ectiveness. gf Methods We collected ETTs of 11 critically ill patients after extubation. Micro-CT scan (SkyScan 1176; Bruker, Belgium) was performed using a resolution of 35 μm. Axial sections of the 20 cm above the cuff were reconstructed, and the volume of secretions was assessed by a density criterion. Microbiological cultures of the ETT lavage fl uid were then obtained. Patient’s demographics and clinical data were recorded. In a diff erent set of bench experiments, we injected 1 ml water-based polymer into new ETTs of diff erent sizes. We measured resistance to airfl ow before and after using an ETT cleaning device (Airway Medix Closed Suction System; Biovo Technologies, Tel Aviv, Israel). We also obtained resistance values of intact ETTs as controls. HoB, head of bed elevation. aCuff pressure values reported as median (fi rst to third quartile). P155 Towards safer airway management in the critically ill: lessons from National Audit Project 4 Bedside airway boxes did not contain a checklist of standardized equipment. A diffi cult airway trolley was not located on the ICU. Following introduction of the changes described, a signifi cant improvement in the use of ETCO2 to confi rm intubation (46% vs. 19%, P <0.01) and continuous use of ETCO2 monitoring (100% vs. 80% P ≤0.0004) was observed. The use of the pre- intubation checklist (7%) and stickers (11%) was poor. All bedside boxes had a checklist and the required equipment. A diffi cult airway trolley is located on the ICU with an equipment list and DAS algorithm attached. No SAEs were recorded in either phase. Early identifi cation of patients at risk of diffi cult intubation in the ICU: development and validation of the MACOCHA score in a multicenter cohort study A De Jong1, N Molinari1, N Terzi2, N Mongardon3, B Jung1, S Jaber1 1Montpellier University Hospital, Montpellier, France; 2Caen University Hospital, Caen, France; 3Cochin University Hospital, Paris, France Critical Care 2013, 17(Suppl 2):P157 (doi: 10.1186/cc12095) A De Jong1, N Molinari1, N Terzi2, N Mongardon3, B Jung1, S Jaber1 1Montpellier University Hospital, Montpellier, France; 2Caen University Hospital, Caen, France; 3Cochin University Hospital, Paris, France Critical Care 2013, 17(Suppl 2):P157 (doi: 10.1186/cc12095) Introduction Diffi cult intubation (DI) in the ICU is a challenging issue, associated with severe life-threatening complications [1,2]. The objective was to develop and validate a simplifi ed score for identifying patients with DI in the ICU and to report related complications. Introduction Diffi cult intubation (DI) in the ICU is a challenging issue, associated with severe life-threatening complications [1,2]. The objective was to develop and validate a simplifi ed score for identifying patients with DI in the ICU and to report related complications. Conclusion Serious airway complications are rare. Safer airway manage ment is essential in the ICU to prevent morbidity and mortality. S59 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Methods Data collected in a prospective multicenter-study from 1,000 consecutive intubations from 42 ICUs were used to develop a simplifi ed score of DI, which was then validated externally in 400 consecutive intubation procedures from 18 other ICUs and internally by bootstrap on 1,000 iterations. common indications of intubation were acute respiratory failure (61.0%) and coma (17.1%). The primary outcome occurred in 138 of 246 intubations (56.1%). Within the fi rst hour, the incidence of severe hypotension was 47.2%, of severe hypoxemia was 20.3%, of cardiac arrest was 5.7% and of death was 4.5%. Results In multivariate analysis, the main predictors of DI (incidence = 11.3%) were related to the patient (Mallampati score III or IV, obstructive sleep apnea syndrome, reduced mobility of cervical spine, limited mouth opening), to pathology (severe hypoxia, coma) and to the operator (non-anesthesiologist). From the β-parameter, a seven- item simplifi ed score (MACOCHA score; Table 1) was built, with an area under the curve (AUC) of 0.89 (95% CI = 0.85 to 0.94). Tracheal intubation for a diffi cult airway using Airway scope®, KingVision® and McGRATH®: a comparative manikin study of inexperienced personnel J Itai1, Y Tanabe2, T Nishida1, T Inagawa1, Y Torikoshi1, Y Kida1, T Tamura1, K Ota1, T Otani1, T Sadamori1, K Une1, R Tsumura1, Y Iwasaki1, N Hirohashi1, K Tanigawa1 J Itai1, Y Tanabe2, T Nishida1, T Inagawa1, Y Torikoshi1, Y Kida1, T Tamura1, K Ota1, T Otani1, T Sadamori1, K Une1, R Tsumura1, Y Iwasaki1, N Hirohashi1, K Tanigawa1 g 1Hiroshima University Hospital, Hiroshima-shi, Japan; 2Hiroshima City Hospital, Hiroshima-shi, Japan Critical Care 2013, 17(Suppl 2):P159 (doi: 10.1186/cc12097) g 1Hiroshima University Hospital, Hiroshima-shi, Japan; 2Hiroshima City Hospital, Hiroshima-shi, Japan Critical Care 2013, 17(Suppl 2):P159 (doi: 10.1186/cc12097) g 1Hiroshima University Hospital, Hiroshima-shi, Japan; 2Hiroshima City Hospital, Hiroshima-shi, Japan Critical Care 2013, 17(Suppl 2):P159 (doi: 10.1186/cc12097) Table 1 (abstract P157). MACOCHA score calculation worksheet Points Factors related to patient Mallampati score III or IV 5 Obstructive apnea syndrome 2 Reduced mobility of cervical spine 1 Limited mouth opening <3 cm 1 Factors related to pathology Coma 1 Severe hypoxemia (<80%) 1 Factor related to operator Non-anaesthesiologist 1 Total 12 Conclusion DI in ICU is strongly associated with severe life-threatening complications. A simple score including seven clinical items discriminates diffi cult and non-DI in ICU. References 1. Jaber et al.: Crit Care Med 2006, 34:2355-2361. 2. Jaber et al.: Intensive Care Med 2010, 36:248-255. P158 Incidence of major complications related to endotracheal intubation in ICUs of nonacademic Brazilian public hospitals AB Cavalcanti1, K Normilio-da-Silva1, JC Acarine Mouro1, F Moreira1, AA Kodama1, R Del-Manto2, O Berwanger1 1Hospital do Coração – HCor, São Paulo, Brazil; 2Hospital Militar de Área de São Paulo – HMASP, São Paulo, Brazil Critical Care 2013, 17(Suppl 2):P158 (doi: 10.1186/cc12096) d b d h d f Table 1 (abstract P157). MACOCHA score calculation worksheet Points Factors related to patient Mallampati score III or IV 5 Obstructive apnea syndrome 2 Reduced mobility of cervical spine 1 Limited mouth opening <3 cm 1 Factors related to pathology Coma 1 Severe hypoxemia (<80%) 1 Factor related to operator Non-anaesthesiologist 1 Total 12 Table 1 (abstract P157). MACOCHA score calculation worksheet Table 1 (abstract P157). MACOCHA score calculation worksheet Introduction The Airway scope® (AWS), the KingVision® (KV) and the McGRATH® (MG) are new indirect video laryngoscopes designed to facilitate eff ective and safe tracheal intubation under various conditions. However, there are few comparative studies as for performance in tracheal intubation attempted by inexperienced personnel. Early identifi cation of patients at risk of diffi cult intubation in the ICU: development and validation of the MACOCHA score in a multicenter cohort study In the validation cohort (prevalence of DI = 8%), the AUC was of 0.86 (95% CI = 0.76 to 0.96), with a sensitivity of 73%, a specifi city of 89%, a negative predictive value of 98% and a positive predictive value of 36%. After internal validation by bootstrap, the AUC was 0.89 (95% CI = 0.86 to 0.93). Severe life-threatening events (severe hypoxia, collapse, cardiac arrest or death) occurred in 38% of the 1,000 cases. Patients with DI (n = 113) had signifi cantly higher severe life-threatening complications than those who had a non-DI (51% vs. 36%, P <0.0001). Conclusion Incidence of major complications associated with endotracheal intubation is unacceptably high in Brazilian public ICUs. There is an urgent need for eff ective measures to increase safety of this common procedure in our environment. Tracheal intubation for a diffi cult airway using Airway scope®, KingVision® and McGRATH®: a comparative manikin study of inexperienced personnel The purpose of this study was to evaluate success rates, time to intubation with use of these devices by inexperienced personnel in a simulated manikin diffi cult airway.i Methods Twenty-nine fi fth-year medical students with no previous experience in tracheal intubation participated in this study. We used an advanced patient simulator (SimMan®; Laerdal Medical, Stavanger, Norway) to simulate normal and diffi cult airway scenarios including cervical spine rigidity, swollen tongue, and pharyngeal edema. The sequences in selecting devices and scenarios were randomized. Success rate for tracheal intubation, and the time required for visualization of the glottis (T1), tracheal intubation (T2), and infl ation of the lungs (T3) were analyzed. Also, numbers of audible dental click during the intubation attempt were recorded. The three diff erent intubation devices were tested in four diff erent scenarios by 29 students. Results All three devices had very high success rates of tracheal intubation (AWS 100%; KV 100%; MG 99%). In the normal airway, T1, T2 and T3 were AWS: 4.4 ± 3.3 seconds, 7.5 ± 3.8 seconds, 11.1 ± 3.8 seconds; KV: 6.9 ± 6.9 seconds, 10.3 ± 7.9 seconds, 14.1 ± 8.3 seconds; MG: 4.6 ± 1.3 seconds, 11.4 ± 5.2 seconds, 16.1 ± 5.4 seconds, respectively (NS). In the three diffi cult airway scenarios, T1, T2 and T3 were AWS: 5.3 ± 4.7 seconds, 10.4 ± 6.9* seconds, 14.2 ± 6.9* seconds; KV: 10.9 ± 12.8 seconds, 21.5 ± 18.1 seconds, 25.4 ± 18.3 seconds; MG: 13.0 ± 15.3 seconds, 26.2 ± 24.6 seconds, 31.0 ± 24.3 seconds, respectively (P <0.05 AWS vs. KV and MG). The number of audible dental click sounds with the MG was greater than with the AWS and KV (AWS 5%; KV 8%; MG 28%; P <0.05 AWS and KV vs. MG). 1. Jaber et al.: Crit Care Med 2006, 34:2355-2361. 2. Jaber et al.: Intensive Care Med 2010, 36:248-255. P159 P159 Tracheal intubation for a diffi cult airway using Airway scope®, KingVision® and McGRATH®: a comparative manikin study of inexperienced personnel J Itai1, Y Tanabe2, T Nishida1, T Inagawa1, Y Torikoshi1, Y Kida1, T Tamura1, K Ota1, T Otani1, T Sadamori1, K Une1, R Tsumura1, Y Iwasaki1, N Hirohashi1, K Tanigawa1 1Hiroshima University Hospital, Hiroshima-shi, Japan; 2Hiroshima City Hospital, Hiroshima-shi, Japan Critical Care 2013, 17(Suppl 2):P159 (doi: 10.1186/cc12097) Incidence of major complications related to endotracheal intubation in ICUs of nonacademic Brazilian public hospitals Conclusion The AWS, KV and MG had very high success rates of tracheal intubation and are suitable as intubation devices for inexperienced personnel. In the diffi cult airway, however, the intubation time with AWS was signifi cantly shorter than with KV and MG. These fi ndings suggested that AWS may be most useful device particularly in diffi cult situations such as emergency settings. Further studies in a clinical setting are needed to confi rm these fi ndings. Introduction Our objective is to determine the incidence of major complications related to endotracheal intubations (death, cardiac arrest, severe hemodynamic instability or severe hypoxemia) per- formed in the ICUs of nonacademic Brazilian public hospitals. Methods This is a report of the baseline phase of a cluster randomized trial with two parallel arms. In this baseline phase, we collected data from sequential patients needing endotracheal intubation in 17 ICUs from Brazil. Patients needing endotracheal intubation after cardiac arrest were excluded. In a second ongoing phase, not reported here, we have randomized ICUs to a multifaceted intervention to prevent complications of endotracheal intubation. Primary outcome was defi ned as the occurrence of death, cardiac arrest, severe cardiovascular instability (systolic blood pressure <60 mmHg at least one time, <90 mmHg and persisting >30 minutes or need to initiate vasopressors) or severe hypoxemia (SpO2 <80%) within 1 hour after intubation. P160 NT-proBNP and cardiac cycle effi ciency changes during extubation process in critically ill patients i Results One hundred patients were surveyed. The ET and LT groups included 49 and 51 patients, respectively. Tracheostomy was performed using a percutaneous procedure in 48 patients (ET: 25, LT: 23) and a surgical procedure in 52 patients (ET: 24, LT: 28). Sixty-two patients (ET: 34, LT: 28) survived to discharge and 16 patients died in the ICU (ET: 7, LT: 9). Fifty-six patients (ET: 31, LT: 25) were weaned from ventilator support and tracheostomy cannula was removed in 20 patients (ET: 11, LT: 9). There were no signifi cant diff erences in type of tracheostomy procedure, period from tracheostomy until ICU and hospital discharge, rate of patients who could be weaned from ventilator and removed tracheostomy cannula, and ICU and hospital mortality between the groups. The length of mechanical ventilation and the time to removal of tracheostomy cannula were signifi cantly shorter in the ET group (5 ± 7 vs. 26 ± 41 and 29 ± 24 vs. 94 ± 83 days, respectively). Introduction Several factors can lead to weaning failure from mechanical ventilation (MV). Among these, cardiac dysfunction is one of the main causes. NT-proBNP has been proposed as a biomarker of cardiovascular function during weaning from MV. Unfortunately it does not provide for a continuous monitoring of cardiac function. Pulse wave analysis may serve as a continuous bedside monitoring tool of cardiovascular performance. Cardiac cycle effi ciency (CCE) is an indirect index of left ventricular performance obtained by the pulse contour method MostCare (Vygon, Padova, Italy). The aim of this study was to evaluate the correlation between NT-proBNP and CCE and the potential usefulness of such variables during the weaning process from MV. Conclusion In this retrospective study, early tracheostomy reduced the length of weaning after tracheostomy and the time to removal of tracheostomy cannula, while there were no diff erences in the length of ICU stay and patient outcome. In critically ill adult patients who require mechanical ventilation, a tracheostomy performed at an earlier stage may shorten the duration of artifi cial ventilation. A further randomized clinical trial is essential to determine the eff ectiveness and safety of early tracheostomy. Methods Twenty-two long-term (>48 hours) mechanically ventilated patients capable of performing a weaning trial of spontaneous breathing (SBT) were enrolled in the study. Inclusion criteria were: age >18 years and equipment with a standard arterial catheter line. Resistance of neonatal endotracheal tubes: a comparison of four commercially available types Introduction In mechanically ventilated neonates the fl ow-dependent resistance of the endotracheal tube (ETT) causes a noticeable pressure diff erence between airway and tracheal pressure [1]. This may potentially lead to retardation of the passive driven expiration and dynamic lung infl ation consecutively but more importantly increases Introduction In mechanically ventilated neonates the fl ow-dependent resistance of the endotracheal tube (ETT) causes a noticeable pressure diff erence between airway and tracheal pressure [1]. This may potentially lead to retardation of the passive driven expiration and dynamic lung infl ation consecutively but more importantly increases Results We evaluated 246 intubations. The mean age was 49 ± 21.9 years; the SAPS 3 admission score was 57.4 ± 15.4. The most S60 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 after extubation (T2, T3) compared with T1; conversely, CCE showed an inverse trend. Overall, a negative correlation was found between NT-proBNP and CCE values (R = –0.81, P <0.001). Signifi cant inverse correlations were found between NT-proBNP and CCE at T1, T2, and T3 (R = –0.91, –0.75 and –0.73 respectively; P < 0.001). The overall correlation between NT-proBNP and CCE was –0.74 in the SB group and –0.86 in the CPAP group. Standard hemodynamic and ventilatory data did not show signifi cant changes during the study. the work of breathing. The aim of this study was to compare the resistive pressure drop of four commercially available neonatal ETTs with internal diameter (ID) of 2.0 mm. the work of breathing. The aim of this study was to compare the resistive pressure drop of four commercially available neonatal ETTs with internal diameter (ID) of 2.0 mm. Methods The pressure–fl ow relationship of neonatal ETTs (internal diameter 2.0 mm) of four diff erent manufacturers (Mallinckrodt, Hi- Contour, Covidien, Dublin, Ireland; Portex, Cole’s Neonatal Tube, Smith Medical, St Paul, MN, USA; Rüsch, Silko Clear, Telefl ex Medical, Kernen, Germany; and Vygon, Pediatric Endotracheal Tube, Ecouen, France) was determined in a physical model consisting of a tube connector, an anatomically curved ETT and an artifi cial trachea. The model was ventilated with a sinusoidal gas fl ow with an amplitude of ±100 ml/ second and a ventilation rate ranging from 40 to 60 cycles/minute. The coeffi cients of an approximation equation considering ETT resistance were fi tted separately to the measured pressure–fl ow curves for inspiration and expiration. Comparison of outcomes between early and late tracheostomy for critically ill patients Guttmann J, et al.: Crit Care Med 2000, 28:1018-1026. Comparison of outcomes between early and late tracheostomy for critically ill patients Results The pressure drop profi les of all ETTs were nonlinearly fl ow dependent. The expiratory pressure drop (Pexp) slightly exceeded the inspiratory one (Pinsp). The ETT of Portex Cole’s Neonatal Tube had a signifi cant lower pressure drop (Pinsp 8.8 ± 0.3 cmH2O vs. Pexp –11.2 ± 0.2 cmH2O at an air fl ow of 100 ml/second) compared with all other ETTs (Mallinckrodt: Pinsp 19.2 ± 0.4 cmH2O vs. Pexp –24.3 ± 0.7 cmH2O; Rüsch: Pinsp 28.3 ± 0.8 cmH2O vs. Pexp –33.0 ± 0.8 cmH2O; Vygon: Pinsp 19.2 ± 0.4 cmH2O vs. Pexp –22.6 ± 0.6 cmH2O, all P <0.05) for all fl ow and ventilation rates, respectively. K Suzuki1, S Kusunoki1, T Yamanoue1, K Tanigawa2 1Hiroshima Prefectural Hospital, Hiroshima, Japan; 2Hiroshima University Hospital, Hiroshima, Japan Critical Care 2013, 17(Suppl 2):P162 (doi: 10.1186/cc12100) Introduction Tracheostomy is one of the more commonly performed procedures in critically ill patients requiring long-term mechanical ventilation. However, the optimal timing or method of performing tracheostomies in this population remains to be established. In the present study, we compared outcomes of early and late tracheostomy in critically adult patients with diff erent clinical conditions. y Conclusion The ETT resistance highly contributes to the total airway resistance in neonatal ventilation. The fl ow-dependent pressure drop of shouldered Cole’s Neonatal Tube (Portex) was up to 70% less in inspiration (resp. 67% in expiration) compared with straight tubes with an internal diameter corresponding to the narrow part of Cole’s tube. We conclude that neonatal intubation with a Cole’s tube can clearly reduce the resistive load due to the endotracheal tube and thus potentially prevent additionally work of breathing. Reference yf Methods All patients needing tracheostomy in the Critical Care Medical Center of Hiroshima Prefectural Hospital from January 2009 to December 2011 were surveyed. Patients with tracheostomy who were not indicated for mechanical ventilation were excluded from the subjects. Early tracheostomy (ET) was defi ned as <10 days after tracheal intubation and late tracheostomy (LT) was defi ned as ≥10 days after intubation. We compared patient characteristics, type of tracheostomy procedure, length of weaning from ventilator and outcomes between the groups. Data are shown as the mean ± SD, with unpaired t test and Mann–Whitney U test used for statistical analyses. Statistical signifi cance was accepted at P <0.05. 1. Guttmann J, et al.: Crit Care Med 2000, 28:1018-1026. 1. NT-proBNP and cardiac cycle effi ciency changes during extubation process in critically ill patients Exclusion criteria were: neuromuscular disease, tracheotomy, renal failure, and traumatic brain injury. During the weaning process, NT- proBNP plasma levels, CCE, and standard hemodynamic and ventilatory data were collected 30 minutes before extubation (T1), 2 hours (T2) and 12 hours later (T3). After removal of tracheal tube, patients with a history of heart failure received continuous positive airway pressure (CPAP group). Patients with normal cardiac function were maintained with spontaneous breathing (SB group). Reference . Arabi Y, et al.: Crit Care 2004, 8:R347-R352. Arabi Y, et al.: Crit Care 2004, 8:R347-R352. Arabi Y, et al.: Crit Care 2004, 8:R347-R352. Resistance of neonatal endotracheal tubes: a comparison of four commercially available types gi g g y Conclusion NT-proBNP correlated well with CCE. The latter seems to be an additional attractive index of cardiovascular state that, in association with NT-proBNP changes, may provide information about cardiac function on a beat-by-beat basis during weaning process from MV. P162 Ultrasound scanning for percutaneous dilatational tracheostomy: a systematic review y R Pugh, A Slater y R Pugh, A Slater y R Pugh, A Slater R Pugh, A Slater Glan Clwyd Hospital, Bodelwyddan, UK Critical Care 2013, 17(Suppl 2):P163 (doi: 10.1186/cc12101) Results Sixty-six paired NT-proBNP and CCE values were obtained. Patients in the SB group and in the CPAP group were 10 and 12, respectively. In both groups there was a trend towards an increase in NT-proBNP values after extubation, an opposite trend was observed regarding CCE values (P <0.05). NT-proBNP levels showed an increase Glan Clwyd Hospital, Bodelwyddan, UK Critical Care 2013, 17(Suppl 2):P163 (doi: 10.1186/cc12101) Introduction Percutaneous dilatational tracheostomy (PDT) remains a frequently performed procedure in the ICU. However, there is great S61 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Figure 1 (abstract P164). Oxygenation time to oxygen alveolar pressure >100 mmHg. Figure 2 (abstract P164). Mean carbon dioxide alveolar pressure at the start and at the end of the test. variability in the course of blood vessels in the pre-tracheal area. A 5% risk of clinically relevant bleeding was recently reported for patients undergoing PDT [1]. We conducted a systematic review of reports evaluating clinical outcomes following use of ultrasound scanning (US) for PDT. variability in the course of blood vessels in the pre-tracheal area. A 5% risk of clinically relevant bleeding was recently reported for patients undergoing PDT [1]. We conducted a systematic review of reports evaluating clinical outcomes following use of ultrasound scanning (US) for PDT. Figure 1 (abstract P164). Oxygenation time to oxygen alveolar pressure >100 mmHg. Methods Two investigators performed a search of the literature using the following databases: CENTRAL, Embase, MEDLINE and SCOPUS. The following eligibility criteria were used: population including adults >16 years managed in the ICU; use of ultrasound to guide decision- making pre-PDT or guide PDT performance; report of clinically relevant outcome measures. Nonrandomised controlled trials were classifi ed according to Cochrane Non-Randomised Study Methods Group criteria [2] and evaluated for risk of bias. [ ] Results An initial search identifi ed 2,043 reports, of which 10 studies met eligibility criteria: eight case series, one randomised controlled trial (RCT) and one prospective cohort study, incorporating 488 patients. Two studies specifi cally reported data on patients with obesity (n = 29 patients) and one study reported data for a group of patients with spinal cord fi xation (n = 6). Reference 1. Cook TM, Nolan JP, Magee PT, Cranshaw JH: Needle cricothyroidotomy. Anaesthesia 2007, 62:289-291. P Persona, P Diana, A Ballin, F Baratto, M Micaglio, C Ori g Clinica di Anestesia e Medicina Intensiva, Padova, Italy y Critical Care 2013, 17(Suppl 2):P164 (doi: 10.1186/cc12102) Introduction Failed airway situations are potentially catastrophic events and require a correct approach with appropriate tools. Recently, Ventrain has been presented as a manual device for emergency ventilation through a small-bore cannula, which can provide expiratory assistance by applying the Venturi eff ect. y R Pugh, A Slater US was used to guide decision to perform PDT or surgical tracheostomy in fi ve studies, with decision to perform surgical tracheostomy ranging from 0 to 27% of cases. US was used to guide insertion point in seven studies, and used real-time in four studies. Times to perform US-guided PDT were reported in four studies (ranging from 8 to 12 minutes). No studies compared time taken with or without US. Data on complications of procedure were reported in nine studies. Minor bleeding was reported for eight cases (1.6% overall). Prolonged bleeding was reported in two cases (0.4%). There were no episodes of catastrophic bleeding among 488 cases. High risk of bias was identifi ed in fi ve studies in terms of patient selection. An intervention protocol was not defi ned in three reports. No attempt was made at blinding any aspect of the 10 studies. Figure 2 (abstract P164). Mean carbon dioxide alveolar pressure at the start and at the end of the test. g y Conclusion Use of US guidance could theoretically help minimise risk of haemorrhagic complications during PDT and perhaps reduce time taken to perform PDT. However, there is currently inadequate evidence from controlled cohort studies or RCTs to suggest that routine use for PDT in selected or unselected groups improves clinically relevant outcome measure. References 1. Delaney et al.: Crit Care 2006, 10:R55. 2. Higgins JPT, Green S (Eds): Cochrane Handbook for Systematic Reviews of Interventions. Version 5.1.0. The Cochrane Collaboration; 2011. [www.cochrane-handbook.org] 2. Higgins JPT, Green S (Eds): Cochrane Handbook for Systematic Reviews of Interventions. Version 5.1.0. The Cochrane Collaboration; 2011. [www.cochrane-handbook.org] vs. Melker –0.3; vs. Quicktrach –5.9) (Figure 2) and moreover the users judged it more favorably. Conclusion In this manikin study, Ventrain seemed to be able to appropriately oxygenate and ventilate a patient in a CICV situation. When compared with the best available choices, it has shown not to be inferior. R f Evaluation of a new device for emergency transcricoid ventilation in a manikin model Reference g CW Carspecken, D Talmor This may represent a paucity of evidence on the subject, suggesting that further clinical trials on the topic are needed to contribute to the evidence base. This also highlights the need for international consensus on the topic, to reduce duplication of eff orts, standardise practice, and improve outcomes. Reference 1. Appraisal of Guidelines for Research and Evaluation: Instrument. AGREE II [http://www.agreetrust.org/] ble 1 (abstract P166). Summary of results returned following systematic review Total Results with Full texts Guidelines AGREE instrument atabase results limits retrieved found compliant bMed 4,685 1,596 33 EDLINE 3,859 1,337 17 26 2 NAHL 711 172 6 edical societies/guideline websites/clearinghouses N/A N/A N/A 54 3 oogle 17,600,000 497 N/A Introduction Protocol-based care of the tracheostomised patient is important, as adverse events confer a high rate of mortality. Little is known regarding the existence of formal evidence-based guidelines on tracheostomy care. The aim of this study was to perform a systematic review for evidence-based guidelines on adult tracheostomy care. (<$11) electronics were designed such that no power supply would be needed apart from a computer or mobile device. Collection of airfl ow signal and calibration was performed with a standard 3 l syringe with fl ow volume measurement on one adult subject. Results Calibration of the assembled device over range of 0.5 to 3 l/ second enabled conversion of pressure diff erence to air fl ow with a mean measured tube resistance of 0.258 (kPasecond)/l. Robust signal responses to an adult subject’s continuous respiratory maneuvers on the tube itself were demonstrated. Subject performance of the pMDI technique with subsequent tidal volume breathing was recorded and analyzed (Figure 1). Methods A systematic search of PubMed, MEDLINE, guideline clearinghouses, centres of evidence-based practice, and professional societies’ guidelines relating to care of adult patients with a tracheostomy was performed by two reviewers. In addition, a Google search of publicly available tracheostomy care guidelines was performed. Search terms: (tracheostom* OR tracheotom) AND (protocol OR guideline* OR standard* OR management OR consensus OR algorithm). Filters: English language, human, from 1 January 1990 to date, adult patients. Guideline appraisal criteria: the quality of guidelines retrieved was assessed using the Appraisal of Guidelines Research and Evaluation II (AGREE II) instrument [1]. y Conclusion Design and calibration of a novel low-cost monitoring device for bronchodilator delivery monitoring enabled detection and recording of characteristic fl ow volume respiratory patterns for point-of-care diagnostics in resource-limited settings. g CW Carspecken, D Talmor Future work will require clinical testing and automated detection of the correct pMDI patient technique. Results The search results are summarised in Table 1. A total of 80 guidelines were identifi ed. Five were found to satisfy the AGREE II criteria and only three related to the entire spectrum of tracheostomy management. The majority was informal and was not published or evidence based. p References References 1. Ari A, et al.: Respir Care 2012, 57:613-626. 2. Riviello E, et al.: Crit Care Med 2011, 39:860. 2. Riviello E, et al.: Crit Care Med 2011, 39:860. Conclusion Five evidence-based guidelines on adult tracheostomy management were identifi ed. This may represent a paucity of evidence on the subject, suggesting that further clinical trials on the topic are needed to contribute to the evidence base. This also highlights the need for international consensus on the topic, to reduce duplication of eff orts, standardise practice, and improve outcomes. g CW Carspecken, D Talmor Harvard Medical School and Beth Israel Deaconess Medical Center, Boston, MA, USA y yf Methods We used the SimulARTI Human Patient Simulator to evaluate Ventrain. Initially, we studied the eff ectiveness and security in ventilating and oxygenating the patient. In a second phase, the Ventrain performance was compared with what is considered to be the present gold standard (Quicktrach II, Portex Mini-Trach II Seldinger Kit, Melker Emergency Cricothyrotomy Catheter Set). Seven anesthesiologists performed an emergency transcricoid ventilation with each device in the same setting. Critical Care 2013, 17(Suppl 2):P165 (doi: 10.1186/cc12103) Critical Care 2013, 17(Suppl 2):P165 (doi: 10.1186/cc12103) Introduction Eff ective delivery of aerosolized bronchodilators for patients with asthma is crucial for adequate therapy in critical care and emergent settings. Often administered with pressure-metered dose inhalers (pMDIs), bronchodilator delivery depends on the correct patient technique during administration [1] and the ability to measure treatment response, which are diffi cult to monitor at the point of care and particularly so in resource-poor settings where standard in- hospital monitoring is unavailable [2].l Results Ventrain provided an average tidal volume of 334 ml and an average minute volume of 2.4 l in the considered situation, with a modifi cation of PAO2 from 32 to 702 mmHg and of PACO2 from 54.5 to 38.8 mmHg. In the second phase, the time needed to obtain an eff ective oxygenation with Ventrain was found to be shorter than other devices (median diff erence; vs. Minitrach –60 seconds; vs. Melker –35 seconds; vs. Quicktrach –25 seconds) (Figure 1); the ability to remove CO2 resulted bigger (average diff erence: vs. Minitrach –11.9; Methods A point-of-care device for airfl ow measurement during bronchodilator delivery was designed and tested for use in resource- limited settings. The handheld device was constructed from a clinical aerosol delivery tube with a bidirectional sensor for pressure diff erential detection about the aerosol element (Figure 1). The custom low-cost S62 Critical Care 2013, Volume 17 Suppl 2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 http://ccforum.com/supplements/17/S2 Figure 1 (abstract P165). Figure 1 (abstract P165). Figure 1 (abstract P165). Figure 1 (abstract P165). $11) electronics were designed such that no power supply would be eeded apart from a computer or mobile device. Collection of airfl ow gnal and calibration was performed with a standard 3 l syringe with l ow volume measurement on one adult subject. g CW Carspecken, D Talmor esults Calibration of the assembled device over range of 0.5 to 3 l/ econd enabled conversion of pressure diff erence to air fl ow with a ean measured tube resistance of 0.258 (kPasecond)/l. Robust signal sponses to an adult subject’s continuous respiratory maneuvers on e tube itself were demonstrated. Subject performance of the pMDI chnique with subsequent tidal volume breathing was recorded and nalyzed (Figure 1). onclusion Design and calibration of a novel low-cost monitoring evice for bronchodilator delivery monitoring enabled detection nd recording of characteristic fl ow volume respiratory patterns for oint-of-care diagnostics in resource-limited settings. Future work will quire clinical testing and automated detection of the correct pMDI atient technique. eferences Ari A, et al.: Respir Care 2012, 57:613-626. Riviello E, et al.: Crit Care Med 2011, 39:860. 166 vidence-based guidelines and protocols for the management of dult patients with a tracheostomy: a systematic review Siddiqui1, PB Sherren2, MA Birchall3 North West London Hospitals NHS Trust, London, UK; 2Barts and The London HS Trust, London, UK; 3The Royal National Throat Nose and Ear Hospital, ndon, UK itical Care 2013, 17(Suppl 2):P166 (doi: 10.1186/cc12104) Introduction Protocol-based care of the tracheostomised patient is important, as adverse events confer a high rate of mortality. Little is known regarding the existence of formal evidence-based guidelines on tracheostomy care. The aim of this study was to perform a systematic review for evidence-based guidelines on adult tracheostomy care. Methods A systematic search of PubMed, MEDLINE, guideline clearinghouses, centres of evidence-based practice, and professional societies’ guidelines relating to care of adult patients with a tracheostomy was performed by two reviewers. In addition, a Google search of publicly available tracheostomy care guidelines was performed. Search terms: (tracheostom* OR tracheotom) AND (protocol OR guideline* OR standard* OR management OR consensus OR algorithm). Filters: English language, human, from 1 January 1990 to date, adult patients. Guideline appraisal criteria: the quality of guidelines retrieved was assessed using the Appraisal of Guidelines Research and Evaluation II (AGREE II) instrument [1]. Results The search results are summarised in Table 1. A total of 80 guidelines were identifi ed. Five were found to satisfy the AGREE II criteria and only three related to the entire spectrum of tracheostomy management. The majority was informal and was not published or evidence based. Conclusion Five evidence-based guidelines on adult tracheostomy management were identifi ed. Can we keep neck breathers safe? A survey on training adequacy of medical staff caring for tracheostomy/laryngectomy patients Results PDTs with LMA were successful in 99.3% of the patients (n = 149). The ratings were 1 or 2 in 96% of cases with regards to ventilation and to blood gas analysis, in 96.6% for identifi cation of relevant structures and tracheal puncture site, and in 93.3% for the view inside the trachea during PDT. A rating of 5 was assigned to one patient requiring tracheal reintubation for inadequate ventilation. There were no damages to the bronchoscope or reports of gastric aspiration. Can we keep neck breathers safe? A survey on training adequacy of medical staff caring for tracheostomy/laryngectomy patients C Tjen, G Rajendran, S Hutchinson Norfolk and Norwich University Hospital, Norwich, UK Critical Care 2013, 17(Suppl 2):P168 (doi: 10.1186/cc12106) C Tjen, G Rajendran, S Hutchinson j j Norfolk and Norwich University Hospital, Norwich, UK y Critical Care 2013, 17(Suppl 2):P168 (doi: 10.1186/cc12106) Introduction A fatal incident related to a blocked tracheostomy tube prompted a review in our Trust. To provide safe tracheostomy care, changes in staffi ng, education and operational policies were recommended. Training of potential fi rst responders to tracheostomy or laryngectomy emergencies remains outstanding. We aim to quantify the training defi cit. Tracheostomies are common in critical care but these patients require ongoing management of an artifi cial airway on discharge to the ward and even the community. In 2010 our critical care unit cared for 108 tracheostomy patients, of which 30 were transferred to the wards. The 4th National Audit Project highlighted complications including hypoxic brain injury and death [1] and the National Patient Safety Agency recognised a number of avoidable aspects [2]. Existing guidelines for management of these patients including emergencies are not widely known. p p g p Conclusion The Blue Dolphin PDT using a LMA showed defi nite advantages regarding inspection of dilation process. This method improves visualization of the trachea and larynx during a video-assisted procedure and prevents the diffi culties associated with the use of an ET such as cuff puncture, tube transection by the needle, accidental extubation, and bronchoscope lesions. The LMA results as an eff ective and successful ventilatory device during PDT. This may be especially relevant in cases of diffi cult patient anatomy where improved structural visualization optimizes operating conditions. The intensivist performing PDT should be scrupulous when deciding which method to use. References 1. Cook T, et al.: Br J Anaesth 2011, 106:632-642. 1. Cook T, et al.: Br J Anaesth 2011, 106:632-642. 2. McGrath B, et al.: Postgrad Med J 2010, 86:522-525. Results All 11 ICUs questioned perform and manage tracheostomies. Of 22 respondents, 10 knew of guidelines covering all of the emergencies described above and their location. Four respondents thought that these guidelines were accessible in an emergency setting, one-half of which were on computer systems requiring a login and search function. With regards to emergency management, 19 respondents felt competent in a tracheostomy emergency; almost exclusively through experience and in-house teaching. No respondents were aware of any formal emergency tracheostomy management courses. 2. McGrath B, et al.: Postgrad Med J 2010, 86:522-525. Laryngeal mask as a safe and eff ective ventilatory device during Blue Dolphin tracheostomy in the ICU The insertion of the LMA, the quality of ventilation, the blood gas values, the view of the tracheal puncture site, and the view of the balloon dilatation were rated as follows: very good (1), good (2), barely acceptable (3), poor (4), and very poor (5). Results PDTs with LMA were successful in 99.3% of the patients (n = 149). The ratings were 1 or 2 in 96% of cases with regards to ventilation and to blood gas analysis, in 96.6% for identifi cation of relevant structures and tracheal puncture site, and in 93.3% for the view inside the trachea during PDT. A rating of 5 was assigned to one patient requiring tracheal reintubation for inadequate ventilation. There were no damages to the bronchoscope or reports of gastric aspiration. 1. Cook TM, et al.: Results of the 4th National Audit Project of the Royal College of Anaesthetists. Br J Anaesth 2011, 106:632-642. College of Anaesthetists. Br J Anaesth 2011, 106:632-642. 2. McGrath BA, et al.: Multidisciplinary guidelines for the management of tracheostomy and laryngectomy airway emergencies. Anaesthesia 2012, 67:1025-1041. p y g g tracheostomy and laryngectomy airway emergencies. Anaesthesia 2012, 67:1025-1041. tracheostomy and laryngectomy airway emergencies. Anaesthesia 2012, 67:1025-1041. Can we keep neck breathers safe? A survey on training adequacy of medical staff caring for tracheostomy/laryngectomy patients In our ICU the Blue Dolphin PDT with LMA has become the procedure of choice. References Can we keep neck breathers safe? A survey on training adequacy of medical staff caring for tracheostomy/laryngectomy patients C Tjen, G Rajendran, S Hutchinson Norfolk and Norwich University Hospital, Norwich, UK Critical Care 2013, 17(Suppl 2):P168 (doi: 10.1186/cc12106) Can we keep neck breathers safe? A survey on training adequacy of medical staff caring for tracheostomy/laryngectomy patients C Tjen, G Rajendran, S Hutchinson Norfolk and Norwich University Hospital, Norwich, UK Critical Care 2013, 17(Suppl 2):P168 (doi: 10.1186/cc12106) P167 Survey of emergency tracheostomy management in the East of England region N Lawrence, L Oakley, C Swanavelder, M Palmer West Suff olk Hospital, Bury St Edmunds, UK Critical Care 2013, 17(Suppl 2):P167 (doi: 10.1186/cc12105) Results We achieved a response rate of 39% (65/168). Respondents comprised: 33% anaesthesia/critical care, 47% medicine and 14% surgery. Over one-half (36/65) had managed tracheostomy/laryngec- tomy emergencies, with 42% (15/36) of these incidents occurring on the wards and one in an outpatient clinic. Only 20% (13/65) had received any formal training on management of a blocked/misplaced tracheostomy tube and only 18% (12/65) were aware of any guidelines. One-third of responders lacked confi dence in management of these emergencies and 88% felt they would benefi t from formal training including simulation. Introduction The 4th National Audit Project of the Royal College of Anaesthetists [1] concluded that the majority of airway-related signifi cant complications in ICUs resulted from displaced or blocked tracheostomies and recommended together with the Intensive Care Society and the National Tracheostomy Safety Project that each ICU in the UK should have an emergency airway management plan and guidelines [2]. The aim of this survey was to establish whether such guidelines exist and are familiar to those working within the ICUs of the East of England (EoE), their ease of availability in an emergency and the degree of emergency tracheostomy training within the region. Conclusion The population of patients with exteriorised tracheas is increasing and represents a high-risk group. Management of airway emergencies in these patients is not part of standard life-support courses. According to our trainees, these scenarios are relatively common and a signifi cant proportion of fi rst responders are poorly equipped to deal with them. Our Trust will be including specifi c training on the emergency management of neck breathers as part of in-house resuscitation training. We would contend that national resuscitation courses should consider doing the same. f Methods Data collection was via a telephone survey of 11 ICUs in the EoE training region during July 2012 with one senior ICU nurse and one ICU trainee questioned per hospital. Questions related to the existence and accessibility of guidelines for tracheostomy emergencies, and to the respondent’s degree of emergency tracheostomy training and their perceived availability of formal training. P166 P166 Evidence-based guidelines and protocols for the management of adult patients with a tracheostomy: a systematic review 1. Appraisal of Guidelines for Research and Evaluation: Instrument. AGREE II [http://www.agreetrust.org/] 1. Appraisal of Guidelines for Research and Evaluation: Instrument. AGREE II [http://www.agreetrust.org/] Table 1 (abstract P166). Summary of results returned following systematic review Total Results with Full texts Guidelines AGREE instrument Database results limits retrieved found compliant PubMed 4,685 1,596 33 MEDLINE 3,859 1,337 17 26 2 CINAHL 711 172 6 Medical societies/guideline websites/clearinghouses N/A N/A N/A 54 3 Google 17,600,000 497 N/A Table 1 (abstract P166). Summary of results returned following systematic review Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S63 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 P167 ENT, thoracics and A&E were approached. All completed forms were included. P167 Survey of emergency tracheostomy management in the East of England region N Lawrence, L Oakley, C Swanavelder, M Palmer West Suff olk Hospital, Bury St Edmunds, UK Critical Care 2013, 17(Suppl 2):P167 (doi: 10.1186/cc12105) 2. De Nicola A, Sucre MJ: Abstract 829. Proceedings Book of 40th Congress of the Society of Critical Care Medicine 2011: January 15–19 2011; San Diego, CA. Mount Prospect, IL: Society of Critical Care Medicine. Laryngeal mask as a safe and eff ective ventilatory device during Blue Dolphin tracheostomy in the ICU A De Nicola1, MJ Sucre1, G Donnarumma1, A Corcione2 1San Leonardo Hospital, Castellammare di Stabia, Italy; 2Monaldi Hospital, Naples, Italy A De Nicola1, MJ Sucre1, G Donnarumma1, A Corcione2 1San Leonardo Hospital, Castellammare di Stabia, Italy; 2Monaldi Hospital, Naples, Italy Critical Care 2013, 17(Suppl 2):P169 (doi: 10.1186/cc12107) g y y g Conclusion Despite national guidance within the UK this survey highlights that implementation and awareness of emergency tracheostomy guidelines in ICUs in the EoE region is poor, and when present they are not readily accessible in an emergency. Emergency training has largely been informal and the availability of formal training courses has not been recognised. In order to improve patient safety there is a need to ensure that emergency tracheostomy management including guidelines, equipment and formalised tracheostomy emergency training are adopted and embraced universally. References Introduction Usually percutaneous tracheostomy is accomplished via the tracheal tube. Some severe complications during percutaneous dilatational tracheostomy (PDT) may be related to poor visualization of tracheal structures. The alternative implies extubation and re- insertion of a laryngeal mask (LMA). An accidental extubation as well as an injuring of the vocal cords (because of the infl ated cuff during dislocation) appears impossible in this method. Subjectively, the bronchoscopic view obtained via a LMA seems to be better than that obtained with an endotracheal tube (ET) [1,2]. Cook TM, et al.: Results of the 4th National Audit Project of the Royal 1. Cook TM, et al.: Results of the 4th National Audit Project of the Royal College of Anaesthetists. Br J Anaesth 2011, 106:632-642. Methods In this prospective observational study, the bedside PDT was performed using the Ciaglia Blue Dolphin method in 150 critically ill patients. The patient’s tracheal tube was exchanged for a LMA Fastrach™ before undertaking PDT. The insertion of the LMA, the quality of ventilation, the blood gas values, the view of the tracheal puncture site, and the view of the balloon dilatation were rated as follows: very good (1), good (2), barely acceptable (3), poor (4), and very poor (5). Methods In this prospective observational study, the bedside PDT was performed using the Ciaglia Blue Dolphin method in 150 critically ill patients. The patient’s tracheal tube was exchanged for a LMA Fastrach™ before undertaking PDT. References 1. Linstedt U, et al.: Anesth Analg 2010, 110:1076-1082. Methods An anonymous online survey was sent to all trainees who may respond to a tracheostomy emergency in our organisation. Trainees in anaesthesia/critical care, general medicine, general surgery, 2. De Nicola A, Sucre MJ: Abstract 829. Proceedings Book of 40th Congress of the Society of Critical Care Medicine 2011: January 15–19 2011; San Diego, CA. Mount Prospect, IL: Society of Critical Care Medicine. Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S64 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 P170 Figure 1 (abstract P171). P170 Real-time ultrasound-guided balloon dilatational percutaneous tracheostomy is a safe procedure in critically ill patients: an evaluation study A Taha, A Shafi e, M Mostafa, P Wallen, H Hon, R Marktanner Sheikh Khalifa Medical City, Abu Dhabi, United Arab Emirates Critical Care 2013, 17(Suppl 2):P170 (doi: 10.1186/cc12108) Introduction Balloon dilatational percutaneous tracheostomy with radial outward dilation minimizes bleeding and injury to tracheal rings [1]. Using the bronchoscope during the puncture of the trachea holds a risk of inaccurate placement, of hypoventilation and of needle puncturing the bronchoscope. This study was conducted in order to evaluate the safety features and feasibility of combining real-time ultrasound-guided puncture of the trachea with visualization of the needle path during tracheal puncture [2]. We provide precise localization of tracheal cartilage and proper site puncture for the stoma by ultrasound, with subsequent performance of balloon dilatational percutaneous tracheostomy using the Blue dolphin technique.i p y g p q Methods Twenty-fi ve patients including 15 males and 10 females, mean age 61 years, with age range from 23 to 102 years, underwent bedside percutaneous tracheostomy combining real-time ultrasound and the Blue Dolphin technique. Results The median time for the procedure was 15 (12 to 20) minutes. Targeted placement for the tracheostoma between the second and third or the third and fourth tracheal ring was achieved in 100%. No signifi cant complications (for example, tracheal bleeding, puncture posterior tracheal wall, misplacement of the tracheal cannula) occurred. One fractured tracheal ring was identifi ed using bronchoscopy after the procedure. No conversion into a bronchoscopically guided or into a surgical open technique was necessary. found, with no signifi cant diff erence between RV systolic function, pulmonary acceleration time and pulmonary velocity time integral between survivors and nonsurvivors. Right ventricular restriction in interventional lung assist for acute respiratory distress syndrome Critical Care 2013, 17(Suppl 2):P172 (doi: 10.1186/cc12110) G Tavazzi1, M Bojan2, S Canestrini2, M White2, S Price2 1University of Pavia Foundation Policlinico San Matteo IRCCS, Pavia, Italy; 2Royal Brompton Hospital, London, UK Introduction Global left ventricular electromechanical dyssynchrony (GLVD) is uncoordinated LV contraction that reduces the extent of intrinsic energy transfer from the myocardium to the circulation leading to a reduction in peak LV pressure rise, prolonged total isovolumic time (t-IVT) and fall in stroke volume [1]. This potentially important parameter is not routinely assessed in critically ill cardiothoracic patients. Introduction Acute cor pulmonale (ACP) is associated with increased mortality in patients ventilated for acute respiratory distress syndrome (ARDS). Interventional lung assist (iLA) allows a lung-protective ventilatory strategy, whilst allowing CO2 removal, but requires adequate right ventricular (RV) function. RV restriction (including presystolic pulmonary A wave) [1] is not routinely assessed in ARDS. p Methods A prospective analysis of retrospectively collected data in cardiothoracic ICU patients who underwent echocardiography was performed. In addition to epidemiological factors, echo data included comprehensive assessment of LV/RV systolic and diastolic function including Doppler analysis of isovolumic contraction/ relaxation, ejection time (ET) and fi lling time (FT). t-IVT was calculated as (60 – (total ET + total FT)) and the Tei Index as (ICT + IRT) / ET. t-IVT >14 second/minute and Tei index >0.48 were used to defi ne GLVD [2]. Data are shown as mean ± SD/median (interquartile range). p y y Methods A prospective analysis of retrospectively collected data in patients with echo during iLA was performed. Data included epidemiologic and ventilatory factors, LV/RV function, evidence of RV restriction and pulmonary hemodynamics. Data are shown as mean ± SD/median (interquartile range). Results Thirty-two patients (45 ± 17 years), 22 male (68%), SOFA score 11.15 ± 2.38 were included. Pulmonary hypertension (PHT) was 53%, and hospital mortality 43%. Mortality was not associated with age, days on iLA, length of ICU stay, inotropic support, nitric oxide or level of ventilatory support, but was associated with pressor requirement (P = 0.005), a worse PaO2:FiO2 ratio (9.4 (7.8 to 12.6) vs. 15.2 (10.7 to 23.9), P = 0.009) and higher pulmonary artery pressures (56.5 mmHg (50 to 60) vs. 44.5 (40.5 to 51.2), P = 0.02). No echo features of ACP were g Results A total of 103 patients (63.5 ± 18.4 years), 65 male (63%), APACHE II score (14.6 ± 7.4) were included. References References 1. Gromann TW, et al.: Anesth Analg 2009, 108:1862-1866. 2. Venkatakrishna Rajajee et al.: Crit Care 2011, 15:R54. 3. Guinot et al.: Crit Care 2012, 16:R40. P172 Left ventricular electromechanical dyssynchrony and mortality in cardiothoracic intensive care G Tavazzi , M Bojan , A Duncan , A Vazir , S Price 1University of Pavia Foundation Policlinico San Matteo IRCCS, Pavia, Italy; 2Necker-Enfants Malades University Hospital, Paris, France; 3Royal Brompton Hospital, London, UK References The incidence of RV restriction was high (43%), and independent of PHT, RV systolic function and level of respiratory support, but correlated with CO2 levels (restrictive 7.1 kPa (7.4 to 8.0) vs. 6.1 (5.8 to 6.8), P = 0.03). See Figure 1. Conclusion This study demonstrates the feasibility and safeness combining real-time ultrasound guidance and balloon dilatational percutaneous tracheostomy (Blue Dolphin technique) in critically ill patients. In our division, this technique has become the standard bedside tracheostomy procedure because it combines excellent patient safety features with avoidance of intraprocedural tube misplacement, hypoventilation and accidental bronchoscopy damage even in technically diffi cult cases [3]. g Conclusion Typical echo features of ACP were not seen in this study, possibly because of the protective ventilatory strategies allowed by use of iLA. The incidence of RV restriction may refl ect more subtle abnormalities of RV function. Further studies are required to elucidate RV pathophysiology in critically ill adult patients with ARDS. Reference 1. Cullen S, et al.: Circulation 1995, 91:1782-1789. P171 P171 Right ventricular restriction in interventional lung assist for acute respiratory distress syndrome G Tavazzi1, M Bojan2, S Canestrini2, M White2, S Price2 1University of Pavia Foundation Policlinico San Matteo IRCCS, Pavia, Italy; 2Royal Brompton Hospital, London, UK Critical Care 2013, 17(Suppl 2):P171 (doi: 10.1186/cc12109) P171 Right ventricular restriction in interventional lung assist for acute respiratory distress syndrome G Tavazzi1, M Bojan2, S Canestrini2, M White2, S Price2 1University of Pavia Foundation Policlinico San Matteo IRCCS, Pavia, Italy; 2Royal Brompton Hospital, London, UK Critical Care 2013, 17(Suppl 2):P171 (doi: 10.1186/cc12109) Right ventricular restriction in interventional lung assist for acute respiratory distress syndrome G Tavazzi1, M Bojan2, S Canestrini2, M White2, S Price2 1University of Pavia Foundation Policlinico San Matteo IRCCS, Pavia, Italy; 2Royal Brompton Hospital, London, UK Critical Care 2013, 17(Suppl 2):P171 (doi: 10.1186/cc12109) Intracardiac ECG for confi rmation of correct positioning of central venous catheters is safe and cost-eff ective M Eriksson, R Dörenberg Surgical Sciences, Uppsala, Sweden Critical Care 2013, 17(Suppl 2):P175 (doi: 10.1186/cc12113) Methods Data for all ultrasound-guided interventions, including complications, are prospectively collected in our department for audit purposes; in this study we involved only CVC insertions in the ICU between February 2011 and November 2012. Electronic medical and laboratory records and paper-based nursing charts were retrospectively studied for all interventions, specifi cally looking for blood results, coagulation abnormalities and intervention-related complications. Introduction About 10 years ago the use of chest radiographs as the golden standard to ensure correct positioning of central venous catheters (CVC) was questioned. The frequent use of CVCs was also challenged. We decided to retrospectively evaluate our routines in a large surgical unit in a Swedish university hospital. Methods All X-rays were centrally registered. Chest X-ray performed in our unit is almost entirely used to confi rm CVC positioning. The Certofi x CVC set for the Seldinger technique in combination with Certodyn – Universaladapter (B Braun, Germany) is now used as the routine equipment and the right jugular vein is our standard approach. Results In the study period, ultrasound guidance was employed for a total of 291 central line insertions in 220 ICU patients. Coagulopathy was detected in 127 cases at the time of CVC placement (43.6%). On the day of CVC insertion, coagulation abnormalities were corrected in 20 cases (15.7%); 33 out of 50 patients with severe coagulopathy (66.0%) and 74 out of 77 patients with coagulopathy of moderate severity (96.1%) had no correction at all. Correction was started only after CVC insertion for reasons unrelated to CVC placement in a further eight and two patients with severe and less severe coagulopathy (16.0% and 2.6%), respectively. No bleeding complications were observed. Results In 2002 the total number of X-rays performed in patients at our unit was 2,306, which corresponds to the approximate number of inserted CVCs at that time, since a confi rmatory X-ray was routine. X-rays were rarely performed on other indications in our unit. X-ray costs were at that time approximately €300,000 (~€130/each). The year after, 1,726 chest X-rays were performed, refl ecting both the use of intracardiac confi rmation of correct CVC position and also a reduced use of CVCs. This trend has continued over time. In 2011 approximately 600 CVCs were inserted at our unit. X-rays were performed in about 20% of these cases. Cyanoacrylate glue prevents early bleeding of the exit site after CVC or PICC placement p G Scoppettuolo, MG Annetta, C Marano, E Tanzarella, M Pittiruti Catholic University, Rome, Italy Critical Care 2013, 17(Suppl 2):P174 (doi: 10.1186/cc12112) p G Scoppettuolo, MG Annetta, C Marano, E Tanzarella, M Pittiruti Catholic University, Rome, Italy C i i l C 2013 17(S l 2) P174 (d i 10 1186/ 12112) y y itical Care 2013, 17(Suppl 2):P174 (doi: 10.1186/cc12112) Intracardiac ECG for confi rmation of correct positioning of central venous catheters is safe and cost-eff ective The cost for a chest X-ray is today ~€200, meaning that X-ray costs were approximately €24,000. We have not experienced any medical problems when intracardiac ECG was used for positioning confi rmation. On the contrary, aspiration of venous blood without apparent p-waves in a patient with sinus rhythm may suggest improper placement of the CVC; for example, the right brachial vein. p y g p Conclusion In patients undergoing CVC insertion in our ICU, coagulopathy is common. We observed uncomplicated CVC placement in all 41 patients with severe uncorrected coagulopathy and in a further 76 patients with coagulopathy of moderate severity. When combined with other studies, our data suggest that ultrasound-guided CVC placement without routine correction of coagulation abnormalities may be safe in the ICU. Safety of ultrasound-guided central venous access in critically ill patients with uncorrected coagulopathy Safety of ultrasound-guided central venous access in critically ill patients with uncorrected coagulopathy G Reusz1, C Langer1, G Egervari1, P Sarkany1, A Csomos2 1Markhot Ferenc Hospital, Eger, Hungary; 2Semmelweis University, Budapest, Hungary Critical Care 2013, 17(Suppl 2):P173 (doi: 10.1186/cc12111) Conclusion Glue is an inexpensive and highly eff ective tool for avoiding the risk of early bleeding of the exit site after catheter placement. We also suggest that in the next future the glue might prove to have benefi cial collateral eff ects on the risk of extraluminal contamination (by reducing the entrance of bacteria in the space between the catheter and the skin), as well as on the risk of dislocation (by increasing the stability of the catheter inside the skin breach). y G Reusz1, C Langer1, G Egervari1, P Sarkany1, A Csomos2 g g y 1Markhot Ferenc Hospital, Eger, Hungary; 2Semmelweis University, Budapest, Hungary g g y 1Markhot Ferenc Hospital, Eger, Hungary; 2Semmelweis University, Budapest, Hungary y Critical Care 2013, 17(Suppl 2):P173 (doi: 10.1186/cc12111) Introduction Correction of coagulopathy before central venous catheter (CVC) insertion is a common practice; however, when ultrasound guidance is used this is controversial as mechanical complications are rare. Studies in oncology patients suggest that CVC placement without prior correction of coagulopathy is safe but no studies are available for critically ill patients and guidelines do not give recommendations [1,2]. We do not routinely correct coagulopathy, even if severe, when ultrasound guidance is used and the purpose of this retrospective study was to evaluate the safety of this practice. P175 Right ventricular restriction in interventional lung assist for acute respiratory distress syndrome Results In 65 consecutive patients (45 PICCs, 11 dialysis catheters and nine CVCs), there was no signifi cant local bleeding at 1 hour or at 24 hours after catheter placement. No local adverse reaction occurred. No damage to the polyurethane of the catheters was detected. Conclusion Glue is an inexpensive and highly eff ective tool for avoiding the risk of early bleeding of the exit site after catheter placement. We also suggest that in the next future the glue might prove to have benefi cial collateral eff ects on the risk of extraluminal contamination (by reducing the entrance of bacteria in the space between the catheter and the skin), as well as on the risk of dislocation (by increasing the stability of the catheter inside the skin breach). Methods The aim of this pilot study was to verify the effi cacy of a cyanoacrylate glue in reducing the risk of early bleeding at the exit site after CVC or PICC placement. We studied a group of adult patients consecutively undergoing placement of polyurethane CVCs or PICCs without reverse tapering in a non-intensive ward of our hospital. All lines were inserted according to the same protocol, which included 2% chlorhexidine antisepsis, maximal sterile barriers, ultrasound guidance, EKG guidance and securement with sutureless device. Two minutes after placement of the glue, the exit site was covered with a temporary gauze dressing, which was replaced by transparent membrane at 24 hours. All patients were assessed at 1 hour and at 24 hours. fi Conclusion GLVD that limits cardiac output is common in the cardiothoracic ICU, and signifi cantly related to mortality. When diagnosed, the underlying cause should be sought and treatment instigated to minimize the t-iVT (pacing optimization/revascularization/ inotrope titration/volaemia optimization). References References 1. Cavanna L, et al.: World J Surg Oncol 2010, 8:91. 2. Lamperti M, et al.: Intensive Care Med 2012; 38:1105-1117. 1. Cavanna L, et al.: World J Surg Oncol 2010, 8:91. 2. Lamperti M, et al.: Intensive Care Med 2012; 38:1105-1117. Conclusion If we had continued to use CVCs at the same frequency as we did 10 years ago, and used X-ray confi rmation in practically all cases, we would have paid approximately €460,000 annually. Reduced use of CVCs, in combination with intracardiac confi rmation of CVC positioning, has not only allowed us to reduce costs associated with CVC insertion by more than €400,000, corresponding to a reduction rate of more than 90%, but also decreased the patient’s exposure to X-ray irradiation. Stas M, et al.: Eur J Surg Oncol 2001, 27:316-320. Joshi A, et al.: Indian Crit Care Med 2008, 12:10-14. p References 1. Duncan A, et al.: J Am Coll Cardiol 2003, 41:121-128. 2. Tei C, et al.: J Am Coll Cardiol 1996, 28:658-664. 1. Duncan A, et al.: J Am Coll Cardiol 2003, 41:121-128. 2. Tei C, et al.: J Am Coll Cardiol 1996, 28:658-664. p Results In 65 consecutive patients (45 PICCs, 11 dialysis catheters and nine CVCs), there was no signifi cant local bleeding at 1 hour or at 24 hours after catheter placement. No local adverse reaction occurred. No damage to the polyurethane of the catheters was detected.f Right ventricular restriction in interventional lung assist for acute respiratory distress syndrome The prevalence of GLVD was high (24/103, 22%) and associated with signifi cantly increased mortality, 7.5% vs. 25% (P = 0.02). There was no diff erence in requirement for cardiorespiratory support between the two populations, but there were signifi cant diff erences (no GLVD vs. GLVD) in requirement for Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S65 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 patient’s care and logistical problems. In our experience, the rate of signifi cant local bleeding after placement of PICCs without reverse tapering may be as high as 40% at 1 hour and 15% at 24 hours, while the rate of bleeding after placement of a large-bore dialysis catheter is above 50% at 1 hour. pacing (35% vs. 62%, P = 0.02), atrial fi brillation (20% vs. 41%, P = 0.03), QRS duration (92.0 (80.0 to 120.0) vs. 116.5 (95.0 to 154.0), P = 0.01) and QTc (460.0 (416.0 to 498.5) vs. 477.5 (451.2 to 541.0), P = 0.02). There was no signifi cant diff erence in ejection fraction (no GLVD 43.0 (35.0 to 49.5) vs. GLVD 39.6 (29.5 to 49.7), P = 0.43), mitral regurgitation (40.5% vs. 62.5%, P = 0.06), or any other measures of LV systolic or diastolic function between the two groups. There was good correlation between the two methods used to assess dyssynchrony (LV t-IVT:LV Tei index correlation coeffi cient = 0.80, P <0.001). Methods The aim of this pilot study was to verify the effi cacy of a cyanoacrylate glue in reducing the risk of early bleeding at the exit site after CVC or PICC placement. We studied a group of adult patients consecutively undergoing placement of polyurethane CVCs or PICCs without reverse tapering in a non-intensive ward of our hospital. All lines were inserted according to the same protocol, which included 2% chlorhexidine antisepsis, maximal sterile barriers, ultrasound guidance, EKG guidance and securement with sutureless device. Two minutes after placement of the glue, the exit site was covered with a temporary gauze dressing, which was replaced by transparent membrane at 24 hours. All patients were assessed at 1 hour and at 24 hours. Is automated brachial cuff measurement of arterial pressure less accurate in cases of arrhythmia? y K Lakhal1, S Faiz2, M Martin1, AS Crouzet1, F Reminiac2, S Ehrmann2, R Cinotti1, X Capdevila3, K Asehnoune4, Y Blanloeil1, B Rozec1, T Boulain2 1Centre Hospitalier Universitaire Laennec, Nantes, France; 2Centre Hospitalier Universitaire Bretonneau, Tours, France; 3Centre Hospitalier Universitaire Lapeyronie, Montpellier, France; 4Centre Hospitalier Universitaire Hotel-Dieu, Nantes, France Introduction In cases of arrhythmia, the beat-to-beat variation of arterial pressure (AP) may impair the accuracy of automated cuff measurements. Indeed, this oscillometric device relies on the detection of arterial wall oscillations. Our aim was to determine, in ICU patients, whether brachial cuff measurements are really less reliable during arrhythmia than during regular rhythm. y g g y Methods Patients with arrhythmia and carrying an intra-arterial catheter were prospectively and consecutively included in this multi- center study. After each arrhythmic inclusion, a regular rhythm patient was included. A second inclusion was possible in case of change in the cardiac rhythm. Three pairs of invasive and brachial cuff (Philips® MP70 monitor) measurements of mean arterial pressure (MAP) were respectively averaged. Some patients underwent a second set of measurements, after a cardiovascular intervention (passive leg raising, volume expansion, initiation of/increase in catecholamine infusion) allowing the assessment of MAP changes. Figure 2 (abstract P177). Use of CO data. Results In the 111 analyzed inclusions (in 103 patients) there was only one failure in displaying a brachial cuff measurement of MAP. Arrhythmic patients (atrial fi brillation 88%, frequent extrasystoles 7%, fl utter 5%) were similar (P >0.3) to patients in regular rhythm for MAP (median 74 (IQR 67 to 80) vs. 75 (69 to 84) mmHg), SAPS II score, BMI, arm circumference, norepinephrine administration (36% vs. 35%), mechanical ventilation (80% vs. 81%), and site of the intra- arterial catheter (radial artery: 80% vs. 85%). Between arrhythmic and regular rhythm patients: the agreement (Bland–Altman analysis) between invasive and brachial cuff measurements of MAP was similar (mean bias –0.4 ± 7.2 (limits of agreement –14/14) mmHg vs. 3.0 ± 8.2 (–13/19) mmHg, respectively); the detection of hypotension (invasive MAP <65 mmHg) by the brachial cuff was of similar reliability (area under the ROC curve (AUC) = 0.91 (95% CI = 0.80 to 0.97) vs. AUC = 0.98 (0.89 to 1), P = 0.2); and the detection of a response (>10% increase in MAP) to therapy was of similar reliability (AUC = 1 (0.85 to 1) (n = 22) vs. Is automated brachial cuff measurement of arterial pressure less accurate in cases of arrhythmia? AUC = 0.99 (0.78 to 1) (n = 17), P = 0.5). Figure 2 (abstract P177). Use of CO data. Results Fifty-three patients (18.5%) had evidence of CO monitoring. LiDCO was the most popular method (Figure 1). A total of 264 (94%) patients received treatment with vasopressors and/or inotropes. CO data were utilised in a variety of ways (Figure 2). Conclusion The majority of potential donors require vasopressors and/or inotropes post BSD, but it seems only a minority currently have their CO monitored. There is variation in how CO data are utilised to direct haemodynamic management. We welcome the development of standardised bundle-driven donor management. Conclusion These preliminary results suggest that arrhythmia does not impair the reliability of automated cuff measurements of MAP. y References Introduction Early bleeding from the exit site after CVC or PICC placement is a very common event that causes diffi culties in the Stas M, et al.: Eur J Surg Oncol 2001, 27:316-320. Joshi A, et al.: Indian Crit Care Med 2008, 12:10-14. Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S66 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Figure 1 (abstract P177). CO device. Figure 2 (abstract P177). Use of CO data. P176 Figure 1 (abstract P177). CO device. P176 Is automated brachial cuff measurement of arterial pressure less accurate in cases of arrhythmia? K Lakhal1, S Faiz2, M Martin1, AS Crouzet1, F Reminiac2, S Ehrmann2, R Cinotti1, X Capdevila3, K Asehnoune4, Y Blanloeil1, B Rozec1, T Boulain2 1Centre Hospitalier Universitaire Laennec, Nantes, France; 2Centre Hospitalier Universitaire Bretonneau, Tours, France; 3Centre Hospitalier Universitaire Lapeyronie, Montpellier, France; 4Centre Hospitalier Universitaire Hotel-Dieu, Nantes, France Critical Care 2013, 17(Suppl 2):P176 (doi: 10.1186/cc12114) Reference 1. Murugan R, et al.; HIDonOR Study Investigators: Preload responsiveness is associated with increased interleukin-6 and lower organ yield from brain- dead donors. Crit Care Med 2009, 37:2387-2393. Cardiac output monitoring in brain-stem-dead potential organ donors: an audit of current UK practice CJ Wright1, A Broderick2, G Mandersloot3 1Glasgow Royal Infi rmary, Glasgow, UK; 2National Health Service Blood and Transplant, London, UK; 3The Royal London Hospital, London, UK Critical Care 2013, 17(Suppl 2):P177 (doi: 10.1186/cc12115) P179 Prediction of 28-day mortality by indocyanine green disappearance rate, other markers of hepatic function and transpulmonary thermodilution parameters: a prospective study in 154 patients W Huber, D Ertekin, T Langer, B Saugel, T Lahmer, M Messer, C Spinner, C Schultheiss, RM Schmid Klinikum rechts der Isar, Technical University of Munich, Germany Critical Care 2013, 17(Suppl 2):P179 (doi: 10.1186/cc12117) Methods A prospective observational study was designed and conducted. Inclusion criteria were: patients who had underwent elective or urgent/emergent cardiac surgery, with cardiac index (CI) <2.5 l/minute/m2 estimated by means of a PAC, left ventricle ejection fraction (LVEF) <40%, lactate >2 mmol/l, age >18 years. A central venous catheter (CVC) and a PAC were inserted for each patient before surgery in the same right internal jugular vein in accordance with standard procedure. Proper position of the PAC was confi rmed with pressure tracings and chest X-ray. Mixed and central venous blood samples were collected from the distal ports of the PAC and CVC respectively 30 minutes after ICU admission, and every 6 hours for a total of three samples in a 24-hour period for each patient. All blood samples were analyzed by a co-oximeter (Radiometer ABL800 fl ex; Radiometer, Copenhagen, Denmark). Statistical analysis was performed by Stats Direct (Ver.2.5.8, Cheshire, UK) and GraphPad (Vers. Prism 4.0; San Diego, CA, USA). All data were tested for normal distribution with the Kolmogorov–Smirnov test. Statistical analysis was performed by linear regression analysis. The agreement between absolute values of ScvO2 and SvO2 were assessed by the mean bias and 95% limits of agreement (LOA) ((mean bias ± 1.96)×standard deviation) according to the method described by Bland and Altman [5]. Introduction Hepatic dysfunction has been associated with outcome of ICU patients. However, most scoring systems including APACHE II only marginally refl ect acute liver dysfunction on admission. Indocyanine green (ICG) is eliminated by hepatobiliary excretion. Therefore, the ICG plasma disappearance rate (ICG-PDR) is used as a dynamic liver function test. ICG-PDR has been associated with mortality in several studies. Methods A prospective study to compare prediction of 28-day mortality by ICG-PDR, other markers of liver function and scoring systems (primary endpoint). In the subgroup of patients with trans- pulmonary thermodilution (TPTD) monitoring (PiCCO device; Pulsion, Munich, Germany), predictive capabilities of ICG-PDR were compared with cardiac index (CI), extravascular lung water index (EVLWI), global end-diastolic volume index (GEDVI) and pulmonary vascular permeability index (PVPI). ICG-PDR (i.v. Indocyanine green plasma disappearance rate for assessment of liver function: re-evaluation of normal ranges and impact of biometric data Introduction Signifi cant changes in haemodynamics occur after brain stem death (BSD) and there is evidence that yield of transplantable organs may be decreased in donors who remain preload responsive prior to donation [1], suggesting that optimisation of the cardiac output (CO) may be benefi cial in potential organ donors. We describe current UK practice with regard to CO monitoring in this group. W Huber, M Kranzmayr, C Schultheiss, W Reindl, A Krug, B Saugel, U Mayr, RM Schmid W Huber, M Kranzmayr, C Schultheiss, W Reindl, A Krug, B Saugel, U Mayr, RM Schmid Klinikum rechts der Isar, Technical University of Munich, Germany Critical Care 2013, 17(Suppl 2):P178 (doi: 10.1186/cc12116) Introduction The indocyanine green plasma disappearance rate (ICG-PDR) is a dynamic liver function test that can be non-invasively measured by pulse densitometry. ICG-PDR is associated with mortality and other markers of outcome. Due to predominant use of ICG-PDR in the ICU setting, the normal range is based on scarce data available outside the ICU and given with 18 to 25%/minute. Methods We reviewed a database of 287 brain-stem-dead potential organ donors collected by specialist nurses in organ donation (SN-OD) over a 6-month period (30 April 2011 to 31 October 2011) across multiple UK centres. The database contained data on donor management in the period from initial SN-OD review to immediately prior to transfer to the operating theatre. We analysed data on CO monitoring and vasopressor/inotrope use. Where information was missing/not recorded in the dataset, the treatment referred to was interpreted as not given/not done. Methods To prospectively re-evaluate the normal range and to analyze the potential impact of biometric data on ICG-PDR, we measured ICG-PDR (i.v. injection of 0.25 mg/kg ICG; LiMON, Pulsion, Munich, S67 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 independently associated with age (P = 0.025), but not with any other biometric parameter (gender, weight and height). Germany) in 95 outpatients with ulcerative colitis (UC). Due to a prevalence of primary sclerosing cholangitis (PSC) of 5% in UC, patients were asked regarding previous diagnosis of PSC. Additionally, serum bilirubin, AST, ALT, INR, AP, γGT and cholinesterase were determined. Association of biometric data (age, gender, height, weight) and diagnosis of PSC on ICG-PDR were evaluated using Spearman corre- lation, ROC and multivariate analysis (statistics: IBM SPSS 20). Indocyanine green plasma disappearance rate for assessment of liver function: re-evaluation of normal ranges and impact of biometric data Conclusion ICG-PDR on admission is an independent predictor of 28-day mortality. Predictive capabilities particularly of APACHE II can be improved by combination with ICG-PDR. Among TPTD-derived parameters, only PVPI provides signifi cant prediction of mortality. y Results A total of 95 patients (54 male, 41 female), age 43.2 ± 15.5 (21 to 76) years, weight 74 ± 16 kg, height 175 ± 10 cm, BMI 23.7 ± 4, bilirubin 0.48 ± 0.27 mg/dl, AST 26.8 ± 11.4 U/l, ALT 26.8 ± 14.9 U/l, previous diagnosis of PSC 8/95 (8.4%). ICG-PDR ranged from 13.8 to 44.0 with a mean of 28.2 ± 6.8 and a median of 27.0%/minute. In univariate analysis ICG-PDR was signifi cantly associated with age (r = –0.480; P <0.001), weight (r = –0.262; P = 0.011) and female gender (r = 0.221; P = 0.032), but not to height (P = 0.681). In multivariate analysis (R = 0.459) including the variables age, gender, height, weight and diagnosis of PSC, only age was independently (P <0.001) associated with ICG-PDR. With each year in age, ICG-PDR decreased by 0.206%/minute. In ROC analysis, ICG-PDR above the upper normal range (>25%/minute) was signifi cantly associated with young age (AUC 0.746; P <0.001). P180 P180 Mixed and central venous oxygen saturation are not interchangeable in patients with cardiogenic shock after cardiac surgery S Romagnoli, P Balsorano, F Landucci, A De Gaudio AOUC Careggi, Florence, Italy Critical Care 2013, 17(Suppl 2):P180 (doi: 10.1186/cc12118) g y S Romagnoli, P Balsorano, F Landucci, A De Gaudio AOUC Careggi, Florence, Italy Introduction Mixed venous oxygen saturation (SVO2) represents a well-recognized parameter of oxygen delivery (DO2)–consumption (VO2) mismatch and its use has been advocated in critically ill patients in order to guide hemodynamic resuscitation [1] and oxygen delivery optimization. Nevertheless, the pulmonary artery catheter (PAC) is not readily available and its use is not devoid of risks. Furthermore, its use has been decreasing in recent years in surgical and cardiac surgical patients as the benefi t of guiding therapy with this device is unclear [2-4]. Central venous oxygen saturation (ScVO2) has been suggested as an alternative to SVO2 monitoring due to its feasibility in several settings. Unfortunately concerns arise from its capability to correlate with SVO2, the relationship being infl uenced by several factors, such as hemodynamic impairment and pathological process. Hemodynamic instability and shock often complicate cardiac surgery, and the SVO2– ScVO2 relationship has not been specifi cally investigated in this setting. The aim of this study is to compare SVO2 and ScVO2 values in patients with cardiogenic shock after cardiac surgery. i Conclusion ICG-PDR (normal) values should be corrected for age. With a decrease in ICG-PDR of 0.206% per year, the range between the upper and lower normal level (25 to 18%/minute) is passed through within about 35 years of life. These fi ndings are in accordance with functional loss of other organ functions (for example, cardiac output, glomerular fi ltration rate) with increasing age. Normal ranges of innovative markers of organ function mainly derived from ICU populations should be re-evaluated outside the ICU. Utility of transesophageal echocardiography in the ICU: a preliminary US perspective References 1. Rivers E, et al.: Early goal-directed therapy in the treatment of severe sepsis and septic shock. N Engl J Med 2001, 345:1368-1377. 2. Edwards JD: Oxygen transport in cardiogenic and septic shock. Crit Care Med 1991, 19:658-663. 3. Hadian M, et al.: Evidence based of the use of the pulmonary artery catheter: impact data and complications. Crit Care 2006, 10(Suppl 3):S11-S18. 4. Connors AF, et al.: The eff ectiveness of right heart catheterization in the initial care of critically ill patients. JAMA 1996, 276:889-897. 5. Bland JM, Altman DG: Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1986, 1:307-310. 6. Walley KR: Use of central venous oxygen saturation to guide therapy. Am J Respir Crit Care Med 2011, 184:514-520. Introduction While TEE is providing a direct assessment of the cardiac function and volume status as a diagnostic tool, until recently it has been impractical to be continuously available for monitoring. A new disposable, monoplanar TEE probe (ImaCor) can remain in the patient for up to 72 hours, allowing repeated measures of ventricular function and volume status, parameters needed to monitor response to therapy. Methods We assessed the benefi t these TEE data provided in the assessment of fi ve domains: hypovolemia, right ventricular dysfunction, left ventricular dysfunction, sepsis, and valvular abnormality. Bedside practitioners listed their diagnoses before and after seeing primary TEE images perform by trained physicians. We used a 0 to 5 Likert scale to assess diff erential diagnosis before and after the TEE, comparing changes using a paired t test. 1. Rivers E, et al.: Early goal-directed therapy in the treatment of severe sepsis and septic shock. N Engl J Med 2001, 345:1368-1377. 1. Rivers E, et al.: Early goal-directed therapy in the treatment of severe sepsis and septic shock. N Engl J Med 2001, 345:1368-1377. 2. Edwards JD: Oxygen transport in cardiogenic and septic shock. Crit Care Med 1991, 19:658-663. 3. Hadian M, et al.: Evidence based of the use of the pulmonary artery catheter: impact data and complications. Crit Care 2006, 10(Suppl 3):S11-S18.f 3. Hadian M, et al.: Evidence based of the use of the pulmonary artery catheter: impact data and complications. Crit Care 2006, 10(Suppl 3):S11-S18.f g g p Results All requests for TEE were to access hemodynamic instability. A total of 18 patients were screened and nine were eligible, in which 16 total TEE studies were performed. Utility of transesophageal echocardiography in the ICU: a preliminary US perspective There were no complications with TEE and all patients tolerated the long-term placement of the probe well. Of the fi ve diagnostic domains studied, right ventricular failure was the most commonly underdiagnosed contributor to the hemodynamic instability among patients prior to TEE (P = 0.054) (Figures 1 and 2). 7. Reinhart K, et al.: Comparison of central-venous to mixed-venous oxygen saturation during changes in oxygen supply/demand. Chest 1989, 95:1216-1221. 7. Reinhart K, et al.: Comparison of central-venous to mixed-venous oxygen saturation during changes in oxygen supply/demand. Chest 1989, 95:1216-1221. P179 During circulatory shock, not homogeneous oxygen extraction and regional blood fl ow Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S68 Figure 1 (abstract P180). Table 1 (abstract P181). Hemodynamic parameters before and after treatment Group 2 compared with Group 1 (P <0.001). The increase in plasma NO level was signifi cantly higher in Group 2 (Table 1). Conclusion L-Arginine i.v. infusion 5 ml/kg during 24 hours increased NO blood level and decreased MPAP in pediatric sepsis, but did not deteriorate hemodynamic system values. Table 1 (abstract P181). Hemodynamic parameters before and after treatment Group 1 Group 1 Group 2 Group 2 Value before after before after MAP (mmHg) 66.9 ± 3.4 68.8 ± 3.0 72.8 ± 2.0 71.8 ± 1.3 MPAP (mmHg) 43.5 ± 2.6 44.3 ± 3.4 50.8 ± 3.0 28.8 ± 2.1 CO (l/minute/m2) 4.2 ± 0.2 5.0 ± 0.4 4.8 ± 0.2 4.8 ± 0.3 SpO2 (%) 93.7 ± 1.2 93.4 ± 1.5 94.6 ± 0.7 98.1 ± 0.3 redistribution make SVO2 a more reliable parameter suggesting the global adequacy of cardiac output rather than ScVO2. In this study we aimed at evaluating SvO2–ScVO2 diff erences in patients with cardiogenic shock, as defi ned by hyperlactatemia, low CI, and LVEF <40%, after cardiac surgery. Our results highlighted a great variability for these two parameters, with a clinically unacceptable mean bias and LOA. As expected, ScVO2 values were consistently higher. References P179 bolus of 0.25 mg/kg ICG; LiMON device, Pulsion) and all other predictors were determined within 48 hours after admission. Statistics: IBM SPSS 20. Results A total of 20 patients were enrolled. In 18 out of 20 cases all three blood samples were collected. In two patients only two blood samples were drawn as they exited the inclusion criteria. Linear regression analysis between the two variables resulted in an r2 of 0.708. Bland–Altman analysis (Figure 1) for the pooled measurements of SvO2 and ScvO2 showed a mean bias and LOA of 6.82% (SD of bias 5.3) and –3.71 to +17.3% respectively. Results A total of 154 patients (46 female, 108 male), age 59 ± 13 years, APACHE II score 16.0 ± 5.7, SOFA score 7.6 ± 4.2. Etiology: sepsis 14.4%, cirrhosis 28.8%, GI bleeding 8.9%, ARDS 17.8%, cardiogenic shock 4.1%, acute renal failure 3.4%, various 22.6%. The 28-day mortality was signifi cantly predicted by APACHE II (ROC-AUC: 0.762; P <0.001) and SOFA (AUC: 0.784; P <0.001). Among markers of hepatic function on admission, ICG-PDR provided the largest AUC (0.742; P <0.001), which was larger than for GOT (AUC: 0.646; P = 0.019), bilirubin (AUC: 0.641; P = 0.023) and INR (AUC: 0.614; P = 0.067). Among TPTD parameters, only PVPI signifi cantly predicted 28-day mortality (AUC: 0.643; P = 0.043), whereas CI, GEDVI and EVLWI were not predictive. Prediction of 28-day mortality by SOFA could not be improved by models including any hepatic parameter. By contrast, ICG-PDR was independently (P = 0.036) associated with mortality when included in a model (R = 0.58) with APACHE II. This model based on APACHE II and ICG-PDR provided the largest of all ROC-AUCs (AUC: 0.804; P <0.001). ICG-PDR itself was y Conclusion ScVO2 has been advocated as an attractive and simple indicator of DO2–VO2 mismatch [6]. Its role as a surrogate of the well- established SVO2 has been investigated in several settings, and it has been purposed in the hemodynamic resuscitation of critically ill septic patients [1]. Nevertheless, the SVO2–ScVO2 relationship can be infl uenced by several factors due to ScVO2 dependency from global blood fl ow redistribution that can occur during hemodynamic impairments. It has been shown previously that in healthy people ScVO2 values tend to underestimate SVO2 values, due to the higher oxygen content from inferior vena cava [7]. P181 Pulmonary hemodynamic disorder in pediatric sepsis and their correction with L-arginine infusion M Georgiyants, V Korsunov Kharkov Medical Academy Post-Graduate Education, Kharkov, Ukraine Critical Care 2013, 17(Suppl 2):P181 (doi: 10.1186/cc12119) g M Georgiyants, V Korsunov Figure 1 (abstract P182). Ventricular function according to the Likert scale. Kharkov Medical Academy Post-Graduate Education, Kharkov, Ukraine Critical Care 2013, 17(Suppl 2):P181 (doi: 10.1186/cc12119) Introduction The acute respiratory distress syndrome and pulmonary hypertension (PH) is one of the factors of septic mortality. Some data demonstrate arginine defi ciency as an important pathogenic factor of septic PH. We suppose that intravenous L-arginine infusion improves NO production and reduce PH. Methods We examined 46 patients with sepsis, severe sepsis and septic shock in accordance with San Antonio Criteria. Organ dysfunction severity was defi ned according to SOFA. The mean pulmonary artery pressure (MPAP) was estimated by Doppler method, cardiac output (CO), and stroke volume (SV) – by ultrasound M-mode. SpO2, mean arterial pressure (MAP), blood gas, and plasma NO level was evaluated. The patients of Group 1 (n = 21, age 22.1 ± 8.5 months) had respiratory and hemodynamic support, and antibiotics. Group 2 (n = 25, age 27.0 ± 11.2 months) had the same treatment with continuous i.v. infusion of 5 ml/kg body weight 4.2% L-arginine solution during 24 hours. Results The patients of Groups 1 and 2 did not have statistical diff erence of SOFA (4.1 ± 1.0 vs. 4.4 ± 0.7), need for ventilator support (57.0 ± 11.0% vs. 44.0 ± 10.0%), and dose of inotropes (P >0.05). The reduction of MPAP and increase of SaO2 was signifi cantly higher in Figure 1 (abstract P182). Ventricular function according to the Likert scale. Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S69 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Figure 2 (abstract P182). Volume status, valve, sepsis according to the Likert scale. ICU in Thailand that was capable of measuring extravascular lung water. None of the Thai ICUs used transcutaneous PCO2, near-infrared spectroscopy, gut mucosal tonometry and sublingual sidestream dark- fi eld for tissue perfusion monitoring. Only four ICUs had transcutaneous PO2. However, measuring the level of lactate as one of the tissue perfusion markers was routinely performed in about 50% of the ICUs. Conclusion There were variations in monitoring performance in Thai ICUs. These vary by type of hospital. Academic ICUs had a tendency for advance monitoring in overall aspects. Establishing a critical care echocardiography laboratory K Yastrebov1, A McLean2 Establishing a critical care echocardiography laboratory K Yastrebov1, A McLean2 1The St George Hospital, Sydney, Australia; 2Nepean Hospital, Sydney, Australia Critical Care 2013, 17(Suppl 2):P184 (doi: 10.1186/cc12122) Introduction Echocardiography is increasingly utilized by inten sive care physicians in everyday practice. Standardization of echocardiographic studies and reporting, quality assurance and medicolegal requirements necessitate establishment of a dedicated system within the critical care setting. We describe the process of setting up a critical care echocardiography (CCE) laboratory based on our experience from three separate ICUs. Figure 2 (abstract P182). Volume status, valve, sepsis according to the Likert scale. Methods A retrospective review and analysis of the process involved in establishment of echocardiography laboratories within ICUs. Methods A retrospective review and analysis of the process involved in establishment of echocardiography laboratories within ICUs. Results Creating a CCE service involves a number of stages and takes several years to achieve. Major components include staffi ng, equipment, quality control, study archiving and networking capability. For staffi ng the objective is to identify and recruit staff with adequate training and expertise in CCE, providing 24/7 specialist cover in addition to supporting and training junior medical and nursing staff . There is further a need to acquire funding for high-quality ultrasound machines and related hardware as well as long-term DICOM-based archiving and reporting systems. This should be based on projections of annual volumes of echo studies and corresponding digital storage. Networking connectivity is highly desirable, including obligatory back-up solutions and site allocations. A business case incorporating all the above should precede any development as identifi able funding sources and administrative approval are essential. The implementation stage requires the presence of a project leader who can organize the trialing of scanners, archiving, reporting and research systems, ensure compatibility with existing hospital and cardiology networks, and who can assist in individualizing archiving and reporting software refl ecting institutional and ICU specifi cs. Coordination with the IT department is very important. Clear contractual vendor obligations for service, maintenance and future upgrades of hardware and software need to be specifi ed. Training and credentialing of staff is best achieved within a systematic framework that includes ongoing competency review, education and QA programs. Partnership with cardiology may benefi t both groups. Major pitfalls are associated with poor initial training, lack of expertise and leadership, and bad vendor contracts. Cardiopulmonary monitoring in Thai ICUs: results of ICU-RESOURCE I surveys K Chittawatanarat1, A Wattanatham2, D Sathaworn2, C Permpikul3, TSCCM Study Group4 1Chiang Mai University, Chiang Mai, Thailand; 2Pramongkudklao Hospital, Bangkok, Thailand; 3Siriraj Hospital, Mahidol University, Bangkok, Thailand; 4Thai Society of Critical Care Medicine, Bangkok, Thailand Critical Care 2013, 17(Suppl 2):P183 (doi: 10.1186/cc12121) Introduction Although rapid progress in ICU monitoring with advanced equipments has been developed, there were limited data on ICU monitoring in Thailand. The objective of this study was to determine the current utilization of monitoring in Thai ICUs. g Methods A self-administered questionnaire was developed by the TSCCM research subcommittee. Data verifi cation was processed by an online medical research tools program (OMERET). p g Results A total of 350 questionnaires were sent to ICUs throughout Thailand. In total, 256 questionnaires were confi rmed after being received at the end of June 2012. Of these, 140 fi lled forms (56.9%) were returned for fi nal analysis. More than 70% of the ICUs had basic hemodynamic monitoring. Less than 10% of general and regional hospitals could perform cardiac output monitoring by thermodilution technique compared with 60% of academic teaching hospitals. New and advanced hemodynamic monitoring techniques such as pulse pressure variation, systolic pressure variation, stroke volume variation, PiCCO, Vigileo-Flo Tract, Pleth variability index device and echocardiography were available only in ICUs of academic teaching hospitals except ultrasound-based techniques including transthoracic and transesophageal echocardiography and USCOM. For respiratory monitoring, all ICUs had a SpO2 monitoring device but only one-half of them had end-tidal CO2 monitoring. Nearly 80% of ventilator support in participating ICUs was capable of displaying graphic waveform monitoring. Only 43.6% of participating ICUs had a ventilator machine that could calculate lung mechanics data. Advanced respiratory monitoring such as EIT and esophageal pressure monitoring are available only in ICUs of academic teaching hospitals. There was no Conclusion Establishment of a CCE laboratory requires careful planning, and allocation of adequate human and fi nancial resources. Many potential problems can be identifi ed and prevented in advance. Strong expert leadership plays an important role. P185 Establishing a critical care echocardiography laboratory K Yastrebov1, A McLean2 Conclusion Our results suggest that having continuously available TEE for monitoring and management of hemodynamically unstable patients increases awareness of right ventricular dysfunction in the ICU. References Conclusion Our results suggest that having continuously available TEE for monitoring and management of hemodynamically unstable patients increases awareness of right ventricular dysfunction in the ICU. References 1. Vieillard-Baron A, et al.: Intensive Care Med 2004, 30:1734-1739. 2. Monnet X, et al.: Intensive Care Med 2005, 31:1195-1201. 1. Vieillard-Baron A, et al.: Intensive Care Med 2004, 30:1734-1739. 2. Monnet X, et al.: Intensive Care Med 2005, 31:1195-1201. P181 Some advance monitoring used in developed countries is also unavailable in Thailand. P187l P187 Infl uence of positive end-expiratory pressure on four-chamber longitudinal strain analysis by speckle tracking echocardiography F Franchi, A Faltoni, M Cameli, S Cecchini, M Lisi, M Contorni, S Mondillo, S Scolletta University of Siena, Italy Critical Care 2013, 17(Suppl 2):P187 (doi: 10.1186/cc12125) Introduction Speckle-tracking echocardiography (STE) has emerged as an ultrasound technique for accurately evaluating myocardial function also in critically ill patients. By tracking the displacement of the speckles during the cardiac cycle, the strain rate can be measured offl ine after adequate image acquisition. The aim of the study was to evaluate the eff ects of the positive end-expiratory pressure (PEEP) on four-chamber longitudinal strain (LS) analysis in critically ill patients. g y y p Methods We enrolled 20 consecutive patients (mean age 64 ± 18) who needed mechanical ventilation and were admitted to the ICU due to heterogeneous causes. Inclusion criteria were: hypoxia requiring PEEP titration, invasive arterial pressure monitoring, age >18. Exclusion criteria were: myocardial dysfunction, cardiac arrhythmias and valvular pathologies. The same operator performed three standard echocardiography measurements (MyLab 70 Xvision; Esaote), each of them after having increased PEEP at 5, 10, and 15 cmH2O (T1, T2, T3, respectively). Blood gas analysis, respiratory, and hemodynamic parameters provided by a pulse contour method were also recorded. STE analysis was performed offl ine (XStrain™MyLab 70 Xvision; Esaote). Results Left peak atrial LS (LA-PALS) was signifi cantly reduced from T1 to T2, and from T2 to T3 (40.2 ± 12%, 35.9 ± 9%, 28.4 ± 8%, T1, T2, T3, respectively; P <0.05). Right peak atrial LS (RA-PALS) and right ventricular (RV)-LS showed a signifi cant reduction only at T3 (RA-PALS: 44.7 ± 48.5% at T1, 35.9 ± 11% at T3; RV-LS: –20.2 ± 2% at T1, –16.3 ± 1.1% at T3; P <0.05). Left ventricular (LV)-LS did not change signifi cantly during titration of PEEP. Cardiac chambers’ volumes and cardiac output (CO) showed a signifi cant reduction at higher levels of PEEP. Pulse pressure variation was signifi cantly aff ected by higher levels of PEEP (P <0.05). Conclusion Contrast-enhanced ultrasonography clearly improves visualization of the perfusion in various tissues. It is very likely to be superior to standard Doppler ultrasound, and is safe and well tolerated in critically ill patients. P185 Contrast-enhanced ultrasonography in the ICU: promising tool or exciting toy?i Contrast-enhanced ultrasonography in the ICU: promising tool or exciting toy?i Contrast-enhanced ultrasonography in the ICU: promising tool or exciting toy? I Göcze, R Herzog, BM Graf, A Agha, HJ Schlitt, K Pfi ster, E Jung, T Bein University Medical Centre Regensburg, Germany Critical Care 2013, 17(Suppl 2):P185 (doi: 10.1186/cc12123) I Göcze, R Herzog, BM Graf, A Agha, HJ Schlitt, K Pfi ster, E Jung, T Bein University Medical Centre Regensburg, Germany Critical Care 2013, 17(Suppl 2):P185 (doi: 10.1186/cc12123) Introduction Contrast-enhanced ultrasonography (CEUS) is a dynamic digital ultrasound-based imaging technique, which allows quantifi cation of the microvascularisation up to the capillary vessels. As a novel method for assessment of tissue perfusion it is ideally designed for use in the ICU. CEUS is cost-eff ective and safe and can be repeatedly performed at the bedside without radiation and nephrotoxicity. S70 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Methods The frequency of CEUS use in the multidisciplinary surgical ICU was retrospectively evaluated for the period from 1 September 2011 to 1 September 2012. Furthermore, contributions of this novel method to the management of critically ill ICU patients as well as its accuracy were assessed. References Krishnagopalan S, Kumar A, Parrillo JE, Kumar A: Myocardial dysfunction in the patient with sepsis. Curr Opin Crit Care 2002, 8:376-388. 2. Parker MM, McCarthy KE, Ognibene FP, et al.: Right ventricular dysfunction and dilatation, similar to left ventricular changes,characterize the cardiac depression of septic shock in humans. Chest 1999, 97:126-131. y Results In total, 33 CEUS studies were performed in critically ill ICU patients. The most frequent indications included: assessment of the liver perfusion, assessment of the pancreas and kidney perfusion after pancreas and kidney transplantation, assessment of the renal perfusion in acute kidney injury (AKI), assessment of active bleeding and assessment of the bowel perfusion. In all studies, the correct diagnosis was achieved and the transport of critically ill patients to the radiology department for further diagnostic procedures as well as application of iodinated contrast agents was avoided. In 16 cases signifi cant new fi ndings were detected. Twelve of them were missed by conventional standard Doppler ultrasound prior to CEUS. In assessment of seven cases with AKI, impaired or delayed perfusion and microcirculation of the kidney was observed in six patients. In three patients urgent surgical intervention was performed because of CEUS results. In three cases active bleeding was excluded at the bedside due to absence of contrast agent extravasation into hematoma (thigh and perihepatic) or into abdominal cavity, without need for complementary CT imaging or angiography. In one case the regular perfusion of intestinal anastomosis was confi rmed with no need for surgical exploration. None of patients undergoing CEUS manifested any adverse reactions or developed any complications associated with the imaging technique. P186 Hemodynamic disclosure of septic shock patients by intensive care ultrasound P Theerawit, Y Sutherasan, T Hongpanat Ramathibodi Hospital, Mahidol University, Bangkok, Thailand Critical Care 2013, 17(Suppl 2):P186 (doi: 10.1186/cc12124) Hemodynamic disclosure of septic shock patients by intensive care ultrasound P Theerawit, Y Sutherasan, T Hongpanat P Theerawit, Y Sutherasan, T Hongpanat Ramathibodi Hospital, Mahidol University, Bangkok, Thailand Critical Care 2013, 17(Suppl 2):P186 (doi: 10.1186/cc12124) Introduction Even though invasive hemodynamic devices are usually used for assessment of septic shock victims, they cannot evaluate the heart function. LV dysfunction as well as right heart syndrome are not uncommon in sepsis and critical patients. Intensive care ultrasound discloses these data and leads to appropriate treatment. Conclusion In hypoxic patients with normal cardiac function, PEEP titration determined a reduction of LA-LS, RA-LS and RV-LS values. LV-LS values were not infl uenced by PEEP changes. The fall in CO, observed with higher values of PEEP, seemed to be related to the impairment of preload and not of myocardial contractility. Whenever interpreting data on cardiac function obtained with longitudinal strain analysis, attention of the clinician should be drawn to diff erent levels of PEEP. The higher the PEEP, the more the probability of misleading interpretation of STE data. Methods The study was a prospective cross-sectional study. The measurement was performed within 24 hours of ICU admission. We excluded patients with history of COPD and pulmonary hypertension from any diseases. Only good-quality images acquired from subjects were included for analysis. The primary objective was to disclose how the hemodynamic changed in septic patients by ICU-US. y g p p y Results A total of 133 septic patients were measured by ICU-US. Good image quality was acquired in 115 cases (86.47%). The mean ages were 57.48 ± 17.87 years. The three major causes of sepsis were pneumonia, unknown source, abdominal infection and bacteremia. Heart failure at admission was found only in 1.79%. Previous history of hypertension, DM, and coronary artery disease was found in 12.17%, 11.30%, and 1.74% of patients. The mean LV ejection fraction (LVEF) was 54 ± 15.43%. The percentages of patients with LVEF <35%, 35 to 40%, 41 to 45% and >45% were 13.2%, 7.5%, 8.5%, and 70.8% respectively. Diastolic dysfunction defi ned by E/A ratio <1 was observed in 47.5% of patients. A total 44.6% of cases had cardiac output under 4 l/minute whereas CO over 6 l/minute was found in 18.1% of cases. The average mean pulmonary artery pressure was 34.75 ± 15.13 mmHg. The proportion of patients with meanPAP over 25 mmHg was 76.2%. RV to LV ratio >1 was found in 42.4% of septic patients. P187l Promising indications for the use of CEUS in the ICU may be the assessment of kidney microcirculation and assessment of liver perfusion in liver transplant and liver trauma patients. P186 P189 Validation of less-invasive hemodynamic monitoring with Pulsiofl ex in critically ill patients: interim results of a multicentre study K Van de Vijver1, C Pigozzi1, L Vervliet1, V Vanbiervliet1, V Brabers1, I Vos1, H Maes1, N Van Regenmortel1, I De laet1, K Schoonheydt1, H Dits1, J Belda2, Z Molnar3, M Malbrain1 1Ziekenhuis Netwerk Antwerpen, ZNA Stuivenberg, Antwerp, Belgium; 2Hospital Clinico Universitario, Valencia, Spain; 3University of Szeged, Hungary Critical Care 2013, 17(Suppl 2):P189 (doi: 10.1186/cc12127) Results A total of 103 measurements in 77 patients were performed. In seven patients measurement with Nexfi n was not possible. For CO (55 paired measurements), values were 6 ± 2.1 l/minute (range 2.6 to 12). Pearson’s correlation coeffi cient comparing Nexfi n-CO with reference CO showed a good correlation (R2 = 0.52). Bland–Altman analysis comparing both CO techniques revealed a mean bias ± 2SD (LA) of 0.3 ± 3.6 l/minute (58% error). The MAP was 84.2 ± 15.6 mmHg (53 to 131.5) and values obtained with the Nexfi n correlated well with the reference method with an R2 of 0.72. Bland–Altman analysis comparing both MAP techniques revealed a mean bias ± 2SD (LA) of –0.3 ± 18 mmHg (20.9% error). However, Nexfi n-MAP did not correlate well with NIBP (R2 = 0.36). Hemoglobin values obtained with Nexfi n Massimo technique did not correlate well with laboratory values (R2 = 0.26, 33% error). The 26 patients that died in the ICU had higher APACHE II (P = 0.017), SAPS II (P <0.0001), SOFA (P <0.0001) and SOS (P = 0.004) scores and signifi cantly lower MAP (P <0.0001), hemoglobin (P = 0.01) and lower dp/dtmax (P = 0.003), a marker for contractility. There were no outcome diff erences with regard to subgroup analysis in patients with either low or high CO or SVR. Introduction Thermodilution (TD) is considered a gold standard for measurement of cardiac index (CI) in critically ill patients. The aim of this study is to compare intermittent bolus TD CI with intermittent automatic calibration CI (AutoCI) and two continuous CIs obtained by pulse contour analysis with PiCCO2 (PiCCI) and Pulsiofl ex (PuCCI). 2l Methods Interim results of an ongoing prospective multicentre study in 53 patients. Age 58.7 ± 15.4, SAPS II score 51.4 ± 14.7 and SOFA score 10 ± 3.2. P188 Evaluation of a new calibration index suggesting recalibration of the pulse contour cardiac index by transpulmonary thermodilution: a prospective study W Huber, K Waldleitner, S Mair, B Saugel, RM Schmid Klinikum rechts der Isar, Technical University of Munich, Germany Critical Care 2013, 17(Suppl 2):P188 (doi: 10.1186/cc12126) Introduction After calibration by thermodilution (TD), the PiCCO device is able to assess cardiac index (CI) using pulse contour (PC) analysis. The manufacturer suggests recalibration by TD after 8 hours. Recently, we suggested a calibration index indicating a certain probability of a relevant bias and triggering the next calibration. With changes of CIpc compared with the previous CItd being a key predictor of the bias of CIpc compared with the following CItd, the manufacturer implemented a soft alarm indicating changes in CIpc compared with the last CItd (thresholds adjustable to 15%, 25% and 35%). The aim of Conclusion Cardiac dysfunction, namely left ventricle and probably right ventricle, was not uncommon in septic shock patients. Without intensive care ultrasound, all crucial information was delayed until patient deterioration and initial treatment may be harmful. Thus cardiac ultrasound should be used initially to disclose hemodynamic features before routine resuscitation is initiated. S71 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Results In total, 940 CCI and 382 TDCI values were obtained: 940 paired PiCCI and PuCCI; 358 paired AutoCI-TDCI measurements. TDCI values ranged from 1.5 to 6.9 l/minute/m2 (mean 3.6 ± 1.1), AutoCI from 1.8 to 7.2 (3.6 ± 0.9), PiCCI from 1.0 to 7.1 (3.5 ± 1.1) and PuCCI from 1.3 to 7.6 (3.6 ± 1). Pearson’s correlation coeffi cient comparing mean PuCCI and PiCCI values per patient had an R2 of 0.79. Comparison between AutoCI and TDCI had an R2 of 0.51. Changes in AutoCI correlated well with changes in TDCI (R2 = 0.44, concordance coeffi cient = 95.7), as did changes in PuCCI versus changes in PiCCI (R2 = 0.99, CC = 93.4%). Changes in PiCCI and PuCCI induced by an intervention correlated well with each other (R2 = 0.86, CC = 100%). The percentage error (PE) obtained by Bland and Altman analysis and R2 for the diff erent comparisons are presented in Table 1. the study was prospective evaluation of predictive capabilities of the 15% alarm regarding a bias of CIpc compared with CItd exceeding critical thresholds of 15%, 20% and 25%. P190 P190 P190 Interim results of an ongoing study on the use of non-invasive hemodynamic monitoring with Nexfi n in critically ill patients K Van de Vijver, V Brabers, C Pigozzi, L Vervliet, V Vanbiervliet, H Maes, I Vos, M Peetermans, N Van Regenmortel, I De laet, K Schoonheydt, H Dits, M Malbrain Ziekenhuis Netwerk Antwerpen, ZNA Stuivenberg, Antwerp, Belgium Critical Care 2013, 17(Suppl 2):P190 (doi: 10.1186/cc12128) Interim results of an ongoing study on the use of non-invasive hemodynamic monitoring with Nexfi n in critically ill patients K Van de Vijver, V Brabers, C Pigozzi, L Vervliet, V Vanbiervliet, H Maes, I Vos, M Peetermans, N Van Regenmortel, I De laet, K Schoonheydt, H Dits, M Malbrain Ziekenhuis Netwerk Antwerpen, ZNA Stuivenberg, Antwerp, Belgium Critical Care 2013, 17(Suppl 2):P190 (doi: 10.1186/cc12128) Conclusion Bias of CIpc compared with the next CItd does not depend on the time since last CItd. Changes in CIpc itself compared with the last CItd are associated with the bias. A 15% change in CIpc trend alarm provided by the new PiCCO algorithm is signifi cantly associated with bias values exceeding 15%, 20% or 25% of CItd, whereas time to last calibration >8 hours is not associated with bias exceeding these critical thresholds. Introduction The Nexfi n monitor (BMEYE, the Netherlands) enables continuous non-invasive analysis of blood pressure (MAP) as well as cardiac output (CO) measurements. The aim of the present study was to validate the Nexfi n in a mixed population of medical ICU patients. Introduction The Nexfi n monitor (BMEYE, the Netherlands) enables continuous non-invasive analysis of blood pressure (MAP) as well as cardiac output (CO) measurements. The aim of the present study was to validate the Nexfi n in a mixed population of medical ICU patients. Introduction The Nexfi n monitor (BMEYE, the Netherlands) enables continuous non-invasive analysis of blood pressure (MAP) as well as cardiac output (CO) measurements. The aim of the present study was to validate the Nexfi n in a mixed population of medical ICU patients. Methods Interim results of a prospective ongoing study in 77 patients admitted to the medical ICU (46 patients mechanically ventilated, M/F ratio 1/1). Age 65.6 ± 15.9, BMI 25.6 ± 4.8, APACHE II score 22.9 ± 10.9, SAPS II 48 ± 20.1, SOFA score 7.5 ± 4.5. A modifi ed outreach score (SOS) was calculated on admission. P190 For all patients, simultaneous recording of arterial pressure by radial line (n = 78), PiCCO (n = 44) or by NIBP with arm cuff (n = 47) was compared with the Nexfi n monitor. Statistical analysis was performed with Student’s t test, Pearson correlation and Bland–Altman analysis. i Methods Interim results of a prospective ongoing study in 77 patients admitted to the medical ICU (46 patients mechanically ventilated, M/F ratio 1/1). Age 65.6 ± 15.9, BMI 25.6 ± 4.8, APACHE II score 22.9 ± 10.9, SAPS II 48 ± 20.1, SOFA score 7.5 ± 4.5. A modifi ed outreach score (SOS) was calculated on admission. For all patients, simultaneous recording of arterial pressure by radial line (n = 78), PiCCO (n = 44) or by NIBP with arm cuff (n = 47) was compared with the Nexfi n monitor. Statistical analysis was performed with Student’s t test, Pearson correlation and Bland–Altman analysis. P188 the study was prospective evaluation of predictive capabilities of the 15% alarm regarding a bias of CIpc compared with CItd exceeding critical thresholds of 15%, 20% and 25%. Methods A prospective analysis of 329 routine TPTD measurements in 70 patients. The CI-trend alarm was set to 15%, and CIpc, trend alarm (yes/no), relative and absolute changes in CIpc were recorded immediately before TD providing exact measurement of CItd. Predictive capabilities of the 15% trend alarm regarding the bias were evaluated using Spearman correlation, chi-square test, ROC analysis and Wilcoxon test (IBM SPSS 20). Results A total of 70 patients (24 female, 46 male), age 62 ± 14 years, APACHE II score 19.5 ± 7.5, SOFA score 8.9 ± 4.4. At 937 ± 904 (50 to 5,795) minutes after the last CItd, CIpc provided a bias of –0.0566 ± 0.761 l/ minutem2 compared with the next CItd. Percentage error was 34.6%. Absolute and relative bias of CIpc compared with the new CItd did not correlate to time to last TD, but correlated (all P <0.001) to CIpc itself (r = 0.333; r = 0.385) and relative (r = 0.531; r = 0.537) and absolute (r = 0.533; r = 0.529) changes in CIpc compared with last CItd. The 15% CIpc trend alarm was indicated before 83/329 measurements (25.2%). The amount of bias exceeded 15% and 20% in 101 (30.7%) and 79 (24%) of TDs. In TDs with indicated trend alarm (≥15% deviation of CIpc to the last CItd), the amount of bias more frequently exceeded 15% (P = 0.019), 20% (P <0.001) and 25% (P <0.001). Time to last calibration ≥8 hours was not associated with bias exceeding 15% (P = 0.735), 20% (P = 0.888) or 25% (P = 281). Conclusion The preliminary results indicate that in unstable critically ill patients, CI can be reliably monitored with Pulsiofl ex technology via a femoral line. Pulsiofl ex was also able to keep track of changes in CI. P191 Cost-eff ectiveness analysis of stroke volume variation guided perioperative hemodynamic optimization J Benes, J Zatloukal, A Simanova, I Chytra, E Kasal The Medical Faculty and Hospital in Plzen – Charles University Prague, Plzen, Czech Republic Critical Care 2013, 17(Suppl 2):P191 (doi: 10.1186/cc12129) P191 Cost-eff ectiveness analysis of stroke volume variation guided perioperative hemodynamic optimization J Benes, J Zatloukal, A Simanova, I Chytra, E Kasal The Medical Faculty and Hospital in Plzen – Charles University Prague, Plzen, Czech Republic Critical Care 2013, 17(Suppl 2):P191 (doi: 10.1186/cc12129) Introduction Perioperative goal-directed therapy (pGDT) can sub- stantially improve the outcome of high-risk surgical patients [1]. But the approach needs an initial investment and increases the staff workload. Economic factors might participate in the weak adherence to the pGDT concept. Some model studies support pGDT cost-eff ectiveness, but real economic data based on a recent clinical trial are lacking. We performed an economic analysis of hemodynamic optimization using the stroke volume variation trial [2] in order to elucidate this issue. Figure 1 (abstract P192). Correlation between PaO2/FiO2 and EVLWIc. Methods The hospital care invoices of all 120 patients included in the trial were retrospectively extracted. Due to the nature of the data we have adopted the healthcare payer’s perspective. We performed a comparison of induced costs between the Vigileo (n = 60) and Control (n = 60) groups and constructed a cost tree using the study group and complications occurrence as distributive parameters. The incremental cost-eff ectiveness ratio per complication avoided was calculated and, fi nally, diff erent reimbursing categories were assessed as potential cost drivers. (EVLWI) refl ects pulmonary edema [2]. The new EV1000/VolumeView (Edwards Lifesciences) can accurately measure EVLWI corrected for the actual volume of lung parenchyma (EVLWIc). The aim of our study is to prove a stronger correlation between EVLWIc and PaO2/FiO2 compared with EVLWI in patients undergoing pulmonary resection. Methods A prospective observational study. Seven patients with lung cancer undergoing pulmonary resection were monitored using the EV1000 plathform. EVLWI was assessed by thermodilution at the following time points: after intubation (t1); during single-lung ventilation (t2); after lung resection (t3); after ICU admission (t4); 12 hours (t5) and 18 hours after ICU admission (t6). EVLWIc values were also collected at t3 and t4. PaO2/FiO2 was measured at the same time points. Results A decreased rate (18 vs. 35 patients) and number of complications (34 vs. 78) were observed in the original trials Vigileo group. The mean cost of intervened patient was lower (€2,877 ± 2,336 vs. €3,371 ± 3,238; P = 0.38). According to the cost-tree analysis, patients with complications (n = 53; 44%) consumed signifi cantly more resources (€235,623; 63%). References 1. Pinsky MR: Clin Chest Med 2003, 24:549-560. 2. Sakka SG, et al.: Chest 2002, 122:2080-2086. 1. Pinsky MR: Clin Chest Med 2003, 24:549-560. 2. Sakka SG, et al.: Chest 2002, 122:2080-2086. Acknowledgements The study was supported by the MSM0021620819 and the Charles University Research Fund (project number P36). References P193 1. Hamilton MA, et al.: Systematic review and meta-analysis on the use of preemptive hemodynamic intervention to improve postoperative outcomes in moderate and high-risk surgical patients. Anesth Analg 2011, 112:1392-1402.l Goal-directed resuscitation therapy in high-risk patients undergoing cardiac surgery (GRICS study): a randomized controlled trial – preliminary results E Osawa1, A Rhodes2, J Vincent3, J Almeida1, J Fukushima1, B Pileggi1, C Park1, L Camara1, J Auler Jr1, R Chan1, M Piccioni1, M Lima1, F Galas1, L Hajjar1 1Heart Institute, São Paulo, Brazil; 2St George’s Healthcare NHS Trust and St George’s University of London, UK; 3Universite Libre de Bruxelles, Brussels, Belgium Critical Care 2013, 17(Suppl 2):P193 (doi: 10.1186/cc12131) Goal-directed resuscitation therapy in high-risk patients undergoing cardiac surgery (GRICS study): a randomized controlled trial – preliminary results P191 Cost-eff ectiveness analysis of stroke volume variation guided perioperative hemodynamic optimization J Benes, J Zatloukal, A Simanova, I Chytra, E Kasal The Medical Faculty and Hospital in Plzen – Charles University Prague, Plzen, Czech Republic Critical Care 2013, 17(Suppl 2):P191 (doi: 10.1186/cc12129) A gain of €634 per avoided complications confi rms that the lower complications rate was the most important cost driver. Both the clinical care for patients costs (€505 vs. 912; P = 0.04) and ward stay costs (€244 vs. 402; P = 0.03) were decreased by the intervention. On the contrary, the intervention increased anaesthesia costs (€880 vs. 688; P = 0.001). Results No signifi cant correlation was found between EVLWI and PaO2/ FiO2 (r = –0.3124, P >0.05), while a signifi cant correlation was seen between EVLWIc and PaO2/FiO2 (r = –0.528, P =0.009; Figure 1). Conclusion Despite the small sample size, this study shows that in patients undergoing pulmonary resection the EVLWIc is more strongly correlated to PaO2/FiO2 than EVLWI. Therefore, the EV1000 may be a valuable tool for more reliable hemodynamic monitoring in this subgroup of patients. R f Results No signifi cant correlation was found between EVLWI and PaO2/ FiO2 (r = –0.3124, P >0.05), while a signifi cant correlation was seen between EVLWIc and PaO2/FiO2 (r = –0.528, P =0.009; Figure 1). 2 2 g Conclusion Despite the small sample size, this study shows that in patients undergoing pulmonary resection the EVLWIc is more strongly correlated to PaO2/FiO2 than EVLWI. Therefore, the EV1000 may be a valuable tool for more reliable hemodynamic monitoring in this subgroup of patients. Conclusion Intraoperative fl uid optimization with the use of stroke volume variation and the Vigileo/FloTrac system showed not only a substantial improvement of morbidity, but was also associated with an economic benefi t. This observed benefi t highly exceed the increased monitoring costs in our trial. P192 P192 EV1000/VolumeView: a new device for a more reliable measurement of extravascular lung water index in patients with lung resections A Donati, C Melia, V Monaldi, R Domizi, E Damiani, A Carsetti, C Scorcella, R Castagnani, P Pelaia Università Politecnica delle Marche, Ancona, Italy Critical Care 2013, 17(Suppl 2):P192 (doi: 10.1186/cc12130) P192 EV1000/VolumeView: a new device for a more reliable measurement of extravascular lung water index in patients with lung resections A Donati, C Melia, V Monaldi, R Domizi, E Damiani, A Carsetti, C Scorcella, R Castagnani, P Pelaia Università Politecnica delle Marche, Ancona, Italy Critical Care 2013, 17(Suppl 2):P192 (doi: 10.1186/cc12130) Goal-directed resuscitation therapy in high-risk patients undergoing cardiac surgery (GRICS study): a randomized controlled trial – preliminary results p y E Osawa1, A Rhodes2, J Vincent3, J Almeida1, J Fukushima1, B Pileggi1, C Park1, L Camara1, J Auler Jr1, R Chan1, M Piccioni1, M Lima1, F Galas1, L Hajjar1 2. Benes J, et al.: Intraoperative fl uid optimization using stroke volume variation in high risk surgical patients: results of prospective randomized study. Crit Care 2010, 14:R118. 2. Benes J, et al.: Intraoperative fl uid optimization using stroke volume variation in high risk surgical patients: results of prospective randomized study. Crit Care 2010, 14:R118. jj 1Heart Institute, São Paulo, Brazil; 2St George’s Healthcare NHS Trust and St George’s University of London, UK; 3Universite Libre de Bruxelles, Brussels, Belgium l l (d ) P189 All patients underwent PiCCO monitoring via a femoral line whilst a radial line was kept in place during four 8-hour time periods (in the fi rst two periods, the Pulsiofl ex was connected to a radial line; in the last two it was connected to a femoral line). In the fi rst and third periods, the Pulsiofl ex was calibrated with TDCI, for the second and fourth periods Pulsiofl ex was calibrated with AutoCI. Simultaneous PiCCI and PuCCI measurements were obtained every 2 hours while simultaneous TDCI and AutoCI were obtained every 8 hours. We also looked at the eff ects of 40 interventions. p g Conclusion In unstable critically ill patients, MAP (and CO) can be monitored with the Nexfi n. The exact patient population for this Table 1 (abstract P189). Results of Bland and Altman analysis Pulsiofl ex AutoCal PE (CCl) (%) n R2 PE (TD-Pi) (%) n R2 PE (TD-Pu) (%) n R2 All All 37.9 940 0.73 22.8 382 0.88 38.5 382 0.66 All Yes 43.4 510 0.50 20.4 210 0.88 42.3 210 0.47 All No 27.8 430 0.83 25.6 172 0.85 32.5 172 0.74 Fem All 30.6 464 0.73 20.2 192 0.88 33.0 192 0.66 Rad All 44.2 476 0.58 25.2 190 0.85 43.7 190 0.56 Table 1 (abstract P189). Results of Bland and Altman analysis S72 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Figure 1 (abstract P192). Correlation between PaO2/FiO2 and EVLWIc. technology has yet to be defi ned and more patients are probably needed for pattern recognition, although the results indicate that low MAP and dp/dtmax are associated with poor outcome. In the future, Nexfi n data could theoretically be incorporated in a new outreach score. P191 Treatment-related cardiac complications associated with goal-directed therapy in high-risk surgical patients: a meta-analysis N Arulkumaran, C Corredor, M Hamilton, M Grounds, J Ball, A Rhodes, M Cecconi Treatment-related cardiac complications associated with goal-directed therapy in high-risk surgical patients: a meta-analysis N Arulkumaran, C Corredor, M Hamilton, M Grounds, J Ball, A Rhodes, M Cecconi St George’s Hospital, London, UK Critical Care 2013, 17(Suppl 2):P195 (doi: 10.1186/cc12133) Treatment-related cardiac complications associated with goal-directed therapy in high-risk surgical patients: a meta-analysis N Arulkumaran, C Corredor, M Hamilton, M Grounds, J Ball, A Rhodes, M Cecconi St George’s Hospital, London, UK Critical Care 2013, 17(Suppl 2):P195 (doi: 10.1186/cc12133) Introduction We hypothesized that goal-directed therapy (GDT) is not associated with an increased risk of cardiac complications in high-risk, noncardiac surgical patients. Patients with limited cardiopulmonary reserve are at risk of mortality and morbidity after major surgery [1]. Augmentation of the oxygen delivery index (DO2I) with a combination of intravenous fl uids and inotropes (GDT) has been shown to reduce the postoperative mortality and morbidity in high-risk patients [2]. However, concerns regarding cardiac complications associated with fl uid challenges and inotropes used to augment cardiac output may deter clinicians from instituting early GDT in the very patients who are more likely to benefi t. Results Twenty patients from the GDT group were compared with 20 control patients. Both groups were comparable concerning baseline characteristics and severity scores, except for a higher prevalence of hypertension and heart failure in the GDT group. Intraoperative data showed no diff erence regarding length of extracorporeal circulation, fl uid balance, transfusion or inotropic requirement. Patients from the GDT group were given more fl uids within the fi rst 8 hours as compared with the conventional group (1,250 ml vs. 500 ml, P <0.001). GDT patients showed a median ICU stay of 3 days (95% CI: 3 to 4) compared with 5 days in control patients (95% CI: 4 to 7). Moreover, hospital stay was less prolonged in GDT patients (10 days vs. 14 days, P = 0.043). Inotropic dependence was lower in the GDT group (29 hours vs. 55 hours, P = 0.003) as well as the cumulative dobutamine dosage (8 vs. 19 μg/kg/day, P = 0.025). Also, GDT group presented a lower incidence of infections, tachyarrithmias and acute renal failure (RIFLE R) when compared with the control group. yi Methods Systematic search of MEDLINE, Embase and CENTRAL databases for randomized controlled trials of GDT in high-risk surgical patients. Studies including cardiac surgery, trauma, and pediatric surgery were excluded to minimize heterogeneity. References 1. Pearse RM, et al.: Lancet 2012, 380:1059-1065. 2. Hamilton MA, et al.: Anesth Analg 2011, 112:1392-1402. 2. Hamilton MA, et al.: Anesth Analg 2011, 112:1392-1402. Results In the group treated with a restrictive volume approach, patients received fl uids at the rate of 7.0 ± 1.0 ml/kg/hour. PaO2/FiO2 was 288 ± 14 after intubation and 270 ± 22 before extubation. In the group treated with a liberal volume approach, fl uids were replaced at 11.0 ± 2.0 ml/kg/hour. PaO2/FiO2 was 259 ± 24 after intubation and 223 ± 43 before extubation. Surgery combined with OLV was found to signifi cantly aff ect the PaO2/FiO2 value (ANOVA, F1,14 = 15.85a, P = 0.001, partial η2 = 0.531). The average PaO2/FiO2 level was signifi cantly higher in the restrictive-replacement group than in the liberal-replacement group (ANOVA, F1,14 = 9.66, P = 0.008, partial η2 = 0.408). There was no interaction between the groups (ANOVA, F1,14 = 1.7a, P = 0.215, partial η2 =0.108). Mean length of stay in the ICU was similar between the restrictive-replacement group (5.2 ± 2.3 days) and the liberal- replacement group (6.3 ± 1.6 days) (ANOVA, F1,14 = 0.814a, P = 0.382, partial η2 = 0.055). Perioperative volume management for esophageal cancer surgery M Karaman Ilic, G Madžarac, J Kogler, D Stančić Rokotov, N Hodoba University Hospital Centre Zagreb, Croatia Critical Care 2013, 17(Suppl 2):P194 (doi: 10.1186/cc12132) Perioperative volume management for esophageal cancer surgery M Karaman Ilic, G Madžarac, J Kogler, D Stančić Rokotov, N Hodoba University Hospital Centre Zagreb, Croatia Critical Care 2013, 17(Suppl 2):P194 (doi: 10.1186/cc12132) Introduction Pulmonary complications are the primary reason for extending a patient’s stay in the ICU. The aim of this study was to investigate whether perioperative volume management can infl uence the PaO2/FiO2 value and total length of stay in the ICU. Conclusion Perioperative, physiologically guided, GDT in high-risk surgical patients is not associated and actually reduces postoperative cardiovascular complications. 2 2 Methods Sixteen patients were divided into two groups: one group was treated with a restrictive approach (≤8 ml/kg/hour), and the other with a liberal approach (> 8 ml/kg/hour). Patients were randomly allocated using sealed envelopes. During the thoracic part of the surgical procedure, all patients received one-lung ventilation (OLV). P195 or extracardiac arteriopathy) were allocated to GDT or conventional hemodynamic therapy. We excluded patients with endocarditis, previous use of dobutamine, need for IABP, high dose of vasopressors and emergency surgery. The GDT protocol involved hemodynamic resuscitation aimed at a target of a cardiac index >3 l/minute/m2 through a three-step approach: fl uid therapy of 250 ml lactated Ringer’s solution, dobutamine infusion up to a dose of 20 μg/kg/minute, and red blood cell transfusion to reach a hematocrit level above 28%. EV1000/VolumeView: a new device for a more reliable measurement of extravascular lung water index in patients with lung resections g Critical Care 2013, 17(Suppl 2):P193 (doi: 10.1186/cc12131) g Critical Care 2013, 17(Suppl 2):P193 (doi: 10.1186/cc12131) Introduction A goal-directed resuscitation therapy (GDT) through optimization of cardiac output reduces complications in noncardiac surgeries. We investigated whether the implementation of a GDT protocol in high-risk cardiac surgery with the use of LiDCO Rapid reduces postoperative complications as compared with the standard of care. Introduction A goal-directed resuscitation therapy (GDT) through optimization of cardiac output reduces complications in noncardiac surgeries. We investigated whether the implementation of a GDT protocol in high-risk cardiac surgery with the use of LiDCO Rapid reduces postoperative complications as compared with the standard of care. A Donati, C Melia, V Monaldi, R Domizi, E Damiani, A Carsetti, C Scorcella, R Castagnani, P Pelaia Università Politecnica delle Marche, Ancona, Italy Critical Care 2013, 17(Suppl 2):P192 (doi: 10.1186/cc12130) Introduction Hemodynamic monitoring is important in high-risk surgical patients in order to detect and correct circulatory instability, thereby improving outcome [1]. The extravascular lung water index Methods We performed a prospective and randomized study whereby consecutive patients fulfi lling one high-risk criteria (EuroSCORE >5, ejection fraction <50%, recent myocardial infarction, unstable angina S73 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 1. Nisanevich V, Felsenstein I, Almogy G, Weissman C, Einav S, Matot I: Eff ect of intraoperative fl uid management on outcome after intraabdominal surgery. Anesthesiology 2005, 103:25-32.l 1. Nisanevich V, Felsenstein I, Almogy G, Weissman C, Einav S, Matot I: Eff ect of intraoperative fl uid management on outcome after intraabdominal surgery. Anesthesiology 2005, 103:25-32. 2. Wei S, Tian J, Song X, Chen Y: Association of perioperative fl uid balance and adverse surgical outcomes in esophageal cancer and esophagogastric junction cancer. Ann Thorac Surg 2008, 86:266-272. Treatment-related cardiac complications associated with goal-directed therapy in high-risk surgical patients: a meta-analysis N Arulkumaran, C Corredor, M Hamilton, M Grounds, J Ball, A Rhodes, M Cecconi We reviewed the rates of all cardiac complications, arrhythmias, acute myocardial ischemia, and acute pulmonary edema. Meta-analyses were performed and forest plots drawn using RevMan software. Data are presented as odd ratios (ORs) (95% CIs), and P values. Conclusion A hemodynamic therapy tailored to an optimized cardiac output reduced the length of ICU stay, vasoactive drug requirement and postoperative complications. Results We identifi ed 23 randomized controlled trials including 2,219 patients, who reported cardiac complications. GDT was associated with a reduction in total cardiovascular complications (OR = 0.55 (0.39 to 0.78), P = 0.0007), and with a signifi cantly reduced incidence of arrhythmias (OR = 0.59 (0.38 to 0.91), P = 0.02). GDT was not associated with an increase in acute pulmonary edema (OR = 0.68 (0.42 to 1.10), P = 0.11) or acute myocardial ischemia (OR = 0.70 (0.38 to 1.27), P = 0.23). Subgroup analysis of overall cardiovascular complications revealed that the benefi t is most pronounced in patients receiving fl uid and inotrope therapy (OR = 0.55 (0.34 to 0.89), P = 0.01) to achieve a supranormal oxygen delivery target (OR = 0.50 (0.32 to 0.79), P = 0.003), guided by the use of minimally invasive cardiac outmonitoring (OR = 0.50 (0.33 to 0.77), P = 0.002). p p p Acknowledgement Grants received from FAPESP 2011/19987-5. Acknowledgement Grants received from FAPESP 2011/19987-5. Estimation of potential cost-savings related to the implementation of perioperative hemodynamic goal-directed therapy COVIG (T1) 0.31 (CI 0.24 to 1.63) –0.49 –3.41 to 2.43 COECHO vs. COMC (T1) 0.71 (CI 0.56 to 1.08) –0.055 –1.19 to 1.08 COECHO vs. COVIG (T2) 0.27 (CI 0.16 to 1.53) –0.49 –3.24 to 2.34 COECHO vs. COMC (T2) 0.79 (CI 0.66 to 1.107) –0.023 –0.94 to 0.9 CI, confi dence interval; LoA, limits of agreement. between 0.35 and 0.55. Importantly, these odd ratios were not related to the morbidity rates. In the US publication [2], extra costs for treating patients with 1+ complication were $17,949. In the Swiss (CH) publication [3], they were $34,446. See Table 1. y Conclusion Depending on the pre-implementation morbidity rate, the degree of pGDT-induced morbidity reduction and the country, cost-savings ranged between $808 and $13,434 per patient. This large variability suggests that local/hospital estimations are desirable before starting pGDT implementation. These tailored evaluations would also allow more precise cost-saving estimations by taking into account the actual and expected pGDT compliance rates. and compared with those calculated with the echocardiographic standard formulation (stroke volume = cross-sectional area×velocity time integral; COECHO = SV×heart rate). In every patient CO was measured twice: at baseline (T1) and after volume loading (500 ml lactate Ringer solution) (T2). Agreements between COVIG, COMC, and COECHO were evaluated by means of simple linear regression (r2) and Bland–Altman analysis. and compared with those calculated with the echocardiographic standard formulation (stroke volume = cross-sectional area×velocity time integral; COECHO = SV×heart rate). In every patient CO was measured twice: at baseline (T1) and after volume loading (500 ml lactate Ringer solution) (T2). Agreements between COVIG, COMC, and COECHO were evaluated by means of simple linear regression (r2) and Bland–Altman analysis. 1. Hamilton MA, et al.: A systematic review and meta-analysis on the use of preemptive hemodynamic intervention to improve postoperative outcomes in moderate and high-risk surgical patients. Anesth Analg 2011, 112:1392-1402. 2. Boltz et al.: Synergistic implications of multiple postoperative outcomes. Am J Med Qual 2012, in press. 3. Vonlanthen R, et al.: The impact of complications on costs of major surgical procedures. Ann Surg 2011, 254:907-913. 3. Vonlanthen R, et al.: The impact of complications on costs of major surgical procedures. Ann Surg 2011, 254:907-913. y Results Twenty patients were enrolled in the study. Values of r2, bias and limit of agreement at T1 and T2 are summarized in Table 1. Estimation of potential cost-savings related to the implementation of perioperative hemodynamic goal-directed therapy p p T Simon, G Marx RWTH University Hospital Aachen, Germany y y Critical Care 2013, 17(Suppl 2):P196 (doi: 10.1186/cc12134) Introduction Many studies have demonstrated the ability of peri- operative hemodynamic goal-directed therapy (pGDT) to decrease post operative morbidity in patients undergoing medium-to-high-risk surgery [1]. As a result, pGDT may be a cost-saving strategy. Our goal was to provide an estimation of potential cost-savings based on recent literature. Methods The largest and most recent meta-analysis [1] on pGDT was used to estimate what could be the reduction of postoperative morbidity if pGDT was to be adopted. Costs related to the treatment of patients developing at least one (1+) postoperative complication were obtained from two recent US [2] and Swiss [3] publications. Potential cost-savings related to the adoption of pGDT were calculated according to the actual morbidity rate, assuming 0% pGDT use so far, and 100% compliance rate after implementation. Conclusion Results from this small sample indicate that esophageal carcinoma surgery by itself had a detrimental eff ect on the PaO2/FiO2 value, which restriction of perioperative volume did not signifi cantly aff ect. Volume restriction also did not aff ect length of stay in the ICU. References 1. Nisanevich V, Felsenstein I, Almogy G, Weissman C, Einav S, Matot I: Eff ect of intraoperative fl uid management on outcome after intraabdominal surgery. Anesthesiology 2005, 103:25-32.l Results The 2011 meta-analysis [1] of 29 RCTs (4,805 patients) showed that pGDT is associated with a reduction in the rate of patients developing 1+ postoperative complications with odd ratios ranging 2. Wei S, Tian J, Song X, Chen Y: Association of perioperative fl uid balance and adverse surgical outcomes in esophageal cancer and esophagogastric junction cancer. Ann Thorac Surg 2008, 86:266-272. Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S74 Actual morbidity rate no pGDT (%) 10 20 30 40 50 60 Expected morbidity rate pGDT (%) 3.5 to 5.5 7 to 11 11 to 17 14 to 22 18 to 28 21 to 33 Expected cost reduction/US patient ($) 808 to 1,167 1,615 to 2,333 2,423 to 3,500 3,231 to 4,667 4,039 to 5,833 4,846 to 7,000 Expected cost reduction/CH patient ($) 1,550 to 2,239 3,100 to 4,478 4,478 to 6,545 6,200 to 8,956 7,578 to 11,023 9,300 to 13,434 Table 1 (abstract P197) Pearson r2 Bias LoA COECHO vs. Cardiac output estimation: Vigileo and Mostcare versus echocardiography S Romagnoli, D Quattrone, AR De Gaudio S Romagnoli, D Quattrone, AR De Gaudio S Romagnoli, D Quattrone, AR De Gaudio Azienda Ospedaliero Universitaria Careggi, Florence, Italy Critical Care 2013, 17(Suppl 2):P197 (doi: 10.1186/cc12135) g Azienda Ospedaliero Universitaria Careggi, Florence, Italy y Critical Care 2013, 17(Suppl 2):P197 (doi: 10.1186/cc12135) Conclusion Vigileo did not prove to be a substitute to the reference system; pre-loaded data, necessary for vascular impedance estimation, may be one of the main limitations that made Vigileo measurements less accurate than the MostCare ones. On the contrary, MostCare, an uncalibrated totally independent system, was shown to properly estimate the vascular impedance in these hemodynamically stable patients. Further comparisons in unstable conditions are needed to confi rm our observations. Introduction In the present study we analyzed the reliability for cardiac output (CO) measurement of Vigileo (Edwards Lifescience, Irvine, CA, USA) and MostCare (pressure recording analytical method; Vygon-Vytech, Padova, Italy) in comparison with transthoracic Doppler echocardiography (as the reference method) in patients undergoing vascular surgery. Estimation of potential cost-savings related to the implementation of perioperative hemodynamic goal-directed therapy CO values ranged from 3.9 and 8.6 l/minute (echo), from 3.4 to 9.9 (Vigileo) and from 4 to 8.3 (MostCare); the Pearson’s and Bland–Altman methods showed poor agreement between COECHO and COVIG, demonstrating a tendency to overestimation (see Figure 1). The percentage of error (PE) was 51.7% at T1 and 49.3% at T2. On the contrary, MostCare measures showed good agreement with echocardiography (see Table 1) with a PE of 22.4% at T1 and of 17% at T2. Computerized decision support system improves consistency of haemodynamic assessment amongst ICU team members AE Aneman, R Ranganatha Liverpool Hospital, Liverpool, Australia Critical Care 2013, 17(Suppl 2):P199 (doi: 10.1186/cc12137) Computerized decision support system improves consistency of haemodynamic assessment amongst ICU team members AE Aneman, R Ranganatha Liverpool Hospital, Liverpool, Australia Critical Care 2013, 17(Suppl 2):P199 (doi: 10.1186/cc12137) Methods The in vitro set-up consisted of a centrifugal pump, a network of tubes with variable volumes, an electromagnetic fl owmeter to measure and adjust the generated fl ow, heating devices to maintain constant temperature (37°C), two thermistors for thermodilution measurement, an ultrasound transducer and a pressure stabilizer. A small bolus of UCA diluted in cold saline (1 mg SonoVue® in 20 ml saline at 4°C) was injected into the system. The cold UCA passage through a fi rst and a second region of interest (ROI) was measured simultaneously with the ultrasound transducer and the thermistors. The measurements were performed at diff erent fl ows and volumes. BVs were estimated using the two diff erent approaches, namely CEUS and thermodilution. The IDCs were processed and fi tted separately with a dedicated model to estimate the ΔMTT of the cold UCA bolus between the two ROIs and the two thermistors. All the measurements were repeated three times. Results A linear relation between BVs estimated by the two techniques was observed with a correlation coeffi cient of 0.94. The bias of CEUS with respect to the true volumes was –40.1 ml; the bias of thermodilution was 84.3 ml. The most prominent diff erences between the two techniques were observed in case of high volume and low fl ow, possibly due to diff erent transport kinetics between UCAs and heat. AE Aneman, R Ranganatha Liverpool Hospital, Liverpool, Australia p p p Critical Care 2013, 17(Suppl 2):P199 (doi: 10.1186/cc12137) Critical Care 2013, 17(Suppl 2):P199 (doi: 10.1186/cc12137) Introduction This study evaluated the potential of the NAVIGATOR (Applied Physiology) haemodynamic monitor to improve consistency of cardiovascular assessments amongst ICU members with diff erent levels of expertise and experience. Methods Patients (n = 20) post cardiac surgery were monitored by NAVIGATOR to display heart effi ciency (Eh), mean systemic fi lling pressure (Pms) and vascular resistance (SVR) against targets for mean arterial pressure and cardiac output set by the clinical team. i References y Methods Both Vigileo and MostCare were connected to the FloTrac transducer (Edwards Lifescience) for CO calculation. The data from Vigileo and MostCare were registered (COVIG and COMC respectively) . Romano SM, et al.: Crit Care 2002, 30:1834-1841. . Romano SM, et al.: Crit Care 2002, 30:1834-1841. 2. Geisen M, et al.: Curr Opin Crit Care 2012, 18:377-384. Figure 1 (abstract P197). Bland–Altman analysis (T1). Figure 1 (abstract P197). Bland–Altman analysis (T1). Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S75 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 P198 Agreement (maximum two steps deviation for each assessment between staff ; number of patients), disagreement (median and interquartile range of steps) and therapeutic agreement (intervention/s to change Eh, Pms and SVR in similar direction/s, number of patients) were recorded and analysed for statistical diff erence BLIND versus OPEN (Fisher’s exact test, Mann–Whitney test, P <0.05). P198 Brachial pulse waveform characteristics predict development of organ failure in septic patients S Kazune, I Jagmane Hospital of Traumatology and Orthopaedics, Riga, Latvia Critical Care 2013, 17(Suppl 2):P198 (doi: 10.1186/cc12136) y Results Eh was commonly overestimated, Pms commonly under- estimated with no clear trend for SVR. Agreement amongst all categories of staff increased and disagreement score decreased for Eh, Pms and SVR (Table 1) comparing BLIND versus OPEN assessments. Agreement for therapeutic interventions also increased signifi cantly from 4/20 (BLIND) to 18/20 patients (OPEN). Introduction Pulse waveform characteristics (pulse wave transit time and augmentation index) are measures of arterial stiff ness. Previous studies have found an association between severity of acute infl ammatory states and increased arterial stiff ness but it is not known whether non-invasive pulse waveform analysis could predict development of multiple organ failure in septic patients. The purpose of this study was to evaluate the photoplethysmographic brachial artery pulse wave transit time and augmentation index and their changes in response to induced forearm ischemia in septic ICU patients and correlate these indices to the development of subsequent end organ damage. Table 1 (abstract P199) BLIND OPEN Eh 6/20, 2 (1 to 3) 17/20, 1 (0 to 1) Pms 6/20, 2.5 (2 to 4) 17/20, 2 (1 to 2) SVR 8/20, 2 (1 to 2) 16/20, 1 (1 to 2) Patients (n/20) and score (median and IQR). P <0.05. Methods A prospective observational study in patients with sepsis within 24 hours of admission. i References Severity of sepsis was assessed with APACHE II score (median 18.5) and SOFA score (median 7.5). Three- minute signal recording was done concurrently from the brachial artery at the elbow and the radial artery at the wrist with an originally designed photoplethysmograph at rest and after 5 minutes of induced forearm ischemia. Recordings were analyzed to obtain the pulse wave transit time and augmentation index at rest and 60 seconds after induced ischemia. The SOFA score was recalculated at 48 hours post recording. Patients (n/20) and score (median and IQR). P <0.05. Conclusion The assessment of heart function, intravascular fi lling and resistance state in postoperative cardiac patients was made signifi cantly more consistent amongst diff erent ICU staff members using the NAVIGATOR haemodynamic monitor. Such enhanced consistency could potentially make the haemodynamic management more eff ective with improved clinical outcomes. Results We studied 14 consecutive general ICU patients. There was a negative linear relationship between the pulse wave transit time (median 22.6 ms) at rest and increase in SOFA score in 48 hours (P = 0.02, r = 0.96). The postischemic pulse wave transit time increased in all patients (median 25.7 ms) but no association was found between the proportion of increase and subsequent change in SOFA. Correlation between rest (median 7.6) and postischemic (median 7.2) augmentation index and 48-hour SOFA scores was not statistically signifi cant (r = 0.57, P = 0.46). Reference Reference 1. Vlachopoulos C, Dima I, Aznaouridis K, Vasiliadou C, Ioakeimidis N, Aggeli C, Toutouza M, Stefanadis C: Acute systemic infl ammation increases arterial stiff ness and decreases wave refl ections in healthy individuals. Circulation 2005, 112:2193-2200. Volume quantifi cation by contrast-enhanced ultrasound and thermodilution: an in vitro comparison gi Conclusion This study indicates that in early sepsis pulse waveform characteristics could predict the risk of developing end organ failure. The pulse wave transit time is more robust than the augmentation index and could be easier to use in patients with poor perfusion. Vascular reactivity indices do not seem to have predictive value in this context. Introduction In clinical practice, blood volumes (BV) are typically measured by thermodilution. Recently, contrast-enhanced ultrasound (CEUS) has been proposed as an alternative minimally invasive approach for BV assessment [1]. This method measures BV using a single peripheral injection of a small bolus of ultrasound contrast agent (UCA) detected by an ultrasound scanner. By measuring the acoustic backscatter, two indicator dilution curves (IDCs) can be derived from two diff erent sites in the circulatory system. IDC analysis permits deriving the mean transit time (MTT) the bolus takes to travel between the injection site and two measurement sites. Assessment of the BV between these sites is obtained by multiplying the diff erence in MTT (ΔMTT) by the blood fl ow. In this study, we compare diff erent volumes in an in vitro set-up by CEUS with true set-up volumes and thermodilution acquired volumes. Introduction In clinical practice, blood volumes (BV) are typically measured by thermodilution. Recently, contrast-enhanced ultrasound (CEUS) has been proposed as an alternative minimally invasive approach for BV assessment [1]. This method measures BV using a single peripheral injection of a small bolus of ultrasound contrast agent (UCA) detected by an ultrasound scanner. By measuring the acoustic backscatter, two indicator dilution curves (IDCs) can be derived from two diff erent sites in the circulatory system. IDC analysis permits deriving the mean transit time (MTT) the bolus takes to travel between the injection site and two measurement sites. Assessment of the BV between these sites is obtained by multiplying the diff erence in MTT (ΔMTT) by the blood fl ow. In this study, we compare diff erent volumes in an in vitro set-up by CEUS with true set-up volumes and thermodilution acquired volumes. Fluid responsiveness in pressure support ventilation: role of asynchrony A Messina, D Colombo, G Cammarota, M De Lucia, F Della Corte, P Navalesi Università Maggiore della Carità A. Avogadro, Novara, Italy Critical Care 2013, 17(Suppl 2):P202 (doi: 10.1186/cc12140) Introduction Pulse pressure variation (PPV) is a dynamic indicator of fl uid responsiveness, which is known to have a low sensibility and specifi city in patients ventilated in pressure support (PS) [1]. We aim to investigate patient–ventilator asynchrony as a potential source of hemodynamic interference in PS. Methods Institutional ethical approval was obtained. Intubated major trauma patients were sequentially enrolled. Exclusions included major thoracic burns and children. Bioreactance fl ow monitoring (NICOM; Cheetah) was applied at the same time as ECG leads and the calibration step performed during handover from the prehospital team. Time to availability of oxygen delivery data was recorded and trauma team members surveyed regarding for perceived benefi ts and concerns from this monitoring. The infl uence of fl ow monitoring on fl uid resuscitation, time to CT and defi nitive disposal (to OR/ICU) was measured and compared with a control population matched for injury severity score, age and sex. y Methods We performed a prospective study including PS ventilated patients who met inclusion criteria for fl uid depletion [1]. Patients who showed an asynchrony index (AI) exceeding 10% were included in the asynchrony group (AG). The remaining patients were included in the synchrony group (SG) [2]. Beat-to-beat hemodynamic variables were recorded through PRAM (Mostcare; Vytech Health srl, Padova, Italy). PPV cutoff of 13% was used to identify fl uid responders/nonresponders. A fl uid challenge of 500 ml normal saline was given in 5 minutes. An increase of 15% of cardiac index after 10 minutes indicated fl uid responsiveness. Results Cardiac index was available at mean 10.6 minutes (median 9 minutes; SD 3.9), fl uid responsiveness at mean 35.9 minutes (median 35; SD 11.3) and oxygen delivery calculation at mean 25.3 minutes (median 25; SD 7.7). Passive leg raise was not performed in 63% of patients due to concerns about pelvic or brain injury. Volume of fl uid infused (mean 738 vs. 925 ml; P = 0.124), time to CT (mean 57.4 vs. 68.8 minutes; P = 0.08), and time to defi nitive disposal (mean 124.9 vs. 146.1 minute; P = 0.069) were all reduced in the fl ow monitored group, although not signifi cantly diff erent when compared with a matched Results So far, eights patients showed an AI >10% while 16 did not. Overall sensitivity was 28.6% versus 50% in SG; overall specifi city was 76.5% versus 91.7% in AG. Computerized decision support system improves consistency of haemodynamic assessment amongst ICU team members AE Aneman, R Ranganatha Liverpool Hospital, Liverpool, Australia Critical Care 2013, 17(Suppl 2):P199 (doi: 10.1186/cc12137) Four categories of staff participated: nine consultants (C), eight senior registrars (SR), nine registrars (R) and 11 nurses (N) (median ICU experience 15, 7, 2 and 10 years, respectively) and were asked to score Eh, Pms, and SVR ranging (discrete steps of one) from –5 (grossly subnormal) to 0 (normal) to 5 (grossly supranormal) fi rst without (BLIND) and then given (OPEN) access to the NAVIGATOR display. Recommendations for therapeutic interventions were noted. S76 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 control group (Mann–Whitney U rank sum). Eighty-four percent of trauma team members surveyed felt the fl ow monitoring data to be useful, and only 11% felt it may impair clinical management.l Conclusion Given the good correlation between BVs estimated with CEUS and thermodilution, CEUS is not inferior to thermodilution with the advantage of being minimally invasive. Reference Conclusion Cardiac index, fl uid responsiveness and oxygen delivery data can be obtained inform a primary survey. Rather than introducing delays, the use of fl ow monitoring was associated with a trend towards decreased time to imaging; less fl uid use pre-damage control point and reduced time to defi nitive disposal. Further research is required to confi rm benefi ts and mechanism. 1. Mischi M, et al.: IEEE Trans UFFC 2004, 51:1137-1141. 1. Mischi M, et al.: IEEE Trans UFFC 2004, 51:1137-1141. P202 Fluid responsiveness in pressure support ventilation: role of asynchrony A Messina, D Colombo, G Cammarota, M De Lucia, F Della Corte, P Navalesi and disposal y , , 3. Dunham M, et al.: J Trauma Acute Care Surg 2012, 73:479-485. y 3. Dunham M, et al.: J Trauma Acute Care Surg 2012, 73:479-485. King’s Health Partners AHSC, London, UK Critical Care 2013, 17(Suppl 2):P201 (doi: 10.1186/cc12139) Fluid responsiveness in pressure support ventilation: role of asynchrony A Messina, D Colombo, G Cammarota, M De Lucia, F Della Corte, P Navalesi Fluid responsiveness in pressure support ventilation: role of asynchrony A Messina, D Colombo, G Cammarota, M De Lucia, F Della Corte, P Navalesi Università Maggiore della Carità A. Avogadro, Novara, Italy Critical Care 2013, 17(Suppl 2):P202 (doi: 10.1186/cc12140) Introduction The use of cardiac output monitoring has been shown to be benefi cial in the setting of perioperative medicine and critical illness [1,2]. More recently, its application in the setting of major trauma has been described [3]. Here, we describe our preliminary experience of embedding bioreactance fl ow monitoring within the major trauma primary survey of severely injured patients and the subsequent eff ect on patient management. P203 Comparison of pulse pressure variation with radial arterial systolic, diastolic and pulse transit time interval variation in pediatric patients undergoing liver transplantation YJ Moon, IG Jun, WJ Shin, BH Sang, GS Hwang Asan Medical Center, Seoul, South Korea Critical Care 2013, 17(Suppl 2):P203 (doi: 10.1186/cc12141) P203 Comparison of pulse pressure variation with radial arterial systolic, diastolic and pulse transit time interval variation in pediatric patients undergoing liver transplantation YJ Moon, IG Jun, WJ Shin, BH Sang, GS Hwang Asan Medical Center, Seoul, South Korea Critical Care 2013, 17(Suppl 2):P203 (doi: 10.1186/cc12141) l g p Methods We measured the eff ects after the administration of 50, 100 and 500 ml bolus colloid infusions on CO (Modelfl ow (COm) and LiDCO (COli)), CVP and MAP in 21 patients on mechanical ventilation after elective cardiothoracic surgery. From the data we analysed the smallest volume that was predictive for the eff ects of 500 ml on cardiac output. Results COli and COm increased after 50, 100 and 500 ml fl uid loading. Best results are observed for changes in COm after 100 ml fl uid loading (area under the ROC 0.86, 95% CI between 0.65 and 1.00). A change in Modelfl ow CO of at least 4.3% has a sensitivity of 67% and a specifi city of 100% after 100 ml fl uid loading. Sensitivity is 60% and specifi city 83% for a similar cutoff in CO measured with the LiDCO device after 100 ml fl uid loading. In our patient population, MAP and COli did not predict responsiveness with more accuracy than mathematical chance. See Figure 1.l Introduction In pediatric patients, dynamic preload indices to predict fl uid responsiveness have confl icting results in comparison with adults. A recent study demonstrated that pulse pressure variation (PPV) ≥16% has provided an accurate method for predicting fl uid responsiveness in pediatric congenital heart surgery. We aimed to compare PPV and respiratory systolic, diastolic and pulse transit time interval variation (STV, DTV and PTTV, respectively) as predictors of fl uid responsiveness during pediatric liver transplantation. g p p Methods A total of 61 data from 16 pediatric patients, median age 5.4 years (range 0.1 to 9 years), were retrospectively evaluated from electrically recorded radial arterial and central venous pressure (CVP) waveform. The time from the onset of systolic upstroke to the dicrotic notch was defi ned as the systolic time interval (STI), and the time from dicrotic notch to the beginning of systolic upstroke was defi ned as the diastolic time interval (DTI). The time from peak R wave on electrocardiography to the onset of systolic upstroke was defi ned as the pulse transit time (PTT) interval. P203 Comparison of pulse pressure variation with radial arterial systolic, diastolic and pulse transit time interval variation in pediatric patients undergoing liver transplantation YJ Moon, IG Jun, WJ Shin, BH Sang, GS Hwang Asan Medical Center, Seoul, South Korea Critical Care 2013, 17(Suppl 2):P203 (doi: 10.1186/cc12141) STV was calculated by averaging of three consecutive respiratory cycles with the following: (STImaximum – STIminimum) / STImean. The same method was used for calculating DTV, PTTV and PPV. STV, DTV and PTTV were corrected by cardiac period. Averaged CVP was used as a static preload index. PPV threshold ≥16% was used to discriminate fl uid responsiveness. Receiver operating characteristic (ROC) curves and Pearson’s correlation analysis were used for the comparison. Conclusion Changes in pulse contour CO can be used in a mini-fl uid challenge to assess fl uid responsiveness in our postcardiac surgery patients. Effi cacy of intraoperatory optimisation of fl uids guided with transoesophageal Doppler monitorisation: a multicentre randomised controlled trial S Maeso1, R Villalba2, J Ripollés3, S Asuero2, J Blasco1, J Calvo3 1Agencia Lain Entralgo, Madrid, Spain; 2Hospital Universitario Ramón y Cajal, Madrid, Spain; 3Hospital Infanta Leonor, Madrid, Spain Critical Care 2013, 17(Suppl 2):P205 (doi: 10.1186/cc1243) Fluid responsiveness in pressure support ventilation: role of asynchrony A Messina, D Colombo, G Cammarota, M De Lucia, F Della Corte, P Navalesi Università Maggiore della Carità A. Avogadro, Novara, Italy Critical Care 2013, 17(Suppl 2):P202 (doi: 10.1186/cc12140) Overall Cohen’s k was 33.3% versus 61.2% in AG (see Figure 1). However, because none of the responders in the AG group was detected by PPV, statistical analysis was not feasible within this subgroup. Figure 1 (abstract P202). Figure 1 (abstract P202). S77 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Conclusion The consistency of PPV in predicting fl uid responsiveness during PS seems to be more reliable in the patients with better patient– ventilator synchrony. References References 1. Heenen S, et al.: Crit Care 2006, 10:R102. 2. Vitacca M, et al.: Chest 2004, 126:851-859. Introduction The mini-fl uid challenge is a widely used strategy to manage fl uid loading in the ICU and OR. Although it might be a rational strategy, data on the mini-fl uid challenge and its reliability are very limited. We investigated the value of changes in pulse contour cardiac output as a result of a mini-fl uid challenge of 50 and 100 ml to predict fl uid loading responsiveness. Mini-fl uid challenge: how much fl uid and what parameter to use? B Geerts, RB De Wilde, JJ Maas, LP Aarts, JR Jansen Leiden University Medical Centre, Leiden, the Netherlands Critical Care 2013, 17(Suppl 2):P204 (doi: 10.1186/cc12142) Mini-fl uid challenge: how much fl uid and what parameter to use? B Geerts, RB De Wilde, JJ Maas, LP Aarts, JR Jansen Leiden University Medical Centre, Leiden, the Netherlands Critical Care 2013, 17(Suppl 2):P204 (doi: 10.1186/cc12142) y y References 1. Heenen S, et al.: Crit Care 2006, 10:R102. 2. Vitacca M, et al.: Chest 2004, 126:851-859. Effi cacy of intraoperatory optimisation of fl uids guided with transoesophageal Doppler monitorisation: a multicentre randomised controlled trial S Maeso1, R Villalba2, J Ripollés3, S Asuero2, J Blasco1, J Calvo3 1Agencia Lain Entralgo, Madrid, Spain; 2Hospital Universitario Ramón y Cajal, Madrid, Spain; 3Hospital Infanta Leonor, Madrid, Spain Critical Care 2013, 17(Suppl 2):P205 (doi: 10.1186/cc1243) Results PPV showed correlations with STV, DTV and PTTV (r = 0.65, 0.57 and 0.60, respectively), but less with CVP (r = –0.30). Area under ROC curves (AUC) of STV, DTV, PTTV and CVP were 0.834, 0.872, 0.832 and 0.613, respectively. Cutoff values of STV, DTV, PTTV and CVP were 7.7% (sensitivity/specifi city, 0.80/0.83), 7.7% (sensitivity/specifi city, 0.70/0.88), 8.7% (sensitivity/specifi city 0.67/1.0) and 3.1% (sensitivity/ specifi city 0.50/0.85), respectively. Introduction The objective is to compare stay in surgery monitored with oesophageal Doppler with unmonitored. Introduction The objective is to compare stay in surgery monitored with oesophageal Doppler with unmonitored. g Methods A randomized trial. We present preliminary results obtained in the fi rst 55 cases. The surgeries were general and urological. ISRCTN93543537. Results There were no diff erences in any of the baseline variables. A total of 69.1% were men. The mean age was 65.80 years. There were 63.6% general surgery and 36.4% urologic surgery. There were 81.5% open surgeries and 18.5% laparoscopic. The results were favorable to the intervention group for most outcomes; these diff erences did not i y y Conclusion This study shows that STV, DTV and PTTV can be used as a surrogate for PPV ≥16%, suggesting that this novel method can be used to predict hemodynamic response during pediatric surgery. Reference 1. Renner J, et al.: Br J Anaesth 2012, 108:108-115. Figure 1 (abstract P204). Cardiac function curve: a fl uid challenge of 50 ml in a (non)responder. gure 1 (abstract P204). Cardiac function curve: a fl uid challenge of 50 ml in a (non)responder. Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S78 Table 1 (abstract P205). Analysis of the outcome variables ODM Control Outcome Units (n = 32) (n = 23) P value Surgery time Minutes 206.20 225.59 0.532 Total stay Days 13.92 18.19 0.292 Postsurgery Days 10.93 14.36 0.280 Time to enteral Days 2.73 3.13 0.567 Time to deambulation Days 2.52 3.00 0.359 Complications % 34.4 21.7 0.377 Table 1 (abstract P205). Effi cacy of intraoperatory optimisation of fl uids guided with transoesophageal Doppler monitorisation: a multicentre randomised controlled trial Analysis of the outcome variables P207 Prediction of stroke volume response to fl uid bolus in 100 children R Saxena, A Durward, I Murdoch, S Tibby Guy’s & St Thomas’ NHS Trust, London, UK Critical Care 2013, 17(Suppl 2):P207 (doi: 10.1186/cc12145) Figure 1 (abstract P206). Figure 1 (abstract P206). reach statistical signifi cance (Table 1). We emphasize the diff erence in postoperative stay of 3.4 days. Only complications results were against the intervention. Conclusion The preliminary results obtained for the postsurgical length of stay, as for most of the outcomes, were favorable to monitoring by oesophageal Doppler. P206 P206 Looking for the infl exion point of the Frank–Starling curve HD Aya, M Geisen, C Ebm, N Fletcher, M Grounds, A Rhodes, M Cecconi St George’s Healthcare NHS Trust, London, UK Critical Care 2013, 17(Suppl 2):P206 (doi: 10.1186/cc12144) Introduction Fluid overload is associated with poor outcome in the critically ill. Thus, an accurate predictor of a positive haemodynamic response (increase in stroke volume) to fl uid challenge is vital.i l Methods We studied the predictive value (positive response defi ned as change in stroke volume >15% after 10 ml/kg fl uid bolus) of a range of haemodynamic variables: static (CVP, active circulating volume, central blood volume, total end diastolic volume), dynamic (systolic pressure variation, stroke volume variation) and contactility (dp/dt), in a group of 100 ventilated children (median weight 10 kg). Variables were measured using transpulmonary ultrasound dilution and PRAM (an arterial pulse contour method). Introduction Fluid responsiveness is defi ned based on an arbitrary increase of cardiac output (CO) or stroke volume (SV) of 10 to 15%. We hypothesise that the variation of heart effi ciency (Eh) and the slope (S) defi ned by the relative increase of CO over the relative increase of mean fi lling pressure (Pmsa) can be used as alternative defi nitions of fl uid responsiveness. l p Methods Patients admitted to the ICU were monitored with a calibrated LiDCOplus (LiDCO, UK) and Navigator (Applied Physiology, Australia) to estimate Pmsa and Eh (Pmsa – central venous pressure/Pmsa). A 250 ml fl uid challenge was performed over 5 minutes. Categorical data were compared by Pearson chi-square test. Correlation was assessed by Kappa test. The infl exion point of S to defi ne responders was obtained by ROC curve analysis. p Results We performed 168 paired measurements (pre-fl uid and post- fl uid challenge), with a SV response rate of 45%. Overall predictive values were poor, but slightly better for static versus dynamic variables (Table 1). When SV response was analysed as a continuous variable, the two predictive multivariable variables were change in TEDVI and baseline dp/dt (r2 = 0.30, both P <0.001). Table 1 (abstract P207). ROC areas for haemodynamic variables AUC 95% CI CVP 0.50 0.41 0.59 ACVI 0.62 0.54 0.71 CBVI 0.59 0.50 0.68 TEDVI 0.65 0.56 0.73 SPV 0.59 0.50 0.69 SVV 0.48 0.39 0.58 dp/dt 0.59 0.50 0.68 Table 1 (abstract P207). ROC areas for haemodynamic variables Table 1 (abstract P207). P207 d P207 Prediction of stroke volume response to fl uid bolus in 100 children R Saxena, A Durward, I Murdoch, S Tibby Guy’s & St Thomas’ NHS Trust, London, UK Critical Care 2013, 17(Suppl 2):P207 (doi: 10.1186/cc12145) P206 ROC areas for haemodynamic variables AUC CI y y Results A total of 104 fl uid challenges were observed in 40 patients. ROC curve analysis reveals an area under the curve of 0.93 (95% CI = 0.85 to 1, P <0.001). The best cutoff for the slope was 0.76 (sensitivity 0.92, specifi city 0.93). The proportions of responders identifi ed by the ΔEh (Table 1) and by the slope method (Table 2) are smaller compared with the relative increase of SV method. Signifi cant correlation was found between both methods and the ΔSV (ΔEh κ = 0.54, P <0.001; S κ = 0.55, P <0.001). See Figure 1. g Conclusion Moderate agreement is observed between new and current defi nitions of fl uid responsiveness. 1. Michard F, et al.: Chest 2002, 121:2000. Table 1 (abstract P206). Distribution of events according to ΔSV and ΔEh Response by ΔSV ≥10% Response by ΔEh ≥0 P value Nonresponder 62 (59.6%) 76 (73.1%) <0.001 Responder 42 (40.4%) 28 (26.9%) <0.001 Table 2 (abstract P206). Distribution of events according to ΔSV and the slope (S) Response by ΔSV ≥10% Response by S ≥0.76 P value Nonresponder 62 (59.6%) 75 (72.1%) <0.001 Responder 42 (40.4%) 29 (27.9%) <0.001 Table 1 (abstract P206). Distribution of events according to ΔSV and ΔEh Conclusion The predictive ability for typical static and dynamic haemodynamic variables, when taken in isolation, is poor. However, improved prediction is seen when baseline contractility is taken into account. SP De Wolf, J Stens, RJ Van der Zwan, N Koning, C Boer VU University Medical Centre, Amsterdam, the Netherlands Critical Care 2013, 17(Suppl 2):P208 (doi: 10.1186/cc12146) Introduction This study compared the eff ect of pulse pressure variation (PPV) and cardiac index (CI)-guided fl uid therapy versus mean arterial S79 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Conclusion SDF imaging is a feasible, non-invasive bedside method to study the postoperative intestinal microcirculation. The established reference ranges are useful for early detection of postoperative local complications and studies of microcirculatory changes induced by systemic pathologies; for example, in sepsis. pressure (MAP)-guided fl uid therapy on microcirculatory perfusion in patients undergoing abdominal surgery. g g g y Methods Patients undergoing elective abdominal surgery were randomized into a PPV/CI-guided group (n = 11) or a MAP-guided (n = 12) group. P206 PPV, CI and MAP were measured using the non-invasive fi nger arterial blood pressure measurement device ccNexfi n (Edwards Lifesciences BMEYE, Amsterdam, the Netherlands). Tidal volumes were ≥8 ml/kg with PEEP ≥8 mmHg. In both groups, MAP of 70 mmHg was maintained. In the PPV/CI group, an intraoperative algorithm was used keeping the PPV under 12% and CI above 2.5 l/minute/ m2 using fl uid therapy and dobutamine and noradrenaline infusion, respectively. Sublingual microvascular perfusion was measured after anesthesia induction, and every subsequent hour using sidestream dark-fi eld imaging (Microscan; Microvision Medical, Amsterdam, the Netherlands). The perfused small vessel density (PVD) values were offl ine quantifi ed.i P210 P210 Monitoring microcirculatory blood fl ow with a new sublingual tonometer in a porcine model of haemorrhagic shock P Palagyi, J Kaszaki, Z Molnar University of Szeged, Hungary Critical Care 2013, 17(Suppl 2):P210 (doi: 10.1186/cc12148) Introduction Tissue capnometry has been used to assess organ perfusion but it is not generally available at the bedside [1,2]. Our aim was to test a new sublingual capillary tonometer in haemorrhagic shock. fl qi Results The fi rst hour during surgery, the PPV/CI-guided group tended to receive more fl uids than the MAP-guided group (1,014 ± 501ml vs. 629 ± 463 ml; P = 0.07). At this time point, the PVD was slightly lower in the PPV/CI-guided group (16.7 ± 3.1 mm/mm2) when compared with the MAP-guided group (17.9 ± 3.9 mm/mm2; P = 0.41). In both groups the PVD remained stable during the fi rst 2 hours of surgery. However, 2 hours after the start of surgery, the PVD in the PPV/CI group restored and tended to be higher than in the MAP-guided group (21.1 ± 1.9 vs. 18.1 ± 3.4 mm/mm2; P = 0.09). After 1 hour of surgery, the administered fl uid volume correlated inversely with PVD (r = –0.59, P = 0.011).l Methods Thirty-six mini-pigs were anaesthetised, ventilated and divided into sham operated and shock groups. Instrumentation included: intestinal and sublingual tonometry, haemodynamic monitoring (PiCCO; Pulsion, Germany) and orthogonal polarization spectroscopy (OPS). After baseline measurements (T0) haemorrhagic shock was induced and maintained by reducing mean arterial pressure (MAP) to ~40 mmHg for 60 minutes. Measurements were repeated every 30 minutes (T1 to T6). Fluid resuscitation started after T2 aiming to increase MAP to 75% of the baseline value. OPS imaging was performed at T0, T2 and T6. P211 Methods For this observational prospective cohort study 77 patients were screened. In total, 165 SDF measurements could be obtained. All patients included had bowel surgery for chronic infl ammatory bowel disease or intestinal malignancies and received an ileostomy. Patients were excluded if they had signs of local infection or bleeding, or if they were admitted to the ICU for sepsis or perioperative complications. The SDF device was gently inserted into the stoma at a depth of 3 to 4 cm, and fi ve real-time images were recorded. All videos recorded were analyzed offl ine using AVA® software (Microvision Medical, Amsterdam, the Netherlands). The following parameters were quantifi ed: microvascular fl ow index (MFI), total vessel density (TVD), perfused vessel density (PVD), and proportion of perfused vessels (PPV). Patients were followed for 3 days post surgery with fi ve images captured every day. References 1. Creteur et al.: Curr Opin Crit Care 2006, 12:272-277. 2. Cammarata et al.: Shock 2009, 31:207-211. 1. Creteur et al.: Curr Opin Crit Care 2006, 12:272-277. 2. Cammarata et al.: Shock 2009, 31:207-211. Postoperative imaging of the intestinal microcirculation I Abd 1 R H ll1 D H l 1 V C 2 CH L h 1 Abdo1, R Hall1, D Henzler1, V Cerny2, CH Lehmann1 1Dalhousie University, Halifax, Canada; 2Charles University, Medical Faculty in Hradec Kralove, Czech Republic Conclusion In this model of haemorrhagic shock, sublingual PCO2 gap showed good correlation with RBCV, suggesting that this new sublingual capillary tonometer may be an appropriate tool for monitoring microcirculation at the bedside. f Introduction Sidestream dark-fi eld (SDF) imaging was introduced recently to study the sublingual microcirculation in humans. Patients with ileostomies off er a unique access to the intestinal microcirculation. To date, no reference ranges for standard microcirculatory parameters of the gut are available. Therefore, the aim of our study was to establish a database for postoperative microcirculatory parameters for ileostomies. P209 Postoperative imaging of the intestinal microcirculation I Abdo1, R Hall1, D Henzler1, V Cerny2, CH Lehmann1 1Dalhousie University, Halifax, Canada; 2Charles University, Medical Faculty in Hradec Kralove, Czech Republic Critical Care 2013, 17(Suppl 2):P209 (doi: 10.1186/cc12147) P206 Data are presented as the median (interquartile range), for statistical analysis Friedmann ANOVA, Mann–Whitney and Spearman tests were used as appropriate. l y Conclusion Goal-directed fl uid management resulted in a higher administered fl uid volume in the beginning of surgery, and this was associated with a slightly reduced microcirculatory perfusion when compared with MAP-guided fl uid management. Microcirculatory perfusion tended to improve as surgery progressed in the goal-directed fl uid therapy group. Our fi ndings suggest that goal-directed and MAP- guided fl uid management are associated with distinct patterns in fl uid resuscitation, which may be of consequence for microvascular perfusion. p pp p Results Bleeding resulted in a signifi cant decrease in MAP and cardiac index, and an increase in heart rate. Macrohaemodynamic changes were accompanied by signifi cant changes in red blood cell velocity (RBCV) and a signifi cant increase in the intestinal and sublingual mucosal-to-arterial carbon dioxide partial pressure diff erence (PCO2 gap): from 4 (2 to 11) to 30 (23 to 37) mmHg in the sublingual, and from 25 (17 to 31) to 50 (33 to 64) mmHg in the ileum. RBCV decreased from 1,075 (945 to 1,139) to 520 (449 to 621) μm/second in the sublingual area, and from 646 (596 to 712) to 419 (350 to 451) μm/second in the ileum. There was signifi cant correlation between RBCV and PCO2 gap in sublingual and intestinal regions alike: r = –0.58; r = –0.71, P <0.0001, respectively. Sublingual microcirculatory changes during transient intra-abdominal hypertension: a study in laparoscopic surgery patients Introduction Microvascular alterations play an important role in development of organ failure [1]. It is not known whether increased intra-abdominal pressure (IAP) is associated with microcirculatory perfusion derangements. Our hypothesis was that transiently increased IAP is related to microcirculatory alterations in laparoscopic cholecystectomy patients. Introduction Microvascular alterations play an important role in development of organ failure [1]. It is not known whether increased intra-abdominal pressure (IAP) is associated with microcirculatory perfusion derangements. Our hypothesis was that transiently increased IAP is related to microcirculatory alterations in laparoscopic cholecystectomy patients. Results We were able to capture clear images of the small intestine microvasculature. Distinct villi were visible with a dense network of microvessels (diameter: 6 to 17 μm). Mean TVD (± 2SD) was 19.3 (± 1.0) mm/mm2, PVD: 18.5 (± 1.1) mm/mm2, PPV: 94.5 (± 5)% and MFI: 2.8 (± 0.1). Patients’ age, sex and comorbidity had no signifi cant impact on postoperative microvascular parameters. No signifi cant changes of the microvascular parameters were observed during the fi rst 3 postoperative days. Methods Sixteen patients (14 female, two male) who underwent laparoscopic cholecystectomy were studied. Sublingual orthogonal polarization spectral (OPS) imaging was used to detect microcirculatory function. OPS was done before surgery, at least 15 minutes after initiation of pneumoperitoneum and 1 hour after the end of pneumo- peritoneum. The microcirculation cutoff value for vessels was 20 μm. Data are presented as medians with interquartile ranges. S80 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Results Patient median age was 54 (39 to 63) years, ASA score 2 (2 to 3), BMI 29.7 (24.9 to 34.7), haemoglobin concentration 138 (133 to 142) g/l, and hematocrit 42 (39 to 43). IAP was held at 12.5 (12 to 13) mmHg, median duration of pneumoperitoneum was 45 (24 to 55) minutes. Median MAP was 86 (69 to 93), abdominal perfusion pressure (APP) 73 (57 to 81) mmHg during the pneumoperitoneum. Median fl uid administration during anesthesia was 1,050 (1,000 to 1,400) ml. Altogether 448 microcirculation videos were taken. Interobserver variability was 24%. The following microcirculatory parameter values describe before, during and after pneumoperitoneum periods. Total vascular density was 19.4 (17.0 to 21.1); 18.5 (17.0 to 20.9); 19.3 (16.9 to 20.9) n/mm2. Perfused vessels density was 13.3 (10.9 to 15.2); 13.8 (8.9 to 18.0); 13.1 (11.0 to 16.0) n/mm2. Microcirculatory response to experimentally-induced whole body heat stress in healthy humans A reduction in glycocalyx thickness, represented by an increased PBR, correlates with impairment of perfused microvascular vessel density. Our data support the use of intraoperative PBR monitoring as a novel clinical indicator of microcirculatory perfusion. g y y Results The perfused vessel density remained stable during off - pump surgery, while the PBR decreased from 2.6 ± 0.1 μm (baseline) to 2.3 ± 0.1 μm (ICU). Nonpulsatile CPB was associated with loss of microcirculatory perfusion during bypass (15.1 ± 2.7 mm/mm2) and ICU admission (15.3 ± 2.6 mm/mm2) compared with baseline (19.8 ± 2.8 mm/mm2). Pulsatile CPB reduced the perfused vessel density from 20.9 ± 2.4 to 16.7 ± 2.6 mm/mm2, but this was restored towards baseline levels upon ICU admission (20.3 ± 2.3 mm/mm2; P = 0.02 between groups). The PBR increased upon initiation of CPB in both groups, but remained elevated in the nonpulsatile fl ow group only. In the pulsatile group, the PBR started at 2.4 ± 0.1 μm, increased to 2.7 ± 0.1 μm during bypass but restored already during CPB towards a PBR value of 2.3 ± 0.1 μm (ICU). The PBR and perfused vessel density showed a good correlation (r = –0.65; P = 0.002), demonstrating loss of perfused vascular density when the glycocalyx is damaged. y y y y Methods Eight healthy men with no history of cold and/or heat injury were recruited to this study. Passive body heating was performed by continuous immersion up to the waist in the water bath at 44°C and continued until rectal temperature reached 39.5°C. Before, at the end of whole body heating and 1 hour after heating was ended, systemic hemodynamics and direct in vivo observation of the sublingual microcirculation were obtained with sidestream dark-fi eld imaging. Assessment of microcirculatory parameters of convective oxygen transport (microvascular fl ow index (MFI), proportion of perfused vessels (PPV)), and diff usion distance (perfused vessel density (PVD) and total vessel density (TVD)) was done using a semiquantitative method. Vessels were separated into large (mostly venules) and small (mostly capillaries) using a diameter cutoff value of 20 μm.i p y g y y g Conclusion The glycocalyx is damaged after initiation of CPB, and restoration is impaired after exposure to nonpulsatile fl ow during the use of the heart–lung machine. A reduction in glycocalyx thickness, represented by an increased PBR, correlates with impairment of perfused microvascular vessel density. Changes in microcirculatory perfusion during cardiac surgery are paralleled by alterations in glycocalyx integrityf Conclusion Defined cold exposure had no effect on the micro- circulation. Introduction Previous studies demonstrate that loss of glycocalyx integrity is associated with impaired microvascular function. We investigated whether glycocalyx dimensions are reduced in patients undergoing cardiac surgery with or without cardiopulmonary bypass (CPB), and are paralleled by loss of microcirculatory perfusion using in vivo microcirculation measurements. Microvascular response to cold stress in healthy humans A Pranskunas1, R Rasimaviciute1, J Paltanaviciute1, L Daniuseviciute2, V Pilvinis1, M Brazaitis2 Microvascular response to cold stress in healthy humans A Pranskunas1, R Rasimaviciute1, J Paltanaviciute1, L Daniuseviciute2, V Pilvinis1, M Brazaitis2 1Lithuanian University of Health Sciences, Kaunas, Lithuania; 2Lithuanian Sports University, Kaunas, Lithuania Critical Care 2013, 17(Suppl 2):P213 (doi: 10.1186/cc12151) Introduction Cold exposure can be adapted for exercise or therapeutic purposes, but its impact on microcirculation in healthy humans has not been well defi ned. We hypothesize that whole body cold stress may impair microcirculation. Introduction Cold exposure can be adapted for exercise or therapeutic purposes, but its impact on microcirculation in healthy humans has not been well defi ned. We hypothesize that whole body cold stress may impair microcirculation. Methods Seven volunteers were recruited for the water immersion procedure. During the cooling protocol the volunteers every 20 minutes of immersion were asked to step out from the bath and rest for 10 minutes in a room environment and then return to the water bath for the next 20 minutes of immersion. This head-out immersion procedure in bath water at 14°C continued until the rectal temperature was dropped to 35.5°C or the time of 180 minutes was terminated. Maximum cold water immersion time was 120 minutes. Before, at the end of whole body cooling and 1 hour after cooling was ended, systemic hemodynamics and direct in vivo observation of the sublingual microcirculation were obtained with sidestream dark-fi eld imaging. Assessment of microcirculatory parameters of convective oxygen transport (microvascular fl ow index (MFI), proportion of perfused vessels (PPV)), and diff usion distance (perfused vessel density (PVD) and total vessel density (TVD)) was done using a semiquantitative method.i Conclusion Microcirculatory alterations are mild during transient increase of intra-abdominal pressure in laparoscopic surgery patients. References 1. De Backer D, et al.: Crit Care Med 2006, 34:1918-1924. 2. Vellinga NAR, et al.: Crit Care Res Pract 2012. doi:10.1155/2012/121752 1. De Backer D, et al.: Crit Care Med 2006, 34:1918-1924. 2. Vellinga NAR, et al.: Crit Care Res Pract 2012. doi:10.1155/2012/121752 1. De Backer D, et al.: Crit Care Med 2006, 34:1918-1924. 1. De Backer D, et al.: Crit Care Med 2006, 34:1918-1924. 2. Vellinga NAR, et al.: Crit Care Res Pract 2012. doi:10.1155/2012/121752 P212 Changes in microcirculatory perfusion during cardiac surgery are paralleled by alterations in glycocalyx integrity C Boer1, NJ Koning1, J Van Teeff elen2, AB Vonk1, H Vink2 1VU University Medical Center, Amsterdam, the Netherlands; 2Maastricht University, Maastricht, the Netherlands Critical Care 2013, 17(Suppl 2):P212 (doi: 10.1186/cc12150) Sublingual microcirculatory changes during transient intra-abdominal hypertension: a study in laparoscopic surgery patients Proportion of perfused vessels (PPV) was 61 (50 to 69); 64 (45 to 76); 60 (54 to 67)%. Microvascular fl ow index was 2.4 (2.0 to 2.5); 2.5 (2.0 to 3.0); 2.3 (2.0 to 2.9) and heterogeneity index was 0.8 (0 to 0.9); 0.6 (0 to 1.0); 0.6 (0 to 0.8). No signifi cant diff erences in microcirculatory parameters were observed between time points. PPV was somewhat less than that of described in healthy volunteers (61% vs. 90%) [2]. Microcirculatory response to experimentally-induced whole body heat stress in healthy humans A Pranskunas1, E Milieskaite1, I Maraulaite1, L Daniuseviciute2, Z Pranskuniene1, V Pilvinis1, M Brazaitis2 1Lithuanian University of Health Sciences, Kaunas, Lithuania; 2Lithuanian Sports University, Kaunas, Lithuania Critical Care 2013, 17(Suppl 2):P214 (doi: 10.1186/cc12152) Methods Patients undergoing on-pump surgery with nonpulsatile (n = 11) or pulsatile (n = 13) CPB or off -pump surgery (n = 13) underwent sublingual sidestream dark-fi eld imaging at baseline, during coronary grafting and upon ICU admission to assess perfused microvascular vessel density. Glycocalyx integrity was evaluated using the GlycoCheck Measurement Software, and expressed as the perfused boundary region (PBR). An increase in PBR represents deeper penetration of erythrocytes into the glycocalyx, and is indicative for compromised glycocalyx thickness. Introduction Vasodilation and increased skin blood fl ow (also sweating) are infl uential in heat dissipation during heat exposure and exercise. It is unclear how heat stress infl uences microcirculation. Side dark-fi eld imaging visualizes the blood fl ow at the capillary level and helps to assess perfusion heterogeneity. Clinical and experimental data show that the sublingual region is clinically relevant for detecting microcirculatory alterations and more represents central microcirculation than cutaneous perfusion. We hypothesize that whole body heat stress may increase capillary density. g y y Results The perfused vessel density remained stable during off - pump surgery, while the PBR decreased from 2.6 ± 0.1 μm (baseline) to 2.3 ± 0.1 μm (ICU). Nonpulsatile CPB was associated with loss of microcirculatory perfusion during bypass (15.1 ± 2.7 mm/mm2) and ICU admission (15.3 ± 2.6 mm/mm2) compared with baseline (19.8 ± 2.8 mm/mm2). Pulsatile CPB reduced the perfused vessel density from 20.9 ± 2.4 to 16.7 ± 2.6 mm/mm2, but this was restored towards baseline levels upon ICU admission (20.3 ± 2.3 mm/mm2; P = 0.02 between groups). The PBR increased upon initiation of CPB in both groups, but remained elevated in the nonpulsatile fl ow group only. In the pulsatile group, the PBR started at 2.4 ± 0.1 μm, increased to 2.7 ± 0.1 μm during bypass but restored already during CPB towards a PBR value of 2.3 ± 0.1 μm (ICU). The PBR and perfused vessel density showed a good correlation (r = –0.65; P = 0.002), demonstrating loss of perfused vascular density when the glycocalyx is damaged. Conclusion The glycocalyx is damaged after initiation of CPB, and restoration is impaired after exposure to nonpulsatile fl ow during the use of the heart–lung machine. Changes in microcirculatory perfusion during cardiac surgery are paralleled by alterations in glycocalyx integrity C Boer1, NJ Koning1, J Van Teeff elen2, AB Vonk1, H Vink2 1VU University Medical Center, Amsterdam, the Netherlands; 2Maastricht University, Maastricht, the Netherlands Critical Care 2013, 17(Suppl 2):P212 (doi: 10.1186/cc12150) Results During cooling and 1 hour after cooling was ended, a signifi cant increase in cardiac output (P = 0.028 and P = 0.043) was observed, but there were no changes in heart rate or mean arterial pressure in comparison with baseline variables. There were no signifi cant changes in PPV, MFI, PVD and TVD of small vessels in comparison with baseline variables during all observational time. Changes in microcirculatory perfusion during cardiac surgery are paralleled by alterations in glycocalyx integrity C Boer1, NJ Koning1, J Van Teeff elen2, AB Vonk1, H Vink2 1VU University Medical Center, Amsterdam, the Netherlands; 2Maastricht University, Maastricht, the Netherlands Critical Care 2013, 17(Suppl 2):P212 (doi: 10.1186/cc12150) P215 Observational study of the eff ects of age, diabetes mellitus, cirrhosis and chronic kidney disease on sublingual microvascular fl ow T Reynolds1, S Jhanji2, A Vivian-Smith1, RM Pearse1 1Royal London Hospital, London, UK; 2Royal Marsden Hospital, London, UK Critical Care 2013, 17(Suppl 2):P215 (doi: 10.1186/cc12153) i Methods In a prospective cohort during 1 year, we followed consecu- tively enrolled patients admitted immediately postoperative to the surgical ICU of Hospital Santa Luzia, Brasília, Brazil. Patients were assigned to two groups: LC >10% and LC ≤10%. The primary outcome measure was mortality at 7 and 28 days. The secondary outcome included hospital and ICU length of stay (LOS). Introduction Sidestream dark-fi eld (SDF) imaging is an important new technology that has been used to demonstrate microcirculatory abnormalities in a variety of critical illnesses [1]. The microcirculation is also aff ected by age and chronic comorbidities. However, the eff ect of these conditions on SDF microcirculatory parameters has not been well described. p g y ( ) Results A total of 417 patients were followed. In total, 50.4% were male and 83% underwent elective surgery. The mean age was 59 ± 16, APACHE II score 8 ± 5, SAPS 2 26 ± 11. The mortality at 7 days was 0.95% (n = 4) and the mortality at 28 days was 2.15% (n = 9), respectively. Hospital mortality was 4.79% (n = 20). Sixty-one percent (n = 255) of the patients had LC >10% versus 39% (n = 162) with LC ≤10%. Those who had LC ≤10% were older (62 ± 16 vs. 57 ± 17, P = 0.00) and had greater APACHE II score (9 ± 6 vs. 7 ± 4, P = 0.00) and SAPS 2 (28 ± 12 vs. 25 ± 10, P = 0.02). There was no diff erence in ICU LOS (5 ± 12 vs. 4 ± 9 days, P = 0.54) and hospital LOS (10 ± 15 vs. 9 ± 11 days, P = 0.48). Initial lactate levels were lower in the group with LC ≤10% (1.1 ± 0.9 vs. 1.9 ± 1.6, P = 0.00); however, mean lactate was higher in 24 hours (2.0 ± 1.8 vs. 1.0 ± 0.7, P = 0.00). P215 All of the patients who died in the fi rst 7 days had LC ≤10% (2.46%, n = 4, P = 0.02); this group also had a higher mortality at 28 days (4.32%, n = 7 vs. 0.78%, n = 2; P = 0.03). The relative risk for mortality LC ≤10% in 7 and 28 days was 1.02 (95% CI: 1.00 to 1.05) and 5.07 (95% CI: 1.17 to 27.09), respectively. Signifi cant diff erence was observed in the Kaplan–Meier survival curves for 7 and 28 days (P = 0.01 and 0.02, respectively). The sensibility of LC ≤10% was 100% (95% CI: 51 to 100%) for 7-day mortality and 78% (95% CI: 45 to 94%) for 28-day mortality. The specifi city was 62% (95% CI: 57 to 66%) for 7-day mortality and 62% (95% CI: 57 to 66%) for 28-day mortality. C l i D i i i i l l l l l l d Methods Sublingual SDF images were obtained from fi ve groups of 20 participants: healthy volunteers under 25 years, healthy volunteers over 55 years, and stable patients over 55 years with one of diabetes mellitus (DM), cirrhosis and stage 5 chronic kidney disease (CKD). Microcirculatory parameters [1] between the groups were then com- pared for signifi cance using ANOVA for parametric data and the Kruskal–Wallis test for nonparametric data. This was approved by the local ethics committee. Results All DM patients were type 2, with mean glycated haemoglobin (HbA1c) of 8.8% (SD 1.7%). Seventeen cirrhotic patients were Child– Pugh–Turcotte score A and one was score B. For CKD, the mean estimated glomerular fi ltration rate was 11.5 ml/minute (SD 2.9). Median microvascular fl ow index (MFI) was 2.85 (IQR 2.75 to 3.0) for participants aged <25, 2.81 (2.66 to 2.97) for those aged >55, 2.88 (2.75 to 3.0) for those with DM, 3.0 (2.83 to 3.0) for those with cirrhosis and 3.0 (2.78 to 3.0) for those with CKD (P for diff erence = 0.14). There were no signifi cant diff erences in the proportion of perfused vessels and perfused vessel density between the groups. See Figure 1. Conclusion Despite initial lactate levels, lactate clearance ≤10% proved to be a good predictor of mortality in 7 and 28 days in surgical patients admitted in the postoperative period to the ICU. References e e e ces 1. Nguyen HB, et al.: Crit Care Med 2004, 32:1637-1642. 2. Jansen TC, et al.: Am J Respir Crit Care Med 2010, 182:752-756. y p y Reference 1. De Backer D, et al.: How to evaluate the microcirculation: report of a round table conference. Crit Care 2007, 11:R101. Figure 1 (abstract P215). Microcirculatory response to experimentally-induced whole body heat stress in healthy humans Our data support the use of intraoperative PBR monitoring as a novel clinical indicator of microcirculatory perfusion. f Results Whole body heating resulted in signifi cantly increased heart rate (P = 0.012) and cardiac output (P = 0.046) in comparison with baseline variables. One hour after heating was ended, the heart rate S81 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 remained increased (P = 0.012), but cardiac output returned to baseline values. During all observational time the mean arterial pressure remain unaltered. There was no signifi cant diff erence in MFI and PPV of small vessels at the end of heating and 1 hour after heating in comparison with baseline variables. One hour after heating we observed signifi cant increase in PVD (P = 0.046) and TVD (P = 0.028) of small vessels regardless of lack of diff erence at the end of heating (P = 0.753 and P = 0.075, respectively) in comparison with baseline variables. C l i Wh l b d h i i d d i d d h i remained increased (P = 0.012), but cardiac output returned to baseline values. During all observational time the mean arterial pressure remain unaltered. There was no signifi cant diff erence in MFI and PPV of small vessels at the end of heating and 1 hour after heating in comparison with baseline variables. One hour after heating we observed signifi cant increase in PVD (P = 0.046) and TVD (P = 0.028) of small vessels regardless of lack of diff erence at the end of heating (P = 0.753 and P = 0.075, respectively) in comparison with baseline variables. P217 Assessment of the mottling score as a mortality predictor in critically ill patients E Bastos de Moura, F Ferreira Amorim, C Darwin Silveira, M Oliveira Maia Hospital Santa Luzia, Brasília, Brazil Critical Care 2013, 17(Suppl 2):P217 (doi: 10.1186/cc12155) P215 y Conclusion Older age, diabetes, and chronic kidney and liver disease need not be considered confounding factors for comparison of SDF microcirculatory parameters in the critically ill. Microcirculatory response to experimentally-induced whole body heat stress in healthy humans Lactate clearance as a determinant of mortality in surgical patients FF Amorim1, EB Moura2, AR Santana1, FB Soares1, LG Godoy1, TA Rodrigues1, LJ Almeida1, GM FIlho1, TA Silva1, AP Amorim3, JA Neto2, MO Maia2 1Escola Superior de Ciências da Saúde, Brasília, Brazil; 2Unidade de Terapia Intensiva Adulto do Hospital Santa Luzia, Brasília, Brazil; 3Liga Academica de Medicina Intensiva do Distrito Federal (LIGAMI), Brasilia, Brazil Critical Care 2013, 17(Suppl 2):P216 (doi: 10.1186/cc12154) Lactate clearance as a determinant of mortality in surgical patients FF Amorim1, EB Moura2, AR Santana1, FB Soares1, LG Godoy1, TA Rodrigues1, LJ Almeida1, GM FIlho1, TA Silva1, AP Amorim3, JA Neto2, MO Maia2 1Escola Superior de Ciências da Saúde, Brasília, Brazil; 2Unidade de Terapia Intensiva Adulto do Hospital Santa Luzia, Brasília, Brazil; 3Liga Academica de Medicina Intensiva do Distrito Federal (LIGAMI), Brasilia, Brazil Critical Care 2013, 17(Suppl 2):P216 (doi: 10.1186/cc12154) Conclusion Whole body heating induced time-dependent changes in capillary density. Introduction Serial measurements of lactate over time may be a better prognosticator than a single lactate concentration [1]. Early lactate- guided therapy also reduces ICU length of stay and ICU and hospital mortality [2]. This study aims to assess the prognostic value of the lactate clearance (LC) in the fi rst 24 hours in surgical patients. Introduction Serial measurements of lactate over time may be a better prognosticator than a single lactate concentration [1]. Early lactate- guided therapy also reduces ICU length of stay and ICU and hospital mortality [2]. This study aims to assess the prognostic value of the lactate clearance (LC) in the fi rst 24 hours in surgical patients. Impact of diff erent haemodynamic resuscitation strategies on brain perfusion and tissue oedema markers in a model of severe haemorrhagic shock pi yf p g y y Results Of the 770 patients included in the analysis, 52 died within 30 days. The standard SI at a cutoff point of 0.7 had the highest predictability and sensitivity of 30-day mortality (area under the curve (AUC) = 0.7, sensitivity = 0.79, specifi city = 0.56). In comparison, one of the modifi ed SIs (pulse/diastolic blood pressure) had 0.65 sensitivity and 0.71 specifi city (AUC = 0.73). A heart rate >100 bpm predicted 30- day mortality with 0.40 sensitivity and 0.82 specifi city (AUC = 0.63). Conclusion To our knowledge, this is the fi rst study to examine the relationship between the SI and mortality in patients with a GI haemorrhage. It appears that the standard SI, when compared with various modifi ed SIs and conventional vital signs, had the highest combined predictability and sensitivity of 30-day mortality in a population of patients suff ering from GI haemorrhage. Further prospective studies are needed to confi rm these fi ndings. Faculdade de Medicina, Universidade de São Paulo, Brazil i yf g y y Results Of the 770 patients included in the analysis, 52 died within 30 days. The standard SI at a cutoff point of 0.7 had the highest predictability and sensitivity of 30-day mortality (area under the curve (AUC) = 0.7, sensitivity = 0.79, specifi city = 0.56). In comparison, one of the modifi ed SIs (pulse/diastolic blood pressure) had 0.65 sensitivity and 0.71 specifi city (AUC = 0.73). A heart rate >100 bpm predicted 30- day mortality with 0.40 sensitivity and 0.82 specifi city (AUC = 0.63).i Faculdade de Medicina, Universidade de São Paulo, Brazil Critical Care 2013, 17(Suppl 2):P218 (doi: 10.1186/cc12156) Critical Care 2013, 17(Suppl 2):P218 (doi: 10.1186/cc12156) Introduction This study aimed to compare the cerebral eff ects of terlipressin (TERLI) with conventional prehospital fl uid resuscitation with lactated Ringer’s solution (LR) in a model of haemorrhagic shock (HS). y y y pi y Conclusion To our knowledge, this is the fi rst study to examine the relationship between the SI and mortality in patients with a GI haemorrhage. It appears that the standard SI, when compared with various modifi ed SIs and conventional vital signs, had the highest combined predictability and sensitivity of 30-day mortality in a population of patients suff ering from GI haemorrhage. Further prospective studies are needed to confi rm these fi ndings. Impact of diff erent haemodynamic resuscitation strategies on brain perfusion and tissue oedema markers in a model of severe haemorrhagic shock Methods Pigs (20 to 30 kg) were randomized into one of the groups: Sham (n = 2), HS (n = 9), LR (3× volume bled; n = 9) or TERLI (2 mg bolus; n = 9). HS induced to target MAP of 40 mmHg was maintained for 30 minutes. Brain tissue oxygen pressure (PbtO2), intracranial pressure (ICP), cerebral perfusion pressure (CPP), haemodynamics and blood gas analyses were assessed prior to HS (baseline) up to 120 minutes after treatment. Tissue markers of brain oedema (aquaporin-4 (AQP4) and Na–K–Cl cotransporter-1 (NKCC1)), apoptosis (pre-apoptotic protein (Bax)) and oxidative stress (thiobarbituric acid reactive substances (TBARS)) were also measured. Results Sham animals had no signifi cant changes in the variables assessed. HS resulted in a signifi cant decrease in CPP (mean varied from 36 to 39 mmHg), PbtO2 (from 23.6 to 26.6 mmHg), ICP (from 1 to 2 mmHg) and haemodynamics (MAP from 38 to 40 mmHg; CI from 1.8 to 2.1 l/minute/m2), and a signifi cant increase in blood lactate (from 6.7 to 8.9 mmol/l) and cerebral AQP4 (mean ± SE; 167 ± 54% of sham), NKCC1 (237 ± 47% of sham), Bax (167 ± 44% of sham) and TBARS. Fluid resuscitation was followed by an increase in ICP (from 7 to 9 mmHg) and a decrease in CPP (from 41 to 52 mmHg), with an increased expression of cerebral AQP4 (210 ± 56% of sham), NKCC1 (163 ± 32% of sham) and Bax (137 ± 24% of sham). Only TERLI restored baseline values of CPP (from 54 to 61 mmHg) and did not change the cerebral expression of AQP4 (100 ± 6% of sham), NKCC1 (100 ± 1% of sham), Bax (102 ± 6% of sham) and TBARS. Both TERLI and LR recovered baseline levels of PbtO2 (TERLI from 30.0 to 34.2 mmHg; LR from 29.4 to 40.7 mmHg) and MAP (TERLI from 53 to 64 mmHg; LR: 48 to 61 mmHg). Blood lactate levels were not recovered in any group (TERLI from 5.7 to 8.1 mmol/l; LR from 4.5 to 7.7 mmol/l). Inhaled carbon monoxide or nebulized sodium nitrite protect against hemorrhagic shock-induced mitochondrial dysfunction H Gomez, D Escobar, B Ataya, L Gordon, O Ogundele, M Pinsky, S Shiva, B Zuckerbraun University of Pittsburgh, PA, USA Critical Care 2013, 17(Suppl 2):P220 (doi: 10.1186/cc12158) Introduction Shock induces mitochondrial damage, which can lead to tissue injury and infl ammation. Accuracy of the shock index and various modifi ed shock indexes to predict early mortality in patients suff ering from gastrointestinal haemorrhage Introduction The shock index (SI) is an easy-to-use clinical tool that rapidly identifi es patients at risk of haemodynamic decompensation. Previous studies focused primarily on patients suff ering from pneumonia, pulmonary embolism, ruptured ectopic pregnancy and traumatic haemorrhagic shock. Modifi ed SIs have also been studied. Could the SI be accurate in patients suff ering from gastrointestinal (GI) haemorrhage? The aim of this study was to compare the performance of the SI with various modifi ed SIs and conventional vital signs in predicting 30-day mortality in a population of patients with a GI haemorrhage. Conclusion The mottling score was an objective reproducible system to bedside use and a good predictor of septic shock mortality. References 1. Hernandez G, et al.: Evolution of peripheral vs metabolic perfusion 1. Hernandez G, et al.: Evolution of peripheral vs metabolic perfusion parameters during septic shock resuscitation. A clinical-physiologic stu 1. Hernandez G, et al.: Evolution of peripheral vs metabolic perfusion parameters during septic shock resuscitation. A clinical-physiologic study. J Crit Care 2012, 27:283-288. parameters during septic shock resuscitation. A clinical-physiologic study. J Crit Care 2012, 27:283-288. 2. Ait-Oufella H, et al.: Mottling score predicts survival in septic shock. Intensive Care Med 2011, 37:801-807. g Methods A single-center post-hoc analysis was conducted of prospectively collected data from patients diagnosed with a GI haemorrhage episode in an academic emergency department (ED) from March 2008 to December 2011. Data were extracted from two databases used at our ED. The SI (pulse/systolic blood pressure) and nine modifi ed SIs were calculated from the available fi rst documented vital signs. ROC curves were used to determine sensitivity and specifi city of the diff erent SIs in predicting 30-day mortality. References 1. Cavus E, et al.: Resuscitation 2009, 80:567-572. 2. Bayram B, et al.: Am J Emerg Med 2011, 30:1176-1182. y Results One hundred and seventeen patients were analyzed; 20 were excluded (18 terminal illness, two with incomplete data). Ninety-seven patients were included; the mean age was 72.8 years, mean SAPS II score was 46.8 (SD ± 15.7), mean APACHE II score was 19.2 (SD ± 8.1); mean norepinephrine dose was 1.25 μg/kg/minute; mean length of stay in ICU was 19.2 days (1 to 176); mottling score distribution was: score 0: 41 patients; score 1: 33 patients; score 2: 14 patients; score 3: two patients; score 4: three patients; score 5: four patients. The sepsis origin was as follows: 65, pulmonary (67%); 18, abdominal (18.5%); nine, urinary (9.5%); two, osseous (2%); one, mediastinal; one, skin and soft tissue; and one, central nervous system (1% each). Comparing the mortality in Groups 1, 2 and 3, we found a signifi cant diff erence (P = 0.042), even greater when considering 28-day mortality (P = 0.004). The Kaplan–Meier survival method showed P = 0.000. P219 P219 Accuracy of the shock index and various modifi ed shock indexes to predict early mortality in patients suff ering from gastrointestinal haemorrhage J St-Cyr Bourque, J Cliche, J Chauny, R Daoust, J Paquet, É Piette Hôpital du Sacré-Coeur de Montréal, Canada Critical Care 2013, 17(Suppl 2):P219 (doi: 10.1186/cc12157) Assessment of the mottling score as a mortality predictor in critically ill patients y E Bastos de Moura, F Ferreira Amorim, C Darwin Silveira, M Oliveira Maia Hospital Santa Luzia, Brasília, Brazil Critical Care 2013, 17(Suppl 2):P217 (doi: 10.1186/cc12155) Introduction The use of peripheral perfusion objective parameters to anticipate successful resuscitation in septic shock has been recently investigated [1]. The mottling score, a perfusion parameter used for decades, has been proposed to correlate with septic shock survival [2], and was tested in this study as a clinical tool in predicting mortality. Methods A prospective observational study was conducted, with patients consecutively admitted to a tertiary hospital ICU in Brasília, Brazil. From July 2011 to May 2012, all patients diagnosed with septic shock were enrolled. Demographic data, diagnoses, shock origin and severity scores were recorded. After initial resuscitation, the score was registered in the fi rst 3 days by the same observer, considering the score on the lower limb without an arterial catheter, or the worst between the lower limbs, and the worst in the 3 days. Exclusion criteria were terminal illness with no intervention decision and incomplete S82 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 contributed to the cerebral apoptosis. None of the treatments caused cerebral oxidative stress. data. The scores are pooled in Group 1 (scores 0 and 1), Group 2 (scores 2 and 3) and Group 3 (scores 4 and 5) to compare mortality. Statistical analysis was made using the chi-square test. contributed to the cerebral apoptosis. None of the treatments caused cerebral oxidative stress. data. The scores are pooled in Group 1 (scores 0 and 1), Group 2 (scores 2 and 3) and Group 3 (scores 4 and 5) to compare mortality. Statistical analysis was made using the chi-square test. P221 Is norepinephrine more eff ective than other vasopressors for septic shock? A systematic review and meta-analysis F Zhou1, Z Peng2, W Zhang3, J Bishop2, Q Song1 1Chinese People’s Liberation Army General Hospital, Beijing, China; 2University of Pittsburgh, PA, USA; 3University of Pittsburgh School of Medicine, PA, USA Critical Care 2013, 17(Suppl 2):P221 (doi: 10.1186/cc12159) Is norepinephrine more eff ective than other vasopressors for septic shock? A systematic review and meta-analysis y y Results A total of 300 patients underwent randomization, were infused with the study drug (148 patients received vasopressin, and 152 norepinephrine), and were included in the analysis. Patients who received vasopressin had a lower rate of morbidity (23.5% vs. 34%, P = 0.001) as compared with the norepinephrine group. The 30-day mortality rate was 6.1% in the norepinephrine group and 4.6% in the vasopressin group (P = 0.570). There were no signifi cant diff erences in the overall rates of serious adverse events (7.4% and 6.6%, respectively; P = 0.772). Introduction Norepinephrine has been widely used in septic shock. However, its eff ect remains controversial. We conduct a systematic review and meta-analysis to compare the eff ect between norepinephrine and other vasopressors. Methods The PubMed, Embase, and Cochrane Library databases from database inception until October 2012 were searched. We selected randomized controlled trials in adults with septic shock and compared norepinephrine with other vasopressors. The quality of each study included was assessed with Jadad score. After assessing for heterogeneity of treatment eff ect across trials using the I2 statistic, we used a fi xed eff ect model (P ≥0.1) or random-eff ects model (P <0.1) and expressed results as the risk ratio (RR) for dichotomous outcomes or the f Conclusion Vasopressin reduces major morbidity after cardiac surgery as compared with norepinephrine among patients with cardiac surgery with vasoplegic shock. p g Acknowledgement Clinical Trials number NCT01505231. p g Acknowledgement Clinical Trials number NCT01505231. P223 standardized mean diff erence (SMD) for continuous data with 95% CI. Results Eighteen trials (n = 2,715) met inclusion criteria, which compared norepinephrine with fi ve diff erent vasopressors (dopamine, vasopressin, epinephrine, terlipressin and phenylephrine). The mean Jadad score was 3.11. Overall, there was no diff erence in mortality in the comparisons between norepinephrine and vasopressin, epinephrine, terlipressin and phenylephrine (P >0.05, respectively). However, norepinephrine had a trend in decreasing mortality compared with dopamine (RR, 0.84; 95% CI, 0.68 to 1.02; P = 0.08). P222 11 mg in PBS×30 minutes, n = 6), and sham (n = 3). CO or NaNO2 were initiated ~30 minutes before resuscitation. Primary endpoints were changes in muscle and platelet mitochondrial respiration between BL and EndObs, quantifi ed by muscle respiratory control ratio (RCR, traditional respirometry), and by the change in proton-leak respiration (PLR) and mitochondrial reserve capacity in platelets. Secondary endpoint was mortality at EndObs. P222 Vasopressin Versus Norepinephrine for the Management of Shock After Cardiac Surgery (VaNCS study): a randomized controlled trial L Hajjar1, JL Vincent2, A Rhodes3, D Annane4, F Galas1, J Almeida1, S Zeferino1, L Camara1, V Santos1, J Pereira1, E Osawa1, E Maciel1, A Rodrigues1, J Jardim1, D Blini1, E Araujo1, F Bergamin5, R Kalil Filho1, J Auler Jr1 1Heart Institute, São Paulo, Brazil; 2Universite Libre de Bruxelles, Brussels, Belgium; 3University of London, UK; 4Faculte Medicine Paris, France; 5Cancer Institute, São Paulo, Brazil Critical Care 2013, 17(Suppl 2):P222 (doi: 10.1186/cc12160) Vasopressin Versus Norepinephrine for the Management of Shock After Cardiac Surgery (VaNCS study): a randomized controlled trial L Hajjar1, JL Vincent2, A Rhodes3, D Annane4, F Galas1, J Almeida1, S Zeferino1, L Camara1, V Santos1, J Pereira1, E Osawa1, E Maciel1, A Rodrigues1, J Jardim1, D Blini1, E Araujo1, F Bergamin5, R Kalil Filho1, J Auler Jr1 1Heart Institute, São Paulo, Brazil; 2Universite Libre de Bruxelles, Brussels, Belgium; 3University of London, UK; 4Faculte Medicine Paris, France; 5Cancer Institute, São Paulo, Brazil Critical Care 2013, 17(Suppl 2):P222 (doi: 10.1186/cc12160) y Results Skeletal muscle RCR decreased in the HSR group (P = 0.04) but not in sham. Decrease in RCR was primarily due to decreased ADP- dependent respiration, without change in state 4 respiration. HSR also resulted in platelet mitochondrial dysfunction as demonstrated by increased PLR and decreased reserve capacity. This correlated with increased platelet activation (%CD62P+ by fl ow cytometry) in HSR. CO or NaNO2 treatment prevented these deleterious changes in both muscle and platelet mitochondrial respiration, as well as limited HSR-induced platelet activation. CO treatment also improved reserve capacity compared with baseline. Mortality was higher in HSR than in CO or NaNO2 (80 vs. 33 and 33%, respectively). Introduction Vasoplegic syndrome is a common complication after cardiac surgery, with negative impact on patient outcomes and hospital costs. Pathogenesis of vasodilatory phenomenon after cardiac surgery remains a matter of controversy. Loss of vascular tone can be partly explained by the depletion of neurohypophyseal arginine vasopressin stores. 1. Jadad AR, et al.: Control Clin Trials 1996, 17:1-12. 2. Higgins JP, et al.: BMJ 2003, 327:557-560. Intraaortic counterpulsation in a second-level institution: indications, management and outcome R Gómez López, P Fernández Ugidos, P Vidal Cortés, M Lorenzo Lage, A Tizón Varela, E Rodríguez Álvarez Complexo Hospitalario Universitario de Ourense, Spain Critical Care 2013, 17(Suppl 2):P223 (doi: 10.1186/cc12161) Introduction Counterpulsation is an important support for patients with cardiac diseases. The use of these devices has been limited typically to hospitals with a cardiac surgery service. The aim of this study is to describe the management of patients with an intraaortic balloon pump (IABP) in our second-level institution. Methods An observational study that includes all patients with IABP in our hospital from January 2010 to September 2012, followed to November 2012. Epidemiological and clinical variables as an indication of IABP, therapeutic management and outcome were collected. Because of the small size sample, statistical analysis was limited to descriptive parameters. Conclusion There is not suffi cient evidence to prove that norepinephrine is superior to vasopressin, epinephrine, terlipressin and phenylephrine in terms of mortality. However, norepinephrine is associated with a decreased HR, cardiac index and an increased SVRI, and appears to have a greater eff ect on decreasing mortality compared with dopamine. Results Seventeen patients (76.5% men, age 66.6 ± 11.6 (43 to 85) years, APACHE score 21.5 ± 14.9 (3 to 46) points) were treated with IABP. The reason for ICU admission was STEMI (n = 6 patients, 35.3%), no-STEMI (n = 5, 29.4%), acute heart failure (n = 4, 23.5%) and out-of-hospital Impact of diff erent haemodynamic resuscitation strategies on brain perfusion and tissue oedema markers in a model of severe haemorrhagic shock Resuscitative adjuncts to limit mitochondrial injury may be eff ective to reduce tissue injury and protect against the sequelae of hemorrhagic shock (HS). Others and we have demonstrated the protective eff ects of inhaled carbon monoxide (CO) or nebulized sodium nitrite (NaNO2) in models of HS. Our aim was to test the hypothesis that CO and NaNO2 protect against hemorrhagic shock-induced tissue injury/infl ammation by limiting mitochondrial damage and preventing bioenergetic failure. Methods Twenty anesthetized female Yorkshire pigs were subjected to severe hemorrhage until unable to compensate or 90 minutes, and were then resuscitated with volume/pressors. Muscle and platelet samples were obtained at baseline (BL) and 2 hours after resuscitation (EndObs). Animals were randomized to: standard of care (HSR, n = 5); HSR+CO (CO; 250 ppm×30 minutes, n = 6); or HSR+NaNO2 (NaNO2; Conclusion TERLI recovered cerebral perfusion and oxygenation with no signifi cant changes in ICP and cerebral markers of oedema, apoptosis and oxidative stress. LR did not recover CPP probably due to a signifi cant increase in ICP caused by brain oedema, which may have S83 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 P222 Vasopressin is commonly used as an adjunct to catecholamines to support blood pressure in refractory septic shock, but its eff ect on vasoplegic shock is unknown. We hypothesized that the use of vasopressin would be more eff ective on treatment of shock after cardiac surgery than norepinephrine, decreasing the composite endpoint of mortality and severe morbidity. 2 y Conclusion In severe HS, mitochondrial injury in platelets and muscle was limited by CO or NaNO2. Although not powered for a secondary endpoint, mortality was double in HSR versus adjunctive therapies. This suggests that CO and NaNO2 may protect mitochondrial function by maintaining ATP-coupled respiration and reserve capacity, and that this may confer a survival advantage. However, further investigations are required. Methods In this prospective and randomized, double-blind trial, we assigned patients who had vasoplegic shock to receive either vasopressin (0.01 to 0.06 U/minute) or norepinephrine (0.01 to 1 μg/ kg/minute) in addition to open-label vasopressors. All vasopressor infusions were titrated and tapered according to protocols to maintain a target blood pressure. The primary endpoint was major morbidity according to STS (30-day mortality, mechanical ventilation >48 hours, mediastinitis, surgical re-exploration, stroke, acute renal failure). Secondary outcomes were time on mechanical ventilation, ICU and hospital stay, new infection, the time to attainment of hemodynamic stability, occurrence of adverse events and safety. P221 There were a decreased heart rate (HR) (SMD, –2.10; 95% CI, –3.95 to –0.25; P = 0.03), cardiac index (SMD, –0.73; 95% CI, –1.14 to –0.03; P = 0.004) and an increased systemic vascular resistance index (SVRI) (SMD, 1.03; 95% CI, 0.61 to 1.45; P <0.0001) with the treatment of norepinephrine compared with dopamine.fi Intraaortic counterpulsation in a second-level institution: indications, management and outcome R Gómez López, P Fernández Ugidos, P Vidal Cortés, M Lorenzo Lage, A Tizón Varela, E Rodríguez Álvarez Complexo Hospitalario Universitario de Ourense, Spain Critical Care 2013, 17(Suppl 2):P223 (doi: 10.1186/cc12161) 3. Mayo Clin Proc 2012, 87:620-628. 1. J Cardiothorac Vasc Anesth 2011, 25:526-535. P224 Results Our urosepsis population contained 364 patients, of which 129 (35.4%) were either using a BB, a CCB or both before their admission. Mean age was 74.9 years and 48.1% of the patients were women. A total of 36 patients died (9.9%) in a 30-day period. The group taking either BB or CCB had a signifi cantly lower mean SI (0.76, 95% CI: 0.72 to 0.81 vs. 0.93, 95% CI: 0.89 to 0.98, P <0.0001). In our urosepsis population, a SI of 0.7 had a sensitivity of 0.76 and a specifi city of 0.27 (area under the curve (AUC): 0.596) for patients taking neither BB nor CCB. In the group taking either or both medications, a SI of 0.7 had a sensitivity of 0.57 and a specifi city of 0.42 (AUC: 0.578). In both groups, lowering the SI to 0.5 increased the sensitivity to more than 0.95 but lowered specifi city signifi cantly.f Impact of left ventricular ejection fraction and elevated fi lling pressures on mortality in mechanically ventilated patients in severe sepsis or septic shock P225 Eff ect of β-blockers and calcium channel blockers on shock index predictability in patients suff ering from urosepsis J Cliche, J St-Cyr Bourque, R Daoust, J Chauny, J Paquet, E Piette Sacré-Coeur Hospital, Montreal, Canada Critical Care 2013, 17(Suppl 2):P225 (doi: 10.1186/cc12163) Eff ect of β-blockers and calcium channel blockers on shock index predictability in patients suff ering from urosepsis Eff ect of β-blockers and calcium channel blockers on shock index predictability in patients suff ering from urosepsis f J Cliche, J St-Cyr Bourque, R Daoust, J Chauny, J Paquet, E Piette Sacré-Coeur Hospital, Montreal, Canada Critical Care 2013, 17(Suppl 2):P225 (doi: 10.1186/cc12163) Introduction In our busy emergency departments the identifi cation of patients at risk of rapid hemodynamic decompensation is crucial. The shock index (SI) (pulse/systolic blood pressure) is a non-invasive clinical sign associated with more complications if higher than 0.7. The eff ect of β-blockers (BB) and calcium channel blockers (CCB) on the SI has not yet been described. Most studies excluded these patients, owing to the medications’ eff ect on the cardiac pulse. Considering that BB and CCB are commonly prescribed, we studied their eff ect on the SI’s predictability on 30-day mortality in a urosepsis population. Methods This single-center post-hoc analysis of prospectively collected data was conducted in an academic Canadian emergency department (ED) between March 2008 and February 2011. Data were extracted from two institutional databases. We included patients with a fi nal diagnosis of urosepsis, sepsis, pyelonephritis and urinary tract infection. Selected patients also had a documented positive urine culture. The SI predictability on 30-day mortality was calculated for patients taking BB and/or CCB as well as patients taking none of these medications. Sensitivity and specifi city were determined using ROC curves. t tests were used to compare mean SI between both groups. g pp y Conclusion In our hospital the IABP was mainly used in refractory cardiogenic shock because of myocardial ischemia, with an all-cause mortality of 41.2%. Low rates of complications were observed. Transfer of patients with IABP was performed safely. 2. Crit Care Med 2009, 37:441-447. P226 Levosimendan in critically ill adults: a utilisation review J Aron1, S Harrison2, A Milne2, S Patel2, R Maharaj2 1Lewisham NHS Trust, London, UK; 2King’s College Hospital NHS Trust, London, UK ii Results All cause 30-day mortality was 50% (n = 29). Forty-six patients (79%) had normal LV function. Forty-four (76%) patients had normal LV fi lling pressures. Patients who survived had lower E/e’ ratio, (median E/e’ 10.6 ± 4 vs. 13.6 ± 7, P = 0.1) but this was not statistically signifi cant. However, when defi ned as E/e’ >15, the mortality was 71%. On the contrary, patients with low LVEF had a mortality of 41% and LVEF was no diff erent between survivors and nonsurvivors (55 ± 16 vs. 58 ± 14, P = 0.34). Introduction Levosimendan improves haemodynamic performance and may have cardioprotective eff ects. Small trials have demonstrated effi cacy in well-defi ned populations [1]. Given the uncertain benefi ts we describe contemporaneous patient selection, utilisation of levosimedan, haemodynamic eff ects and outcomes. f Methods The study was performed at a single centre’s three ICUs. Data from 38 consecutive admissions, from 2010 to 2012, treated with levosimendan were collected retrospectively. Demographics, illness severity, comorbidity, haemodynamic, metabolic, biochemical, resource utilisation, organ support and hospital outcomes were analysed. Conclusion Myocardial dysfunction is a well-known entity in patients with septic shock. The clinical spectrum of this entity is broad, including LV, RV and diastolic dysfunction. Although the E/e’ ratio has been a known prognostic indicator in other cardiac conditions, its role in these patients is less clear. This study demonstrated that when septal mitral annulus E/e’ was >15, it was a better marker for mortality than LVEF in mechanically ventilated patients with severe sepsis or septic shock. Larger studies incorporating diastolic evaluation and TDI should be performed to further clarify this fi nding. Results Our cohort had a mean age of 58 (95% CI = 53.4 to 64.4). Only 18% underwent cardiothoracic surgery: sepsis, pulmonary emboli and myocardial/pericardial diseases were also treated with levosimendan. Admission characteristics included mean PaO2/FiO2 ratio (PFR) of 186.7 mmHg (95% CI = 160 to 212) and mean cardiac index (CI) of 2.3 (95% CI 1.9 to 2.8). The median APACHE II score was 24 (interquartile range 18.5 to 30). Levosimendan was initiated at a variable point during ICU treatment (days 0 to 14) and was usually the third inotrope References S84 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 cardiac arrest (n = 2, 11.8%). The indications of IABP were refractory cardiogenic shock (n = 12, 70.6%), high-risk percutaneous coronary intervention (n = 2, 11.8%), refractory angina (n = 1, 5.9%), refractory pulmonary edema (n = 1, 5.9%) and electrical storm (n = 1, 5.9%). Five IABPs (27.4%) were inserted in the ICU and the rest in the catheterization laboratory. Six patients (35.3%) suff ered a cardiac arrest prior to hemodynamic stabilization. Three patients (17.6%, the electrical storm and the out-of-hospital cardiac arrest) died before coronary catheterization was performed and the other three were treated with mild therapeutic hypothermia. Thirteen patients (76.5%) needed invasive mechanical ventilation. In six patients (35.3%) invasive hemodynamic monitoring was performed (one pulmonary artery catheter, fi ve PICCO). Transthoracic echocardiography was performed in all patients and transesophageal in six (35.3%). Six of the patients (35.3%) were transferred to the reference centre for immediate coronary artery bypass grafting (CABG). No complications were reported during the transfer. During the ICU stay, femoral artery pseudoaneurysm was reported in one of the patients and inguinal hematoma after IABP withdrawal in two (no transfusion required). Four patients (27.4%) died because of refractory cardiogenic shock despite revascularization. The length of mechanical support was 1.67 ± 1.5 days (0 to 6). Impact of left ventricular ejection fraction and elevated fi lling pressures on mortality in mechanically ventilated patients in severe sepsis or septic shock p p S Gillespie, J Pulido Mayo Clinic, Rochester, MN, USA Critical Care 2013, 17(Suppl 2):P224 (doi: 10.1186/cc12162) S Gillespie, J Pulido Mayo Clinic, Rochester, MN, USA Introduction Myocardial dysfunction in septic shock is common and the presentation is broad. There are confl icting data regarding the prognostic implications of low left ventricular (LV) ejection fraction and elevated E/e’ ratio on mortality in this patient population. We sought to assess the impact of LV dysfunction and elevated E/e’ ratio on 30- day mortality in mechanically ventilated patients with severe sepsis or septic shock. Conclusion To our knowledge this is the only study analysing the eff ect of BB and CCB on the SI predictability of 30-day mortality. Our results indicate that SI cannot be used to accurately predict mortality with patients suff ering from urosepsis. In both our groups, SI performance was poor, as shown by the ROC curves. BB or CCB did not infl uence these results. Methods Fifty-eight mechanically ventilated patients with severe sepsis or septic shock admitted from 1 August 2007 to 31 January 2009 were prospectively evaluated with transthoracic echocardiogram within 24 hours of admission. Left ventricular ejection fraction was assessed using the modifi ed Simpson method as recommended by the American Society of Echocardiography. Normal LV function was defi ned as LVEF 40%. Mitral infl ow pulsed wave Doppler of peak E waves and tissue Doppler imaging (TDI) of the septal mitral annulus peak velocities were measured, the E/e’ ratio was obtained. Elevated LV fi lling pressures was defi ned as E/e’ >15. P227 P227 Levosimendan versus dobutamine in Tako-tsubo cardiomyopathy FR Righetti, MP Parolini, GC Castellano St. Boniface Hospital Verona, Italy Critical Care 2013, 17(Suppl 2):P227 (doi: 10.1186/cc12165) and their interaction. Data are reported as mean ± standard deviation. Results Ivabradine decreased the HR from 223 ± 18 to 196 ± 15 bpm (P <0.05). Contractility and relaxation remained unchanged. Ivabradine reduced the positive chronotropic eff ect at all doses of dobutamine (10 nM: 232 ± 37 vs. 187 ± 19, 100 nM: 265 ± 37 vs. 211 ± 40, 1 μM: 316 ± 35 vs. 250 ± 39, 10 μM: 320 ± 33 vs. 235 ± 40 bpm; P <0.05). It shifted the maximum positive inotropic action of dobutamine to lower dose ranges (100 nM: 2,924 ± 841 vs. 2,978 ± 955, 300 nM: 3,743 ± 925 vs. 4,795 ± 1,298, 1 μM: 4,138 ± 935 vs. 4,896 ± 1,861 mmHg/second; P <0.05). A comparable shift was seen for relaxation (100 nM: –2,178 ± 686 vs. –2,520 ± 742, 300 nM: –2,615 ± 726 vs. –3,150 ± 888, 1 μM: –2,903 ± 752 vs. –2,972 ± 967 mmHg/second; P <0.05). Levosimendan increased the HR only at high doses. With ivabradine, no positive chronotropic eff ect of levosimendan was observed (100 nM: 185 ± 30 vs. 162 ± 22, 1 μM: 208 ± 28 vs. 166 ± 29, 10 μM: 242 ± 27 vs. 168 ± 36 bpm; P <0.05). Ivabradine attenuated the positive inotropic eff ect of levosimendan (100 nM: 2,303 ± 303 vs. 1,737 ± 262, 1 μM: 2,977 ± 481 vs. 1,940 ± 449, 10 μM: 3,480 ± 941 vs. 2,189 ± 542 mmHg/ second; P <0.05) but did not signifi cantly alter its lusitropic eff ect. Introduction Tako-tsubo cardiomyopathy is a clinical entity charac- terized by a dysfunction of the left ventricle, usually transient, which manifests itself with symptoms that can mimic an acute coronary syndrome. It is characterized by transient ballooning modifi cation of the left ventricular apex, probably due to neurogenic stimuli resulting in low cardiac output syndrome. The aim of this prospective randomized study was to evaluate, by serial transesophageal echocardiography (TEE), what is the best drug treatment between levosimendan and dobutamine to restore a satisfactory cardiac function in the case of low cardiac output. Methods Twelve adult patients, aged 18 years, were admitted to the ICU with Tako-tsubo cardiomyopathy at entrance. P228 Combined eff ects of ivabradine with dobutamine or levosimendan in isolated perfused hearts D Konrad, R Vicenzi-Moser, M Vicenzi, U Aldenhoff , E Schwarzl, W Toller Medical University Graz, Austria Critical Care 2013, 17(Suppl 2):P228 (doi: 10.1186/cc12166) (range 0 to 5) commenced. Treatment with levosimendan (Table 1) resulted in improved CI and PFR with reductions in EVLW, BE, lactate and creatinine. The ICU length of stay was 15.4 days (95% CI = 13.2 to 17.6) and the hospital mortality was 54%. No signifi cant adverse eff ects were reported. (range 0 to 5) commenced. Treatment with levosimendan (Table 1) resulted in improved CI and PFR with reductions in EVLW, BE, lactate and creatinine. The ICU length of stay was 15.4 days (95% CI = 13.2 to 17.6) and the hospital mortality was 54%. No signifi cant adverse eff ects were reported. Introduction Dobutamine and levosimendan improve cardiac contractility in patients with heart failure, septic cardiomyopathy or cardiac surgery. Tachycardia in these cases is undesired because of aggravating ischemia. Ivabradine, a selective sinus node inhibitor, does not aff ect contractility. A combination of ivabradine with positive inotropic drugs might be favorable. Thus, we compared the cardiac eff ects of dobutamine or levosimendan alone, and combined with ivabradine using the Langendorff method of isolated perfused hearts. Methods Isolated guinea pig hearts (n = 37) were perfused with incremental doses of dobutamine (10 nM to 10 μM) or levosimendan (30 nM to 10 μM) either alone or combined with 3 μM ivabradine. Heart rate (HR), left ventricular pressures, contractility (+dLVP/dt) and relaxation (–dLVP/dt) were recorded. Data for each drug (dobutamine or levosimendan) were analyzed by two-way ANOVA for repeated measures including the two main eff ects of ivabradine and drug dose and their interaction. Data are reported as mean ± standard deviation. Results Ivabradine decreased the HR from 223 ± 18 to 196 ± 15 bpm (P <0.05). Contractility and relaxation remained unchanged. Ivabradine reduced the positive chronotropic eff ect at all doses of dobutamine (10 nM: 232 ± 37 vs. 187 ± 19, 100 nM: 265 ± 37 vs. 211 ± 40, 1 μM: 316 ± 35 vs. 250 ± 39, 10 μM: 320 ± 33 vs. 235 ± 40 bpm; P <0.05). It shifted the maximum positive inotropic action of dobutamine to lower dose ranges (100 nM: 2,924 ± 841 vs. 2,978 ± 955, 300 nM: 3,743 ± 925 vs. 4,795 ± 1,298, 1 μM: 4,138 ± 935 vs. 4,896 ± 1,861 mmHg/second; P <0.05). A comparable shift was seen for relaxation (100 nM: –2,178 ± 686 vs. –2,520 ± 742, 300 nM: –2,615 ± 726 vs. P227 The patients were divided randomly into two groups: levosimendan (six patients) treated with levosimendan and standard treatment, and the control group (six patients) with dobutamine and standard treatment. In all patients, serial TEE was performed studying the systolic function, by ejection fraction of the left ventricle with Simpson’s method. The TEEs were performed at the entrance of the patient, after 12 and 24 hours of treatment. The results were expressed as mean with standard deviation. For the comparison between the two groups we used the Fisher test, considered signifi cant with P <0.05. Conclusion Addition of ivabradine to dobutamine attenuates its chronotropic actions without diminishing its inotropic eff ects. A combination of levosimendan with ivabradine does not seem to provide benefi t. Clinical studies are necessary to confi rm these experimental results. P228 P228 Combined eff ects of ivabradine with dobutamine or levosimendan in isolated perfused hearts D Konrad, R Vicenzi-Moser, M Vicenzi, U Aldenhoff , E Schwarzl, W Toller Medical University Graz, Austria Critical Care 2013, 17(Suppl 2):P228 (doi: 10.1186/cc12166) References Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S85 Table 1 (abstract P226). Treatment outcomes Variable Mean 95% CI CI Δd5 – d0 +1.34 0.4 to 2.2 PFR Δd5 – d0 +284 223 to 345 BE Δd5 – d0 +2.9 0.85 to 5 Lac Δd5 – d0 –1 –2 to 0.2 d0, treatment commenced. Table 1 (abstract P226). Treatment outcomes control group there remains a dysfunction in systolic function. We have shown the drug therapy based on levosimendan contributes to improving the systolic function of the left ventricle compared with treatment with dobutamine despite the initial cardiac stunning. Reference 1. Landoni G, et al.: Eff ects of levosimendan on mortality and hospitalization. A metanalysis of randomized controlled studies. Crit Care Med 2012, 40:634-646. 1. Landoni G, et al.: Eff ects of levosimendan on mortality and hospitalization. A metanalysis of randomized controlled studies. Crit Care Med 2012, 40:634-646. P228 Combined eff ects of ivabradine with dobutamine or levosimendan in isolated perfused hearts D Konrad, R Vicenzi-Moser, M Vicenzi, U Aldenhoff , E Schwarzl, W Toller Medical University Graz, Austria Critical Care 2013, 17(Suppl 2):P228 (doi: 10.1186/cc12166) –3,150 ± 888, 1 μM: –2,903 ± 752 vs. –2,972 ± 967 mmHg/second; P <0.05). Levosimendan increased the HR only at high doses. With ivabradine, no positive chronotropic eff ect of levosimendan was observed (100 nM: 185 ± 30 vs. 162 ± 22, 1 μM: 208 ± 28 vs. 166 ± 29, 10 μM: 242 ± 27 vs. 168 ± 36 bpm; P <0.05). Ivabradine attenuated the positive inotropic eff ect of levosimendan (100 nM: 2,303 ± 303 vs. 1,737 ± 262, 1 μM: 2,977 ± 481 vs. 1,940 ± 449, 10 μM: 3,480 ± 941 vs. 2,189 ± 542 mmHg/ second; P <0.05) but did not signifi cantly alter its lusitropic eff ect. Conclusion Levosimendan is usually used in patients with cardiogenic shock unresponsive to conventional inotropes or mechanical augmentation. The mortality of this group is high but represents patients with shock refractory to conventional treatment. Its use in sepsis, myocarditis and pulmonary embolism is not well established. In this group levosimendan appears to have a favourable eff ect on gas exchange, renal function and tissue perfusion. Limitations include retrospective analysis and missing data. p y g Reference 1. Landoni G, et al.: Eff ects of levosimendan on mortality and hospitilisation. A meta-analysis of RCT. Crit Care Med 2012, 40:634-646. Intraabdominal hypertension and abdominal compartment syndrome in patients admitted to a medical and surgical ICU at a third referral university hospital Intraabdominal hypertension and abdominal compartment syndrome in patients admitted to a medical and surgical ICU at a third referral university hospital y p N Chindavech1, S Yenarkart2, S Kongsayreepong2 1Siriraj Hospital, Mahidol University, Bangkok, Thailand; 2Division of Critical Care Medicine, Bangkok, Thailand Critical Care 2013, 17(Suppl 2):P231 (doi: 10.1186/cc12169) Introduction Intraabdominal hypertension (IAH), especially abdominal compartment syndrome (ACS), can aff ect organ function leading to multiple organ failure. Appropriate and prompt management could improve survival. Less recognition of this problem in critically ill patients has been reported. The aim of this study was to study the prevalence, predictive factors and clinical outcome of IAH and ACS in a mixed population of critically ill patients by intermittent measuring bladder pressure during the ICU stay. Results The positive inotropic action of dobutamine was least at 37°C, more pronounced at 40°C and best at 34°C (37°C vs. 40°C vs. 34°C: 300 nM 280 ± 71 vs. 301 ± 94 vs. 345 ± 99%, 1 μM 310 ± 74 vs. 327 ± 92 vs. 389 ± 144%, 10 μM 297 ± 69 vs. 339 ± 96 vs. 359 ± 175%; P <0.05). Dobutamine’s positive lusitropic eff ect was not signifi cantly altered by temperature. The positive inotropic action of levosimendan was best at 37°C, in hyperthermia and hypothermia only the three highest doses of levosimendan increased contractility (37°C vs. 40°C vs. 34°C: 100 nM 121 ± 21 vs. 134 ± 15 vs. 87 ± 12%, 300 nM 135 ± 22 vs. 137 ± 17 vs. 94 ± 15%, 1 μM 153 ± 22 vs. 147 ± 21 vs. 108 ± 21%, 3 μM 172 ± 19 vs. 150 ± 24 vs. 115 ± 23%, 10 μM 173 ± 31 vs. 156 ± 28 vs. 117 ± 28%; P <0.05). The positive lusitropic eff ect of levosimendan at 37°C was almost absent in hypothermia and hyperthermia (P <0.05). g y Methods This prospective observational study was done in 130 adult patients (age >18 years) admitted to a medical and general surgical ICU at a third referral university hospital during June to November 2011. Variables about the patient’s profi le laboratory data and clinical outcome as ICU and hospital length of stay, and ICU, in-hospital and 28-day mortality were recorded. y y Results There were 33 (25.4%) medical and 93 (74.6%) surgical ICU patients in this study. Intraabdominal hypertension and abdominal compartment syndrome in patients admitted to a medical and surgical ICU at a third referral university hospital Surgical patients had higher prevalence of IAH than medical patients (57.8% vs. 33.3%, P = 0.015). Medical patients were admitted with severe sepsis/septic shock, AKI, pneumonia and ARDS. Surgical patients were more acutely ill with high ASA (III to IV), severity score, underwent emergency abdominal surgery and received more transfusion but were no diff erent in type of fl uid replacement. Signifi cant risk factors of IAH were coagulopathy (OR = 2.09, 95% CI = 1.62 to 2.69), intraabdominal infection (OR = 1.87, 95% CI = 1.40 to 2.48), retroperitonium hematoma (OR = 1.82, 95% CI = 1.36 to 2.44), marked ascites (OR = 1.76, 95% CI = 1.32 to 2.36), acidosis (pH <7.2) (OR = 1.82, 95% CI = 1.37 to 2.43), severe sepsis/septic shock (OR = 1.63, 95% CI = 1.14 to 2.33), and massive transfusion (OR = 1.51, 95% CI = 1.10 to 2.08). Patients with IAH had more reopened surgeries and had higher ICU, hospital and 28-day mortality. Sixteen (12.3%) patients had ACS, 15 patients underwent emergency surgery, two patients had temporary abdominal closured and one-half of the patients had severe abdominal sepsis and massive transfusion. Fourteen patients died despite temporary abdominal closure. Delayed release abdominal tamponade were most causes of death. Conclusion In isolated perfused hearts, dobutamine has its best positive inotropic eff ect in hypothermia whereas levosimendan increases contractility best at normothermic conditions. Clinical studies are necessary to confi rm these experimental results. Confl icting results between V/Q SPECT and angioscan in pulmonary embolism: what to do? Confl icting results between V/Q SPECT and angioscan in pulmonary embolism: what to do? JM Martel, MN Nadeau, RD Daoust, JP Paquet, JC Chauny Hôpital du Sacré-Coeur de Montréal, Canada Critical Care 2013, 17(Suppl 2):P230 (doi: 10.1186/cc12168) Confl icting results between V/Q SPECT and angioscan in pulmonary embolism: what to do? JM Martel, MN Nadeau, RD Daoust, JP Paquet, JC Chauny Hôpital du Sacré-Coeur de Montréal, Canada Critical Care 2013, 17(Suppl 2):P230 (doi: 10.1186/cc12168) Introduction The objective of this study is to determine the fi nal clinical diagnosis of patients who underwent the double investigation for pulmonary embolism with confl icting results. Pulmonary embolism is a prevalent pathology in the emergency department (ED). A certain proportion of patients undergo a double radiological investigation (V/Q SPECT and angioscan), which incurs higher costs and X-ray doses. However, no study to date has addressed this issue. p Conclusion The prevalence and morbidity/mortality of IAH and ACS were high in this institute. Early appropriate and prompt management, especially fl uid and releasing a tamponade eff ect, were important. P232 P231 Intraabdominal hypertension and abdominal compartment syndrome in patients admitted to a medical and surgical ICU at a third referral university hospital N Chindavech1, S Yenarkart2, S Kongsayreepong2 1Siriraj Hospital, Mahidol University, Bangkok, Thailand; 2Division of Critical Care Medicine, Bangkok, Thailand Critical Care 2013, 17(Suppl 2):P231 (doi: 10.1186/cc12169) Intraabdominal hypertension and abdominal compartment syndrome in patients admitted to a medical and surgical ICU at a third referral university hospital N Chindavech1, S Yenarkart2, S Kongsayreepong2 1Siriraj Hospital, Mahidol University, Bangkok, Thailand; 2Division of Critical Care Medicine, Bangkok, Thailand Critical Care 2013, 17(Suppl 2):P231 (doi: 10.1186/cc12169) P229l i Results Patients in the two groups were statistically comparable with respect to sex (P = 0.31) and age (P = 0.53). The causes of the syndrome of Tako-tsubo were: subarachnoid hemorrhage (six patients) after coronary artery bypass graft (four patients), and polytrauma (two patients). All patients had low cardiac output. In the levosimendan group the ejection fraction at entrance was 25 ± 6%, after 12 hours 36 ± 10%, and 47 ± 5% after 24 hours. In the control group the ejection fraction at entrance was 24 ± 7%, after 12 hours 28 ± 6% and after 24 hours 33 ± 4%. Comparing the two groups we reached statistical signifi cance, P = 0.026. Infl uence of temperature on the cardiac action of dobutamine and levosimendan in isolated perfused hearts U Aldenhoff , R Vicenzi-Moser, M Vicenzi, D Konrad, E Schwarzl, W Toller Medical University Graz, Austria Critical Care 2013, 17(Suppl 2):P229 (doi: 10.1186/cc12167) Introduction Inotropic agents (catecholamines or calcium-sensitizers) are frequently used in hypothermic as well as hyperthermic patient conditions. Divergent results from animal experiments raise doubt as to whether they act to the same extent at diff erent body temperatures. Thus, we studied the infl uence of clinically relevant temperatures on the hemodynamic eff ects of dobutamine and levosimendan using the Langendorff model of isolated perfused hearts. Conclusion Comparing the two groups, we noticed that both started from a low cardiac output. However, in the group who used the drug therapy based on levosimendan we saw a return of systolic function of the left ventricle to near-normal levels within 24 hours, while in the S86 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Methods Isolated guinea pig hearts (n = 60) were perfused with incremental doses (10 nM to 10 μM) of dobutamine or levosimendan either at normothermic (37°C), hyperthermic (40°C) or hypothermic (34°C) perfusion conditions. Contractility (+dLVP/dt), relaxation (–dLVP/dt), left ventricular pressures and heart rate were recorded. Data with increasing drug dosage were calculated in percent from baseline for each temperature tested. Data for each drug were analysed by two- way ANOVA for repeated measures including the two main eff ects of temperature and drug dose and their interaction. Data are reported as mean ± standard deviation. P232 Methods This retrospective study included patients who underwent a double investigation in the ED of Hôpital du Sacré-Coeur de Montréal from April 2008 to October 2012. Patients were selected from a computerized database of medical prescriptions (MediaMed Technologie). Data were then extracted from the patient’s fi les: patient characteristics, radiology report, diagnosis and treatment. Descriptive statistics were conducted. 3 Red cell distribution width predicts cardiovascular complications after high-risk surgery M Geisen, HD Aya, C Ebm, MA Hamilton, J Ball, M Grounds, A Rhodes, M Cecconi St George’s Healthcare NHS Trust, London, UK Critical Care 2013, 17(Suppl 2):P232 (doi: 10.1186/cc12170) Red cell distribution width predicts cardiovascular complications after high-risk surgery M Geisen, HD Aya, C Ebm, MA Hamilton, J Ball, M Grounds, A Rhodes, M Cecconi St George’s Healthcare NHS Trust, London, UK Critical Care 2013, 17(Suppl 2):P232 (doi: 10.1186/cc12170) Myocardial injury in critically ill patients admitted with noncardiac diagnosesfi Hunziker H, et al.: Crit Care 2012, 16:R89. 1. Bazick HS, et al.: Crit Care Med 2011, 39:1913-1921. 2. Hunziker H, et al.: Crit Care 2012, 16:R89. 1. Bazick HS, et al.: Crit Care Med 2011, 39:1913-1921. 2. Hunziker H, et al.: Crit Care 2012, 16:R89. References 1. Jin R, et al.: Circulation 2005, 111:3359-3365. 2. Stamous SC: Ann Thorac Surg 2011, 91:42-48. 1. Jin R, et al.: Circulation 2005, 111:3359-3365. 2. Stamous SC: Ann Thorac Surg 2011, 91:42-48. Myocardial injury in critically ill patients admitted with noncardiac diagnosesfi g J Lo1, K Lei2, I Webb2, J Coutts2, J Chambers2, A Griffi ths2, J Smith2, E Connell3, P Collinson3, J Peacock1, D Treacher2, M Ostermann2 1King’s College London, London, UK; 2Guys & St Thomas Foundation Hospital, London, UK; 3St George’s Hospital, London, UK Critical Care 2013, 17(Suppl 2):P234 (doi: 10.1186/cc12172) Introduction In the critically ill, the incidence of raised cardiac troponin T (cTnT) levels is high. Although the mechanisms of myocardial injury are not well understood, raised cTnT levels are associated with increased mortality. The aim of our study was to determine the incidence, prevalence and outcome of silent myocardial injury as determined by raised cTnT levels and concomitant ECG changes in critically ill patients admitted for noncardiac reasons. Results A total of 119 patients were included. Seventy-six (63%) patients developed complications postoperatively. Thirty patients (25.2%) developed cardiovascular complications. These patients had a higher median RDW than patients without cardiovascular compli- cations (14.7 vs. 13.8%, P <0.05). RDW on ICU admission was associated with serum lactate concentration on ICU admission: receiver operating characteristic analysis showed an area under the curve of 0.68 (SE = 0.06, P = 0.005; see Figure 1). RDW >14.35% predicted hyperlactataemia with a sensitivity of 76.0% and a specifi city of 71.1%. ad tted o o ca d ac easo s. Methods ECGs were taken and cTnT was measured daily during the fi rst week and on alternate days during the second week until discharge from the ICU or death. After completion of the study, all cTnT levels and ECGs were analysed independently and patients were classifi ed into four groups: defi nite MI (cTnT ≥15 ng/l and defi nite ECG changes of MI), possible MI (cTnT ≥15 ng/l and ischaemic changes on ECG), troponin rise alone (cTnT ≥15 ng/l with no ischaemic ECG changes), or normal. All medical notes were reviewed independently by two ICU clinicians. Results A total of 144 patients were included in the analysis (42% female; mean age 61.9 (SD 16.9); mean APACHE II score 19.4). In total, 121 patients (84%) had at least one cTnT level ≥15 ng/l during their stay in the ICU. Twenty patients (14%) fulfi lled criteria for a defi nite MI, of whom 65% were septic and 50% were on noradrenaline at the time (ICU and hospital mortality: 25% and 30%, respectively). P234 Myocardial injury in critically ill patients admitted with noncardiac diagnoses J Lo1, K Lei2, I Webb2, J Coutts2, J Chambers2, A Griffi ths2, J Smith2, E Connell3, P Collinson3, J Peacock1, D Treacher2, M Ostermann2 1King’s College London, London, UK; 2Guys & St Thomas Foundation Hospital, London, UK; 3St George’s Hospital, London, UK Critical Care 2013, 17(Suppl 2):P234 (doi: 10.1186/cc12172) Consequences of obesity in outcomes after cardiac surgery: analysis of the ARIAM registry EC Curiel-Balsera1, J Muñoz-Bono1, MJ Delgado-Amaya1, , , g y , R Hinojosa-Pérez2, A Reina-Toral3, A Gordillo-Brenes4, R Rivera-Fernández1 1Hospital Regional Carlos Haya, Málaga, Spain; 2Virgen del Rocio Hospital, Seville, Spain; 3Virgen de las Nieves Hospital, Granada, Spain; 4Puerta del mar Hospital, Cádiz, Spain Conclusion Eighty-four per cent of critically ill patients had a raised cTnT level at some stage during their stay in the ICU. More than 40% of patients fulfi lled criteria for a possible or defi nite MI, of whom only 20% were recognised clinically. ICU and hospital outcome were signifi cantly worse in patients with a cTnT rise. The proportion of patients with sepsis was similar between the patients with a defi nite, possible or no MI. Critical Care 2013, 17(Suppl 2):P233 (doi: 10.1186/cc12171) Critical Care 2013, 17(Suppl 2):P233 (doi: 10.1186/cc12171) Introduction Obesity is a disease that aff ects a large part of the population and has been associated with worse outcomes after cardiac surgery. The aim of our study is to evaluate the consequences of obesity related to postoperative complications, hospital length of stay and mortality. Red cell distribution width predicts cardiovascular complications after high-risk surgery Results In all, 125 patients underwent the double investigation. Our sample had a mean age of 63.1 years (SD ±18.6) and was composed of 82 (65.6%) women. One hundred and fi fteen patients (92%) underwent the V/Q SPECT fi rst. The result of 66 (52.8%) SPECT was intermediate or indeterminate. The fi nal diagnosis was pulmonary embolism for 23 patients (18.4%). A signifi cant proportion of patients (19, 38.0%) had confl icting results with the two tests. In this subpopulation, four (21.1%) had a fi nal diagnosis of pulmonary embolism. In the 16 patients with a result of high probability V/Q SPECT and negative angioscan, one (6.3%) had a fi nal diagnosis of pulmonary embolism, but three (100%) with low probability SPECT and positive angioscan were given this fi nal diagnosis. Introduction The red-cell distribution width (RDW) is associated with cardiovascular morbidity and mortality and is a predictor of ICU survival. It was the aim of this study to investigate the potential of RDW to predict postoperative cardiovascular complications (new onset of treated arrhythmias, myocardial ischaemia or initiation of vasopressor support) and its association with markers of tissue perfusion (serum lactate >2.5 mmol/l). Methods Analysis of prospectively recorded data for a register of patients admitted to an 18-bed ICU in a large teaching hospital after undergoing high-risk surgery. Haemodynamic and laboratory parameters during the fi rst 12 hours of ICU stay were recorded as well as demographic characteristics. Assessment for postoperative complications was performed using the postoperative morbidity survey and the Clavien-Dindo classifi cation. In addition, clinical outcome data (hospital mortality, length of ICU stay, length of hospital stay, readmission to the ICU) were recorded. i Conclusion Most patients underwent the double investigation because of intermediate or indeterminate V/Q SPECT results. In case of confl icting results, clinicians based their decision on the angioscan. In future studies, it would be useful to identify contributing factors for this discordance. Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 S87 http://ccforum.com/supplements/17/S2 Figure 1 (abstract P232). Prediction of hyperlactataemia according to RDW. in the development of overall postoperative complications (33% in obese and 35.8% in nonobese, P = 0.07), although less appreciated were reoperation rate or stroke, as well as further development of postoperative renal failure. Red cell distribution width predicts cardiovascular complications after high-risk surgery After adjusting for severity and length of cardio bypass time, obese patients had lower mortality without being statistically signifi cant, OR = 0.94 (0.79 to 1.04). There were no diff erences in ICU length of stay, but obese patients had greater ward length of stay, 9.04 (10.43) versus 1.18 (9.2) days, P = 0.01. in the development of overall postoperative complications (33% in obese and 35.8% in nonobese, P = 0.07), although less appreciated were reoperation rate or stroke, as well as further development of postoperative renal failure. After adjusting for severity and length of cardio bypass time, obese patients had lower mortality without being statistically signifi cant, OR = 0.94 (0.79 to 1.04). There were no diff erences in ICU length of stay, but obese patients had greater ward length of stay, 9.04 (10.43) versus 1.18 (9.2) days, P = 0.01. Conclusion Obese patients undergoing cardiac surgery have mortality, rate of complications and length of stay similar to nonobese patients. Obese patients required less reoperation but developed more frequent postoperative renal failure. R f Myocardial injury in critically ill patients admitted with noncardiac diagnosesfi Thirty-nine patients (27%) had a possible MI, of whom 69% were septic and on noradrenaline (ICU and hospital mortality: 31% and 41%, respectively). Sixty-two patients (43%) had a raised troponin without ECG, of whom 69% were septic and 50.7% were on noradrenaline (ICU and hospital mortality: 23% and 31%, respectively). Twenty-three patients had normal cTnT results and serial ECGs, of whom 61% had sepsis. ICU and hospital mortality was 4%. Only 25% of defi nite MIs and 18% of possible MIs were recognised by the clinical teams at the time. Methods ECGs were taken and cTnT was measured daily during the fi rst week and on alternate days during the second week until discharge from the ICU or death. After completion of the study, all cTnT levels and ECGs were analysed independently and patients were classifi ed into four groups: defi nite MI (cTnT ≥15 ng/l and defi nite ECG changes of MI), possible MI (cTnT ≥15 ng/l and ischaemic changes on ECG), troponin rise alone (cTnT ≥15 ng/l with no ischaemic ECG changes), or normal. All medical notes were reviewed independently by two ICU clinicians. y pi y Conclusion RDW is a potential parameter for perioperative risk stratifi cation. Further studies will have to elucidate the ability of acute elevations in RDW to refl ect impaired tissue perfusion. References p y y Results A total of 144 patients were included in the analysis (42% female; mean age 61.9 (SD 16.9); mean APACHE II score 19.4). In total, 121 patients (84%) had at least one cTnT level ≥15 ng/l during their stay in the ICU. Twenty patients (14%) fulfi lled criteria for a defi nite MI, of whom 65% were septic and 50% were on noradrenaline at the time (ICU and hospital mortality: 25% and 30%, respectively). Thirty-nine patients (27%) had a possible MI, of whom 69% were septic and on noradrenaline (ICU and hospital mortality: 31% and 41%, respectively). Sixty-two patients (43%) had a raised troponin without ECG, of whom 69% were septic and 50.7% were on noradrenaline (ICU and hospital mortality: 23% and 31%, respectively). Twenty-three patients had normal cTnT results and serial ECGs, of whom 61% had sepsis. ICU and hospital mortality was 4%. Only 25% of defi nite MIs and 18% of possible MIs were recognised by the clinical teams at the time. 1. Bazick HS, et al.: Crit Care Med 2011, 39:1913-1921. 2. P235 Methods An observational, prospective, multicenter study of patients included in the ARIAM registry of adult cardiac surgery between March 2008 and March 2011. We analyzed clinical variables, the surgical procedure, postoperative complications and mortality, comparing the group of patients with body mass index (BMI) greater or less than 30 kg/m2. P235 Validation of the GRACE score risk in our population L Sayagues1, J Sieira1, E Abbu2, J Chico3, C Pena3, J Gonzalez Juantey2 1HULA, Lugo, Spain; 2CHUS, Santiago de Compostela, Spain; 3CHUVI, Vigo, Spain Critical Care 2013, 17(Suppl 2):P235 (doi: 10.1186/cc12173) P235 Validation of the GRACE score risk in our population L Sayagues1, J Sieira1, E Abbu2, J Chico3, C Pena3, J Gonzalez Juantey2 1HULA, Lugo, Spain; 2CHUS, Santiago de Compostela, Spain; 3CHUVI, Vigo, Spain Critical Care 2013, 17(Suppl 2):P235 (doi: 10.1186/cc12173) 35 Validation of the GRACE score risk in our population Validation of the GRACE score risk in our population L Sayagues1, J Sieira1, E Abbu2, J Chico3, C Pena3, J Gonzalez Juantey2 1HULA, Lugo, Spain; 2CHUS, Santiago de Compostela, Spain; 3CHUVI, Vigo, Spain C iti l C 2013 17(S l 2) P235 (d i 10 1186/ 12173) p p L Sayagues1, J Sieira1, E Abbu2, J Chico3, C Pena3, J Gonzalez Juantey2 1HULA, Lugo, Spain; 2CHUS, Santiago de Compostela, Spain; 3CHUVI, Vigo, Spain Critical Care 2013, 17(Suppl 2):P235 (doi: 10.1186/cc12173 Results The study included 4,712 patients with a mean age of 64.03 (SD ±12.08) years, BMI 28.53 (SD ±4.7) and EuroSCORE 5.58 (SD ±2.91). In 1,940 patients (35.7%) BMI was >30 kg/m2. There were no diff erences Introduction The Global Registry of Acute Coronary Events (GRACE) risk score provides an estimation of the probability of death within S88 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 6 months of hospital discharge in patients with acute coronary syndrome (ACS). Our aim was to assess the validity of this risk score in our contemporary cohort of patients admitted to our third-level hospital. Figure 2 (abstract P236). Incomplete and complete revascularization. p Methods The study involved 1,185 consecutive patients with ACS evaluated between February 2004 and February 2009. Their virtual status was determined 6 months after hospital discharge and the validity of the GRACE risk score was evaluated. y Results In total, 450 (38.8%) patients were admitted for STEMI and 725 (61.2%) for NSTEMI. Percutaneous revascularisation was performed in 846 (71.5%). p Critical Care 2013, 17(Suppl 2):P236 (doi: 10.1186/cc12174) Introduction This is a prospective study in which all ACS cases attending a level 3 hospital were collected consecutively from February 2004 to 2010, and were clinically followed-up until 2012. Introduction In patients with severe and inoperable aortic stenosis, some trials have proven that TAVI excels standard procedures and has also been proven less cost-eff ective. We aim at reviewing short-term results after 2 years since the implementation of this technique in our premises. y Methods Of a total 4,589 cases, only 2,515 were revascularized with ICP (1,742 complete and 907 incomplete; 160 failed). We split the cases into two main groups: STEMI and NSTEMI. p Methods A study of a retrospective cohorts of patients who underwent isolated aortic valve replacement (AVR) by either conventional surgery or TAVI (CoreValve device) from June 2010 to December 2011 at the University Hospital Carlos de Haya (Málaga, Spain). Clinical epidemiologic, complication and short-term outcome variables were registered. Qualitative variables are expressed as percentages, while quantitative variables are expressed as means and SD. Fisher’s exact test and Mann–Whitney’s U-test were used where necessary (5% maximum error ratio). g p Results We observed a 0.6 higher accumulative survival rate in patients with complete ICP over patients who underwent surgery, incomplete ICP or mixed treatment. Those with conservative measures solely have, obviously, worst prognosis. See Figures 1 and 2. Figure 1 (abstract P236). FEVI and prognosis. Results A total number of 27 TAVI and 154 isolated AVR procedures were completed. Intervention typology was chosen according to the recommendations of scientifi c societies, apart from patients’ fulfi llment of the anatomic criteria required for percutaneous implant. Mean age was 67 ± 11 years (54% males) in AVR and 80 ± 6 years (44% males) in TAVI (P <0.05). The additive EuroSCORE in AVR was 7 ± 2 and 9 ± 2 in TAVI (P <0.05). However, 55.6% of the percutaneous-valve patients presented previous coronary-tree alterations with stent implantation, while only 7% of AVR patients showed these alterations (P <0.001). ICU mortality in TAVI and AVR patients was 3.7% and 8.2%, respectively (P = NS). Regarding complications, 48.1% of TAVI patients showed altered heart rhythm and 33% required a permanent pacemaker. Electrical disorders were observed in 4% of AVR patients, while 1.9% of these patients required a permanent pacemaker (P <0.001 for both). Reoperation was necessary in 14.8 and 1.9% of TAVI and AVR patients, respectively (P <0.001). P235 The median GRACE risk score was 121 (interquartile range 96/144). Mortality after discharge was 4.4%. The calibration of the GRACE score was Hosmer–Lemeshow P >0.2 and its discriminatory capacity was excellent. Area under the ROC curve was 0.86, 95% CI 0.807 to 0.916, in all patients. See Table 1. Table 1 (abstract P235) GRACE risk Total STEMI NSTEMI Low 270 (22.7) 177 (38.3) 93 (12.8) Medium 334 (28.2) 141 (30.7) 193 (26.7) High 580 (49) 142 (30.9) 438 (60.5) Figure 2 (abstract P236). Incomplete and complete revascularization. Figure 2 (abstract P236). Incomplete and complete revascularization. Conclusion In our study patients undergoing ICP have higher survival rates in comparison with cardiac surgery except those >65 years old and diabetic groups in which cardiac surgery has higher ratios than ICP. In the rest of the groups, no matter how many coronary arteries were aff ected, only those with complete ICP present higher survival rates. It would be important to repeat this study in a multicentric cohort. Conclusion The GRACE risk score for predicting death within 6 months of hospital discharge was validated and can be used in patients with ACS. It would be perfect in the future to include the GRACE risk score in the medical records of this type of patients. Also it would be very interesting to validate this in a multicentric study. Non-invasive valve implantation with TAVI versus conventional aortic valve replacement Non-invasive valve implantation with TAVI versus conventional aortic valve replacement E Trujillo-García, C Joya-Montosa, MJ Delgado-Amaya, E Curiel-Balsera Hospital Regional Carlos Haya, Málaga, Spain Critical Care 2013, 17(Suppl 2):P237 (doi: 10.1186/cc12175) p p L Sayagues1, J Sieira1, E Abbu Assi2, J Chico3, C Pena3, J Gonzalez Juanatey2 1HULA, Lugo, Spain; 2CHUS, Santiago de Compostela, Spain; 3CHUVI, Vigo, Spain aortic valve replacement E Trujillo-García, C Joya-Montosa, MJ Delgado-Amaya, E Curiel-Balsera Hospital Regional Carlos Haya, Málaga, Spain Critical Care 2013, 17(Suppl 2):P237 (doi: 10.1186/cc12175) P238 Time to recover from shock is determinant of a positive fl uid balance in septic shock S Lobo, AL Cunha Faculdade de Medicina de São José do Rio Preto, Brazil Critical Care 2013, 17(Suppl 2):P238 (doi: 10.1186/cc12176) Introduction Excess fl uids may be harmful in critically ill patients. We aimed to evaluate the cumulative fl uid balance during 7 days in patients with septic shock after recovery from shock. Methods A prospective and observational study in septic shock patients. Patients with MAP >65 mmHg and lactate <2.0 mEq/l were included <12 hours after weaning from vasopressors. Daily fl uid balance was registered during 7 days after the enrollment. Patients were divided into two groups according to the full cohort’s median cumulative fl uid balance administered during the period of shock (use of vasopressors) calculated on study day 1: Group 1 ≤4.4 l (n = 20) and Group 2 >4.4 l (n = 20). i Results Seventy-nine patients were enrolled. Fifty-one patients were with moderate UGIB, and 28 patients were with severe UGIB. As indicated in Tables 1 and 2, there was a signifi cant relationship between CLac and sustained bleeding in moderate UGIB (P = 0.02). On the other hand, there was no signifi cant relationship between CLac and sustained bleeding in severe UGIB (P = 0.58). p Results Cumulative fl uid balance was 1.6 ± 1.8 l in Group 1 and 10.2 ± 4.1 l in Group 2 and 8.5 ± 5.3 l in Group 1 and 18.5 ± 7.9 l in Group 2 on study day 8 (P <0.001 for both). Time for recovery from shock was predictive of receiving larger volume of fl uids (OR: 1.38, 95% CI: 1.08 to 1.75, P = 0.009). After zeroing fl uid balance on study day 2, 7 days cumulative fl uid balance continues to increase in both groups (Figure 1). Patients in Group 2 had more prolonged length of stay in the ICU and in hospital than patients in Group 1. Table 1 (abstract P239). Relationship between lactate clearance and sustained bleeding in moderate UGIB (n = 51) Table 1 (abstract P239). Relationship between lactate clearance and sustained bleeding in moderate UGIB (n = 51) Sustained bleeding Nonsustained bleeding (n = 19) (n = 32) P value CLac <0.5 18 (95%) 21 (65%) 0.02 CLac ≥0.5 1 (5%) 11 (34%) Conclusion After recovery from septic shock we notice a huge accu- mulated fl uid balance. A more positive fl uid balance was associated with a more prolonged length of stay in the ICU and in the hospital. P239 Lactate clearance is a predictor of sustained bleeding in emergency room patients with moderate upper gastrointestinal bleeding T Wada1, A Hagiwara2, N Yahagi1, A Kimura2 1University of Tokyo, Japan; 2National Center for Global Health and Medicine, Tokyo, Japan Critical Care 2013, 17(Suppl 2):P239 (doi: 10.1186/cc12177) Figure 1 (abstract P238). Figure 1 (abstract P238). Table 2 (abstract P239). Relationship between lactate clearance and sustained bleeding in severe UGIB (n = 28) Sustained bleeding Nonsustained bleeding (n = 6) bleeding (n = 22) P value CLac <0.5 4 (67%) 18 (82%) 0.58 CLac ≥0.5 2 (33%) 4 (18%) Conclusion If an initial fl uid therapy for moderate UGIB results in high CL h bl di i h l d h d F h i Table 2 (abstract P239). Relationship between lactate clearance and sustained bleeding in severe UGIB (n = 28) Conclusion If an initial fl uid therapy for moderate UGIB results in high CLac, the bleeding might already have stopped. For such a patient, we may save urgent endoscopy. p Critical Care 2013, 17(Suppl 2):P236 (doi: 10.1186/cc12174) y Conclusion Even with our limited experience, TAVI patients are observed to be older, to involve higher surgical risk, and to have undergone previous coronary-tree interventions. Although no Figure 1 (abstract P236). FEVI and prognosis. S89 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 signifi cant diff erences were found regarding mortality, a higher rate of complications was observed in TAVI patients. With no short-term diff erences, a signifi cant rate of postsurgical complications, and a cost- effi ciency handicapped technique, analysis of long-term outcomes seems necessary to assess TAVI’s advantages over conventional AVR. UGIB patient needs an intervention or not. However, the intervention which the GBS mentions includes not only endoscopy but also blood transfusion. Therefore, we cannot determine whether a UGIB patient needs urgent endoscopy or just blood transfusion by GBS alone. We hypothesized that high lactate clearance (CLac) would decrease the likelihood of sustained UGIB. Methods This is a retrospective study. UGIB patients, who visited the emergency department (ED) of the National Center for Global Health and Medicine from April 2011 to March 2012 and received urgent endoscopy in the ED, were enrolled. We collected for each patient the GBS, the blood lactate value on arrival in the ED, the blood lactate value after bolus administration of 20 to 40 ml/kg Ringer’s acetate (initial fl uid therapy) and the report of urgent endoscopy. We classifi ed the severity of UGIB according to GBS. A score ≤12 was classifi ed as moderate, and a score ≥13 was classifi ed as severe. CLac was defi ned as the percentage decrease in blood lactate from the time of arrival in the ED to the time when an initial fl uid therapy was fi nished. CLac <50% was defi ned as low, and CLac ≥50% was defi ned as high. Whether a patient had sustained bleeding or not was determined based on the report of urgent endoscopy. The relationship between CLac and sustained bleeding was examined by Fisher’s exact test, and P <0.05 was considered statistically signifi cant. P238 P238 Time to recover from shock is determinant of a positive fl uid balance in septic shock S Lobo, AL Cunha Faculdade de Medicina de São José do Rio Preto, Brazil Critical Care 2013, 17(Suppl 2):P238 (doi: 10.1186/cc12176) P240 P240 Muscular glucose assessed by microdialysis and blood glucose can predict mortality in septic shock A Massoudi, M Belhadj Amor, C Romdhani, A Ben Gabsia, I Labbene, M Ferjani Military Hospital of Tunis, Tunisia Critical Care 2013, 17(Suppl 2):P240 (doi: 10.1186/cc12178) predict mortality in septic shock A Massoudi, M Belhadj Amor, C Romdhani, A Ben Gabsia, I Labbene, M Ferjani Military Hospital of Tunis, Tunisia Critical Care 2013, 17(Suppl 2):P240 (doi: 10.1186/cc12178) Conclusion After recovery from septic shock we notice a huge accu- mulated fl uid balance. A more positive fl uid balance was associated with a more prolonged length of stay in the ICU and in the hospital. Introduction The aim of our study was to assess the muscular glucose by microdialysis and its association with mortality in septic shock patients. Introduction The aim of our study was to assess the muscular glucose by microdialysis and its association with mortality in septic shock patients. P239 Lactate clearance is a predictor of sustained bleeding in emergency room patients with moderate upper gastrointestinal bleeding T Wada1, A Hagiwara2, N Yahagi1, A Kimura2 1University of Tokyo, Japan; 2National Center for Global Health and Medicine, Tokyo, Japan Critical Care 2013, 17(Suppl 2):P239 (doi: 10.1186/cc12177) Methods We conducted a preliminary prospective study. We included septic shock patients hemodynamically optimized according to international recommendations. A microdialysis catheter was inserted in the femoral quadriceps. Interstitial fl uid samples were collected every 6 hours for 5 days. The determination of muscular glucose was performed by the CMA 600 analyzer (CMA/Microdialysis AB, Sweden). We also performed a dosage of concomitant blood glucose. The study population was divided into two groups according to hospital mortality. Statistic analysis: Mann–Whitney test and chi-squared test: Introduction There is no useful predictor of sustained upper gastro- intestinal bleeding (UGIB). Glasgow–Blatchford scoring (GBS) is based on simple clinical and laboratory variables and can predict whether a S90 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 comparisons between groups. Quantitative variables were expressed as mean ± standard deviation or median (interquartile range) as appropriate. Nasogastric tube dislodgement: a problem on our ICU B Morton, R Hall, T Ridgway, O Al-Rawi Liverpool Heart & Chest, Liverpool, UK Critical Care 2013, 17(Suppl 2):P242 (doi: 10.1186/cc12180) Results We included 12 patients with septic shock. The mortality rate was 50%. P240 Demographics were comparable between groups except for age (66 ± 9 vs. 41 ± 12, dead patients vs. survivors, respectively; P = 0.002). Pneumonia was the major cause of septic shock (10 patients). We analysed 167 blood samples and 166 muscular glucose samples. We found a positive association between muscular glucose, blood glucose and mortality. Tissue glucose was signifi cantly higher among dead patients compared with survivors at the 54th hour. Comparing all data, muscular glucose (P = 0.02) and blood glucose (P = 0.007) were signifi cantly higher in dead patients (Table 1). Introduction It was noted on our unit that dislodgement of nasogastric tubes occurred commonly. This can lead to an increased risk of aspiration, interruptions in nutritional support, skin breakdown and radiographic exposure [1]. It is recommended that the position of nasogastric tubes should be confi rmed by aspiration and pH testing, with radiographic confi rmation used only when this is not possible [2]. Methods We performed a retrospective review of chest X-ray (CXR) requests for the 3-month period June to August 2012 using the trust radiology information system. The proportion of CXR requests for confi rmation of position and patient demographics were measured with an estimation of the fi nancial cost performed. Table 1 (abstract P240). Association between muscular glucose, blood glucose and mortality Dead patients Survivors (n = 6) (n = 6) P value Muscular glucose (mmol/l) 9.4 (5.66; 13.71) 7.87 (5.62; 10.41) 0.02 Blood glucose (mmol/l) 10.9 (8.3; 15.5) 8.6 (6.9; 11.4) 0.007 Table 1 (abstract P240). Association between muscular glucose, blood glucose and mortality i Results There were 541 patients admitted to the critical care area in the study period. In total, 207 out of 2,340 (8.8%) CXRs performed were for confi rmation of position. Repeated X-rays were required in some patients (see Table 1); these patients were older and tended to have a longer length of stay. A mobile CXR costs £25 in our trust, if one CXR is accepted per patient with a nasogastric tube; there was an excess of 160 images with a cost of £4,000 in the 3-month period. Conclusion Our data suggest that muscular glucose assessed by microdialysis and blood glucose are associated with mortality in septic shock patients. Therefore, muscular glucose may refl ect the metabolic alterations and microcirculatory dysfunction induced by septic shock. y References References 1. Lorente L, et al.: Accidental catheter removal in critically ill patients: a 1. Lorente L, et al.: Accidental catheter removal in critically ill patients: a prospective and observational study. Crit Care 2004, 8:R229-R233. 2. National Patient Safety Agency: Reducing Harm Caused by the Misplacement of Nasogastric Feeding Tubes. NPSA/2011/PSA002. NHS; 2011. [http://www.nrls.npsa.nhs.uk/alerts/?entryid45=129640] Conclusion There was a high prevalence of constipation among critical care patients with poor adherence to the bowel protocol, which requires improvements in staff awareness and new recommendations (drug chart amendment, and so forth). Duration of mechanical ventilation and failure to feed were greater in constipated than nonconstipated patients. This may indicate a common correlation and further studies are warranted. Due to the possible signifi cant impact of constipation on patients’ recovery, each critical care unit should introduce a bowel protocol or ensure compliance with the existing one before the evidence is clearly established. f Handling of dietary protein in critically ill patients Handling of dietary protein in critically ill patients Handling of dietary protein in critically ill patients F Liebau1, O Rooyackers1, L Van Loon2, J Wernerman1 1Karolinska University Hospital Huddinge, Stockholm, Sweden; 2NUtrIm Handling of dietary protein in critically ill patients F Liebau1, O Rooyackers1, L Van Loon2, J Wernerman1 1Karolinska University Hospital Huddinge, Stockholm, Sweden; 2NUtrIm, Maastricht University, Maastricht, the Netherlands Critical Care 2013, 17(Suppl 2):P243 (doi: 10.1186/cc12181) g y y Liebau1, O Rooyackers1, L Van Loon2, J Wernerman1 , y , , 1Karolinska University Hospital Huddinge, Stockholm, Sweden; 2NUtrIm M i h U i i M i h h N h l d Maastricht University, Maastricht, the Netherlands Critical Care 2013, 17(Suppl 2):P243 (doi: 10.1186/cc12181) y Critical Care 2013, 17(Suppl 2):P243 (doi: 10.1186/cc12181) Introduction Protein turnover measurements by stable isotope techniques can be applied to assess the nutritional/metabolic status in critically ill patients and their response to feeding. Because of uncertain gastrointestinal transport and uptake of nutrients, their contribution needs to be measured separately. We studied whole-body protein kinetics, with special emphasis on the contribution of dietary protein, in ICU patients and healthy controls. Introduction Protein turnover measurements by stable isotope techniques can be applied to assess the nutritional/metabolic status in critically ill patients and their response to feeding. Because of uncertain gastrointestinal transport and uptake of nutrients, their contribution needs to be measured separately. We studied whole-body protein kinetics, with special emphasis on the contribution of dietary protein, in ICU patients and healthy controls. 2. Palácio de Azevedo R: Intestinal constipation in intensive care unit. Rev Bras Ter Intensiva 2009, 21:324-331. P241 Constipation in critically ill patients and its relationship to feeding and weaning from respiratory support g p E Spodniewska, A Guha p Royal Liverpool University Hospital, Liverpool, UK y p y p p Critical Care 2013, 17(Suppl 2):P241 (doi: 10.1186/cc12179) Critical Care 2013, 17(Suppl 2):P241 (doi: 10.1186/cc12179) Introduction Constipation can be defi ned as a failure of the bowels to open for 3 consecutive days. It is a common problem in intensive care settings but not many studies have so far raised the subject. Methods The audit had the Trust Audit Committee’s approval. The existing protocol was used as the benchmark. Patients were studied prospectively to assess compliance with the local bowel protocol, incidence of constipation and relationship to weaning from respiratory support and feeding. All HDU and all mechanically ventilated ICU patients who stayed on the ward for more than 3 days were included, except for patients after bowel surgery and patients with encephalopathy. Conclusion An excess of CXRs were performed for confi rmation of nasogastric tube in our patient population. The recommended methods for position confi rmation were reinforced amongst medical staff . The high number of repeated imaging for some patients indicates that dislodgement of tubes was also a problem. We propose that nasogastric tubes should be bridled after fi rst dislodgement or at tracheostomy insertion to minimise dislodgement in the future. Results Among the 24 HDU and 21 ICU patients audited in the Royal Liverpool University Hospital, 67% and 57% respectively were constipated. Laxatives were prescribed when patients had not opened their bowels for 3 days in 25% HDU and 75% ICU cases. Taking into consideration that the median age, APACHE II score and length of stay for constipated and nonconstipated patients were similar, the relationship to feeding and respiratory support were assessed. Constipated patients required mechanical ventilation for an average of 6.8 days and nonconstipated for 4.3 days. Failure to feed was observed at least once in 58% constipated and 44% nonconstipated ICU patients and 19% constipated and 12.5% nonconstipated HDU patients. 1. Mostafa SM: Constipation and its implications in the critically ill patient. Br J Anaesth 2003, 91:815-819. P240 Table 1 (abstract P242) Table 1 (abstract P242) Mean days CXR for Total NG Total from fi rst to Mean NG Patients CXR CXR Proportion last CXR age 1 47 47 321 0.15 8.45 67.55 2 16 32 181 0.18 15.94 69.63 3 7 21 87 0.24 17.86 70.00 4 10 40 149 0.27 15.90 69.20 5 3 15 52 0.29 23.33 66.00 6 3 18 61 0.30 40.00 76.67 8 2 16 52 0.31 39.50 80.00 9 2 18 46 0.39 29.50 83.00 Confi rmation of nasogastric tube placement in critical care M Moore, R Thomson Confi rmation of nasogastric tube placement in critical care M Moore, R Thomson St Georges Healthcare NHS Trust, London, UK Critical Care 2013, 17(Suppl 2):P244 (doi: 10.1186/cc12182) St Georges Healthcare NHS Trust, London, UK Critical Care 2013, 17(Suppl 2):P244 (doi: 10.1186/cc12182) Conclusion In this population of ICU patients, routine/daily checks of NG pH aspirate appear to be limited. This is almost certainly due to the use of continuous NG feed together with PPIs. The usefulness of pH testing in newly placed NG tubes, however, appears more reliable. Introduction Placement of nasogastric tubes (NGTs) is commonplace in critical care. Misplacement of NGTs is rare and considered a never event [1]. Strategies to avoid never events (by confi rming NGT position) include pH analysis of gastric secretions or chest X-ray confi rmation of tube position. For this reason the authors set out to establish the effi ciencies surrounding safe placement of NGTs in a 17-bed adult cardiothoracic critical care unit in a large teaching hospital. Figure 2 (abstract P245). pH values – new NG tubes. Figure 1 (abstract P245). pH values – routine checks. Figure 1 (abstract P245). pH values – routine checks. Methods This small-scale study of 25 NGT placements during a 5-week period collated data supplied by questionnaire by healthcare workers responsible for NGT placements.i Results Analysis of Adverse Incident Reports identifi ed no never events of misplaced NGTs within the previous 10 years. This audit revealed that the commonest type of NGT was a radio-opaque tube with stylet (corfl o) (92% of placements), with occasional use of the electromagnetic placement system (cortrak) (8% of placements). Sizes 10 (40%) and 12 (56%) were most common. Tube placement was confi rmed by: X-ray (72%); pH of aspirates (35%); electromagnetic tube placement (one patient). The time taken from decision to place NGT to use varied (range 15 to 510 minutes). Little distinction was seen in the time taken to use and NGT confi rmed by aspirate alone (205 minutes) or by X-ray (220 minutes), although the shortest interval was seen in electromagnetic NGT placement (15 minutes). The cost of NGTs confi rmed by aspirate alone was low (approximately £10.00), higher with X-ray confi rmation/electromagnetic placement (approximately £45.00). Figure 1 (abstract P245). pH values – routine checks. Figure 2 (abstract P245). pH values – new NG tubes. pH testing to confi rm nasogastric tube position on the ICU: are we wasting our time? p Results Protein net balance was lower in patients than in the reference group at baseline (–1.8 ± 1.7 vs. 0.6 ± 0.6 mg/kg BW/hour, P = 0.003), and after enteral feeding (–1.1 ± 1.5 vs. 0.6 ± 0.6 mg/kg BW/hour, P = 0.049). Recovery of labelled Phe from enteral feeding into the systemic circulation was higher in the reference group as compared with patients (20.3 + 11.2% vs. 7.0 + 4.8%, P = 0.005). Enteral feeding did not aff ect protein metabolism in the reference group. In patients, protein breakdown became slightly lower during enteral feeding (10.6 ± 3.3 vs. 11.2 ± 3.3 mg/kg BW/hour, P = 0.086) and protein net balance became slightly higher (–1.1 ± 1.5 vs. –1.8 ± 1.7 mg/kg BW/ hour, P = 0.086). P Temblett, S George Introduction For such a simple procedure, the insertion and position checking of nasogastric (NG) tubes can be very problematic. The UK’s National Patient Safety Agency declared that ‘Placement of NG tubes together with confi rmation of correct placement can carry signifi cant risks’ and recommends that measuring the pH of NG aspirate should be the fi rst-line method of determining correct NG position (safe range ≤5.5) [1]. Methods In order to assess the usefulness of pH testing of NG aspirates in critical care patients, a prospective survey of pH testing of NG tube aspirate was carried out. This was undertaken both in patients who had a newly placed NG tube and in patients who were having regular/ routine checks of their existing NG tube. Conclusion Intrinsically isotope-labelled casein can be used to quantify dietary contribution to protein metabolism in critically ill patients. Hypocaloric enteral feeding marginally improved protein balance in these patients. The low recovery of enterally administered labelled amino acid underlines the need to quantify uptake from the gastrointestinal tract when protein turnover measurements are performed in critically ill patients on enteral nutrition. Results A total of 168 separate pH readings in 41 ICU patients receiving continuous enteral nutrition were recorded. In total, 137/168 patients were receiving proton pump inhibitors (PPIs). Eighteen readings were from newly placed NG tubes and 150 readings from old NG tubes. Fifty- three per cent of routine pH readings were falsely high; that is, pH 6 or above despite the NG tube being in the stomach (Figure 1). References 1. Mostafa SM: Constipation and its implications in the critically ill patient. Br J Anaesth 2003, 91:815-819. 2. Palácio de Azevedo R: Intestinal constipation in intensive care unit. Rev Bras Ter Intensiva 2009, 21:324-331. S91 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Methods Mechanically ventilated, not enterally fed ICU patients (n = 9) were recruited from an interdisciplinary ICU. Healthy, overnight-fasted volunteers (n = 6) served as reference. A primed constant i.v. infusion of 2H-labeled phenylalanine (Phe) and tyrosine was used to quantify whole-body protein metabolism. Patients remained on parenteral nutrition (PN) as clinically indicated; controls received PN starting 2.5 hours before starting enteral feeding. Intrinsically 13C-Phe-labeled casein was infused for 6 hours by nasogastric tube at 0.75 g protein/ hour, together with maltodextrin at 2.73 g/hour. Protein breakdown, synthesis, net balance, and Phe splanchnic extraction were calculated before and at the end of the enteral feeding period, using equations for steady-state whole-body protein kinetics. Comparisons were made by Wilcoxon matched pairs and Mann–Whitney U tests; values are reported as mean ± SD. confi rmations. Even if the frequency and volume of gastric aspiration were greater, there is a belief that pH testing may not be suffi ciently accurate (since many factors alter patients’ gastric pH). It is possible that new technologies such as electromagnetic NGT placement may allow faster/equally safe practices. Further study including cost/benefi t analysis will be needed to confi rm this. Reference 1. National Patient Safety Agency: Patient Safety Alert (5): Reducing the Harm Caused By Misplaced Nasogastric Feeding Tubes. London: NPSA; 2005. [http://www.nrls.npsa.nhs.uk/resources/?EntryId45=59794] pH testing to confi rm nasogastric tube position on the ICU: are we wasting our time? Twenty- eight per cent of newly placed NG tubes had falsely high pH readings (Figure 2). Reference Reference 1. National Patient Safety Agency: Reducing the Harm Caused by Misplaced Nasogastric Feeding Tubes. NPSA/PSA001./PSA002. NPSA; 2005, 2011. [www.nrls.npsa.nhs.uk] P246 Eff ect of probiotic containing lactobacillus, bifi dobacterium and streptococcus thermophilus in critically ill patients M Ebrahimi Mamaghani, S Sanaie, A Mahmoudpour, H Hamishehkar Tabriz University of Medical Sciences, Tabriz, Iran Critical Care 2013, 17(Suppl 2):P246 (doi: 10.1186/cc12184) Introduction Sepsis is the most common cause of death in ICUs [1]. Destruction of intestinal barrier function and increased translocation of bacteria to systemic blood fl ow can lead to sepsis [2]. Probiotics may have benefi cial eff ects in improvement of critically ill patients by modulating intestinal barrier and reduction of infl ammation [3]. The aim of this trial was to determine the eff ect of probiotic on infl ammatory biomarkers and mortality rate of sepsis in critically ill patients in the ICU. p Methods This double-blind, randomized controlled trial was conducted on 40 critically ill patients admitted to the ICU. They were randomly assigned to receive placebo or probiotic for 7 days. The APACHE score, Sequential Organ Failure Assessment (SOFA) and systemic concentrations of IL-6, procalcitonin (PCT) and protein C were measured before initiation of the study and on days 4 and 7. Also, 28- day mortality was evaluated for each patient. y y p Results IL-6 and PCT levels decreased and protein C levels increased signifi cantly in probiotic group over the treatment period (P <0.001). There was a signifi cant diff erence in IL-6, PCT and protein C levels of the 7th day between two groups (P = 0.001, 0.006 and <0.001, respectively). Compared with controls, probiotic was eff ective in improving APACHE and SOFA scores in 7 days (P <0.001). There was signifi cant diff erence between the probiotic and control group in the 28-day mortality rate (20% vs. 55% respectively, P = 0.048).l y Conclusion Probiotics reduce infl ammation and mortality rate in critically ill patients and might be considered as an adjunctive therapy to sepsis. Figure 1 (abstract P247). Selenium levels before and after 10 days of admission. p References Confi rmation of nasogastric tube placement in critical care M Moore, R Thomson Conclusion Despite the small dataset the results demonstrate a concerning delay in the application of enteral feeding and/or drug administration. Whilst reassuring in the steps taken to avoid never events, this study demonstrates that there may be delays in time-critical administration of enteral medicine or optimal nutritional practices. This study reveals a signifi cant problem with aspirating gastric contents for pH testing, necessitating a large number of X-ray position Figure 2 (abstract P245). pH values – new NG tubes. S92 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Table 1 (abstract P247). Mean selenium levels in diff erent sources of sepsis Source Quantity Level (mg/dl) Abdominal 12 0.43 Chest 12 0.71 Skin 3 0.55 CNS 2 0.53 GU 2 0.58 Figure 1 (abstract P247). Selenium levels before and after 10 days of admission. Table 1 (abstract P247). Mean selenium levels in diff erent sources of sepsis Selenium levels in patients with diff erent sources of sepsis L French, P Temblett Morriston Hospital, Swansea, UK Critical Care 2013, 17(Suppl 2):P247 (doi: 10.1186/cc12185) Selenium levels in patients with diff erent sources of sepsis L French, P Temblett Morriston Hospital, Swansea, UK Critical Care 2013, 17(Suppl 2):P247 (doi: 10.1186/cc12185) p References 1. Angus DC, Linde-Zwirble WT, Lidicker J, Clermont G, Carcillo J, Pinsky MR: Epidemiology of severe sepsis in the USA: analysis of incidence, outcome, and associated costs of care. Crit Care Med 2001, 29:1303-1310. 1. Angus DC, Linde-Zwirble WT, Lidicker J, Clermont G, Carcillo J, Pinsky MR: Epidemiology of severe sepsis in the USA: analysis of incidence, outcome, and associated costs of care. Crit Care Med 2001, 29:1303-1310. Conclusion All patients admitted with early septic shock had subnormal selenium levels. Patients with an abdominal source of septic shock had the lowest levels. This survey supports previous studies indicating early supplementation may be benefi cial in septic shock patients. References 2. Hassoun HT, Kone BC, Mercer DW: Post-injury multiple organ failure: the role of gut. Shock 2001, 15:1-10. 2. Hassoun HT, Kone BC, Mercer DW: Post-injury multiple organ failure: the role of gut. Shock 2001, 15:1-10. 3. Morrow LE: Probiotics in the intensive care unit. Curr Opin Crit Care 2009, 15:144-148. 3. Morrow LE: Probiotics in the intensive care unit. Curr Opin Crit Care 2009, 15:144-148. References 1. Berger MM: Nutr Clin Pract 2006, 21:438-449. 2. Angstwurm MW, et al.: Crit Care Med 2007, 35:118-126. 3. Andrews PJ, et al.: BMJ 2011, 342:d1542. References 1. Berger MM: Nutr Clin Pract 2006, 21:438-449. 2. Angstwurm MW, et al.: Crit Care Med 2007, 35:118-126. 3. Andrews PJ, et al.: BMJ 2011, 342:d1542. 1. Berger MM: Nutr Clin Pract 2006, 21:438-449. 1. Berger MM: Nutr Clin Pract 2006, 21:438-449. 2. Angstwurm MW, et al.: Crit Care Med 2007, 35:118-126. 3. Andrews PJ, et al.: BMJ 2011, 342:d1542. P248 Selenium levels in patients with diff erent sources of sepsis L French, P Temblett Morriston Hospital, Swansea, UK Critical Care 2013, 17(Suppl 2):P247 (doi: 10.1186/cc12185) P248 Is a combined i.v. and enteral glutamine regimen superior to a single i.v. glutamine regimen in severe thoracic trauma? D Pavelescu, I Grintescu, I Luca-Vasiliu, L Mirea Emergency Hospital Floreasca, Bucharest, Romania Critical Care 2013, 17(Suppl 2):P248 (doi: 10.1186/cc12186) Morriston Hospital, Swansea, UK Morriston Hospital, Swansea, UK D Pavelescu, I Grintescu, I Luca-Vasiliu, L Mirea D Pavelescu, I Grintescu, I Luca Vasiliu, L Mirea Emergency Hospital Floreasca, Bucharest, Romania g y p Critical Care 2013, 17(Suppl 2):P248 (doi: 10.1186/cc12186) Introduction The aim of this study is to establish whether diff erent types of sepsis have an impact on selenium levels. Selenium is an essential trace element involved in antioxidant and immunological reactions. Selenium levels have been shown to be low in patients with systemic infl ammatory response syndrome and sepsis. Selenium replacement has been recommended in patients with sepsis [1,2]. Greater than 5 days of supplementation may also help to prevent the development of new infections on ICUs [3]. Introduction Glutamine regulates many biological functions in preserving the cell, acts as a key respiratory fuel and nitrogen donor for rapidly dividing cells, and modulates the expression of many genes associated with metabolism, cell defences and repair, and cytokine production. In severe thoracic trauma, glutamine supplementation is essential because the body consumes more than it produces and glutamine eff ects become dependent on its route of delivery. Methods This is a prospective survey where selenium levels were collected from patients admitted with septic shock to a tertiary ICU, for 6 months from October 2010 to March 2011. f Methods Fifty-two patients 19 to 78 years old with surgery for severe thoracic trauma were assessed in two groups: Group A received 0.3 to 0.5 g/kg/day i.v. glutamine + 20 g enteral glutamine for 7 days, supplementation to enteral nutrition; Group B receive only i.v. glutamine supplementation to enteral nutrition 0.3 to 0.5 g/kg/day for 7 days. Weaning time, the duration of p.o. ileus, incidence and time to resolution of VAP, glycemic level and the percentage decrease of CRP at 96 hours were assessed in both groups. Results Selenium levels were measured in 31 patients with septic shock. Abdominal and chest sepsis were the main sources of infection. Those with an abdominal source of sepsis had the lowest levels, as shown in Table 1. All septic shock patients who had selenium levels taken within the fi rst 10 days of admission had subnormal levels (<0.8 mg/dt), and after 10 days had levels within the normal range, as shown in Figure 1. P248 Is a combined i.v. and enteral glutamine regimen superior to a single i.v. glutamine regimen in severe thoracic trauma? D Pavelescu, I Grintescu, I Luca-Vasiliu, L Mirea Emergency Hospital Floreasca, Bucharest, Romania Critical Care 2013, 17(Suppl 2):P248 (doi: 10.1186/cc12186) Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S93 Critical Care 2013, Volume 17 Suppl 2 pp http://ccforum.com/supplements/17/S2 Results Weaning time and the duration of p.o. ileus were signifi cantly lower in Group A; although the incidence of VAP is similar in both groups, the time of VAP resolution is lower, the glycemic control is better in Group A. The percentage of CRP decrease is higher in Group A. See Figure 1. Conclusion Glutamine becomes an essential amino acid in severe thoracic trauma and when the patients are fed other than TPN (enteral P249 Clinical eff ect of altered lipid emulsion composition containing fi sh oil in postoperative patients following abdominal surgery: a prospective, randomized, open-label, comparative, multicenter phase 3 clinical study M Keum1, S Hong1, H Han2, D Yoon3, J Seo4, I Yun5 1Asan Medical Center Seoul South Korea; 2Seoul National University Figure 1 (abstract P248). Results. tract P248). Results Results Weaning time and the duration of p.o. ileus were signifi cantly lower in Group A; although the incidence of VAP is similar in both groups, the time of VAP resolution is lower, the glycemic control is better in Group A. The percentage of CRP decrease is higher in Group A. See Figure 1. Conclusion Glutamine becomes an essential amino acid in severe thoracic trauma and when the patients are fed other than TPN (enteral, oral); although hard evidence is lacking, both administration routes may be effi cient as soon as possible. Results Weaning time and the duration of p.o. ileus were signifi cantly lower in Group A; although the incidence of VAP is similar in both groups, the time of VAP resolution is lower, the glycemic control is better in Group A. The percentage of CRP decrease is higher in Group A. See Figure 1. P249 P249 Clinical eff ect of altered lipid emulsion composition containing fi sh oil in postoperative patients following abdominal surgery: a prospective, randomized, open-label, comparative, multicenter phase 3 clinical study M Keum1, S Hong1, H Han2, D Yoon3, J Seo4, I Yun5 1Asan Medical Center, Seoul, South Korea; 2Seoul National University Bundang Hospital, Seongnam, South Korea; 3Gangnam Severance Hospital, Seoul, South Korea; 4Samsung Medical Center, Seoul, South Korea; 5Konkuk University Medical Center, Seoul, South Korea Critical Care 2013, 17(Suppl 2):P249 (doi: 10.1186/cc12187) P249 Clinical eff ect of altered lipid emulsion composition containing fi sh oil in postoperative patients following abdominal surgery: a prospective, randomized, open-label, comparative, multicenter phase 3 clinical study g Conclusion Glutamine becomes an essential amino acid in severe thoracic trauma and when the patients are fed other than TPN (enteral, oral); although hard evidence is lacking, both administration routes may be effi cient as soon as possible. Introduction Omega-3 fatty acids have been shown to decrease infl ammatory responses after trauma and surgery resulting in S94 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 vs. 16.2 ± 19.4 IE, P = 0.155; EN: 52.9 ± 25.6 vs. 61.7 ± 25%, P = 0.304; day 3: gluc: 129 ± 17 vs. 133 ± 21 mg/dl, P = 0.578; ins: 35.1 ± 30.9 vs. 32.3 ± 30.5 IE, P = 0.802; EN: 78.3 ± 28.9 vs. 83.4 ± 29%, P = 0.617; day 5: gluc: 124 ± 16 vs. 132 ± 17 mg/dl, P = 0.276; ins: 25.7 ± 37 vs. 33 ± 26.4 IE, P = 0.562; EN: 73.9 ± 41.4 vs. 87.1 ± 23%, P = 0.354). Interestingly, the calorie achievement was not associated with insulin demand (day 1: R = 0.241, P = 0.156) or average blood glucose (day 1: R = 0.248, P = 0.14) throughout the study. reduction of morbidity and mortality in postoperative patients. The n-6:n-3 polyunsaturated fatty acid (PUFA) ratio in parenteral nutrition (PN) also infl uences the immune modulation. The aim of this study was to evaluate the optimal ratio of PUFA in postoperative patients following abdominal surgery. Methods In a prospective, randomized, open-label, comparative, multicenter, phase 3 clinical study, we compared the safety and effi cacy of a 2.1:1 ratio of n-6:n-3 fatty acid compared with 2.5:1 in postoperative patients requiring PN. Eff ect of hypocaloric versus normocaloric parenteral nutrition on whole body protein kinetics in critically ill neurosurgical patients O Rooyackers, A Berg, J Wernerman y O Rooyackers, A Berg, J Wernerman y , g, Karolinska Institutet and University Hospital, Huddinge, Sweden Critical Care 2013, 17(Suppl 2):P251 (doi: 10.1186/cc12189) y , g, arolinska Institutet and University Hospital, Huddinge, Sweden y g Karolinska Institutet and University Hospital, Huddinge, Sweden Critical Care 2013, 17(Suppl 2):P251 (doi: 10.1186/cc12189) y ritical Care 2013, 17(Suppl 2):P251 (doi: 10.1186/cc12189) Table 1 (abstract P249). Lipid emulsion composition of CFO and KAB Table 1 (abstract P249). Lipid emulsion composition of CFO and KAB CFO KAB Table 1 (abstract P249). Lipid emulsion composition of CFO and KAB CFO KAB Table 1 (abstract P249). Lipid emulsion composition of CFO and KAB CFO KAB LCT (g/l) 60 60 MCT (g/l) 50 60 Olive oil (g/l) 50 50 Fish oil (g/l) 40 30 n-6:n-3 2.1:1 2.5:1 Introduction The optimal feeding of critically ill patients treated in the ICU is controversial. Present guidelines for protein feeding are based on weak evidence obtained with suboptimal methods. Whole body protein kinetics is an attractive technique to assess optimal protein intake by measuring the eff ect of protein feeding strategies on protein synthesis rates, protein degradation rates and protein balance. Here protein kinetics were measured in critically ill neurosurgical patients during hypocaloric and normocaloric parenteral nutrition. g y Methods Neurosurgical patients on mechanical ventilation (n = 16) were studied. Energy expenditure was measured with indirect calorimetry. After that, the patients were randomized to receiving 24 hours of 50% of measured energy expenditure followed by 24 hours of 100% or 100% before 50%. Whole body protein kinetics were measured during the last half hour of the feeding periods using stable isotope-labeled phenylalanine as a tracer. During a continuous infusion of labeled phenylalanine and tyrosine, plasma samples were obtained and later analyzed for the content of the labeled amino acids using mass spectrometry. Protein kinetics were calculated using standard steady-state kinetics. In addition, amino acid concentrations were analyzed by HPLC. Student’s t test was used for statistical analyses. Conclusion CFO containing low n-6:n-3 PUFA ratio was comparable with KAB in safety and effi cacy in postoperative patients requiring PN and also had advantages with regard to lipid metabolism aspect. References References 1. Simopoulos AP: Biomed Pharmacother 2002, 56:365-379. 2. Heller AR, et al.: Crit Care Med 2006, 34:972-979. References 1. Simopoulos AP: Biomed Pharmacother 2002, 56:365-379. 2. Heller AR, et al.: Crit Care Med 2006, 34:972-979. P249 Fifty-four patients were assigned to receive PN with Combifl ex Omega peripheral® (CFO, low ratio group, n = 28) or SMOFKabiven peripheral® (KAB, high ratio group, n = 26) for 3 days after abdominal surgery. Safety and effi cacy were monitored daily with laboratory parameters, vital signs, and adverse events from the operation day (day 0) until the end of the study (day 4). Conclusion Medical critically ill patients with fat-based or glucose- based EN achieve similar glucose control. EN was not associated with glucose concentrations or insulin demand. P250 Infl uence of fat-based versus glucose-based enteral nutrition formulas on glucose homeostasis y y y Results The patients received 0.5 ± 0.1 and 1.1 ± 0.2 g amino acids/ kg/day (P <0.001) on the days with 50 and 100% of measured energy expenditure respectively. Energy expenditures were 23.4 ± 2.7 and 24.5 ± 2.3 kcal/kg/day (P = 0.05) on the 50 and 100% days respectively. Plasma amino acids concentrations were 2.8 ± 0.5 and 2.9 ± 0.4 mM (P = 0.085) on the 2 days respectively. Whole body protein synthesis was 12% lower when 50% of energy expenditure was given, 11.7 ± 3.0 versus 13.3 ± 2.2 mg/kg/hour (P = 0.025), whilst protein degradation was unaltered 13.6 ± 3.5 versus 14.0 ± 2.6 mg/kg/hour (P = 0.56). Also protein oxidation was unaltered 3.0 ± 2.1 versus 2.9 ± 1.4 mg/kg/hour (P = 0.85). This resulted in a 60% higher whole body protein balance with the normocaloric nutrition, –1.9 ± 2.1 versus –0.7 ± 1.3 mg/kg/ hour (P = 0.014). M Wewalka, A Drolz, C Zauner Introduction Trauma patients who receive fat-based parenteral nutrition (PEN) achieve better glucose control compared with those fed with glucose-based PEN [1,2]. Therefore, we determined whether fat- based enteral nutrition (EN) has the same benefi t on glucose control and exogenous insulin demand in medical intensive care patients compared with glucose-based EN. Here we present preliminary data for this ongoing randomized controlled cohort study. Methods Medical critically ill patients with need for mechanical ventilation and without contraindications for EN are included in the study. Patients are randomly assigned to receive either fat-based (n = 30) or glucose-based (n = 30) EN. To evaluate the individual calorie demand, indirect calorimetry is performed after an overnight fast. The determined amount of EN is administered continuously for 5 days. Glucose concentrations are measured at least three times per day and averaged. Furthermore, exogenous insulin demand per 24 hours and calorie achievement per 24 hours are evaluated daily. Conclusion The protein kinetics measurements and the protocol used were useful to assess the effi cacy of nutritional support in critically ill patients. In the critically ill neurosurgical patients treated in the ICU, hypocaloric feeding was associated with a more negative protein balance, while the amino acid oxidation was not diff erent. References 1. Tappy L, et al.: Crit Care Med 1998, 26:860-867. 1. Tappy L, et al.: Crit Care Med 1998, 26:860-867. 2. Huschak G, et al.: Intensive Care Med 2005, 31:1202-1208. 2. Huschak G, et al.: Intensive Care Med 2005, 31:1202-1208. Results Total cholesterol (TC) and low-density lipoprotein-cholesterol (LDL-C) levels were less changed signifi cantly in the low ratio group (3 ± 18 vs. 16 ± 23 mg/dl, P = 0.027 for TC, 4 ± 12 vs. 12 ± 18 mg/dl, P = 0.026 for LDL-C) compared with the high ratio group in postoperative patients. Other laboratory parameters and adverse events did not show statistically signifi cant diff erences between the groups. See Table 1. P252 Withholding parenteral nutrition for 1 week reduces ICU-acquired weakness Results So far, 37 patients, 16 with fat-based, 21 with glucose-based EN have been included in the study. Both groups had similar age (62 ± 10 vs. 58 ± 16 years, P = 0.44), body mass index (26.7 ± 5.8 vs. 28.4 ± 4.4 kg/ m2, P = 0.302), SAPS II score (62.4 ± 12.7 vs. 64 ± 12.3, P = 0.697), and fasting plasma glucose (132 ± 34 vs. 121 ± 26 mg/dl, P = 0.269). Furthermore, resting energy expenditure was similar in both groups (1,522 ± 365 vs. 1,573 ± 313 kcal/day, P = 0.647). Throughout the entire study period, average blood glucose, exogenous insulin demand, and calorie achievements per day were similar between the groups (day 1: gluc: 139 ± 30 vs. 127 ± 20 mg/dl, P = 0.143; ins: 27.8 ± 28.4 G Hermans, B Clerckx, T Vanhullebusch, F Bruyninckx, M Casaer, P Meersseman, D Mesotten, S Vancromphaut, P Wouters, R Gosselink, A Wilmer, G Van den Berghe UZ Leuven, Belgium Critical Care 2013, 17(Suppl 2):P252 (doi: 10.1186/cc12190) G Hermans, B Clerckx, T Vanhullebusch, F Bruyninckx, M Casaer, P Meersseman, D Mesotten, S Vancromphaut, P Wouters, R Gosselin A Wilmer, G Van den Berghe Critical Care 2013, 17(Suppl 2):P252 (doi: 10.1186/cc12190) Critical Care 2013, 17(Suppl 2):P252 (doi: 10.1186/cc12190 Introduction ICU-acquired weakness (ICUAW) is a frequent and important complication of critical illness [1]. A large randomized S95 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 intake. In the abdominal muscle compartments, changes were similar, albeit smaller. Femoral and abdominal subcutaneous adipose tissue compartments were unaff ected by disease and nutritional strategy. Conclusion Early PN did not prevent the pronounced wasting of skeletal muscle observed over the fi rst week of critical illness. Moreover, early PN increased the amount of adipose tissue within the muscle compartments. R f controlled trial (EPaNIC: clinicaltrials.gov: NCT00512122) [2] showed that withholding parenteral nutrition during the fi rst week of ICU stay whereby tolerating substantial caloric defi cit (late PN) accelerated recovery and shortened weaning time as compared with early parenteral substitution for defi cient enteral feeding (early PN). We examined the impact of late PN, as compared with early PN, on incidence and recovery of ICUAW. y Methods A preplanned subanalysis of adult patients included in the EPaNIC trial. P254 P254 Withholding parenteral nutrition during the fi rst week of critical illness increases plasma bilirubin but lowers the incidence of cholestasis and gallbladder sludge YM Vanwijngaerden1, L Langouche1, M Gielen1, Y Debaveye1, M Casaer1, C Liddle2, S Coulter2, R Brunner1, P Wouters1, A Wilmer1, G Van den Berghe1, D Mesotten1 1University Hospitals KU Leuven, Belgium; 2University of Sydney, Australia Critical Care 2013, 17(Suppl 2):P254 (doi: 10.1186/cc12192) 5 Withholding parenteral nutrition during the fi rst week of critical illness increases plasma bilirubin but lowers the incidence of cholestasis and gallbladder sludge YM Vanwijngaerden1, L Langouche1, M Gielen1, Y Debaveye1, M Casaer1, C Liddle2, S Coulter2, R Brunner1, P Wouters1, A Wilmer1, G Van den Berghe1, D Mesotten1 1University Hospitals KU Leuven, Belgium; 2University of Sydney, Australia Critical Care 2013, 17(Suppl 2):P254 (doi: 10.1186/cc12192) Introduction Cholestatic liver dysfunction (CLD) during critical illness, defi ned by hyperbilirubinemia, often occurs and is associated with poor outcome. Parenteral nutrition (PN) is assumed to aggravate CLD. However, hyperbilirubinemia more frequently occurred when the start of PN was delayed until day 8 in the ICU (late PN) [1]. Late PN accelerated recovery as compared with early initiation of PN. Results Clinical ICUAW evaluation was performed in 600 patients (matched n = 558), electrophysiological testing in 730 (matched n = 684). Late PN reduced the incidence of ICUAW at fi rst evaluation from 43.1% to 34.4%, P = 0.03 (matched: early PN 41.6%, late PN 33.3%, P = 0.04). Signifi cantly fewer patients in the late PN group developed weakness at any time during ICU stay (late PN 37.0%, early PN 46.4%, P = 0.02; matched: late PN 36.2%, early PN 45.2%, P = 0.03). ICUAW may have recovered faster with late PN than with early PN (P = 0.05, matched P = 0.06). Other outcomes were not diff erent. y y Methods This was a preplanned subanalysis of a large randomized controlled trial on early versus late initiation of PN (n = 4,640) [1]. Plasma total bilirubin was quantifi ed in all patients daily while in the ICU. Liver enzymes ALT, AST, GGT and ALP were quantifi ed twice weekly in all patients while in the ICU. In a random predefi ned subset of patients, circulating bile salts were also quantifi ed with MS-HPLC at baseline and on day 3, day 5 and the last day in the ICU (n = 280). Impact of early parenteral nutrition on muscle and adipose tissue compartments during critical illness L Langouche1, MP Casaer2, W Coudyzer2, D Vanbeckevoort2, B De Dobbelaer2, FG Güiza1, PJ Wouters2, D Mesotten2, G Van den Berghe2 1KU Leuven, Belgium; 2University Hospitals Leuven – KU Leuven, Belgium Critical Care 2013, 17(Suppl 2):P253 (doi: 10.1186/cc12191) g y Conclusion Tolerating substantial caloric defi cit by withholding PN until day 8 of critical illness increased circulating levels of bilirubin but reduced the occurrence of gallbladder sludge and lowered GGT, ALP and ALT levels. These results suggest that hyperbilirubinemia during critical illness dies not necessarily refl ect cholestasis and instead may be an adaptive response. Additional analyses on a propensity score- matched patient population are ongoing. Introduction The goal of enhanced nutrition in critically ill patients is to improve outcome by reducing lean tissue wasting. However, such eff ect has not been proven. This study aimed to assess the eff ect of early administration of parenteral nutrition (PN) on muscle volume and composition by repeated quantitative computer tomography (qCT). Introduction The goal of enhanced nutrition in critically ill patients is to improve outcome by reducing lean tissue wasting. However, such eff ect has not been proven. This study aimed to assess the eff ect of early administration of parenteral nutrition (PN) on muscle volume and composition by repeated quantitative computer tomography (qCT). p y p q p g p y (q ) Methods We performed a preplanned substudy of a randomized controlled trial (EPaNIC) that compared early initiation of PN when enteral nutrition was insuffi cient (early PN) with tolerating a pronounced nutritional defi cit for 1 week in the ICU (late PN) [1]. Late PN prevented infections and accelerated recovery. We studied 15 EPaNIC study neurosurgical patients requiring prescheduled repeated follow-up CT scans and six healthy volunteers matched for age, gender and BMI. Repeated abdominal and femoral qCT images were obtained in a standardized manner on median ICU day 2 (IQR 2 to 3) and day 9 (8 to 10). Intramuscular, subcutaneous and visceral fat compartments were delineated manually. Muscle and adipose tissue volume and composition were quantifi ed using standard Hounsfi eld Unit ranges. Results Critical illness evoked substantial loss of femoral muscle volume in 1 week, irrespective of the nutritional regimen. Early PN reduced the quality of the muscle tissue, as refl ected by the attenuation, revealing increased intramuscular water/lipid content. Early PN also increased the volume of adipose tissue islets within the femoral muscle compartment. P252 Withholding parenteral nutrition for 1 week reduces ICU-acquired weakness The study was performed between October 2008 and November 2010 and included those patients who required intensive care for ≥8 days as well as a computer-generated, admission category- matched, random sample of short-stay ICU patients, the latter to correct for possible bias evoked by earlier ICU discharge in one of the two study groups. Assessors blinded for treatment allocation evaluated muscle strength clinically three times weekly from awakening onward and performed nerve conduction studies and electromyography (NCS and EMG) weekly. The primary outcome was the incidence of ICUAW, diagnosed clinically by the Medical Research Council (MRC) sum score (<48/60) [3] at fi rst evaluation. Secondary outcomes included ICUAW at worst and last MRC evaluation, recovery from ICUAW and incidence of abnormal fi ndings on NCS and EMG. All analyses were performed on the total dataset and on a for-baseline characteristics propensity score-matched sample to correct for possible imbalances between the groups. Reference 1. Casaer MP, et al.: N Engl J Med 2011, 365:506-517. 1. Casaer MP, et al.: N Engl J Med 2011, 365:506-517. 1. Casaer MP, et al.: N Engl J Med 2011, 365:506-517. Impact of early parenteral nutrition on muscle and adipose tissue compartments during critical illness These changes in skeletal muscle integrity correlated with caloric p y p q p g p y q Methods We performed a preplanned substudy of a randomized controlled trial (EPaNIC) that compared early initiation of PN when enteral nutrition was insuffi cient (early PN) with tolerating a pronounced nutritional defi cit for 1 week in the ICU (late PN) [1]. Late PN prevented infections and accelerated recovery. We studied 15 EPaNIC study neurosurgical patients requiring prescheduled repeated follow-up CT scans and six healthy volunteers matched for age, gender and BMI. Repeated abdominal and femoral qCT images were obtained in a standardized manner on median ICU day 2 (IQR 2 to 3) and day 9 (8 to 10). Intramuscular, subcutaneous and visceral fat compartments were delineated manually. Muscle and adipose tissue volume and composition were quantifi ed using standard Hounsfi eld Unit ranges. Reference 1. Casaer et al.: N Engl J Med 2011, 365:506-516. 1. Casaer et al.: N Engl J Med 2011, 365:506-516. P254 Gallbladder sludge was evaluated by ultrasound on ICU day 5 by blinded assessors (n = 776). f Conclusion As compared with early PN, late PN reduced the incidence of ICUAW and may have accelerated recovery thereof. References Results From day 1 after randomization until the end of the 7-day intervention window, plasma bilirubin was higher in the late PN than in the early PN group (all P <0.001). In the late PN group, as soon as PN was started on day 8, plasma bilirubin also fell and the two groups became comparable. Maximum levels of GGT, ALP and ALT during the ICU stay were higher in the early PN group (all P <0.01). Compared with baseline, the circulating glycine and taurine conjugated primary bile salts were elevated on day 3, day 5 and last day of the ICU stay (P <0.01 for all). However, there was no diff erence between the two groups. More patients in the early PN than in the late PN group had gallbladder sludge on day 5 (45% vs. 37%; P = 0.04).i 1. Stevens et al.: Int Care Med 2007, 33:1876-1891. 2. Casaer et al.: N Engl J Med 2011, 365:506-516. 3. De Jonghe et al.:, JAMA 2002, 288:2859-2867. 1. Stevens et al.: Int Care Med 2007, 33:1876-1891. 2. Casaer et al.: N Engl J Med 2011, 365:506-516. 3. De Jonghe et al.:, JAMA 2002, 288:2859-2867. References the duration of renal replacement therapy (RRT) [1]. The impact of the intervention on early markers of catabolism has not been investigated. Methods We studied the impact of early versus late PN on daily markers of catabolism in the ICU in the total study population and in propensity score-matched subgroups of long-stay patients. In addition, we calculated the net incorporation rate of the extra amino acids supplied by early PN. 1. Debaveye, Van den Berghe: Annu Rev Nutr 2006, 26:513-538. 2. Masiero et al.: Cell Metab 2009, 10:507-515. 3. Vanhorebeek et al.: J Clin Endocrinol Metab 2011, 96:E633-E645. 4. Derde et al.: Endocrinology 2012, 153:2267-2276. 5. Casaer et al.: N Engl J Med 2011, 365:506-517. Results Plasma urea, the urea/creatinine ratio and nitrogen excretion increased over time in the ICU. Early PN further increased these markers of catabolism, from the fi rst day of amino acid infusion onward, and only marginally improved the nitrogen balance. Also in the group that received PN only after the fi rst week in the ICU, ureagenesis was increased by infusing amino acids. Over the fi rst 2 weeks, approximately two-thirds of the extra amino acids supplied by early PN were net wasted in urea. The above fi ndings were confi rmed in propensity score- matched subgroups of long-stay patients. The higher urea levels with early PN, rather than the kidney function as such, may have driven the observed longer duration of RRT, as supported by multiple regression analysis. P255 Impact of early parenteral nutrition on catabolism J Gunst1, I Vanhorebeek1, MP Casaer1, G Hermans1, PJ Wouters1, J Dubois2, K Claes1, M Schetz1, G Van den Berghe1 1KU Leuven, Belgium; 2Jessa Hospital, Hasselt, Belgium Critical Care 2013, 17(Suppl 2):P255 (doi: 10.1186/cc12193) Introduction Prolonged critically ill patients enter a state of hypercatabolism and muscle weakness, which has been associated with increased morbidity and mortality. Early, full feeding of ICU patients has been advocated to counteract catabolism. However, a large, multicenter study found that early parenteral nutrition (PN) had no signifi cant impact on mortality and even increased dependency on intensive care with, among others, a signifi cant prolongation of Introduction Prolonged critically ill patients enter a state of hypercatabolism and muscle weakness, which has been associated with increased morbidity and mortality. Early, full feeding of ICU patients has been advocated to counteract catabolism. However, a large, multicenter study found that early parenteral nutrition (PN) had no signifi cant impact on mortality and even increased dependency on intensive care with, among others, a signifi cant prolongation of S96 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Overcrowding estimation in the emergency department: is the simplest score the best? Overcrowding estimation in the emergency department: is the simplest score the best? V Winkin, V Pinckaers, V D’Orio, A Ghuysen CHU Liège, Belgium Critical Care 2013, 17(Suppl 2):P258 (doi: 10.1186/cc12196) p V Winkin, V Pinckaers, V D’Orio, A Ghuysen CHU Liège, Belgium Critical Care 2013, 17(Suppl 2):P258 (doi: 10.1186/cc12196) V Winkin, V Pinckaers, V D’Orio, A Ghuysen CHU Liège, Belgium Critical Care 2013, 17(Suppl 2):P258 (doi: 10.1186/cc12196) Results As compared with controls, muscle from critically ill patients showed reduced myofi ber density, a shift to smaller (especially type I) myofi bers, lower myosin and actin mRNA, upregulated mRNA of the ubiquitin ligases muscle-ring-fi nger-1 and atrogin-1, a small increase in the autophagosome formation marker LC3-II/LC3-I, and increases in the autophagic substrates ubiquitin and p62 (all P ≤0.006). Late-PN, resulting in a larger caloric defi cit than early-PN, had no substantial impact on atrophy markers. In contrast, late-PN increased LC3-II/LC3-I (P = 0.02), which coincided with less accumulation of ubiquitinated proteins/aggregates (P = 0.05). Fewer patients on late-PN developed muscle weakness as compared with early-PN (42% vs. 68%, P = 0.05). In multivariable analysis, a lower LC3-II/LC3-I ratio (P = 0.05) and higher myofi ber density (P = 0.04) were independently associated with muscle weakness. Introduction Emergency department (ED) overcrowding is a major international problem with a negative impact on both patient care and providers. Among validated methods of measurement, emergency physicians have to choose between simple and complex scores [1,2]. The aim of the present study was to compare the complex National Emergency Department Overcrowding Scale (NEDOCS) with the simple occupancy rate (OR) determination. We further evaluated the correlation between these scores and a qualitative assessment of crowding. Methods The study was conducted in two academic hospitals and one county hospital in Liège, Chênée and Verviers; each with an ED census of over 40,000 patient visits per year. Samplings occurred over a 2-week period in January 2011, with fi ve sampling times each day.f Methods The study was conducted in two academic hospitals and one county hospital in Liège, Chênée and Verviers; each with an ED census of over 40,000 patient visits per year. Samplings occurred over a 2-week period in January 2011, with fi ve sampling times each day. Results ED staff considered overcrowding as a major concern in the three EDs. Median OR ranged from 68 to 100, while the NEDOCS ranged from 64.5 to 76.3. P256 f Results In the period studied, the gross attendance fi gure increased by 20,480 (33.72%), whilst the admission rate rose from 22 to 27%. Following closure of the neighbouring ED, the proportion of high- acuity patients attending our institution increased dramatically, with the proportion of category one and two patients (Manchester Triage Scale) increasing by 8.33% (P = 0.076) and 18.80% (P <0.001), respectively. Likewise, ambulance arrivals increased out of proportion to the total increase in attendances (P = 0.016). Admissions from the ED to the ICU increased by 63.04%. Consultants workloads now include 50% more category 1 and 2 patients (P = 0.001).i g p Reference g p Reference 1. Casaer et al.: N Engl J Med 2011, 365:506-517. P257 Impact of closing an emergency department on a neighbouring teaching hospital: the concentrate eff ect J Millar, R Wilson, P O’Connor, R McLaughlin Royal Victoria Hospital, Belfast, UK Critical Care 2013, 17(Suppl 2):P257 (doi: 10.1186/cc12195) P257 Impact of closing an emergency department on a neighbouring teaching hospital: the concentrate eff ect J Millar, R Wilson, P O’Connor, R McLaughlin Royal Victoria Hospital, Belfast, UK Critical Care 2013, 17(Suppl 2):P257 (doi: 10.1186/cc12195) Introduction Closure of an acute hospitals emergency department (ED) has important ramifi cations for those centres expected to take up the resultant workload. The continued reconfi guration of emergency care is likely to produce an increasing number of these scenarios. Little evidence is available to support planning of such initiatives and thus the implications are diffi cult to anticipate. This study aims to demonstrate one hospital’s experience of the rationalisation of emergency care and its eff ect on workload. y Conclusion The extra amino acids supplied by early PN appeared ineffi cient to reverse the negative nitrogen balance, not because of insuffi cient amino acid delivery, but rather because of insuffi cient incorporation with, instead, increased degradation into urea. The substantial catabolism of the extra amino acids, leading to pronounced urea generation, may have prolonged the duration of RRT in the early PN group. Methods This retrospective study was conducted in a large teaching hospital. Activity data were analysed for a 12-month period following the closure of a neighbouring ED. The results were subsequently compared against the year prior to closure. Attendance, triage data, admission rates and waiting times were compared across the two periods, as were workload data for all grades of physician. The chi- squared test was used to examine diff erences between groups.i Impact of early versus late parenteral nutrition on morphological and molecular markers of atrophy and autophagy in skeletal muscle of critically ill patients Impact of early versus late parenteral nutrition on morphological and molecular markers of atrophy and autophagy in skeletal muscle of critically ill patients I Vanhorebeek, MP Casaer, F Güiza, S Derde, I Derese, PJ Wouters, Y Debaveye, J Gunst, G Hermans, G Van den Berghe KU Leuven, Belgium Critical Care 2013, 17(Suppl 2):P256 (doi: 10.1186/cc12194) Introduction Muscle weakness of critical illness is associated with prolonged dependency on ventilatory support and delayed rehabilitation. Muscle wasting related to poor nutrition has long been considered a major determinant, whereas the importance of myofi ber integrity only recently emerged [1-4]. We hypothesized that nutrient restriction early during illness aggravates atrophy while preserving myofi ber integrity by activating the crucial cellular quality control pathway autophagy. The latter could be important to preserve muscle function. Conclusion Reconfi guration of emergency care can have dramatic implications for existing services; these may not always be anticipated. Rationalisation of ED’s may result in a concentration of high-acuity patients accompanied by a downturn in the numbers of patients whose presentations are amenable to care delivered in other settings. This abrupt change in case mix requires a re-examination of existing workforces and their seniority. Methods Critically ill patients (n = 122) were randomized to early (early- PN) or late (late-PN) initiation of parenteral nutrition to complete failing enteral nutrition, while maintaining normoglycemia (80 to 110 mg/ dl) with insulin, in the EPaNIC study [5]. Vastus lateralis biopsies were harvested after 1 week and compared with matched controls (n = 20). P259 GP-led walk-in centre in the UK: another way for urgent healthcare provision M Arain, J Nicholl, M Campbell The University of Sheffi eld, UK Critical Care 2013, 17(Suppl 2):P259 (doi: 10.1186/cc12197) M Arain, J Nicholl, M Campbell The University of Sheffi eld, UK Critical Care 2013, 17(Suppl 2):P259 (doi: 10.1186/cc12197) Introduction It is evident that accident and emergency departments are overloaded with patients, which results in delays in healthcare provision [1]. A large proportion of patients consist of patients with minor illness that can be seen by a healthcare provider in a primary care setting. The aim of the study was to determine the characteristics of patients using GP walk-in services, patients’ satisfaction and the eff ect on emergency department (ED) services. Introduction It is evident that accident and emergency departments are overloaded with patients, which results in delays in healthcare provision [1]. A large proportion of patients consist of patients with minor illness that can be seen by a healthcare provider in a primary care setting. The aim of the study was to determine the characteristics of patients using GP walk-in services, patients’ satisfaction and the eff ect on emergency department (ED) services. Figure 1 (abstract P260). GP patient visits before and after the reform of emergency services. has a negative impact on patient safety [1]. The aim of this study was to analyse whether a reform of emergency care can reduce patient fl ow into the ED. y Methods The survey was conducted in Sheffi eld and Rotherham walk-in centres over 3 weeks during September and October 2011. A self-reported, validated questionnaire was used to conduct survey on the patients presenting at these centres. We estimated that a sample size of around 400 patients from each centre was required to achieve statistically robust results. A post-visit, short questionnaire was also sent to those who agreed for the second questionnaire and provided contact details. ED data were also obtained from April 2008 to March 2010, 1 year before and 1 year after the opening of the GP walk-in centre. Data were entered and analysed in PASW Statistics 18. Ethical approval of the study was obtained from the NHS ethical review committee. Methods A substantial reform of emergency care took place in the province of Kanta-Häme in Southern Finland. Three separate out-of- hours services in primary healthcare (PHC) and one ED in the hospital were combined into one large ED in April 2007. P259 GP-led walk-in centre in the UK: another way for urgent healthcare provision Basic principles of the new ED were: the ED is only for those patients who are seriously ill or injured, and need immediate care; PHC (healthcare centres) take care of acute ordinary illnesses and nonserious injuries during offi ce hours. To achieve these principles a regional fi ve-scale triage system was planned and implemented. The information plan was established. Citizens were systematically informed about the principles of the new ED by mail, articles in the newspapers and interviews in the radio and television. The ED’s Internet pages were planned and established. The number of patient visits (Hämeenlinna region) was analyzed 2 years before and after establishing the new ED. Results A total of 1,030 patients participated in the survey (Rotherham 501; Sheffi eld 529). The mean age of the participants was 32.1 years at Sheffi eld and 30.88 years at Rotherham. A higher proportion of users were female, around 59% at both centres. Most of the patients rated high for convenience of the centre opening hours and location (above 85%, apart from the location of Sheffi eld centre, which was rated high by around 72% of the research participants). Overall 93% patients were satisfi ed with the service at Rotherham centre and around 86% at the Sheffi eld centre. Based on the estimation of the monthly counts of patients attending ED and the GP walk-in centre, around 14% monthly reduction in minor attendances at ED was expected. However, ED routine data did not show any signifi cant reduction in minor attendances as a result of the opening of the GP walk-in centre. g Results During the 2-year period before the establishment of the new ED the mean number of GP patient visits was 1,845 ± 43/month. During the 2-year period after the reform the number was diminished to 1,364 ± 21/month. This change was not associated with the increase of the patient visits taken care of by specialists and hospital residents. See Figure 1. Conclusion An extensive reform of the emergency services can notably reduce patient fl ow into the ED. Reference 1. Boyle A, et al.: Emerg Med Int 2012:838610. [Epub ahead of print] 1. Boyle A, et al.: Emerg Med Int 2012:838610. [Epub ahead of print] p g Conclusion These walk-in centres have been shown to increase accessibility to healthcare service through longer opening hours and walk-in facility. P259 P259 GP-led walk-in centre in the UK: another way for urgent healthcare provision M Arain, J Nicholl, M Campbell The University of Sheffi eld, UK Critical Care 2013, 17(Suppl 2):P259 (doi: 10.1186/cc12197) Reference 1. Gerard K, Lattimer V: Preferences of patients for emergency services available during usual GP surgery hours: a discrete choice experiment. Fam Pract 2005, 22:28-36. Introduction Rio de Janeiro’s Fire Squad is responsible for EMS in the city. During 2010 we implemented 10 ambulances with EKG transmission capability in our city. Our intention was to access the prevalence of acute myocardial infarction in the prehospital setting. Methods We used the Aerotel HeartView EKG system to acquire patient examination and a blackberry phone to transmit and receive the PDF EKG trace. The PDF comes with the cardiologist’s interpretation from a remote hospital, the HCOR São Paulo. Introduction Rio de Janeiro’s Fire Squad is responsible for EMS in the city. During 2010 we implemented 10 ambulances with EKG transmission capability in our city. Our intention was to access the prevalence of acute myocardial infarction in the prehospital setting. P259 GP-led walk-in centre in the UK: another way for urgent healthcare provision Although the eff ect on the reduction of patients’ load at the ED is not visible as these centres cover a fraction of the population, the centre has a potential to divert patients from the ED. P261 Prehospital EKG evaluation in Rio de Janeiro ambulances RV Vasconcellos, FE Erthal, RV Vargas Instituto Nacional de Cardiologia, Rio de Janeiro, Brazil Critical Care 2013, 17(Suppl 2):P261 (doi: 10.1186/cc12199) P260 P260 Establishing a new emergency department: eff ects on patient fl ow V Rautava1, T Valpas1, M Nurmikari2, A Palomäki1 1Kanta-Häme Central Hospital, Hämeenlinna, Finland; 2City of Hämeenlinna, Finland Critical Care 2013, 17(Suppl 2):P260 (doi: 10.1186/cc12198) P260 Establishing a new emergency department: eff ects on patient fl ow V Rautava1, T Valpas1, M Nurmikari2, A Palomäki1 1Kanta-Häme Central Hospital, Hämeenlinna, Finland; 2City of Hämeenlinna, Finland Critical Care 2013, 17(Suppl 2):P260 (doi: 10.1186/cc12198) Overcrowding estimation in the emergency department: is the simplest score the best? We found a signifi cant correlation between Conclusion Early-PN did not counteract muscle atrophy whereas it suppressed autophagy and aggravated weakness. Statistically, muscle weakness was not explained by atrophy or wasting but rather by impaired autophagy and preservation of muscle density. Thus, tolerating nutrient restriction early during critical illness may preserve myofi ber integrity by activating autophagy. i Results ED staff considered overcrowding as a major concern in the three EDs. Median OR ranged from 68 to 100, while the NEDOCS ranged from 64.5 to 76.3. We found a signifi cant correlation between Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S97 the OR and the NEDOCS (Pearson R = 0.973, 0.974 and 0.972), as well as between the OR, the NEDOCS and the subjective evaluation (P = 0.001). Conclusion Crowding evaluation in the ED requires validated, easy-to- use, scores. Our study indicates that the simple OR is as accurate as the complex NEDOCS, allowing continuous crowding assessment. References Figure 1 (abstract P260). GP patient visits before and after the reform of emergency services. the OR and the NEDOCS (Pearson R = 0.973, 0.974 and 0.972), as well as between the OR, the NEDOCS and the subjective evaluation (P = 0.001). Conclusion Crowding evaluation in the ED requires validated, easy-to- use, scores. Our study indicates that the simple OR is as accurate as the complex NEDOCS, allowing continuous crowding assessment. References 1. Hwang U, McCarthy ML, Bernstein SL, et al.: Measures of crowding in the emergency department: a systematic review. Acad Emerg Med 2011, 18:527-538. 1. Hwang U, McCarthy ML, Bernstein SL, et al.: Measures of crowding in the emergency department: a systematic review. Acad Emerg Med 2011, 18:527-538. 2. Hoot NR, Zhou C, Jones I, Aronsky D: Measuring and forecasting emergency department crowding in real time. Ann Emerg Med 2007, 49:747-755. 2. Hoot NR, Zhou C, Jones I, Aronsky D: Measuring and forecasting emergency department crowding in real time. Ann Emerg Med 2007, 49:747-755. Establishing a new emergency department: eff ects on patient fl ow V Rautava1, T Valpas1, M Nurmikari2, A Palomäki1 y Methods We used the Aerotel HeartView EKG system to acquire patient examination and a blackberry phone to transmit and receive the PDF EKG trace. The PDF comes with the cardiologist’s interpretation from a remote hospital, the HCOR São Paulo. Establishing a new emergency department: eff ects on patient fl ow V Rautava1, T Valpas1, M Nurmikari2, A Palomäki1 1Kanta-Häme Central Hospital, Hämeenlinna, Finland; 2City of Hämeenlinna, Finland Critical Care 2013, 17(Suppl 2):P260 (doi: 10.1186/cc12198) p Results We realized 503 EKG examinations in total. Of these, 248 (49%) had as the chief complaint chest pain, 101 (20%) shortness of breath, 47 (9%) syncope, 36 (7%) palpitation; other complaints were 15%. We detected 32 examinations (6.36%) with ST elevation MI and Critical Care 2013, 17(Suppl 2):P260 (doi: 10.1186/cc12198) Introduction Overcrowding in emergency departments (EDs) is a widely known problem. It causes problems and delays in the ED and S98 Critical Care 2013, Volume 17 Suppl 2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum com/supplements/17/S2 http://ccforum.com/supplements/17/S2 Figure 1 (abstract P261). Acute inferior MI with complete heart block. Figure 2 (abstract P261). Paroxysmal supraventricular tachycardia. Figure 1 (abstract P261). Acute inferior MI with complete heart block. Figure 1 (abstract P261). Acute inferior MI with complete heart block. Figure 1 (abstract P261). Acute inferior MI with complete heart block. Figure 2 (abstract P261). Paroxysmal supraventricular tachycardia. Figure 2 (abstract P261). Paroxysmal supraventricular tachycardia. Figure 2 (abstract P261). Paroxysmal supraventricular tachycardia. S99 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 leg and patient satisfaction were used to assess the outcome. The assessments were made before the FIB, at 15 minutes, 30 minutes and 45 minutes. 44 examinations (8.75%) with ST depression. Atrial fi brillation was detected in 43 examinations (8.5%). See Figures 1 and 2. 44 examinations (8.75%) with ST depression. Atrial fi brillation was detected in 43 examinations (8.5%). See Figures 1 and 2. Conclusion This experience gave us an idea of the prevalence for acute ST elevation MI in the prehospital setting, so that we can better develop our prehospital thrombolysis protocol and focus our training for cardiology care. Results A convenience sample of 19 patients was enrolled in this study. The compartment block was placed by three mergence ultrasound trainers. The mean age was 58.5 years. There were 13 females and six males. P264 Abdominal pain in adolescent females: a single-centre audit and review of management Abdominal pain in adolescent females: a single-centre audit and review of management Introduction Abdominal pain in adolescent females has undergone recent changes with regards to its management under various specialities. The authors report a single-centre audit looking at the correct investigation and management of 12-year-old to 16-year-old girls with abdominal pain in the emergency department setting. Introduction Abdominal pain in adolescent females has undergone recent changes with regards to its management under various specialities. The authors report a single-centre audit looking at the correct investigation and management of 12-year-old to 16-year-old girls with abdominal pain in the emergency department setting. Methods A single-centre audit and retrospective analysis of patients took place using case notes and computerised records. Documentation was analysed using statistical analysis and minimum standards were set and reviewed. Results In 1 year, 76 long peripheral catheters were inserted in emergency conditions, using ultrasound guidance. The success rate was 100%; most lines lasted >1 week and were used for diff erent purposes, including contrast medium injection. Results After exclusion criteria 62 females between the ages of 12 and 16 presented to the paediatric emergency department in Leicester with abdominal pain as the predominant admission symptom during a 12-month period. Documentation of the gynaecological history was poor (menstrual history 47%, sexual history 14%, contraception 8%), as was the performance of basic investigations (urine dipsticks 65%, pregnancy test 42%). Documentation was analysed with regard to discharge diagnosis. Ultrasound investigation was performed on seven of the patients but only once admitted to various specialities. No ultrasound was undertaken upon admission. p p g j Conclusion The direct Seldinger technique allows a rapid and safe placement of the catheter in a vein of the arm or of the forearm, even when the vessel cannot be palpated or seen, as long as it can be visualized by ultrasound and it is not deeper than 2 cm. The long life of this type of peripheral line (up to 2 weeks) is guaranteed by the material (polyurethane being more biocompatible than Tefl on) and by the length of the catheter (which reduces the risk of dislodgment). Also, these catheters are particularly cost-eff ective if compared with a central line or with a midline catheter, since a complete kit including catheter, 20 G needle and 20 cm guidewire costs between €15 and €20. Conclusion Improvement in documentation of minimum standards for these patients is needed. P265 Introduction The lumbar plexus block provides excellent analgesia after hip and knee surgery. One approach to the three major nerves (femoral, lateral cutaneous and obturator) of the lubar plexus is the fascia iliaca compartment block, fi rst described by Dalens and colleagues. Using this blind approach, complete nerve block in the distribution of the lumbar plexus may be achieved only in 38% of cases. Ultrasound-guided regional techniques off er a number of advantages including real-time needle guidance and direct observation of local anesthetic spread within tissue planes. Establishing a new emergency department: eff ects on patient fl ow V Rautava1, T Valpas1, M Nurmikari2, A Palomäki1 The mean pain score was 9.5 at time 0. The mean pain score had improved to 5.5 at 15 minutes, 4.5 at 30 minutes and 3 at 45 minutes, respectively. The patient satisfaction was scored 4.5 on a scale of 1 to 5 where 5 was the most satisfi ed. No patient required any further analgesia up to 90 minutes and no issues were raised about the pain or discomfort upon patient transfer. We have no documented complications or side eff ects. Technique of ultrasound-guided peripheral venous access in the emergency room f Conclusion FIB is now widely used by non-anaesthetic trainees to combat pain in preoperative care due to its safe landmarks. Our results have shown that it can be used safely and eff ectively for pain management in hip fracture by emergency physicians, who are trained in the technique, on the shop fl oor. MG Annetta, G Scoppettuolo, M Biancone, F Toni, M Pittiruti Catholic University, Rome, Italy MG Annetta, G Scoppettuolo, M Biancone, F Toni, M Pittiruti Catholic University, Rome, Italy Critical Care 2013, 17(Suppl 2):P262 (doi: 10.1186/cc12200) Catholic University, Rome, Italy Critical Care 2013, 17(Suppl 2):P262 (doi: 10.1186/cc12200) Critical Care 2013, 17(Suppl 2):P262 (doi: 10.1186/cc12200) Introduction In emergency situations, patients may need a fast and reliable peripheral venous access, which sometimes may be diffi cult to obtain, because of poor visualization of the superfi cial veins due to edema, obesity, hypovolemia or local abnormalities. In such cases, insertion of a central line is potentially time consuming and possibly associated with complications. Furthermore, central lines inserted in emergency are known to be at high risk of infection, so guidelines recommend that they should removed within 24 to 48 hours. In this setting, ultrasound-guided placement of a peripheral venous access might be more rapid, safer and more cost-eff ective than a central line. Methods We have reviewed retrospectively our experience with the emergency use of 18 G or 20 G polyurethane catheters, 8 to 10 cm long, inserted by direct Seldinger technique. P264 Reference 1. Ioannidis JP, Salem D, Chew PW, Lau J: Accuracy and clinical eff ect of out-of- hospital electrocardiography in the diagnosis of acute cardiac ischemia: a meta-analysis. Ann Emerg Med 2001, 37:461-470. P264 Abdominal pain in adolescent females: a single-centre audit and review of management A multidisciplinary care pathway could improve outcome. Consideration should be given to whether early ultrasound investigation is appropriate and there is a further need for investigation as to whether this would improve longer term outcomes. P263 Multidisciplinary approach to improving documentation of visua acuity in patients presenting with ocular trauma L Low, M Johnston NHS Tayside, Dundee, UK Critical Care 2013, 17(Suppl 2):P265 (doi: 10.1186/cc12203) Critical Care 2013, 17(Suppl 2):P265 (doi: 10.1186/cc12203) Introduction This study aimed to implement a multidisciplinary quality improvement project in Perth Royal Infi rmary A&E department to improve documentation of visual acuity (VA) in patients presenting with ocular injury. Methods We hypothesized that real-time UFIB could be successfully performed in the ED and would provide an excellent adjunct or alternative to repeat doses of IVMS for pain control in patients with HFx. The study was conducted at University Hospital, Birmingham, where we see about 90,000 patients every year. All patients with confi rmed femoral (neck/shaft) fractures and pain score >7 were included in the study. Patients with local wounds, or suspected signifi cant pelvic injury were excluded. A combination of 15 ml lignocaine 1% and 15 ml bupvivcaine 0.25% was used. VAS (0 to 10), pain on movement of the Methods The improvement project involves a three-pronged multi- disciplinary approach: ensure that equipment required for VA testing (Snellen chart and pinhole mask) was readily available; encourage VA testing at fi rst point of contact with A&E staff , both nursing/medical staff ; and refresher online course on how to test for VA quickly and accurately, in the 6/X format. We compared the pre-intervention (2 September to 2 October 2012) and post-intervention (11 October to 19 November 2012) rates of VA documentation using the chi-square test. S100 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Figure 1 (abstract P265). Table 1 (abstract P266). Comparison of children receiving BiPAP for status asthmaticus by severity of illness Table 1 (abstract P266). Multidisciplinary approach to improving documentation of visua acuity in patients presenting with ocular trauma L Low, M Johnston NHS Tayside, Dundee, UK Critical Care 2013, 17(Suppl 2):P265 (doi: 10.1186/cc12203) Comparison of children receiving BiPAP for status asthmaticus by severity of illness Moderate Severe (n = 206) (n = 197) P value Age (years) 6.8 ± SD 2.8 7.1 ± SD 4.2 X Gender 62% male 61% male X History of asthma 92% 87% X Prior PICU admission 21% 32% X Prior history of BiPAP use 19% 24% X Prior history of intubation 6% 7% X Mean triage for BiPAP start (hours) 2.8 ± SD 2.1 1.2 ± SD 1.25 <0.0001 Mean time on BiPAP (hours) 3.9 ± SD 3.6 5.6 ± SD 5.2 <0.0001 Median time on BiPAP (hours) 2.7 3.8 X Admission to PICU 58% 75% X Total length of hospital stay 42 ± SD 26 50 ± SD 33 <0.06 Serious complications None None X 72-hour returns n = 1 (0.5%) – n = 4 (2%) – X no admits no admits Results During the pre-intervention period, of a total of 44 patients who presented to A&E with eye injury, only 36 patients (95%) had their VA tested. Following intervention, there was signifi cant improvement in VA testing, where all 43 patients presenting to A&E with eye injuries had their VA tested (100%, P = 0.02). Documentation of VA in the correct (6/X) format increased from 82 to 84% following intervention. There was a 15% improvement in documentation of best-corrected VA, from 48 to 63% post intervention. See Figure 1. Results During the pre-intervention period, of a total of 44 patients who presented to A&E with eye injury, only 36 patients (95%) had their VA tested. Following intervention, there was signifi cant improvement in VA testing, where all 43 patients presenting to A&E with eye injuries had their VA tested (100%, P = 0.02). Documentation of VA in the correct (6/X) format increased from 82 to 84% following intervention. There was a 15% improvement in documentation of best-corrected VA, from 48 to 63% post intervention. See Figure 1. g Conclusion Through a multidisciplinary approach, we were successful in achieving our target of 100% VA documentation rate in all patients presenting with eye injury to PRI A&E. P267 Report of an outbreak of toxicity from a novel drug of abuse in the UK: Eric-3 C Morden, S Haig, C Kelly Conclusion All patients recovered without any sequelae. Three patients’ hair started to grow 3 months from ingestion of thallium, and after half a year their hair was restored to their former state. We had diffi culty ordering iron(III)hexacyanoferrate(II) because this is also known as an antidote for cesium. On 11 March 2011 a megathrust earthquake and tsunami hit Japan and the giant tsunami gave rise to an accident at a nuclear power generation plant. Because the rumor of radioactive substances including cesium might be spread was the talk in the city near the nuclear power plant, the authorities put the antidote under heavy supervision. We could also collect the data for the course of thallium concentration. Thallium concentration of the patients who had an antidote was reduced more rapidly but these patients had a loose stool, thought to be a side eff ect of this antidote. Great Western Hospital, Swindon, UK Critical Care 2013, 17(Suppl 2):P267 (doi: 10.1186/cc12205) Introduction We present a case series of toxicity due to a novel sub- stance in the UK: Eric-3. Novel drugs of abuse are becoming more common throughout the world, and they represent particular diffi culties in their acute management. A recent report from the European Monitoring Centre for Drugs and Drug Addiction and Europol has reported 49 new psychoactive substances reported via its early warning system. Methods This was a retrospective case-note review over a 6-month period. Patients were included if their presentation was due to recent ingestion of Eric-3. Physiological data, symptoms, outcome and destination of the patient from the emergency department were collected. Postmortem toxicological analysis was obtained for one of the two patients who died.i Reference 1. Centers for Disease Control and Prevention: Thallium poisoning from eating contaminated cake – Iraq, 2008. MMWR Morb Mortal Wkly Rep 2008, 57:1015-1018. 1. Centers for Disease Control and Prevention: Thallium poisoning from eating contaminated cake – Iraq, 2008. MMWR Morb Mortal Wkly Rep 2008, 57:1015-1018. Results Forty-one attendances were identifi ed from 18 patients. Two patients died and fi ve were admitted to the ICU. Heart rate and temperature on arrival tended to be above normal (mean heart rate was 112 bpm, with an SD of 18; mean temperature was 37.45°C with an SD of 0.95). In total, 63.4% of attendances included agitation and 34.1% choreiform movements. α-Methyltryptamine and 3-/4-fl uoroephedrine were found in the blood of one of the patients who died. P270 Heart rate variability in children with tricyclic antidepressant intoxication BiPAP for treating moderate and severe asthma exacerbations in a PED A Williams, T Abramo A Williams, T Abramo Introduction BiPAP utilization for the treatment of severe refractory status asthmaticus patients has become an accepted therapy but is not well described for moderate exacerbations. We sought to analyze outcomes from our BiPAP quality database for children presenting in status asthmaticus at varying levels of severity. Methods PED status asthmaticus patients requiring BiPAP from 1 January 2010 to 31 August 2012 had a bedside interview and documentation of information at the time therapies were given. Incomplete data were collected retrospectively. All data were stored and analyzed using a RedCap database. Subjects were stratifi ed into severity groups based on asthma score at the time of BiPAP placement. Results There were 206 subjects in the moderate severity group and 197 in the severe group. Table 1 shows the groups were well matched and compares other pertinent data. Children with severe presentations were placed on BiPAP sooner (P <0.001) and remained on BiPAP longer (P <0.001). The moderate group had a longer wait until BiPAP placement. Tables 2 and 3 demonstrate higher initial BiPAP (IPAP/EPAP) settings with increasing age and severity. Figure 1 trends initiation and termination asthma scores stratifi ed by severity at BiPAP matched and compares other pertinent data. Children with severe presentations were placed on BiPAP sooner (P <0.001) and remained on BiPAP longer (P <0.001). The moderate group had a longer wait until BiPAP placement. Tables 2 and 3 demonstrate higher initial BiPAP (IPAP/EPAP) settings with increasing age and severity. Figure 1 trends initiation and termination asthma scores stratifi ed by severity at BiPAP Figure 1 (abstract P266). Initiation and termination asthma scores grouped by severity. Figure 1 (abstract P266). Initiation and termination asthma scores grouped by severity. S101 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 placement. More of the severe group was admitted to the PICU and had overall longer hospitalizations (P <0.06). None experienced severe complications. Conclusion BiPAP is a benefi cial therapy for children presenting to the PED with severe asthma exacerbations. It may have utility for less severe asthma exacerbations. Table 2 (abstract P266). BiPAP for treating moderate and severe asthma exacerbations in a PED Three patients consented to oral administration of an antidote. Two patients rejected administration because their symptoms were mild and getting better. All symptoms of all patients gradually disappeared by August. We also followed up the course of blood concentration of thallium. The concentration in three patients who took the antidote was reduced more rapidly than the two patients who did not take it. p Conclusion BiPAP is a benefi cial therapy for children presenting to the PED with severe asthma exacerbations. It may have utility for less severe asthma exacerbations. P267 BiPAP for treating moderate and severe asthma exacerbations in a PED Mean settings by age: moderate group Age (years) IPAP EPAP <3 (n = 22) 12.1 ± SD 2.1 6.4 ± SD 1.3 3 to 4 (n = 48) 12.1 ± SD 1.6 6.4 ± SD 1.2 5 to 6 (n = 51) 13.0 ± SD 2.3 6.6 ± SD 1.3 7 to 8 (n = 24) 13.9 ± SD 2.1 7.2 ± SD 1.2 9 to 10 (n = 28) 13.9 ± SD3.2 7.4 ± SD 2.0 11 to 12 (n = 25) 13.8 ± SD 1.8 7.0 ± SD 1.3 13 to 14 (n = 10) 13.8 ± SD 3.2 7.4 ± SD 1.4 15 to 17 (n = 8) 17.0 ± SD 2.4 9.5 ± SD 0.9 Table 3 (abstract P266). Mean settings by age: severe group Age (years) IPAP EPAP <3 (n = 21) 12.7 ± SD 2.0 6.8 ± SD 1.3 3 to 4 (n = 50) 13.3 ± SD 2.8 6.8 ± SD 1.4 5 to 6 (n = 26) 13.9 ± SD 2.2 7.3 ± SD 1.3 7 to 8 (n = 29) 15.0 ± SD 2.4 7.7 ± SD 1.5 9 to 10 (n = 26) 15.7 ± SD 3.5 8.0 ± SD 1.8 11 to 12 (n = 22) 14.5 ± SD 3.2 7.6 ± SD 1.6 13 to 14 (n = 9) 15.3 ± SD 3.2 8.0 ± SD 1.7 15 to 17 (n = 14) 17.5 ± SD 3.0 9.5 ± SD 1.8 References 1. EMCDDA: Europol Annual Report on the Implementation of Council Decision 2005/387/JHA. Lisbon: EMCDDA/Europol; 2011. 2. Brandt SD, et al.: Drug Test Anal 2010, 2:377-382. 3. Sikk K, et al.: Acta Neurol Scand 2007, 115:385-389. 4. Stepens A, et al.: N Engl J Med 2008, 358:1009-1017. 5. Murphree HB, et al.: Clin Pharmacol Ther 1961, 2:722-726. P268 Abstract withdrawn P269 Thallium group poisoning incident in Japan 2011 Y Namba, R Suzuki, J Sasaki, M Takayasu, K Watanabe, D Kenji, M Hayashi, Y Kitamura, M Kawamo, H Masaki, E Kyuuno, M Hayashi, M Yamaguchi, A Maeda Showa University Fujigaoka Hospital, Yokohama, Japan Critical Care 2013, 17(Suppl 2):P269 (doi: 10.1186/cc12207) Introduction Thallium is an odorless, tasteless, heavy metal that has been often used for intentional poisonings. In severe patients, thallium poisoning produces neuromuscular symptoms such as extreme pain and muscle weakness. Introduction Thallium is an odorless, tasteless, heavy metal that has been often used for intentional poisonings. BiPAP for treating moderate and severe asthma exacerbations in a PED In severe patients, thallium poisoning produces neuromuscular symptoms such as extreme pain and muscle weakness. Methods Five case reports. p Results All patients worked at a pharmaceutical factory. They joined a tea party held at their workplace at the end of April 2011. The fi ve patients drank tea from a teapot someone had put thallium in. A few days later, they complained of femoral numbness and pain caused by pressure. About a week later, three of fi ve patients had profound hair loss. Three weeks after the party, they came to our ER. We thought that their symptoms might be caused by some chemicals. We searched the keywords: ‘lower extremity pain’, ‘hair loss’ and ‘poison’ in the Internet. As a result, thallium, mercury, lead, and so forth, were suspicious metals. In those metals, thallium was most likely because it was used in their factory. We immediately examined the blood concentration of several metals and ordered iron(III)hexacyanoferrate(II) that is known as the antidote for thallium poisoning. Only thallium was positive in the blood metal concentration test. Three patients consented to oral administration of an antidote. Two patients rejected administration because their symptoms were mild and getting better. All symptoms of all patients gradually disappeared by August. We also followed up the course of blood concentration of thallium. The concentration in three patients who took the antidote was reduced more rapidly than the two patients who did not take it. Results All patients worked at a pharmaceutical factory. They joined a tea party held at their workplace at the end of April 2011. The fi ve patients drank tea from a teapot someone had put thallium in. A few days later, they complained of femoral numbness and pain caused by pressure. About a week later, three of fi ve patients had profound hair loss. Three weeks after the party, they came to our ER. We thought that their symptoms might be caused by some chemicals. We searched the keywords: ‘lower extremity pain’, ‘hair loss’ and ‘poison’ in the Internet. y y p p As a result, thallium, mercury, lead, and so forth, were suspicious metals. In those metals, thallium was most likely because it was used in their factory. We immediately examined the blood concentration of several metals and ordered iron(III)hexacyanoferrate(II) that is known as the antidote for thallium poisoning. Only thallium was positive in the blood metal concentration test. Heart rate variability in children with tricyclic antidepressant intoxication Heart rate variability in children with tricyclic antidepressant intoxication EC Dinleyici, Z Kilic, S Sahin, R Tutuncu-Toker, M Eren, ZA Yargic, P Kosger B Ucar Eskisehir Osmangazi University Faculty of Medicine, Eskisehir, Turkey Critical Care 2013, 17(Suppl 2):P270 (doi: 10.1186/cc12208) EC Dinleyici, Z Kilic, S Sahin, R Tutuncu-Toker, M Eren, ZA Yargic, P Kosger, B Ucar Conclusion In this outbreak in the UK, Eric-3 gave symptoms similar to other stimulants known as legal highs, including death. It may have been a novel substance, 3-/4-fl uoroephedrine. This underlines the need for prospective data collection and early national and international information sharing. Eskisehir Osmangazi University Faculty of Medicine, Eskisehir, Turkey Critical Care 2013, 17(Suppl 2):P270 (doi: 10.1186/cc12208) Eskisehir Osmangazi University Faculty of Medicine, Eskisehir, Turkey Critical Care 2013, 17(Suppl 2):P270 (doi: 10.1186/cc12208) Introduction Tricyclic antidepressant (TCA) intoxication is among the most common causes of poisonings in our country. The TCA group of Introduction Tricyclic antidepressant (TCA) intoxication is among the most common causes of poisonings in our country. The TCA group of S102 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 demonstrated edematous wall thickening of gastric mucosa, and ascites was observed in 44.4% (12/27) of these patients. The patients with small intestinal anisakiasis demonstrated limited edematous wall thickening of the intestinal tract, and ascites was observed in 90.9% (40/44) of these patients. Furthermore, phlegmon of mesentery fat was observed in 72.7% (32/44) of the small intestinal anisakiasis patients. Conclusion When the cause of abdominal pain cannot be determined by initial assessment, anisakiasis should be considered, especially if the patient has a history of raw or undercooked seafood ingestion. In the ED, certain methods of diagnosis are evaluation of the time to develop symptoms and CT imaging, and a history of raw or undercooked seafood ingestion should be considered in the diagnosis. References drugs aff ects the central nervous and cardiovascular systems, resulting in severe arrhythmia and death. Heart rate variability (HRV) analysis is a non-invasive assessment method that allows evaluation of the cardiac autonomic (sympathetic and parasympathetic) activity. The aim of this study was to evaluate HRV in children requiring ICU stay due to TCA poisoning. Methods Twenty children with isolated TCA poisoning aged between 3 and 16 years who were hospitalized in the pediatric ICU, between March 2009 and July 2010, and 20 healthy children as a control group were enrolled. Diagnosing anisakiasis in the emergency department St. Luke’s International Hospital, Tokyo, Japan p y p Critical Care 2013, 17(Suppl 2):P271 (doi: 10.1186/cc12209) Introduction Humans can be incidentally parasitized by third-stage Anisakis larvae after ingestion of raw or undercooked seafood. Although the clinical symptom of anisakiasis is abdominal pain, the clinical fi nding is nonspecifi c and may be misdiagnosed as appendicitis, ileus, and so forth. Every year, >40,000 patients visit our ED and our hospital near the Tsukiji market (famous seafood market in Tokyo). We often observed cases of anisakiasis in patients who visited the hospital with abdominal pain as the chief complaint. Thus, we researched to determine factors useful to diagnose anisakiasis. Methods We retrospectively reviewed data of 83 patients (58 men, 25 women) diagnosed with anisakiasis in our ED (22 July 2003 to 22 July 2012) and examined the usefulness of clinical history, blood test, diagnostic imaging, and so forth, for anisakiasis diagnosis. Diagnosis was made after (A) endoscopically proven Anisakis polypide and (B) a hematologically positive Anisakis antibody (IgG, IgA) and CT diagnostic imaging. y Conclusion The implementation of the TM Project at a community hospital has a major impact in the eICU and eED Sepsis Management Program, and improved compliance with recommended care bundles. f Heart rate variability in children with tricyclic antidepressant intoxication Clinical and electrocardiographic (ECG) fi ndings were noted in the TCA poisoning group. In both groups, 24-hour time domain HRV analysis (SDNN, SDANN, SDNNi, RMSDD, NN50, and pNN50) was performed. We also recorded frequency domain analysis results at the fi rst 5 minutes and the last 5 minutes of the 24-hour record (VLF, nLF, nHF, LF/HF ratio). 1. Valle et al.: J Med Case Rep 2012, 6:114. 2. David Hwang et al.: Chonnam Med J 2012, 48:73-75. 1. Valle et al.: J Med Case Rep 2012, 6:114. Results The average level of TCA in the study group was 1,116 ± 635 and TCA levels were positively correlated with the duration of QRS interval (P <0.01). In time-domain nonspectral evaluation, SDNN (P <0.001), SDNN (P <0.05), RMSDD (P <0.01), and pNN50 (P <0.01) were found signifi cantly lower in the TCA intoxication group compared with the control group, while NN50 (P <0.01) was signifi cantly higher in value. The spectral analysis (frequency domain) of data recorded at fi rst 5 minutes after intensive care admission showed that the values of the nLF (P <0.05) and LF/HF ratio (P = 0.001) were signifi cantly higher in the TCA intoxication group than the controls, while nHF (P = 0.001) values were signifi cantly lower. The frequency domain spectral analysis of data recorded at the last 5 minutes showed a lower nHF (P = 0.001) in the TCA intoxication group than the controls, and the LF/HF ratio was signifi cantly higher (P <0.05) in the intoxication group. SDNN (P <0.001), RMSDD (P <0.01), SDNNi (P <0.01), and pNN50 (P <0.01) levels were higher in patients with positive ECG fi ndings than those without positive ECG fi ndings. The LF/HF ratio was higher in seven children with seizures (P <0.001).i P272 Implementation of a sepsis protocol in a community hospital using a Telemedicine Program M Steinman1, CA Abreu Filho1, A Andrade2, R Cal1, N Akamine1, J Teixeira2, E Silva1, A Kanamura1, M Cenderoglo1, C Lottenberg1 1Hospital Israelita Albert Einstein, São Paulo, Brazil; 2Hospital Municipal Dr. Moysés Deutsch, São Paulo, Brazil Critical Care 2013, 17(Suppl 2):P272 (doi: 10.1186/cc12210) Implementation of a sepsis protocol in a community hospital using a Telemedicine Program Introduction Sepsis has high incidence around the world, approximately 400,000 new cases occur annually in Brazil. In developing countries it is still an important cause of death due to poor adherence to best practice medicine protocols, mainly in community hospitals. The Brazilian mortality rate of septic shock is around 60%. The aim of this study is to describe the fi rst Brazilian initiative of implementation of a Telemedicine (TM) Project for therapeutic support of septic patients in a community hospital in São Paulo, Brazil. Conclusion Existing fi ndings give us an idea about HRV’s value to determine arrhythmia and predict convulsion risk in TCA poisonings. HRV can be used as a non-invasive method in determining the treatment and prognosis of TCA poisoning. y p , Methods Since May 2012, a TM Program has been implemented at two hospitals in São Paulo – Hospital Municipal Dr. Moysés Deutsch (HMMD), a public, secondary hospital, and Hospital Israelita Albert Einstein (HIAE), a tertiary private philanthropic entity – due to a partnership with Brazilian Health Ministry. A TM Central Command was located at HIAE with Endpoint 97 MXP Cisco® Solution and a mobile Intern MXP ISDN/IP Cisco® for the remote hospital (HMMD) via dedicated GB/sec connection. Imaging examinations were evaluated using PACS technology. At HMMD, the Sepsis Protocol, based on the Surviving Sepsis Campaign, was started for every recruited patient admitted to the emergency department (ED) or the ICU, and assessed by the Central Command through TM with an experienced consultant. Results Over a 6-month period, 33 patients with diagnosis of sepsis (including severe sepsis and septic shock) admitted to HMMD were evaluated by skilled doctors of HIAE via TM, and the Surviving Sepsis Campaign protocol was instituted. In total, 87.8% of the consultations originated from the ICU and 12.2% from the ED; 16 patients were male (48.4%), mean age was 44.2 years old. TM improved diagnosis in 12.1% and infl uenced clinical management in 87.9% of the cases; in 11 patients (33.3%) TM consultation led to antibiotic change. Hospital mortality rate was 33.3%. P271 Diagnosing anisakiasis in the emergency department T Takabayashi, S Ishimatsu, N Otani, T Mochizuki, R Miyamichi St. Luke’s International Hospital, Tokyo, Japan Critical Care 2013, 17(Suppl 2):P271 (doi: 10.1186/cc12209) Reference 1. Beale R, et al.: Promoting Global Research Excellence in Severe Sepsis (PROGRESS): lessons from an international sepsis registry. Infection 2009, 37:222-232. Results Of the 83 patients, 39 had gastric anisakiasis and 44 had small intestinal anisakiasis. All gastric and small intestinal anisakiasis patients were diagnosed by methods A and B, respectively. A blood test was unable to show the specifi c infl ammatory reaction. A history of raw or undercooked seafood ingestion was noted in 95.2% (79/83) of the patients. This was observed in 100% (39/39) of the gastric anisakiasis patients and 91% (40/44) of the small intestinal anisakiasis patients. With regard to the development of symptoms, symptoms for gastric anisakiasis developed within 48 hours and reached a peak in less than 6 hours, whereas the symptoms for small intestinal anisakiasis reached a peak in 48 hours and persisted for a maximum of 5 days. Diagnostic CT imaging revealed that all the patients with gastric anisakiasis P273 Reform of emergency services: immediate eff ects on cardiac care unit and ICU patient intake AO Alaspää, T Valpas, V Rautava, A Palomäki Kanta-Häme Central Hospital, Hämeenlinna, Finland Critical Care 2013, 17(Suppl 2):P275 (doi: 10.1186/cc12213) Introduction Early onset eff ective care in the emergency department (ED) has been reported to have a great infl uence on the intensive care patients’ morbidity and mortality [1]. Little is known about the infl uence of the reorganisation of the ED on patient intake to the ICUs. The aim of this study was to analyse monthly intake of patients from the ED to the cardiac care unit (CCU) and ICU before and after the reform of emergency services. Results HMMD receives an average of 30 cases of stroke monthly, and thrombolysis did not occur before the implementation of the TM Project, because of the lack of neurologists available to conduce the cases. After implementation of the TM Program, six cases of ischemic stroke were thrombolyzed with alteplase; only one case (16%) progressed to death from septic shock, and one case (16%) presented symptomatic intracranial hemorrhage. g y Methods In Kanta-Häme Central Hospital, a new ED started on 1 April 2007. Four older emergency rooms were combined into one bigger emergency department and an observation ward was introduced with continuous follow-up of vital signs. This study is a retrospective analysis of the patient intake to the CCU and ICU 1 year before and after the reorganisation. Using as data the Finnish Intensive Care Quality Consortium (Intensium, Finland) database and the cardiac database of the hospital, patient transfer from ED to the ICU and CCU was collected and analysed. Monthly pre/post comparisons were carried out statistically by a nonparametric Wilcoxon signed-rank test. Conclusion Thrombolysis in ischemic stroke reduces 30% the risk of disability and 18% the mortality rate. This procedure has been only feasible to be done in the community setting because of the implementation of the TM Project, which permits the presence of a real time consultation with a specialized neurological team from a tertiary center. P274 P274 Analysis of emergency calls achieved in a French emergency dispatching centre: what resources for which patients O Tilak, J Cuny, N Assez, P Goldstein, E Wiel Lille University Hospital Center, Lille Cedex, France Critical Care 2013, 17(Suppl 2):P274 (doi: 10.1186/cc12212) Figure 1 (abstract P275). Combined patient fl ow to the ICU and CCU before and after the reformation of the ED. Initial Brazilian experience of Telestroke for thrombolysis in a community hospital CA Abreu Filho1, A Caluza1, M Steinman1, G Silva1, R Cal1, N Akamine1, J Teixeira2, A Andrade2, E Silva1, A Kanamura1, M Cenderoglo1, C Lottenberg1 CA Abreu Filho1, A Caluza1, M Steinman1, G Silva1, R Cal1, N Akamine1, J Teixeira2, A Andrade2, E Silva1, A Kanamura1, M Cenderoglo1, C Lottenberg1 1Hospital Israelita Albert Einstein, São Paulo, Brazil; 2Hospital Municipal Dr. Moysés Deutsch, São Paulo, Brazil C l C (S l ) (d / ) S103 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 analyses, and then returned home. In total, 25.50% of patients were hospitalized in a medical or surgical department, and 12.42% in the short-term hospitalised unit of the emergency department (stay duration <24 hours). Some 5.10% of patients worsened and were oriented in the ICU. A total 3.77% of patients in a cardiac ICU. In total, 73.84% of patients had stay duration less than 6 hours in the ED, 24.45% <24 hours. Forty percent of patients supported by fi remen and 54% supported by private ambulance left the hospital after a single medical consultation. Introduction Stroke is the leading cause of death in Brazil, the number of stroke patients receiving thrombolysis therapy in the developing world is extremely low. Prehospital delay, fi nancial constraints, and lack of infrastructure are the main barriers for thrombolysis therapy in developing countries. In this way, telemedicine (TM) allows knowledge transfer of medical expertise to remote community hospitals, where there is a shortage of specialized doctors. The aim of this study is to describe the fi rst Brazilian initiative of a Telestroke Program for therapeutic support and monitoring cases of acute stroke, and venous thrombolysis of eligible cases in a community hospital in São Paulo, Brazil. analyses, and then returned home. In total, 25.50% of patients were hospitalized in a medical or surgical department, and 12.42% in the short-term hospitalised unit of the emergency department (stay duration <24 hours). Some 5.10% of patients worsened and were oriented in the ICU. A total 3.77% of patients in a cardiac ICU. In total, 73.84% of patients had stay duration less than 6 hours in the ED, 24.45% <24 hours. Forty percent of patients supported by fi remen and 54% supported by private ambulance left the hospital after a single medical consultation. Conclusion Nearly 70% of patients calling the French emergency medical dispatching centre are sent to hospital. Initial Brazilian experience of Telestroke for thrombolysis in a community hospital Those transportations are supported for two-thirds of cases by a private ambulance or fi remen ambulance. One out of two patients only receive a simple medical consultation in the ED, and go back home. This may concur to the defi ciency of using general medicine in town. They prefer using emergency services for free. Only one patient out of four was hospitalized more than 24 hours. Methods Since May 2012 a TM Program has been implemented at two hospitals in São Paulo – Hospital Municipal Dr. Moysés Deutsch (HMMD), a public, secondary hospital, distant about 60 minutes from the nearest tertiary center, and Hospital Israelita Albert Einstein (HIAE), a tertiary private philanthropic entity – due to a partnership with Brazilian Health Ministry. Patients admitted to the community hospital’s emergency department with acute ischemic stroke as possible diagnosis are submitted to brain imaging examinations, and considered for remote consultation. A TM Central Command was located at HIAE with Endpoint 97 MXP Cisco® Solution and a mobile Intern MXP ISDN/IP Cisco® for the remote hospital (HMMD). Imaging examinations were evaluated using PACS technology. At HIAE the TM Center has skilled doctors, including neurologists, available for consultation 24 hours, 7 days a week. They discussed the neurological cases via TM, and selected stroke patients for intravenous thrombolysis when timely (3 hours symptom onset). P275 P275 Reform of emergency services: immediate eff ects on cardiac car unit and ICU patient intake AO Alaspää, T Valpas, V Rautava, A Palomäki Kanta-Häme Central Hospital, Hämeenlinna, Finland Critical Care 2013, 17(Suppl 2):P275 (doi: 10.1186/cc12213) P277 1. Rivers E, et al.: N Engl J Med 2001, 345:1368-1377. Reform of emergency services: immediate eff ects on cardiac care unit and ICU patient intake Introduction Following patients referred by the French emergency dispatching centre (SAMU 15) in diff erent hospital services (emergency or ICU) is not always easy. The purpose of this study was to analyze each call arriving in the emergency dispatching centre, defi ne resources for transportation, the host hospital department, and analyze the short- term outcome of these patients. p Methods We conducted a prospective, observational study, by collecting all data recorded at the emergency dispatching centre in a continuous 24-hour period. Each call creating a medical fi le was included. We identifi ed the type of call, various administrative information, the purpose of call, resources triggered, medical advice, hospital department admission, and clinical evolution. p p Results A total of 877 patients were included; 67.80% calling from home, 15.63% calling from a public place. Firemen ambulances were sent in 38.89%, private ambulance in 24.65% and an emergency medical ambulance (SMUR) in 11.23%. Simple medical advice without transportation was given in 13.19% of cases. In total, 22.18% of patients were entrusted to the family; 2.89% of patients refused transportation; 69.94% of triggered fi remen ambulances were done by a centre 18 call, without any medical regulation by an emergency physician. A total of 68.59% patients were referred to the emergency department, 1.2% in ICU, and 1.8% in cardiac ICU. In the emergency room, 50.78% of patients received a simple medical consultation with biological Figure 1 (abstract P275). Combined patient fl ow to the ICU and CCU before and after the reformation of the ED. S104 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Results The total decrease in monthly patient infl ow from ED to the ICU and CCU was 30.1% (P = 0.003); that is, from the mean of 47.7 ± 8.2 to 33.3 ± 8.3 patients (Figure 1). The 7.1% decrease in patients taken into the ICU (12.9 ± 4.1 to 12.0 ± 4.6 patients) was not statistically signifi cant. However, the intake from the ED to the ICU decreased by 38.6% (from 34.8 ± 6.5 to 21.3 ± 5.1 patients) (P = 0.002). Conclusion Overall there were no statistical diff erences between the observation years either for overtriage or undertriage. L Escobar Alvaro1, J Jimenez Gonzalez1, A Pelaez Ballesta1, A Corbatón Anchuelo2 Overtriage and undertriage in a prehospital system over 7 years L Carenzo, F Barra, A Messina, D Colombo, T Fontana, F Della Corte Universitè del Piemonte Orientale A. Avogadro, Novara, Italy Critical Care 2013, 17(Suppl 2):P276 (doi: 10.1186/cc12214) Introduction On 11 May 2011 two moderate magnitude earthquakes hit the city of Lorca, southeast Spain. They caused 11 deaths, including two pregnant women and their babies, more than 350 injured, and moderate or severe damage to 80% of the building in the city, including Lorca’s public hospital that had to be evacuated.i Introduction The Novara 118 emergency medical system (EMS) dispatch center manages medical emergency calls coming from a region that spreads out over 1,400 km2 and includes 88 towns and a population of 385,000 people; inhabitant density is 275 inhabitants/km2. p p y Methods Data collection from 1 January 2005 to 31 December 2011 was obtained (EMS software SaveOnLine® Suite 4; REGOLA s.r.l., Turin, Italy). Triage used was the Medical Priority Dispatch System and severity was defi ned by color codes. Methods A descriptive study of the fi les of Lorca’s hospital and the clinical records of patients that attended our service in the 20 hours following the second earthquake on 11 May. i y Results During the study period the Novara 118 dispatch center managed a total of 122,384 EMS interventions. Median (interquartile) overestimation (regardless of severity) was 10.0% (2.4 to 14.1%) while median underestimation was 1.5% (0.8 to 2.5%). See Tables 1 and 2. Results A total of 225 patients were relocated. Simultaneously 224 patients were treated by the emergency service, until the evacuation was completed and after then, from 10:00 pm to 3:00 pm the next day, 47 more in a fi eld hospital placed just outside the hospital building. See Figures 1 and 2. Table 1 (abstract P276). Overtriage (%) Overtriage 2005 2006 2007 2008 2009 2010 2011 Other 3.03 2.47 5.30 4.95 4.06 4.58 4.17 Cardiac 13.23 15.30 27.06 23.72 19.28 19.17 20.14 Drunk 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Poisoning 10.19 18.92 17.91 8.47 8.85 15.20 11.81 Oncological 0.00 2.78 1.05 1.03 3.03 2.47 0.00 Neurologic 9.97 10.05 15.59 15.18 14.57 13.32 13.32 Not identifi ed 13.03 13.15 11.84 11.07 10.33 11.79 7.23 Mental 1.30 0.41 2.09 2.34 2.30 1.27 2.35 Respiratory 10.67 10.89 22.09 18.63 16.29 18.23 14.12 Trauma 12.97 12.68 10.42 8.59 6.78 7.74 7.56 Table 2 (abstract P276). Reform of emergency services: immediate eff ects on cardiac care unit and ICU patient intake When observed individually, trauma showed the only signifi cant overtriage reduction over time; there were no individual modifi cations in undertriage over time. Reference Reference Reference 1. Neely KW, Eldurkar JA, Drake ME: Acad Emerg Med 2000, 7:174-180. 1. Neely KW, Eldurkar JA, Drake ME: Acad Emerg Med 2000, 7:174-180. Conclusion According to our results the reform of the ED may have a signifi cant role in the total infl ow of patients to the CCU and ICU. Reference P278 P278 War injuries and civilian accidents in Afghan children R Kedzierewicz1, P Ramiara2, M Puidupin3, S Mérat4 1CMA des Alpes, Barby, France; 2HIA Sainte Anne, Toulon, France; 3HIA Desgenettes, Lyon, France; 4HIA Begin, Saint Mandé, France Critical Care 2013, 17(Suppl 2):P278 (doi: 10.1186/cc12216) P278 War injuries and civilian accidents in Afghan children R Kedzierewicz1, P Ramiara2, M Puidupin3, S Mérat4 1CMA des Alpes, Barby, France; 2HIA Sainte Anne, Toulon, France; 3HIA Desgenettes, Lyon, France; 4HIA Begin, Saint Mandé, France Critical Care 2013, 17(Suppl 2):P278 (doi: 10.1186/cc12216) p Conclusion The large number of casualties during a major disaster is a global problem, even in the developed countries. When the role of the intensivist is reviewed, many roles were verifi ed to be important; that is, as a leader of a medical team or triage offi cer as well as a professional in the fi eld of specifi c intensive care. However, there are many problems to be solved in the fi elds of disaster medicine. In order to solve the diversifi cation or the various medical problems, it is necessary to compile or systematize a disaster medicine of the world version. The concept of the compendium and our process of trial are shown in relation to intensive care. Introduction We intended to compare paediatric traumatic injuries due to war and others related to civilian accidents (CA). Methods We conducted an observational epidemiologic retrospective study on paediatric cases (<18 years old) seen at the emergency room of Kabul International Airport NATO role 3 medical treatment facility between 2009 and 2012. Introduction We intended to compare paediatric traumatic injuries due to war and others related to civilian accidents (CA). Methods We conducted an observational epidemiologic retrospective study on paediatric cases (<18 years old) seen at the emergency room of Kabul International Airport NATO role 3 medical treatment facility between 2009 and 2012. Results During 3 years, 341 children were admitted to the emergency room (ER) with a mean age of 10 years (9.49 to 10.51). Eighty-eight per cent of children (301) were traumatized, 39% of them (118) due to war injuries (WI) and 61% (183) due to CA. Forty-three per cent of WI (gunshot wound 34%, explosion 66%) but only 19% of injuries due to CA (falls 24%, transport accidents 56%, burns 9%, penetrating injuries 9%, unknown cause 2%) involved polytraumas defi ned as a New Injury Severity Score (NISS) >15 (P <0.001). References Intensive care and disaster medicine: the role of a compendium Y Haraguchi1, Y Tomoyasu1, H Nishi1, M Hoshino1, T Tsubata2, M Sakai1, E Hoshino1 1Disaster Medicine Compendium Team, Japan, Tokyo, Japan; 2Keiyo Hospital, Tokyo, Japan Critical Care 2013, 17(Suppl 2):P279 (doi: 10.1186/cc12217) 1. Hill AD, Vingilis E, Martin CM, Hartford K, Speechley KN: Interhospital transfer of critically ill patients: demographic and outcomes comparison with nontransferred intensive care unit patients. J Crit Care 2007, 22:290-295. 2. Flabouris A, Hart GK, George C: Outcomes of patients admitted to tertiary intensive care units after interhospital transfer: comparison with patients admitted from emergency departments. Crit Care Resusc 2008, 10:97-105. 3. Duke GJ, Green JV: Outcome of critically ill patients undergoing interhospital transfer. Med J Aust 2001, 174:122-125. 1. Hill AD, Vingilis E, Martin CM, Hartford K, Speechley KN: Interhospital transfer of critically ill patients: demographic and outcomes comparison with nontransferred intensive care unit patients. J Crit Care 2007, 22:290-295. 1. Hill AD, Vingilis E, Martin CM, Hartford K, Speechley KN: Interhospital transfer of critically ill patients: demographic and outcomes comparison with nontransferred intensive care unit patients. J Crit Care 2007, 22:290-295. p 2. Flabouris A, Hart GK, George C: Outcomes of patients admitted to tertiary intensive care units after interhospital transfer: comparison with patients admitted from emergency departments. Crit Care Resusc 2008, 10:97-105. 3. Duke GJ, Green JV: Outcome of critically ill patients undergoing interhospital transfer. Med J Aust 2001, 174:122-125. 3. Duke GJ, Green JV: Outcome of critically ill patients undergoing interhospital transfer. Med J Aust 2001, 174:122-125. Introduction There were several catastrophes in the last decades. The make-up of systematic measuring and life-saving medical systems, including intensive care, is thought to be an urgent and essential issue. Our eff orts for establishing a disaster medicine and education system are presented. L Escobar Alvaro1, J Jimenez Gonzalez1, A Pelaez Ballesta1, A Corbatón Anchuelo2 Undertriage (%) Undertriage 2005 2006 2007 2008 2009 2010 2011 Other 1.01 0.77 2.87 1.66 1.44 1.48 1.31 Cardiac 2.16 1.44 1.64 1.60 2.15 1.60 1.14 Drunk 0.00 1.89 0.00 0.00 0.00 0.00 0.00 Poisoning 8.33 0.00 0.00 1.69 0.00 1.60 0.00 Oncological 95.74 0.56 0.00 1.03 3.03 2.47 0.00 Neurologic 2.42 2.32 2.96 3.67 3.05 1.40 2.46 Not identifi ed 1.72 2.13 1.87 2.20 2.04 2.74 3.50 Mental 1.74 0.83 1.05 1.17 0.00 0.42 0.39 Respiratory 2.30 4.16 3.76 3.75 2.57 2.72 2.46 Trauma 1.07 0.92 1.50 1.41 1.36 1.31 1.46 Table 1 (abstract P276). Overtriage (%) Table 1 (abstract P276). Overtriage (%) g Figure 1 (abstract P277). Hospital evacuation. Figure 2 (abstract P277). Emergency service taking care of patients during evacuation. Table 1 (abstract P276). Overtriage (%) Overtriage 2005 2006 2007 2008 2009 2010 2011 Other 3.03 2.47 5.30 4.95 4.06 4.58 4.17 Cardiac 13.23 15.30 27.06 23.72 19.28 19.17 20.14 Drunk 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Poisoning 10.19 18.92 17.91 8.47 8.85 15.20 11.81 Oncological 0.00 2.78 1.05 1.03 3.03 2.47 0.00 Neurologic 9.97 10.05 15.59 15.18 14.57 13.32 13.32 Not identifi ed 13.03 13.15 11.84 11.07 10.33 11.79 7.23 Mental 1.30 0.41 2.09 2.34 2.30 1.27 2.35 Respiratory 10.67 10.89 22.09 18.63 16.29 18.23 14.12 Trauma 12.97 12.68 10.42 8.59 6.78 7.74 7.56 Table 2 (abstract P276). Undertriage (%) Figure 1 (abstract P277). Hospital evacuation. Figure 1 (abstract P277). Hospital evacuation. Figure 1 (abstract P277). Hospital evacuation. Figure 2 (abstract P277). Emergency service taking care of patients during evacuation. Figure 2 (abstract P277). Emergency service taking care of patients during evacuation. Undertriage 2005 2006 2007 2008 2009 2010 2011 Other 1.01 0.77 2.87 1.66 1.44 1.48 1.31 Cardiac 2.16 1.44 1.64 1.60 2.15 1.60 1.14 Drunk 0.00 1.89 0.00 0.00 0.00 0.00 0.00 Poisoning 8.33 0.00 0.00 1.69 0.00 1.60 0.00 Oncological 95.74 0.56 0.00 1.03 3.03 2.47 0.00 Neurologic 2.42 2.32 2.96 3.67 3.05 1.40 2.46 Not identifi ed 1.72 2.13 1.87 2.20 2.04 2.74 3.50 Mental 1.74 0.83 1.05 1.17 0.00 0.42 0.39 Respiratory 2.30 4.16 3.76 3.75 2.57 2.72 2.46 Trauma 1.07 0.92 1.50 1.41 1.36 1.31 1.46 Figure 2 (abstract P277). Emergency service taking care of patients during evacuation. L Escobar Alvaro1, J Jimenez Gonzalez1, A Pelaez Ballesta1, A Corbatón Anchuelo2 S105 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Results Our research indicated that disaster medicine should be established systematically or it is necessary to compile a compendium of disaster medicine from a broad perspective or from a bird’s-eye and long-term view. The Japanese version was tentatively completed with 22 volumes as of the fi nancial year 2005, of which nearly three-quarters are written in Japanese. Although this worked partly during the above- shown catastrophe in Japan 2011, several problems are left to be solved; that is, the insuffi cient operation system of the Japan DMAT or Disaster Medical Assistant Team that seemed to have caused a large number of preventable deaths. Results Our research indicated that disaster medicine should be established systematically or it is necessary to compile a compendium of disaster medicine from a broad perspective or from a bird’s-eye and long-term view. The Japanese version was tentatively completed with 22 volumes as of the fi nancial year 2005, of which nearly three-quarters are written in Japanese. Although this worked partly during the above- shown catastrophe in Japan 2011, several problems are left to be solved; that is, the insuffi cient operation system of the Japan DMAT or Disaster Medical Assistant Team that seemed to have caused a large number of preventable deaths. Conclusion In less than 3 hours 225 patients were evacuated and 224 were given attention by the emergency service of Lorca’s hospital, with the support of personnel from other services of the hospital. The emergency service of the hospital continues to be operative in the building until evacuation is completed and in a fi eld hospital later. P278 Patients experiencing a traumatic injury related to war had lower Paediatric Trauma Scores (PTS) than those injured in a CA (PTS = 6.4 (5.7 to 7.0) vs. PTS = 9.0 (8.7 to 9.4) respectively, P <0.00001), higher ISS (ISS = 16.7 (13.8 to 19.6) vs. ISS = 8.9 (7.5 to 10.2) respectively, P <0.00001), NISS (P <0.0001), mortality rate predicted by the Trauma Injury Severity Score (TRISS, P <0.02) or A Severity Characterization of Traumas (ASCOT, P <0.001). The result is longer overall hospitalization of patients having WI (P < 0.001) and a higher number of surgeries (P <0.02). After the ER, 54% of patients with WI were hospitalized in the ICU (86% of them after surgery) but only 26% of patients involved in a CA (71% after surgery). As many patients with WI as involved in a CA (40%) were admitted to the ward (89% of patients with WI after surgery but only 63% of patients with injuries due to a CA). Thirty-three per cent of patients involved in a CA returned home and one was transferred, whereas only three patients with WI returned home after being in the ER, three patients were transferred and one died in the operating room. Observed paediatric mortality in our medical treatment facility was 2.9% (10 children out of 341): three children died of WI, three due to a CA and one of septic shock due to a medical cause. Factors involved in ICU mortality following medical retrieval: identifying diff erences between ICU survivors and nonsurvivors M Kennedy1, P Visser1, L Harriss2 y 1Ambulance Victoria, Melbourne, Australia; 2Monash University, Melbourne, Australia y 1Ambulance Victoria, Melbourne, Australia; 2Monash University, Melbourne, Australia l l d Critical Care 2013, 17(Suppl 2):P280 (doi: 10.1186/cc12218) Introduction The study aimed to determine factors related to ICU mortality in critically ill patients transferred by Adult Retrieval Victoria (ARV) medical staff . Patients who died in the ICU after interhospital transfer were compared with those who survived. Methods This is a retrospective cohort study of ARV cases between 1 January 2009 and 30 June 2010. Retrieval data were linked with data from the ANZICS CORE APD (Australia and New Zealand Intensive Care Society Centre for Outcome and Resource Evaluation Adult Patient Database). Victoria Data Linkage (VDL) performed linkage of the data. Data included demographic and clinical data obtained during transfer and data related to outcome measures in the ICU. Results Of the 601 cases transferred by ARV during the study period, 549 were eligible for linkage to 25,543 ANZICS APD case records for the same period. VDL matched 460 of these cases (83.8%). Logistic regression analysis revealed the variables associated with mortality were advanced age (OR = 1.02, 95% CI = 1.00 to 1.04, P = 0.02), cardiac principal referral problems (OR = 1.84, 95% CI = 1.02 to 3.32, P = 0.04), lower mean arterial blood pressure (OR = 0.97, 95% CI = 0.95 to 0.99, P = 0.005) and tachycardia (OR = 1.02, 95% CI = 1.00 to 1.03, P = 0.008) on arrival at the destination hospital. Conclusion War injuries are more prone to cause polytrauma than CA. According to the PTS, ISS, NISS, TRISS and ASCOT, children experiencing WI have higher severity scores and predicted mortality rate than others, stay longer in the hospital and have more surgeries. Conclusion Advanced age, lower mean arterial blood pressure and tachycardia towards the completion of transfer were associated with increased ICU mortality in this population. Clinicians should be aware of the additional risk for cardiac patients. P282 P282 Are physicians required during HEMS winch rescue missions? PB Sherren, C Hayes-Bradley, C Reid, B Burns, K Habig Greater Sydney Area HEMS, Sydney, Australia Critical Care 2013, 17(Suppl 2):P282 (doi: 10.1186/cc12220) Introduction A winch-capable helicopter emergency medical service (HEMS) off ers several advantages over standard rescue operations. Little is known about the benefi t of physician winching in addition to a highly trained paramedic. We analysed the mission profi les and interventions performed during rescues involving the winching of a physician in the Greater Sydney Area HEMS (GSA-HEMS). y g y p y p Methods A suggested algorithm for TCA was developed based on the Greater Sydney Area Helicopter Emergency Medical Service’s standard operating procedures and current available evidence. p g p Results An algorithm for the general management of TCA can be seen in Figure 1. In TCA, priority should be given to catastrophic haemorrhage control (tourniquets, direct pressure, haemostatic agents, pelvic and long bone splintage) and volume resuscitation. Simultaneous oxygena tion optimisation should occur with proactive exclusion of tension pneumothoraces with bilateral open thoracostomies. Cardiac ultrasound (US) should be used to help exclude cardiac tamponade and assist in prognostication. The US presence of true cardiac standstill versus low pressure state/pseudo-PEA, and an ETCO2 <1.3 kPa carries a grave prognosis in TCA. Given the high incidence of hypovolaemia, hypoxia and obstructive shock prior to TCA, the role of adrenaline and chest compressions are limited. Figure 2 shows a suggested algorithm for the management of penetrating TCA requiring prehospital thoracotomy. y y y Methods All winch missions involving a physician from August 2009 to January 2012 were identifi ed from the prospectively completed GSA-HEMS electronic database. A structured case-sheet review for a predetermined list of demographic data and physician-only interventions (POI) was conducted. Results We identified 130 missions involving the winching of a physician, of which 120 case sheets were available for analysis. The majority of patients were traumatically injured (90%) and male (85%) with a median age of 37 years. Seven patients were pronounced life extinct on the scene. A total of 63 POI were performed on 48 patients. Administration of advanced analgesia was the most common POI making up 68.3% of interventions. Patients with abnormal RTSc2 scores were more likely to receive a POI when compared with those with normal RTSc2 (P = 0.03). The performance of POI had no effect on median scene times (45 vs. 1. Lockey D, et al.: Ann Emerg Med 2006, 48:240-244. Algorithm for the resuscitation of traumatic cardiac arrest patients in a physician-staff ed helicopter emergency medical service PB Sh C R d b B B Methods Mega-disasters or catastrophes are researched basically on actual medical experience; that is, the 9/11 attack in 2001, Hurricane Katrina in 2005, Indian Ocean earthquake with tsunami in 2004, the Chernobyl incident in 1986 as well as the Higashinihon earthquakes with tsunami, which was followed by the severest degree of nuclear disaster in 2011. f PB Sherren, C Reid, K Habig, B Burns g Greater Sydney Area HEMS, Sydney, Australia y y , y y, Critical Care 2013, 17(Suppl 2):P281 (doi: 10.1186/cc12219) Introduction Survival rates following traumatic cardiac arrest (TCA) are known to be poor but resuscitation is not universally futile [1]. S106 Critical Care 2013, Volume 17 Suppl 2 htt // f / l t /17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 http://ccforum.com/supplements/17/S2 Figure 1 (abstract P281). Traumatic cardiac arrest algorithm. Figure 1 (abstract P281). Traumatic cardiac arrest algorithm. There are distinct diff erences in the pathophysiology between medical cardiac arrests and TCA. Traumatic pathologies associated with an improved chance of successful resuscitation include hypoxia, tension pneumothorax and cardiac tamponade [1]. The authors believe a separate algorithm is required for the management of out-of-hospital TCA attended to by a highly trained physician and paramedic team. P282 43 minutes; P = 0.51). See Tables 1 and 2. Conclusion The suggested algorithm is designed for a highly trained physician-led prehospital team and aims to maximise the number of neurologically intact survivors in out-of-hospital TCA. Reference 1. Lockey D, et al.: Ann Emerg Med 2006, 48:240-244. Conclusion Our high POI rate of 40% coupled with long rescue times and the occasional severe injuries supports the argument for winching doctors. Not doing so would deny a signifi cant proportion of patients time-critical interventions, advanced analgesia and procedural sedation. S107 Critical Care 2013, Volume 17 Suppl 2 http://ccforum com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 http://ccforum.com/supplements/17/S2 Figure 2 (abstract P281). Thoracotomy algorithm. Figure 2 (abstract P281). Thoracotomy algorithm. Figure 2 (abstract P281). Thoracotomy algorithm. P283 Improving maintenance of critical care land and aeromedical transfer equipment D Ashton-Cleary, N Boyd Royal Cornwall Hospital, Truro, UK Critical Care 2013, 17(Suppl 2):P283 (doi: 10.1186/cc12221) Introduction The aim was to assess the content and state of repair of equipment carried for transfer of critical care patients to other hospitals. By chance, several items of date-expired stock were identifi ed in the transfer kit whilst moving a patient to a tertiary centre. This raised the possibility of a more extensive problem with the equipment bags. Due to the geographical location of our district general hospital we undertake around 70 transfers of critical care patients to other hospitals per year (16% by air) and it is clearly important that our equipment is well maintained for these journeys. Table 1 (abstract P282). Demographic data, timings and Coded Revised Trauma Score (RTSc2) Total (n = 120) Male 102 (85) Median (IQR) age (years) 37 (26 to 53) Received a physician-only intervention 48 (40) Pronounced life extinct on arrival 7 (5.8) RTSc2 7.8408 104 (86.7) 7.0001 to 7.8407 2 (1.7) 6.0001 to 7.0000 4 (3.3) 5.0001 to 6.0000 4 (3.3) 4.0001 to 5.0000 0 (0) <4.0001 6 (5) Table 2 (abstract P282). Interventions performed Physician-only intervention Number of interventions (n = 63) Analgesia/procedural sedation Intravenous ketamine 42 (66.7) Intravenous fentanyl 1 (1.6) Fascia iliaca compartment block 1 (1.6) Airway management Rapid sequence induction and intubation 4 (6.3) Surgical airway 1 (1.6) Circulatory support Adult intraosseous access 1 (1.6) Blood transfusion 2 (3.2) Orthopaedic manipulation of joint/limb 6 (9.5) Thoracostomy 1 (1.6) Diagnostic ultrasound 1 (1.6) Hypertonic saline administration 3 (4.8) Data presented as n (%). Safety standards for intrahospital transfer of critical care patients D Ashton-Cleary Royal Cornwall Hospital, Truro, UK Critical Care 2013, 17(Suppl 2):P284 (doi: 10.1186/cc12222) Introduction The aim was to assess care of patients during intrahospital transfer. The UK Royal College of Anaesthetists has defi ned auditable standards for the care of patients and the training of escorting medical and nursing staff in this context [1]. p y y Conclusion Prehospital hyperoxemia did not infl uence the functional neurological outcome. One of the reasons for this fi nding could be the short arrival time to the trauma center where repeated analyses of arterial blood gases were performed. Therefore, correction of fraction of inspired oxygen according to the arterial blood gas analysis shortens the time of hyperoxemia, thus reducing neuronal brain damage. References f Methods Patients in a 27-bed combined general and neurosurgical critical care unit were studied in January 2011 and May 2012. Patients undergoing radiology department imaging or intervention were identifi ed from the electronic imaging library. Records of these transfers were sought in the critical care electronic notes and the standards of documentation graded on a fi ve-point scale (very good, good, average, minimal, absent). Documentation of the grade and training of escorting staff was also sought. Between the two study periods, a transfer safety checklist was introduced. 1. Beynon et al.: Brain tissue oxygen monitoring and hyperoxic treatment in patients with traumatic brain injury. J Neurotrauma 2012, 29:2109-2123. 1. Beynon et al.: Brain tissue oxygen monitoring and hyperoxic treatment in patients with traumatic brain injury. J Neurotrauma 2012, 29:2109-2123. 1. Beynon et al.: Brain tissue oxygen monitoring and hyperoxic treatment in patients with traumatic brain injury. J Neurotrauma 2012, 29:2109-2123. 2. Brenner et al.: Association between early hyperoxia and worse outcomes after traumatic brain injury. Arch Surg 2012, 16:1-5. p j y , 2. Brenner et al.: Association between early hyperoxia and worse outcomes after traumatic brain injury. Arch Surg 2012, 16:1-5. Results A total of 20.9% of 143 patients underwent one or more transfers in January 2011 (40 transfers). In May 2012, 26.4% of 151 patients underwent 57 transfers. In the fi rst period, documentation was graded as minimal (limited to a statement that the patient had left the critical care unit) or absent in 77.5% of transfers. In the 62.5% of patients transferred whilst on invasive ventilation, 88.0% had no documentation by the doctor and in 84.0% it was not known which doctor had escorted the patient. P285 Prehospital hyperoxemia does not infl uence the functional neurological outcome in polytraumatized patients with traumatic head injury V Vujanovic Popovic1, T Pelcl1, M Spindler1, Z Klemenc Ketis2, M Strnad1 1ZD dr. Adolfa Drolca Maribor, Slovenia; 2University of Maribor, Slovenia Critical Care 2013, 17(Suppl 2):P285 (doi: 10.1186/cc12223) g g g Results A total of 13.9% of drug items and 29.2% of equipment items had expired or would do so within 30 days of the initial assessment. The combined weight of one equipment and one drug bag was reduced from 14 to 9 kg (36% reduction) by introducing the new inventory. At reassessment in November 2012, only 10 items of equipment (3.2%) were expired or near to expiry and there were no expired drug items (4.1% near to expiry). In total, 0.3 kg (26 small items) of extraneous equipment had been added through over-restocking and was removed. Conclusion These bags are designed for a clinician to manage a patient when an emergency arises during transfer of a critical care patient. By the introduction of simple measures, the risks posed by expired items or cluttered equipment bags have almost been eradicated. Signifi cant weight savings have been made; this off ers improved ergonomics for staff and is also an important consideration for aeromedical operations. Our department was surprised to discover the extent of decline of our equipment and it may be that other departments would fi nd themselves in a similar position. The anaesthetic registrars who routinely escort the transfer patients have a vested interest to maintain this equipment and this has secured their buy-in to the new checking procedure with clear results. P284 Safety standards for intrahospital transfer of critical care patients D Ashton-Cleary Royal Cornwall Hospital, Truro, UK Critical Care 2013, 17(Suppl 2):P284 (doi: 10.1186/cc12222) P286 Innate immune response-mediated late increase in SuPAR in multi-trauma patients K Timmermans, M Kox, M Vaneker, P Pickkers Radboud University Nijmegen Medical Center, Nijmegen, the Netherlands Critical Care 2013, 17(Suppl 2):P286 (doi: 10.1186/cc12224) P282 Table 2 (abstract P282). Interventions performed Physician-only intervention Number of interventions (n = 63) Analgesia/procedural sedation Intravenous ketamine 42 (66.7) Intravenous fentanyl 1 (1.6) Fascia iliaca compartment block 1 (1.6) Airway management Rapid sequence induction and intubation 4 (6.3) Surgical airway 1 (1.6) Circulatory support Adult intraosseous access 1 (1.6) Blood transfusion 2 (3.2) Orthopaedic manipulation of joint/limb 6 (9.5) Thoracostomy 1 (1.6) Diagnostic ultrasound 1 (1.6) Hypertonic saline administration 3 (4.8) Data presented as n (%). Table 2 (abstract P282). Interventions performed Table 1 (abstract P282). Demographic data, timings and Coded Revised Trauma Score (RTSc2) transfer kit whilst moving a patient to a tertiary centre. This raised the possibility of a more extensive problem with the equipment bags. Due to the geographical location of our district general hospital we undertake around 70 transfers of critical care patients to other hospitals per year (16% by air) and it is clearly important that our equipment is well maintained for these journeys. Introduction The aim was to assess the content and state of repair of equipment carried for transfer of critical care patients to other hospitals. By chance, several items of date-expired stock were identifi ed in the S108 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Methods We maintain two identical sets of equipment (syringes, fl uid, airway management items, and so forth) and drug bags to take on transfers; one equipment and one drug bag taken on each trip. The contents of all four bags were checked and itemised. By careful consideration of the aims of the bags (to provide emergency equipment and drugs for managing one patient during an en-route emergency) a new inventory was devised. Excess items were removed to lighten the bags and improve accessibility to the essential items. Expired stock was removed. A daily checking procedure and tamper-proof seals on the bags were instigated and the bags were reassessed 12 months later. hospital protocols and specifi c training courses to improve standards of transfer medicine locally. The study also identifi ed our portable head CT scanner to have the potential to reduce transfers by 52% and so this has been strongly promoted. Reference Safety standards for intrahospital transfer of critical care patients D Ashton-Cleary Royal Cornwall Hospital, Truro, UK Critical Care 2013, 17(Suppl 2):P284 (doi: 10.1186/cc12222) There was only slight improvement in the second period (71.9% minimal or absent documentation, 80.0% no documentation by the doctor, 72.0% not known which doctor escorted). In the documentation available, six severe complications were noted during the second period (including episodes of severe bradycardia, hypotension and pupil dilatation). 3. Davis et al.: Both hypoxemia and extreme hyperoxemia may be detrimental in patients with severe traumatic brain injury. J Neurotrauma 2009, 26:2217-2223. Prehospital hyperoxemia does not infl uence the functional neurological outcome in polytraumatized patients with traumatic head injury y V Vujanovic Popovic1, T Pelcl1, M Spindler1, Z Klemenc Ketis2, M Strnad1 1ZD dr. Adolfa Drolca Maribor, Slovenia; 2University of Maribor, Slovenia Critical Care 2013, 17(Suppl 2):P285 (doi: 10.1186/cc12223) Introduction The association between hyperoxemia and neurological outcome in trauma patients is not clear. We examined the association between prehospital hyperoxemia and neurological outcome in polytraumatized patients. Introduction The association between hyperoxemia and neurological outcome in trauma patients is not clear. We examined the association between prehospital hyperoxemia and neurological outcome in polytraumatized patients. p y p Methods This was a retrospective study of polytraumatized patients with traumatic head injury who were endotracheal intubated and ventilated with supplemental oxygen (100%) in the prehospital emergency setting. Arterial partial oxygen pressure (PaO2) was measured after arrival at the hospital trauma center. We included patients with initial PaO2 above 160 mmHg (hyperoxemia group). The severity of the trauma was determined upon the admission to the hospital by the Injury Severity Scale (ISS) and the outcome was assessed at the discharge from the hospital using the Glasgow Coma Scale (GCS), Glasgow Outcome Scale (GOS) and Cerebral Performance Categories scale (CPC). Mann–Whitney’s test was used for data analysis. Results Sixty patients were involved in the study. Forty-eight (80%) of them were men and 86.7% sustained blunt trauma. Hyperoxemia was present in 41.6% of patients. Initial average ISS was 38, in patients with normoxemia 32.5 and in patients with hyperoxemia 35.4. Discharge GCS, GOS and CPC in the hyperoxemia group compared with the normoxemia group were 9.86 versus 9.33 (P = 0.503), 2.52 versus 2.24 (P = 0.613) and 3.10 versus 3.19 (P = 0.936) with the duration of hospitalization of 26.64 days versus 27.72 days (P = 0.984).l Reference 1. Morgan G: Critical care services. In Raising the Standard: A Compendium of Audit Recipes. 2nd edition. London: Royal College of Anaesthetists; 2006. P287 Single-dose estrogen infusion can amplify brain levels of Sonic hedgehog, a signal protein for neuro stem cells and repair following the indirect brain injury resulting after severe torso burns PE Pepe1, JG Wigginton1, JW Gatson1, J Simpkins2, D Maass1, K AbdelFattah1, AH Idris1, V Warren1, JP Minei1 1University of Texas Southwestern Medical Center, Dallas, TX, USA; 2University of North Texas, Fort Worth, TX, USA Critical Care 2013, 17(Suppl 2):P287 (doi: 10.1186/cc12225) Introduction Many proposed resuscitative therapies for cardiac arrest and trauma will require the earliest possible intervention and would occur under volatile circumstances, making true informed consent for clinical trials unfeasible. The purpose here was to report our experience using exception to informed consent during the inaugural pilot study of infusing estrogen for acute injury, the so-called RESCUE Shock study. Methods Fifty patients were enrolled in RESCUE Shock in which estrogen or placebo was infused as soon as possible in the emergency department for trauma patients with a low systolic blood pressure (<90 mmHg) at two level I trauma centers. They were all treated with a single-dose estrogen or placebo infusion within 2 hours using exception from informed consent following US federal guidelines. Introduction Severe burn patients are often noted to have subsequent neurocognitive problems. Experimentally, we have found striking, prolonged elevations of infl ammatory markers in the brain (for example, IL-6) even when the injury occurs in a remote anatomic location. This neuroinfl ammatory response can also be signifi cantly blunted by a single post-burn dose of estrogen. Sonic hedgehog (SHH), an important signaling protein found in the brain, controls and directs diff erentiation of neural stem cells, infl uencing brain regeneration and repair by generating new neurons throughout life. As estrogens not only blunt infl ammation but also exert an infl uence on a variety of stem cells, we hypothesized that 17β-estradiol (E2) might aff ect levels of SHH in the post-burn rat brain. Results Investigator-initiated exception from informed consent studies is feasible, with our FDA IND approval obtained in 31 days, IRB 1 approval in 25 days, and IRB 2 approval in 24 days. Community consultation/notifi cation was successfully accomplished with no one opting out and 47/50 enrolled patients or their legal representatives were notifi ed of participation (one died unidentifi ed, two died with no known contact). Innate immune response-mediated late increase in SuPAR in multi-trauma patients p K Timmermans, M Kox, M Vaneker, P Pickkers Radboud University Nijmegen Medical Center, Nijmegen, the Netherlands Critical Care 2013, 17(Suppl 2):P286 (doi: 10.1186/cc12224) K Timmermans, M Kox, M Vaneker, P Pickkers Radboud University Nijmegen Medical Center, Nijmegen, the Netherlands Critical Care 2013, 17(Suppl 2):P286 (doi: 10.1186/cc12224) Introduction The soluble form of urokinase-type plasminogen activator (suPAR) has been identifi ed as a marker for immune activation and is demonstrated to accurately predict outcome in patients with sepsis or infectious diseases. In multi-trauma patients a considerable immunological response also occurs that is related to multiple organ failure and patient outcome. We investigated the kinetics of suPAR, correlation with the immune response and outcome in multi-trauma patients. Conclusion On average our unit conducts nearly two critical care transfers each day. Severe complications seem to complicate at least 10% of these, stressing the risk, need for good care and ongoing training. The intervention made in this audit had little impact on the standard of documentation. However, it has raised the issue within the consciousness of the staff . It is important to identify interventions that have failed to reach a gold standard to provide the impetus to seek other solutions. As a result of this study, the author has devised new Methods Blood was obtained from adult multi-trauma patients (n = 63) on arrival at the emergency room (ER) of the Radboud University S109 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Nijmegen Medical Centre and days 1, 3, 5, 7, 10 and 14 following trauma. Plasma concentrations of TNFα, IL-6, IL-10, IFNγ, IL-8 and MCP-1 were determined by Luminex, and SuPAR concentrations using ELISA. Clinical data were collected from electronic patient fi les. Concentrations, areas under the curve (AUC) and regression coeffi cients were statistically analyzed. Spearman correlation coeffi cients were calculated and diff erences between survival/nonsurvival groups were analyzed using unpaired Student t tests. Methods Male rats (n = 44) were assigned randomly into three groups: controls/no burn (n = 4); burn/placebo (n = 20); and burn/E2 (n = 20). Burned rats received a 40% 3° TBSA dorsal burn, fl uid resuscitation and one dose of E2 or placebo (0.5 mg/kg intraperitoneally) 15 minutes post burn. Innate immune response-mediated late increase in SuPAR in multi-trauma patients Eight animals from each of the two burn groups (burn/placebo and burn/E2) were sacrifi ced at 24 hours and at 7 days, respectively (sham group at 7 days only), with four each of the two burn groups sacrifi ced at 45 days. Brain tissue samples were analyzed by ELISA for SHH. p Results SuPAR values at admission to the ER were higher in nonsurvivors compared with survivors (n = 16, mean ± SEM 4.1 ± 0.6 ng/ml vs. n = 40, 3.0 ± 0.2 ng/ml, P = 0.03). SuPAR levels increased in time. An increase of suPAR did not predict or precede death, however. SuPAR AUC from ER to day 5 tended to correlate with injury severity score (r = 0.5, P = 0.07). Plasma cytokines in the ER did not correlate with suPAR measured at the same time (for example, TNFα: r = 0.2, P = 0.37, IL-10: r = –0.02, P = 0.91), while cytokine concentrations at the ER did correlate with suPAR levels at days 3 (TNFα: r = 0.6, P <0.01, IL-10: r = 0.5, P = 0.02) and 5 (TNFα: r = 0.7, P <0.01). Results Mean levels of SHH levels were signifi cantly elevated within 24 hours as much as 45 days post injury in burned animals receiving the 17β-estradiol (>1,200 pcg/mg) as compared with the placebo- treated burned animals (<700 pg/mg) and controls (<300 pcg/mg). See Figure 1. g Conclusion Early, single-dose estrogen administration following severe burn injury signifi cantly elevated levels of SHH in brain tissue. This fi nding may represent an extremely novel and important pathway for both neuroprotection and neuroregeneration in burn patients. Conclusion Plasma concentrations of SuPAR measured at admission to the ER are associated with overall survival of multi-trauma patients. Furthermore, suPAR concentrations increased during hospital admission, with most pronounced increases found in patients that suff ered more serious injury and related to the innate immune response determined in the ER. These results indicate that suPAR is an innate immune response-induced late mediator in multi-trauma patients. Feasibility and experience of using exception from informed consent in a pilot study of immediate estrogen infusion for hypotensive trauma patients hypotensive trauma patients JG Wigginton1, PE Pepe1, V Warren1, K AbdelFattah1, JW Gaston1, J Simpkins2, JP Minei1, D Maass1, AH Idris1 1University of Texas Southwestern Medical Center, Dallas, TX, USA; 2University of North Texas, Fort Worth, TX, USA Critical Care 2013, 17(Suppl 2):P288 (doi: 10.1186/cc12226) P288 Feasibility and experience of using exception from informed consent in a pilot study of immediate estrogen infusion for hypotensive trauma patients JG Wigginton1, PE Pepe1, V Warren1, K AbdelFattah1, JW Gaston1, J Simpkins2, JP Minei1, D Maass1, AH Idris1 1University of Texas Southwestern Medical Center, Dallas, TX, USA; 2University of North Texas, Fort Worth, TX, USA Critical Care 2013, 17(Suppl 2):P288 (doi: 10.1186/cc12226) P287 The average number of days to verbal notifi cation of patients or advocates was 6.55 days (range 0 to 51 days) as the study team began notifi cation only after the patient or family was able to reasonably understand information about the study. No one decided against continued follow-up. Overall, patients and their families were very enthusiastic about participation and the data safety monitoring board had no safety concerns after reviewing all study data. Figure 1 (abstract P287). y y Conclusion Although delayed notice of participation occurs for many justifi able reasons, the use of exception from informed consent for novel, time-sensitive resuscitation studies is not only crucial, but can be feasible, and well accepted by patients, their advocates and communities at large. Level of lipopolysaccharide-binding protein and microbial landscape with account of severity of sepsis syndromes in polytrauma Level of lipopolysaccharide-binding protein and microbial landscape with account of severity of sepsis syndromes in polytrauma β Methods A total of 168 male rats were assigned randomly to one of three groups: (1) Sham burn (no actual injury) group (n = 8); (2) Placebo (no treatment) burn group (n = 80); and (3) E2 (estrogen treatment) burn group (n = 80). Groups 2 and 3 had 40% TBSA third-degree dorsal burns, early fl uid resuscitation and, 15 minutes post burn, 0.5 mg/kg intraperitoneal estrogen (Group 3) or placebo (Group 2). From each group of 80, eight animals were sequentially sacrifi ced (and cardiac tissue was sampled for IL-6, TNFα, IL-1β) at one of 10 respective time points: 0, 0.5, 1, 2, 4, 6, 8, 18, 24 hours and day 7 post burn (at day 7 only for the eight shams). The markers were measured by ELISA method.i Introduction The aim was estimation of the clinical and predictive signifi cance of the level of lipopolysaccharide-binding protein (LBP) in blood serum and examination of microbial landscape with account of severity of sepsis syndromes in polytrauma. Methods Clinical examination was performed, which included 99 patients with polytrauma according to sepsis syndrome: SIRS (n = 18), local infection (n = 36), sepsis (n = 27), severe sepsis (n = 12), septic shock (n = 6) to the criteria of АССР/SССМ. The microorganism identifi cation was performed using iEMS Reader MF (Labsystems) with the La Chema multimicrotests. The content of LBP in blood serum was assessed with IMMULITE ONE (USA) with the reagents DPC (USA). Statistical analysis of the data was performed with Statistica 6.0. The numerical characteristics of variables are presented as Me (LQ to UQ). The analysis of diff erences was carried with the Kruskal–Wallis test for multiple comparison of independent groups, with Friedman one-way analysis of variance. The diff erences were statistically signifi cant with P <0.05. g y Results In the burned rats, 17β-estradiol signifi cantly decreased the cardiac levels of TNFα at all time points through 45 days post burn, with the sham animal levels (30 pg/mg) more comparable with the estradiol group (70 pg/mg), and signifi cantly less than the placebo animals (332 pg/mg). Similarly, IL-6 levels in the sham animals (70 pg/mg) were comparable with the estradiol group (86.5 pg/mg), and signifi cantly less than the placebo group (730 pg/mg), even at 45 days post burn. Risk factors for the development of complications following blunt chest trauma: a new risk stratifi cation tool C Battle1, H Hutchings2, S Lovett2, P Evans2 1Morriston Hospital, ABMU Health Board, Swansea, UK; 2Swansea University, Swansea, UK Critical Care 2013 17(Suppl 2):P292 (doi: 10 1186/cc12230) C Battle , H Hutchings , S Lovett , P Evans 1Morriston Hospital, ABMU Health Board, Swansea, UK; 2Swansea University, Swansea, UK , Critical Care 2013, 17(Suppl 2):P292 (doi: 10.1186/cc12230) p p Results One hundred patients with BAT presented; 71 had complete data. The accuracy of FAST in BAT was 59.2%; in these, 31 (43.7%) were confi rmed by CT and 11 (15%) by laparotomy. There were 29 (40.8%) inaccurate FAST scans, all confi rmed by CT. FAST had a specifi city of 94.7% (95% CI: 0.75 to 0.99) and sensitivity of 46.2% (95% CI: 0.33 to 0.60). A positive predictive value of 0.96 (0.81 to 0.99) and negative predictive value of 0.39 (0.26 to 0.54). Fisher’s exact test shows positive FAST is signifi cantly associated with intra-abdominal pathology (P = 0.001). Cohen’s chance corrected agreement was 0.3. Twenty- one out of 28 who underwent laparotomies had positive FAST results, indicating accuracy of 75% (95% CI: 57 to 87%). Critical Care 2013, 17(Suppl 2):P292 (doi: 10.1186/cc12230 Introduction The aim of the study was to investigate the risk factors for the development of complications following blunt chest trauma and to develop a risk stratifi cation tool to assist in the management of this patient group. The diffi culties in the management of this patient group in the emergency department (ED) due to the development of late complications are well recognised in the literature [1]. g Methods Between 2009 and 2011 a total of 276 patients were admitted to hospital from the ED of a regional trauma centre in Wales, with the primary diagnosis of blunt chest trauma. Patients with immediate life- threatening injuries were excluded. Data were collected retrospectively and included risk factors (age, number of rib fractures, comorbidity, pre-injury anticoagulant use, smoking status, oxygen saturations and respiratory rate on initial assessment in the ED), and outcomes (mortality, any pulmonary morbidity, length of stay of 7 days or more and need for ICU admission). Development of complications was defi ned as the occurrence of one or more of the outcomes investigated. Multivariable logistic regression using fractional polynomials was used to identify Conclusion Patients with false negative scans requiring therapeutic laparotomy is concerning. In unstable patients, FAST may help in triaging and identifying those requiring laparotomy. Negative FAST scans do not exclude abdominal injury. Further randomised control trials are recommended if the role of FAST is to be better understood. Reference 1. P289 Early administration of a single dose of parenteral estrogen decreases infl ammation in the heart for 45 days after severe burns PE Pepe, JG Wigginton, JW Gatson, AH Idris University of Texas Southwestern Medical Center, Dallas, TX, USA Critical Care 2013, 17(Suppl 2):P289 (doi: 10.1186/cc12227) Introduction Patients with severe burn injury experience a rapid elevation in multiple circulating proinfl ammatory cytokines, with the levels correlating with both injury severity and outcome. In animal S110 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Level of lipopolysaccharide-binding protein and microbial landscape with account of severity of sepsis syndromes in polytrauma f y gi Results In 81% of the critically ill patients with polytrauma the post- traumatic period was accompanied with development of infectious complications, Gram-negative (K. pneumoniae, Acinetobacter spp., E. coli) and Gram-positive (S. Epidermidis, S. aureus). Sepsis was diagnosed on 8 to 10 days in 45% of the patients. The signifi cant increase of LPS-BP was found in the fi rst 3 days of the follow-up, compared with the control values (6.7 times higher in SIRS group (χ2(n = 18, df = 3) = 52.8666, P <0.001); 9.9 times higher in the group with local infection (χ2(n = 36, df = 3) = 91.6629, P <0.001); 15.2 times higher in the sepsis group; 20.5 times higher in the severe sepsis group; 47.3 times higher in the septic shock group (χ2(n = 6, df = 3) = 11.0339, P = 0.0115)), whereas the fi rst positive results of the microbiological examination were obtained only on 5 to 7 days. The diagnostic sensitivity of threshold concentration of LBP in blood serum (335 mkg/ml) was 84%, diagnostic specifi city was 88% (ROC curve: 0.88). Conclusion Following severe burn injury in an animal model, an early single dose of estrogen can decrease the prolonged let alone the early onset of cardiac infl ammation. Based on these data, clinical studies of estrogen infusions should be seriously entertained as estrogen may not only be an inexpensive, simple adjunctive therapy in burn management, it may also obviate the need for many subsequent interventions altogether and even diminish mortality. P290 P290 Accuracy of FAST scan in blunt abdominal trauma in a major London trauma centre S Fleming1, R Bird2, K Ratnasingham3, S Sarker3, M Walsh2, B Patel3 1Royal National Orthopaedic Hospital Stanmore, London, UK; 2Royal London Hospital, London, UK; 3Barts Cancer Institute, London, UK Critical Care 2013, 17(Suppl 2):P290 (doi: 10.1186/cc12228) Conclusion The high incidence of the diagnostic levels of LBP in blood serum in patients with sepsis in the early term, before microbiological confi rmation of infection, allows one to use this parameter as an early marker of development of purulent septic complications conditioned by Gram-negative microfl ora. Introduction Blunt abdominal trauma (BAT) is a leading cause of morbidity and mortality. Rapid diagnosis and treatment with the Advanced Trauma Life Support guidelines are vital, leading to the development of focused assessment with sonography in trauma (FAST). Methods A retrospective study carried out from January 2007 to 2008 on all patients who presented with BAT and underwent FAST scan. All patients subsequently had a CT scan within 2 hours of admission or a laparotomy within 2 days. The presence of intraperitoneal free fl uid was interpreted as positive. P291 models, accumulations of these cytokines have been observed in remote organs, including the heart, brain and lungs. However, data are lacking regarding the long-term levels of cytokines in the heart following severe burn injury and also how infusion of parenteral estrogen, a powerful anti-infl ammatory agent, would aff ect these levels. Using a rat model, we studied the eff ects of a full-thickness third- degree burn on cardiac levels of IL-6 and TNFα over 45 days with and without 17β-estradiol infusion. Level of lipopolysaccharide-binding protein and microbial landscape with account of severity of sepsis syndromes in polytrauma IM Ustyantseva, OI Khokhlova, OV Petukhova, YA Zhevlakova Federal Scientifi c Clinical Center of Miners’ Health Protection, Leninsk- Kuznetsky, Russia Critical Care 2013, 17(Suppl 2):P291 (doi: 10.1186/cc12229) Prediction score of indications for whole body computed tomography in blunt trauma patients Prediction score of indications for whole body computed tomography in blunt trauma patients H Oda, R Sasaki, A Hagiwara, A Kimura National Center For Global Health and Medicine, Tokyo, Japan Critical Care 2013, 17(Suppl 2):P294 (doi: 10.1186/cc12232) Prediction score of indications for whole body computed tomography in blunt trauma patients g p y p H Oda, R Sasaki, A Hagiwara, A Kimura National Center For Global Health and Medicine, Tokyo, Japan Critical Care 2013, 17(Suppl 2):P294 (doi: 10.1186/cc12232) g p y p H Oda, R Sasaki, A Hagiwara, A Kimura National Center For Global Health and Medicine, Tokyo, Japan Critical Care 2013, 17(Suppl 2):P294 (doi: 10.1186/cc12232) Introduction Whole body computed tomography (WBCT) appears to be useful for the early detection of clinically occult injury, although its indications have been controversial. The purpose of this study was to develop a clinical prediction score to clarify the indications for blunt trauma patients with multiple injuries (MI) who require WBCT. g g p p Conclusion The results of this study highlight the risk factors for the development of complications following blunt chest trauma. A risk stratifi cation tool has also been developed that could assist in the prediction of poor outcomes in this patient group. The next stage is to complete a prospective validation study. p p j q Methods We conducted a retrospective study of 173 patients with blunt trauma who underwent WBCT at our emergency center between June 2011 and July 2012. We chose the presence or absence of MI (Injury Severity Score ≥15) in need of surgical intervention as the outcome variable. We used bivariate analyses to identify variables potentially predicting the presentation of MI. The predictor variables were confi rmed by multivariate logistic regression analyses. We assigned a score based on the corresponding coeffi cients. Reference g gfi Results Among the 173 patients enrolled, 53 were in the MI group. Four predictors were found to be independently signifi cant by the logistic analysis: (1) body surface wound ≥3 regions, (2) positive focused assessment with sonography for trauma, (3) white blood cell count ≥11,000/μl, and (4) D-dimer ≥8 μg/ml. Score 1 was assigned to predictor (1), score 2 was assigned to predictors (2), (3) and (4). A prediction score was calculated for each patient by adding these scores. The area under the receiver operating characteristic curve was 0.89. Prediction score of indications for whole body computed tomography in blunt trauma patients No patients with a score of 1 or less had MI (Figures 1 and 2). P293 P293 Guidelines for chest drain insertion do not protect relevant anatomical structures J Bowness1, P Kilgour1, S Whiten1, I Parkin1, J Mooney2, P Driscoll1 1St Andrews University, St Andrews, Fife, UK; 2University of Salford, UK Critical Care 2013, 17(Suppl 2):P293 (doi: 10.1186/cc12231) Guidelines for chest drain insertion do not protect relevant anatomical structures Conclusion In patients with a score of 1 or 0, the presence of MI is less likely. These patients may not require WBCT, and selective CT scans of body parts based on clinical presentation should be considered. Figure 1 (abstract P294). Prediction versus MI probability (validation group). Figure 2 (abstract P294). Clinical scorings. Multivariate, logistic regression analyses. Introduction We have reported the risk of chest drain insertion inferior to the diaphragm when using current international guidelines [1]. Another complication is damage to signifi cant peripheral nerves, such as the long thoracic nerve causing winging of the scapula [2]. We assessed these risks using: the European Trauma Course method, a patient’s handbreadth below their axilla just anterior to the midaxillary line; the British Thoracic Society safe triangle [3]; and the Advanced Trauma Life Support (ATLS) course guidance [4]. Figure 1 (abstract P294). Prediction versus MI probability (validation group). Methods We used the above guidelines to place markers (representing chest drains) in the thoracic wall of 16 cadavers bilaterally (32 sides), 1 cm anterior to the midaxillary line. Subsequent dissection identifi ed the course and termination of the long thoracic nerve, the site of lateral cutaneous branches of intercostal nerves, and their relation to the markers. Results The long thoracic nerve was found in the fi fth intercostal space in 16 of 32 cases, always in or posterior to the midaxillary line. Contrary to the description in Grays’ Anatomy (40th edition) it terminated before the inferior border of serratus anterior. Most commonly it was found to end by branching in the fourth (right) or fi fth (left) intercostal space (range third to sixth). Lateral cutaneous branches of intercostal nerves were found in the fi fth intercostal space in 25 of 32 cases. Contrary to the description in Last’s Anatomy (12th edition) they always passed anterior to the midaxillary line (and marker). Figure 1 (abstract P294). Prediction versus MI probability (validation group). Figure 2 (abstract P294). Clinical scorings. Multivariate, logistic regression analyses. P294 risk factors and develop a risk stratifi cation tool. The signifi cant risk factors in the model were selected using backward elimination with the Akaike Information Criterion (AIC) at a signifi cance level of 0.05. The c index and the Hosmer–Lemeshow (H–L) test were calculated to assess discrimination and calibration of the risk stratifi cation tool respectively. Results A total of 161 patients out of the 276 admitted developed complications following blunt chest trauma. Implementation of backward elimination using AIC values resulted in a fi nal model based on the signifi cant risk factors; age, oxygen saturations, number of rib fractures, presence of chronic lung disease and pre-injury anticoagulant use (all P <0.05). The c index for the tool was 0.80 and the H–L score was 9.22 (P = 0.32), indicating good predictive capabilities of the tool. Risk factors for the development of complications following blunt chest trauma: a new risk stratifi cation tool C Battle1, H Hutchings2, S Lovett2, P Evans2 1Morriston Hospital, ABMU Health Board, Swansea, UK; 2Swansea University, Swansea, UK Critical Care 2013 17(Suppl 2):P292 (doi: 10 1186/cc12230) Smith J: Focused assessment with sonography in trauma (FAST): should its role be reconsidered? Postgrad Med J 2010, 86:285-291. S111 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 1. Ahmad MA, et al.: Assessment of severity of chest trauma. Injury 2010, 41:981-983. P296 P296 Severe hyperkalemia is prevalent and can be detected by quantitative electrocardiographic fi ndings and medication history of calcium channel blocker among the patients with symptomatic or extreme bradycardia SB Chon Kangwon National University Hospital, Chuncheon, South Korea Critical Care 2013, 17(Suppl 2):P296 (doi: 10.1186/cc12234) P296 Severe hyperkalemia is prevalent and can be detected by quantitative electrocardiographic fi ndings and medication history of calcium channel blocker among the patients with symptomatic or extreme bradycardia SB Ch Severe hyperkalemia is prevalent and can be detected by quantitative electrocardiographic fi ndings and medication history of calcium channel blocker among the patients with symptomatic or extreme bradycardia h Conclusion Despite the controversies over the role of fl uids in the prehospital environment, the carriage and use of blood is both feasible and safe in a physician-led helicopter emergency medical service. Reference Kangwon National University Hospital, Chuncheon, South Korea Critical Care 2013, 17(Suppl 2):P296 (doi: 10.1186/cc12234) Introduction Severe hyperkalemia (serum potassium ≥6.0 mmol/l with electrocardiography (ECG) change) should be detected and treated immediately [1,2]. Among symptomatic or extreme bradycardia patients, we sought its prevalence and prediction rule by history, vital sign, and ECG [3,4]. 1. Davenport R, et al.: J Trauma 2011, 70:90-95. Table 1 (abstract P295). Demographics of patients receiving a prehospital blood transfusion Table 1 (abstract P295). P296 Demographics of patients receiving a prehospital blood transfusion Total (n = 147) Male (%) 102 (69.3) Age (years), median (IQR) 34.5 (22 to 52) Mechanisms of injury (%) Motor vehicle collision 87 (59.1) Motor bike collision 20 (13.6) Pedestrian versus car 9 (6.1) Gunshot wound/stabbing 9 (6.1) Fall from a height 5 (3.4) Recreational 6 (4.1) Other 11 (7.5) Number of patients trapped on arrival (%) 45 (30.6) Scene time (minutes), mean (SD) 49.9 (27.8) Time from tasking to arrival at hospital (minutes), mean (SD) 126.5 (51.3) Heart rate, median (IQR) 115 (90 to 130) Systolic blood pressure (mmHg), median (IQR) 80 (65 to 105) RTSo 2, median (IQR) 5.967 (4.083 to 6.904) Total number of PRBC units transfused 382 Total number of PRBC units wasted 66 Volume of crystalloid (ml), median (IQR) 500 (0 to 1,500) Pronounced life extinct on scene 22 (15.0) Methods A retrospective cross-sectional study was performed on patients with symptomatic (heart rate (HR) ≤50/minute with dyspnea, chest pain, altered mentality, dizziness/syncope/presyncope, general weakness, oliguria, or shock) or extreme (HR ≤40/minute) bradycardia at an ED of an academic hospital from June 2008 to March 2012. Risk factors of severe hyperkalemia were chosen by multiple logistic regression analysis among history (gender, age, comorbidity, and current medication), vital sign, and ECG (maximal precordial T wave amplitude, PR and QRS intervals). Scoring index was derived by summing up of simplifi ed regression coeffi cients of independent risk factors. Results A total of 169 cases were enrolled. Mean age was 71.2 years (SD, 12.5 years). Females numbered 87 (51.5%). Thirty-six cases (21.3%) had severe hyperkalemia. Four variables were independent risk factors of severe hyperkalemia (simplifi ed scores in parentheses): medication of calcium channel blocker (CCB: 2); maximal precordial T ≥8.5 mV (2); PR (atrial fi brillation or junctional bradycardia: 1); and HR ≤40/minute (1). (Nagelkerke R2 = 0.503, AUC = 0.849 (95% CI 0.786 to 0.899).) Sensitivity and specifi city reached 0.75 and 0.83 when total score was ≥3. For score ≥4, positive likelihood ratio reached 5.54 (sensitivity 0.50, specifi city 0.91). Conclusion Severe hyperkalemia is prevalent among symptomatic or extreme bradycardia patients and could be detected immediately by a scoring index composed of quantitative ECG parameters and history of medication of CCB. Prediction score of indications for whole body computed tomography in blunt trauma patients Conclusion Placement 1 cm anterior to the midaxillary line minimises risk to the long thoracic nerve and lateral cutaneous branches of intercostal nerves. We therefore conclude that not all areas of the British Thoracic Society safe triangle are indeed safe, and anteroposterior placement should follow the European Trauma Course and ATLS guidelines: just anterior to the midaxillary line (for example, 1 cm). References 1. Bowness J, et al.: It’s higher than you think: chest drains and the 5th ICS. Clin Anatomy, in press. 2. Kesienne EB, et al.: Tube thoracostomy: complications and its management. Pulm Med 2012:256878. 3. Laws D, et al.: BTS guidelines for the insertion of a chest drain. Thorax 2003, 58:ii53-ii59. 4. American College of Surgeons Committee on Trauma: Advanced Trauma Life Support for Doctors, Course Manual. Chicago: American College of Surgeons; 2008:108. Figure 2 (abstract P294). Clinical scorings. Multivariate, logistic regression analyses. Figure 2 (abstract P294). Clinical scorings. Multivariate, logistic regression analyses. S112 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Table 2 (abstract P295). Interventions performed P295 Prehospital blood transfusion: 5-year experience of an Australian helicopter emergency medical service PB Sherren, B Burns Greater Sydney Area HEMS, Sydney, Australia Critical Care 2013, 17(Suppl 2):P295 (doi: 10.1186/cc12233) Introduction There is an emerging body of evidence suggesting that early packed red blood cell transfusion accompanied by fresh frozen plasma, while limiting crystalloids, confers a survival benefi t in major trauma [1]. Prehospital blood transfusion has been infrequently described, and concerns over expense, transfusion reactions, risk of disease transmission, short shelf half-life and diffi cult storage have limited the interest of prehospital providers. p p p Methods All Greater Sydney Area HEMS (GSA-HEMS) prehospital missions involving a blood transfusion over a 66-month period were identifi ed and reviewed. The prospectively completed GSA-HEMS electronic database was utilised to identify patients and extract data.i Results We identifi ed 158 missions involving a prehospital blood transfusion, of which 147 patient datasets were complete. The majority of patients had a blunt mechanism of injury (93.9%) and were male (69.3%) with a median (IQR) age of 34.5 (22 to 52) years (Table 1). The majority of patients were haemodynamically unstable, with a median (IQR) heart rate and systolic blood pressure of 115 (90 to 130) and 80 (65 to 105) mmHg, respectively. Twenty-two patients (15.0%) were pronounced life extinct on the scene. A total of 382 units of packed red blood cells were transfused, with a median of 3 units (range 1 to 6). No early transfusion reactions were noted. A variety of prehospital interventions accompanied the transfusions, ranging from rapid sequence intubation through to thoracotomies (Table 2).l References References 1. Huber-Wgner S, et al.: Lancet 2009, 373:1455-1461. 2. Kimura A, et al.: Acad Emerg Med 2012, 19:734-735. References 1. Huber-Wgner S, et al.: Lancet 2009, 373:1455-1461. 2. Kimura A, et al.: Acad Emerg Med 2012, 19:734-735. Table 2 (abstract P295). Interventions performed Intervention Total (n = 147) Rapid sequence intubation 96 (65.3) Cold endotracheal intubation 15 (10.2) Surgical airway 1 (0.7) Thoracostomy (open or tube) 59 (40.1) Thoracotomy 3 (2.0) Pelvic binder or fracture splintage 89 (60.5) Intraosseous insertion sites 22 (15.0) Humerus 19 Tibia 10 Femur 1 Tourniquet application 15 (10.2) Hypertonic saline administration 16 (10.9) E-FAST performed 27 (18.4) Negative results 9 Positive results (fi ndings) 18 Abdominal free fl uid 14 Pneumothorax 2 Haemothorax 1 Thoracic ultrasound only; all negative 2 (1.3) Cardiac ultrasound only; no cardiac motion 1 (0.7) Focused cardiac ultrasound in resuscitation E Fish, L Fuchs, G Gulati, D Talmor, A Oren-Grinberg E Fish, L Fuchs, G Gulati, D Talmor, A Oren Grinberg Beth Israel Deaconess Medical Center, Boston, MA, USA g Beth Israel Deaconess Medical Center, Boston, MA, USA Results Multivariate logistic regression analysis indicated the following three prehospital variables as signifi cant high-ranking factors for predicting favorable 1-month outcomes: shockable initial rhythm (odds ratio (OR), 5.87; 95% CI, 5.23 to 6.60), witnessed arrest (OR, 3.05; 95% CI, 2.83 to 3.28), and age (≥18 to <71 years; OR, 3.24; 95% CI, 2.97 to 3.53). Using recursive partitioning analysis for development cohort data (2005 to 2008, n = 307,896), we stratifi ed prehospital risk: if OHCA with shockable initial rhythm was witnessed, the probability of CPC 1 to 2 was 20.0% (age, ≥18 to <71 years; grade 1) or 10.3% (age, ≥71 years; grade 2); if OHCA with shockable initial rhythm was unwitnessed, the probability was 6.8% (age, <81 years; grade 3) or 1.8% (age, ≥81 years; grade 4a); if OHCA with unshakable initial rhythm was witnessed, the probability was 1.4% (grade 4b); and if OHCA with unshakable initial rhythm was unwitnessed, the probability was 0.3% (grade 5). The c-statistics for risk stratifi cation of the development and validation cohorts (2009 external data, n = 82,330) were 0.853 (95% CI, 0.846 to 0.859) and 0.875 (95% CI, 0.865 to 0.885), respectively. The odds ratios for CPC 1 to 2 at 1 month between patients at very high and very low were 79.5 (95% CI, 72.5 to 87.4) and 122.1 (95% CI, 102.0 to 147.3), respectively, in these cohorts. , , , Critical Care 2013, 17(Suppl 2):P299 (doi: 10.1186/cc12237) Introduction Focused cardiac ultrasound is recognized as a vital tool in critical care medicine. Few studies, however, have examined the utility of this modality in resuscitation. While integration of ultrasound can supplement resuscitation by potentially establishing the etiology of cardiac arrest, it must be done safely to avoid interruption of compressions. The aim of this study was to examine the integration of focused cardiac ultrasound into resuscitations in our hospital. Methods We performed a retrospective observational study of patients undergoing resuscitation at Beth Israel Deaconess Medical Center, Boston, USA, from 2009 to 2012. Inclusion criteria were age >17 years Figure 1 (abstract P299). Ultrasound fi ndings. References . Nyirenda MJ, et al.: Hyperkalaemia. BMJ 2009, 339:b4114. . Alfonzo AV, et al.: Potassium disorders – clinical spectrum and emergency Alfonzo AV, et al.: Potassium disorders – clinical spectrum and emergency S113 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 management. Resuscitation 2006, 70:10-25. management. Resuscitation 2006, 70:10-25. Methods We performed a prospective study between January 2011 and December 2012 at a university cancer reference hospital in Brazil. Consecutive patients undergoing cardiac arrest and receiving CPR were identifi ed, and a database was used to register baseline characteristics of patients, data for CPR and 30-day survival. During the fi rst 12 months of the study (phase 1), patients received CPR according to the institution’s protocol guided by diff erent physicians. During the last 11 months of the study (phase 2), a multidisciplinary Medical Emergency Team System consisting of a specialized team of ICU nurses, physiotherapists and physicians using an electronic calling system was responsible for the CPR. management. Resuscitation 2006, 70:10 25. 3. Neumar RW, et al.: Part 8: adult advanced cardiovascular life support: 2010 American Heart Association Guidelines for Cardiopulmonary Resuscitation and Emergency Cardiovascular Care. Circulation 2010, 122:S729-S767. 4. Severi S, et al.: Heart rate response to hemodialysis-induced changes in potassium and calcium levels. J Nephrol 2001, 14:488-496. Focused cardiac ultrasound in resuscitation p y Conclusion Prehospital risk stratifi cation (grade 1 to 5) of patients after OHCA using three prehospital factors (shockable initial rhythm, witnessed arrest, and age) accurately classifi es the severity of OHCA and may help clinicians guide in-hospital strategies. Reference 1. Rerisfeld GM, et al.: Survival in cancer patients undergoing in-hospital cardiopulmonary resuscitation: a meta-analysis. Resuscitation 2006, 71:152-160. i p Methods We analyzed data for 390,226 adult patients (age ≥18 years) after nontraumatic OHCA, from a prospectively recorded nationwide Utstein-style Japanese database for 2005 to 2009. The endpoint was 1-month survival with favorable neurological outcome (Cerebral Performance Category (CPC) 1 to 2). Prehospital risk stratifi cation following out-of-hospital cardiac arrest Prehospital risk stratifi cation following out-of-hospital cardiac arrest Y Goto1, T Maeda1, Y Goto2 1Kanazawa University Hospital, Kanazawa, Japan; 2Yawata Medical Center, Komatsu, Japan Critical Care 2013, 17(Suppl 2):P297 (doi: 10.1186/cc12235) p Results A total of 182 patients experienced in-hospital cardiac arrest and received CPR. Most patients were in hospital due to medical admission (82%), had solid tumors (85%), and had localized disease (60%). Pulseless electric activity was the predominant arrest rhythm (54%) and 45% of patients were resuscitated on the ICU. Overall 30-day survival was 9.3%. In the phase 1 of the study, 30-day survival was 3.7%, and in phase 2, survival was 14% (P = 0.017). There were no diff erences in the two phases of the study regarding baseline characteristics of patients, rhythm and place of CPR. Introduction Multivariate analyses have identifi ed factors that have enabled the development of sophisticated equations and scoring models with the ability to predict outcomes following out-of-hospital cardiac arrest (OHCA). However, implementation of such outcome prediction models in research and clinical practice has been slow. The more crucial aspect of these predictions is the lack of prehospital risk stratifi cation for OHCA patients. Prehospital risk stratifi cation for patients after OHCA may help clinicians guide in-hospital strategies, particularly in the emergency department. The purpose of this study was to develop a simple and generally applicable prehospital risk- stratifi cation scheme for patients after OHCA. Conclusion Overall survival from CPR in cancer patients compares favorably with survival rates in noncancer patients. A multidisciplinary Medical Emergency Team System increases survival in cancer patients undergoing cardiopulmonary resuscitation. P298 P298 Multidisciplinary Medical Emergency Team System increases survival in cancer patients undergoing cardiopulmonary resuscitation L Hajjar1, F Galas2, S Vieira2, J Almeida2, E Osawa2, C Park2, J Fukushima2, E Angelo2, A Pinheiro2, J Auler Jr2 1Heart Institute, São Paulo, Brazil; 2Cancer Institute of University of São Paulo, Brazil Critical Care 2013, 17(Suppl 2):P298 (doi: 10.1186/cc12236) Prehospital epinephrine administration and survival among patients with unshockable initial rhythm after out-of-hospital cardiac arrest g Methods Our patient, with a medical history of a laparoscopic repair of a symptomatic diaphragmatic hernia 6 months prior, presented herself at the emergency department with pain in the upper abdomen and nausea. The physical examination, laboratory tests and X-ray of the thorax were normal and she was sent home. Twenty-four hours later paramedics were summoned to our patient because of increased complaints. On arrival of the paramedics she had a normal electrocardiogram (ECG) and during the transfer from her bed to the stretcher she collapsed due to pulseless electric activity (PEA), for which cardiopulmonary resuscitation was started. Sinus rhythm and output was regained after several minutes and the patient was transported to the hospital. At arrival in the hospital, the X-ray of the thorax showed an intrathoracic stomach and a signifi cant mediastinal shift to the right. Results After emergency laparotomy, which concerned correcting the gastric herniation and resection of an ischemic part of stomach, the patient remained hemodynamically stable. Cardiac ischemia was ruled out based on ECG, laboratory fi ndings, cardiac ultrasound and cardiac computed tomography. The ultrasound in the emergency department did show a distended right ventricle and normal left function, which disappeared later (after repositioning the stomach), which is evidence for the mediastinal shift as a cause for the PEA.i Y Goto1, T Maeda1, Y Goto2 Y Goto1, T Maeda1, Y Goto2 Y Goto1, T Maeda1, Y Goto2 1Kanazawa University Hospital, Kanazawa, Japan; 2Yawata Medical Center, Komatsu, Japan 1Kanazawa University Hospital, Kanazawa, Japan; 2Yawata Medical Center, Komatsu, Japan Critical Care 2013, 17(Suppl 2):P302 (doi: 10.1186/cc12240) Critical Care 2013, 17(Suppl 2):P302 (doi: 10.1186/cc12240) Introduction Epinephrine has been a cornerstone of cardiac resuscitation and advanced cardiac life support since the 1960s. However, there is little evidence from clinical trials that epinephrine administration after out-of-hospital cardiac arrest (OHCA) improves long-term survival. There would be subsets of patients for whom epinephrine administration is in fact benefi cial. Our objective was to determine whether prehospital epinephrine administration would improve survival at 1 month in OHCA patients with unshockable initial rhythm. y Methods We analyzed data for 383,045 adult OHCA patients with unshockable initial rhythm, from a prospectively recorded nationwide Utstein-style Japanese database for 2007 to 2010. We divided these patients into two cohorts: prehospital epinephrine administration cohort (n = 30,237) and non-epinephrine administration cohort (n = 352,808). Multidisciplinary Medical Emergency Team System increases survival in cancer patients undergoing cardiopulmonary resuscitation Methods We designed a before-and-after study based on a 3-month retrospective assessment of the adult victims of OOH-CA in 2009, before the implementation of the ALERT protocol in the Liege dispatching centre, and the prospective evaluation of the same 3-month period in 2011, immediately after the implementation of this protocol. Data were extracted from ambulance, paramedical and medical intervention teams fi les, as well as the audio recordings of the dispatching centre. Results There were 233 OOH-CAs detected in the fi rst period and 235 in the second. Victims were predominantly male (59%, both periods), aged 66 and 64 years, respectively. Callers were family members in 52% in 2009 and 64% in 2011. In 2009, only 9.9% victims benefi ted from bystander CPR, while there were 22.5% in 2011 (P < 0.0002). Reasons for protocol underuse were: assistance not off ered (42.3%), caller remote from the victim (20.6%) or emotionally distressed (15.5%). Mean no- fl ow time decreased from 253 seconds in 2009 to 168 seconds in 2011 (NS). Ten victims were admitted in ROSC to hospital in 2009 and 13 in 2011 (P = 0.09). p p g p g Methods We designed a before-and-after study based on a 3-month retrospective assessment of the adult victims of OOH-CA in 2009, before the implementation of the ALERT protocol in the Liege dispatching centre, and the prospective evaluation of the same 3-month period in 2011, immediately after the implementation of this protocol. Data were extracted from ambulance, paramedical and medical intervention teams fi les, as well as the audio recordings of the dispatching centre.i i g p g Results There were 233 OOH-CAs detected in the fi rst period and 235 in the second. Victims were predominantly male (59%, both periods), aged 66 and 64 years, respectively. Callers were family members in 52% in 2009 and 64% in 2011. In 2009, only 9.9% victims benefi ted from bystander CPR, while there were 22.5% in 2011 (P < 0.0002). Reasons for protocol underuse were: assistance not off ered (42.3%), caller remote from the victim (20.6%) or emotionally distressed (15.5%). Mean no- fl ow time decreased from 253 seconds in 2009 to 168 seconds in 2011 (NS). Ten victims were admitted in ROSC to hospital in 2009 and 13 in 2011 (P = 0.09). Multidisciplinary Medical Emergency Team System increases survival in cancer patients undergoing cardiopulmonary resuscitation Conclusion Focused cardiac ultrasound is a feasible adjunct to ALS resuscitation and may assist in the early identifi cation of reversible causes of cardiac arrest. Care must be taken to ensure no interruptions to cardiac compressions are made by performance during pulse checks. Further studies are needed to examine the outcomes associated with its integration into resuscitations. Further studies are needed to examine the outcomes associated with its integration into resuscitations. Conclusion Using the ALERT protocol in the Liege dispatching centre signifi cantly improved the numbers of patients in whom bystander CPR was attempted. Dispatchers must embrace this new opportunity to help callers and be encouraged to accept the responsibility of initiating such assistance. Reference Acute intrathoracic gastric herniation as a rare cause of cardiac arrest DW Hoelen, AL Van Duijn, CL Meuwese, JP Ruurda, MA Sikma UMC Utrecht, the Netherlands Critical Care 2013, 17(Suppl 2):P300 (doi: 10.1186/cc12238) Multidisciplinary Medical Emergency Team System increases survival in cancer patients undergoing cardiopulmonary resuscitation L Hajjar1, F Galas2, S Vieira2, J Almeida2, E Osawa2, C Park2, J Fukushima2, E Angelo2, A Pinheiro2, J Auler Jr2 1Heart Institute, São Paulo, Brazil; 2Cancer Institute of University of São Paulo, Brazil Critical Care 2013, 17(Suppl 2):P298 (doi: 10.1186/cc12236) Introduction In the last years, ICU admission has been high in cancer patients due to increased survival related to advances in treatment. Patients with cancer reportedly have poor outcomes from cardiopulmonary resuscitation (CPR). The goal of this study was to evaluate the eff ectiveness of a multidisciplinary Medical Emergency Team System in the CPR outcomes in cancer patients. Figure 1 (abstract P299). Ultrasound fi ndings. S114 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Introduction Early bystander cardiopulmonary resuscitation (CPR) is a key factor in improving survival from out-of-hospital cardiac arrest (OOH-CA). The ALERT algorithm, a simple and eff ective compression-only telephone CPR protocol, has the potential to help bystanders initiate CPR. This study evaluates the eff ectiveness of the implementation of this protocol in the Liege dispatching centre. and performance of focused cardiac ultrasound during resuscitation. Recorded variables included admission data, code demographics and diagnosis, therapy outside standard ALS protocol, time to return of spontaneous circulation (ROSC), and outcomes data. p Results Of 33 eligible patients, 12 were excluded due to incomplete cardiac ultrasound reports; 21 patients were enrolled. Cohort demographics included: 57.1% male, average age 64.3 years, average BMI 28.5 kg/ m2, average Charlson score 5.5. Resuscitations took place on the wards (52.4%), ICU (42.9%), or operating room (4.8%). Most patients had an initial unshockable rhythm (71.5%). The most common ultrasound fi nding was cardiac standstill (47.6%) (Figure 1). The most common intervention as a result of the ultrasound was initiation of a pressor infusion (33.3%), of which 71.4% were ionotropes. Additional therapies included blood transfusion (4.8%), heparin (9.5%), tPA (4.8%), cardiac catheterization (4.8%), and surgery (9.5%). ROSC was achieved in 37.5% of patients; average time to ROSC was 13 minutes. A total 33.3% of patients who underwent ALS were alive at hospital discharge and 28.6% at 1 year. p e e tat o o t s p otoco t e ege d spatc g ce t e. References 1. Deakin CD, et al.: Resuscitation 2010, 81:1305-1352. 1. Deakin CD, et al.: Resuscitation 2010, 81:1305-1352 2. Robson R. Resuscitation 2010, 81:1453-1454. 3. Breitkreutz R. et al.: Crit Care Med 2007, 35:S150-S161. Acute intrathoracic gastric herniation as a rare cause of cardiac arrest DW Hoelen, AL Van Duijn, CL Meuwese, JP Ruurda, MA Sikma UMC Utrecht, the Netherlands Critical Care 2013, 17(Suppl 2):P300 (doi: 10.1186/cc12238) 1. Ghuysen A, Collas D, Stipulante S, Donneau AF, Hartstein G, Hosmans T, Vantroyen B, D’Orio V: Dispatcher-assisted telephone cardiopulmonary resuscitation using a French-language compression-only protocol in volunteers with or without prior life support training: a randomized trial. Resuscitation 2011, 82:57-63. Introduction In this case report, we describe a patient who presented with a cardiac arrest as a result of an obstructive shock, which progressed into cardiac arrest, caused by an acute para-esophageal gastric herniation. P302 Prehospital epinephrine administration and survival among patients with unshockable initial rhythm after out-of-hospital cardiac arrest The endpoints were 1-month survival after OHCA, prehospital return of spontaneous circulations (ROSCs), and 1-month survival with favorable neurological outcome (Cerebral Performance Category (CPC) scale, categories 1 to 2) at 1 month. Conclusion We are the fi rst to describe a patient requiring cardiopulmonary resuscitation for progressive obstructive shock, due to an intrathoracic stomach. Especially after a laparoscopic repair of a diaphragmatic hernia, this is a rare cause for shock and cardiac arrest, which requires a diff erent medical approach. y Results The rate of 1-month survival was 3.72% for the epinephrine administration cohort and 2.49% for the non-epinephrine adminis- tration cohort, 17.9% versus 3.0% for prehospital ROSC, and 0.57% versus 0.77% for CPC 1 to 2 (all P <0.0001). Positive associations were observed between epinephrine administration and 1-month survival (adjusted odds ratio (aOR), 1.18; 95% CI, 1.11 to 1.27), and prehospital ROSC (aOR, 5.50; 95% CI, 5.29 to 5.72; all P <0.0001). Negative association was observed between epinephrine administration and CPC 1 to 2 (aOR, 0.56; 95% CI, 0.48 to 0.66; P <0.0001). Multivariate logistic analysis revealed that age (<66 years; aOR, 4.31; 95% CI, 2.47 to 8.01), total dose Impact of termination of resuscitation for out-of-hospital cardiopulmonary arrest in Japan Results A total of 430 cardiopulmonary arrest events occurred in- hospital between January 2010 and December 2011. A total of 326 patients were excluded because 252 occurred in the ICU, 70 in the coronary care unit and four in the stork unit. In total, 104 patients were enrolled; 50.9% where female and 49.1% male. Median age was 61 years. A total 41.3% of the arrests were due to respiratory arrest and 58.7% due to cardiac arrest. Out of 104 patients who developed cardiopulmonary arrest, 47 (45.19%) occurred during regular working hours, and 57 (54.81%) occurred after regular working hours (P = 0.0081). The event survival was 87.23% during regular working hours compared with 47.37% for the patients who developed cardiopulmonary arrest after regular working hours (P <0.0001). In total, 53.19% of those who developed cardiopulmonary arrest during regular working hours were discharged alive from the ICU compared with 26.32% of those who developed cardiopulmonary arrest after regular working hours.i Introduction What can one anticipate from the introduction of termination of resuscitation (TOR) for patients suff ering out-of-hospital cardiopulmonary arrest (OHCA) in Japan? Irrespective of whether patients have made a living will requesting that medics do not attempt resuscitation, eff orts are made to resuscitate over 90% of OHCA patients in Japan [1,2]; the number of people resuscitated exceed 120,000 every year. The 2010 American Heart Association (AHA) Guidelines for cardiopulmonary resuscitation (CPR) and emergency cardiovascular care (ECC) defi ned the criteria for TOR; this initiative may help reduce the number of unnecessary hospital transports by 40 to 60% and hold down medical costs [3]. p p y g g Conclusion Event survival and survival to discharge were signifi cantly higher in patients who developed cardiopulmonary arrest during regular working hours. Methods This was a single-center retrospective cohort study of patients who suff ered OHCA and were transported to our hospital between April 2009 and March 2011. We investigated the patients’ characteristics, whether they met the TOR criteria, and their outcome at the time of hospital discharge. g Reference 1. Peberdy MA,Ornato JP, Larkin L, et al.: Survival from in-hospital cardiac arrest during nights and weekends. JAMA 2008, 299:785-792. P305 Results A total of 195 patients (mean age, 69 years), 67% of whom were male, were transported to our hospital after suff ering OHCA. Cardiopulmonary arrest was witnessed in 52 cases (27%). P301 Implementation of dispatcher-assisted cardiopulmonary instructions using the ALERT protocol: preliminary results in Belgium A Ghuysen1, S Stipulante1, M El FAssi1, A Donneau2, V D’Orio1, R Tubes1 1CHU – Ulg Liège, Belgium; 2Liège University, Liège, Belgium Critical Care 2013, 17(Suppl 2):P301 (doi: 10.1186/cc12239) A Ghuysen1, S Stipulante1, M El FAssi1, A Donneau2, V D’Orio1, R Tubes1 1CHU – Ulg Liège, Belgium; 2Liège University, Liège, Belgium Critical Care 2013, 17(Suppl 2):P301 (doi: 10.1186/cc12239) S115 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 of epinephrine (1 mg; aOR, 3.65; 95% CI, 2.61 to 5.18), call-response time (<5 minutes; aOR, 3.58; 95% CI, 1.98 to 6.69), witnessed arrest (aOR, 2.17; 95% CI, 1.51 to 3.16), and pulseless electrical activity as an initial rhythm (aOR, 2.02; 95% CI, 1.46 to 2.80) were signifi cantly associated with CPC 1 to 2 at 1 month in the epinephrine administration cohort. of epinephrine (1 mg; aOR, 3.65; 95% CI, 2.61 to 5.18), call-response time (<5 minutes; aOR, 3.58; 95% CI, 1.98 to 6.69), witnessed arrest (aOR, 2.17; 95% CI, 1.51 to 3.16), and pulseless electrical activity as an initial rhythm (aOR, 2.02; 95% CI, 1.46 to 2.80) were signifi cantly associated with CPC 1 to 2 at 1 month in the epinephrine administration cohort. detection and resuscitation of cardiopulmonary arrest are crucial for better clinical outcome. We conducted our study to evaluate event survival of in-hospital cardiopulmonary arrest after regular working hours in nonmonitored areas of a tertiary-care center. of epinephrine (1 mg; aOR, 3.65; 95% CI, 2.61 to 5.18), call-response time (<5 minutes; aOR, 3.58; 95% CI, 1.98 to 6.69), witnessed arrest (aOR, 2.17; 95% CI, 1.51 to 3.16), and pulseless electrical activity as an initial rhythm (aOR, 2.02; 95% CI, 1.46 to 2.80) were signifi cantly associated with CPC 1 to 2 at 1 month in the epinephrine administration cohort. Conclusion In OHCA patients with unshockable initial rhythm, prehospital epinephrine administration signifi cantly increased the rate of survival at 1 month after cardiac arrest. The best single predictor for favorable neurological outcomes at 1 month following prehospital epinephrine administration after cardiac arrest was age (<66 years) followed by total dose of epinephrine (1 mg) and then by call-response time (<5 minutes). p p References 1. Berdowski J, et al.: Resuscitation 2010, 81:1479-1487. 2. Fukuda T: J Jpn Assoc Acute Med 2010, 23:101-108. 3. Morrison LJ, et al.: N Engl J Med 2006, 355:478-487. 4. Fukuda T: J Jpn Soc Intensive Care Med 2012, 19:275. Results TH was initiated in 93% (64/69) of OOHCA patients and 87% (40/46) with ischaemic cardiac aetiology underwent PCI. ICU survival was 58% (40/69); 65% (26/40) scoring CPC 1 to 2 and 35% (14/40) CPC 3 to 4 at ICU discharge. Two patients with CPC 2 improved to CPC 1 during their hospital stay. All patients with CPC 1 to 2 survived to hospital discharge; two required general rehabilitation before returning home. Only 43% (6/14) of CPC 3 to 4 patients survived to hospital discharge; none returned home. Two went into hospice care, one was repatriated to another hospital and three went to neuro-rehab. No CPC 3 to 4 patients improved CPC scores after ICU discharge. Overall hospital survival was 46%. See Table 1. Cognitive performance and cap out-of-hospital cardiac arrestl Introduction Before the introduction of primary percutaneous coronary intervention (PCI) and therapeutic hypothermia (TH) to out-of-hospital cardiac arrest (OOHCA) management, survival to hospital discharge with intact neurological function was poor [1,2]. We aimed to quantify the survival and degree of neurological impairment in OOHCA patients admitted to our ICU since the adoption of post-OOHCA bundles. Conclusion In our study, 65% of the patients who were transported to the hospital after OHCA met the criteria for TOR. Outcomes for patients who met the TOR criteria were signifi cantly worse than those who did not meet the criteria (2.4% vs. 14.5%, P <0.005). In Japan, eff orts are made to resuscitate almost all individuals who suff er OHCA, but 75% of those patients die within a day. In light of the fact that even the medical cost for each of these patients who die within a day amounts to US$1,500 [4], the introduction of TOR will have a particularly strong impact in Japan. Methods Sixty-nine consecutive OOHCA patients admitted to the ICU at Hammersmith Hospital from 1 January 2011 to 30 June 2012 were identifi ed and reviewed from hospital databases. Cognitive status was scored using Cerebral Performance Category (CPC); 1 to 2 normal–mild and 3 to 4 moderate–severe neurological impairment. Scores were determined from ICU summaries, occupational and physiotherapy reports. Hospital discharge outcomes were determined from hospital databases. Impact of termination of resuscitation for out-of-hospital cardiopulmonary arrest in Japan The 2010 AHA Guidelines for CPR and ECC regarding the criteria for TOR were applied in 126 cases (65%), of whom 113 (90%) were dead on arrival, and 13 were successfully resuscitated and admitted. The outcomes for these 13 patients were as follows: 10 died in the hospital, two patients were discharged with a Glasgow Pittsburgh Cerebral Performance Category (CPC) score of 1, and one patient was transferred to another hospital with a CPC score of 3. P301 Methods A retrospective chart review of all adult patients who developed in-hospital cardiopulmonary arrest between January 2010 and December 2011. Working hours are defi ned as 07:00 to 17:00 Saturday to Wednesday. Event survival is defi ned as return of spontaneous circulation (ROSC) for more than 20 minutes. Adult patients 18 years and above who suff ered from in-hospital cardiopulmonary arrest were included. Patients were excluded if they had cardiopulmonary arrest in the emergency department, had implantable cardiovascular device, arrested in monitored areas or had pre-existing DNR orders. Data analysis was accomplished using SAS, version 9.3 (SAS institute, Inc., Cary, NC, USA). Conclusion In OHCA patients with unshockable initial rhythm, prehospital epinephrine administration signifi cantly increased the rate of survival at 1 month after cardiac arrest. The best single predictor for favorable neurological outcomes at 1 month following prehospital epinephrine administration after cardiac arrest was age (<66 years) followed by total dose of epinephrine (1 mg) and then by call-response time (<5 minutes). P303 Impact of termination of resuscitation for out-of-hospital cardiopulmonary arrest in Japan T Fukuda, N Ohashi, M Gunshin, T Matsubara, S Nakajima, Y Kitsuta, N Yahagi Graduate School of Medicine, The University of Tokyo, Japan Critical Care 2013, 17(Suppl 2):P303 (doi: 10.1186/cc12241) References 1. Stub et al.: Am J Cardiol 2011, 107:522-527. 2. Girotra et al.: N Engl J Med 2012, 367:1912-1920. 3. Herlitz et al.: Heart 2003, 89:25-30. 4. Cronier et al.: Crit Care 2011, 15:122. 1. Stub et al.: Am J Cardiol 2011, 107:522-527. 2. Girotra et al.: N Engl J Med 2012, 367:1912-1920. 3. Herlitz et al.: Heart 2003, 89:25-30. 4. Cronier et al.: Crit Care 2011, 15:122. 2. MacLeod et al.: J Cardiothorac Vasc Anesth 2012, 26:1007-1014. patients: does it mean something? p g C Genbrugge, I Meex, S Scheyltjens, J Dens, C De Deyne ZOL, Genk, Belgium Critical Care 2013, 17(Suppl 2):P306 (doi: 10.1186/cc12244) p g C Genbrugge, I Meex, S Scheyltjens, J Dens, C De Deyne ZOL, Genk, Belgium Critical Care 2013, 17(Suppl 2):P306 (doi: 10.1186/cc12244) p g C Genbrugge, I Meex, S Scheyltjens, J Dens, C De Deyne Introduction During cardiopulmonary resuscitation (CPR) monitoring possibilities are limited. Parnia and colleagues investigated the feasibility and role of near-infrared spectroscopy (NIRS) during CPR in cardiac arrest patients (CA) [1]. NIRS could have a role in predicting return of spontaneous circulation (ROSC). Recently, the Equanox® with four wavelengths sensor was validated to provide absolute data on regional cerebral saturation [2]. We measured cerebral oxygenation (rSO2) during CPR with NIRS technology and analyzed the diff erences between initial cerebral saturations in patients achieving ROSC compared with patients without ROSC. p g y Conclusion As previously reported [2], IHCA was associated with a worse prognosis than OHCA. The OHCA survival rate was better than reported elsewhere [3]. The percentage of IHCA attributed to MI was low. Only one OHCA patient was referred for emergency PCI. Routine coronary angiography with ad hoc PCI in VF OHCA has been associated with increased survival [4]. Greater availability of PCI post OHCA could further improve mortality in patients with a primary cardiac pathology. Further investigation should include management of noncardiogenic cardiac arrest. p p Methods With IRB approval, rSO2 was measured with NIRS during resuscitation in 18 out-of hospital CA patients. The Equanox® Advance (NONIN), a NIRS monitoring device that measures absolute rSO2 values, was applied on the right side of the patient’s forehead when the medical emergency team arrived in a resuscitation setting. Placement of the probe did not interfere with the advanced life support algorithm. The sensor remained on the patient’s forehead during resuscitation, and if ROSC was reached the probe was removed on arrival at the emergency department. If ROSC was not achieved, the probe was removed prehospital. ROSC was defi ned as ROSC during more than 20 minutes. The Mann–Whitney test was utilized for comparison of survivor and nonsurvivor data. Student’s t test was performed to compare the initial rSO2. 1. Parnia et al.: Resuscitation 2012, 83:982-985. P307 P307 Intensive care admission after cardiac arrest: cardiac versus noncardiac causes and consequences for treatment T Hargreaves1, H Kingston1, M Crews1, M Mogk2, I Welters3 1Royal Liverpool University Hospital, Liverpool, UK; 2Moredata GmbH, Giessen, Germany; 3Liverpool University, Liverpool, UK Critical Care 2013, 17(Suppl 2):P307 (doi: 10.1186/cc12245) Intensive care admission after cardiac arrest: cardiac versus noncardiac causes and consequences for treatment T Hargreaves1, H Kingston1, M Crews1, M Mogk2, I Welters3 1Royal Liverpool University Hospital, Liverpool, UK; 2Moredata GmbH, Giessen, Germany; 3Liverpool University, Liverpool, UK Critical Care 2013, 17(Suppl 2):P307 (doi: 10.1186/cc12245) Introduction In-hospital (IHCA) and out-of-hospital cardiac arrest (OHCA) are associated with high mortality [1]. Studies suggest that up to 68% of OHCA is due to acute coronary syndrome, with 38% requiring percutaneous coronary intervention (PCI) [1]. However, revascularisation may not always be available or address the underlying pathology. This study aimed to establish the prevalence of diff erent aetiologies for IHCA and OHCA, and the use of emergency treatment for these patients. disability (CPC 3 to 4) was seen in 19%, greater than previously reported [1]. A higher proportion (35%) of patients discharged from the ICU had moderate–severe neurological disability; most subsequently died in hospital (62%). These fi gures may represent better ICU outcomes subsequent to adoption of OOHCA bundles but suggest further work is required in neuro-disabled survivors. Methods A retrospective case-note review of all patients admitted between 2008 and 2011 to the ICU of an inner-city university hospital after OHCA or IHCA. Biometric data, presenting cardiac rhythm, presumed cause of arrest, management and outcomes were recorded. The Kruskal–Wallis test was used for numerical data analysis and chi- square test for categorical data. Survival from cardiopulmonary arrest after regular working hours in a tertiary-care hospital: retrospective study Introduction Detection and treatment of cardiopulmonary arrest and their antecedents may be less eff ective at night and weekend than weekdays because of hospital staffi ng and response factors [1]. Early Conclusion OOHCA patients admitted to our ICU had a 46% chance of surviving to hospital discharge. Most patients left hospital with good neurological status (CPC 1 to 2); moderate–severe neurological S116 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 disability (CPC 3 to 4) was seen in 19%, greater than previously reported Table 1 (abstract P305). Discharge and neurological status of OOHCA patients Discharge Hospital discharge destination Neuro- Score ICU Hospital Home rehab Repatriation Hospice CPC 1 20 22 19 0 3 0 CPC 2 6 4 4 0 0 0 CPC 3 4 3 0 3 0 0 CPC 4 10 3 0 0 1 2 Total 40 32 23 3 4 2 disability (CPC 3 to 4) was seen in 19%, greater than previously reported [1]. A higher proportion (35%) of patients discharged from the ICU had moderate–severe neurological disability; most subsequently died in hospital (62%). These fi gures may represent better ICU outcomes subsequent to adoption of OOHCA bundles but suggest further work is required in neuro-disabled survivors. References 1. Cobbe SM, et al.: BMJ 1999, 312:1633-1637. 2. Holzer M, et al.: N Engl J Med 2002, 346:549-556. P306 Start value of cerebral saturation in prehospital cardiac arrest patients: does it mean something? C Genbrugge, I Meex, S Scheyltjens, J Dens, C De Deyne ZOL, Genk, Belgium Critical Care 2013, 17(Suppl 2):P306 (doi: 10.1186/cc12244) Table 1 (abstract P305). Discharge and neurological status of OOHCA patients Discharge Hospital discharge destination Neuro- Score ICU Hospital Home rehab Repatriation Hospice CPC 1 20 22 19 0 3 0 CPC 2 6 4 4 0 0 0 CPC 3 4 3 0 3 0 0 CPC 4 10 3 0 0 1 2 Total 40 32 23 3 4 2 Table 1 (abstract P305). Discharge and neurological status of OOHCA patients Discharge Hospital discharge destination 1. Parnia et al.: Resuscitation 2012, 83:982-985. References 1. Cobbe SM, et al.: BMJ 1999, 312:1633-1637. 1. Cobbe SM, et al.: BMJ 1999, 312:1633-1637. 2. Holzer M, et al.: N Engl J Med 2002, 346:549-556. q g Results Data were analysed for 64 patients – 44 (69%) following OHCA and 20 (31%) after IHCA. The median APACHE score for OHCA was 17 and for IHCA was 23.5 (P = 0.001). Hospital survival rate was 10% (n = 2) for IHCA and 38.6% (n = 17) for OHCA (P <0.02). A total of 34.1% (n = 15) OHCA were due to myocardial infarction (MI) compared with 10% (n = 2) of IHCA (P <0.05). The most prevalent aetiologies were MI (n = 17), hypoxia (n = 10), cardiac other (n = 5), sepsis (n = 4), arrhythmia (n = 3) and PE (n = 3). In two IHCA patients more than one likely cause of arrest was reported and in 19 cases no cause was identifi ed. The presenting rhythm was ventricular fi brillation (VF) in 45.3% (n = 29), pulseless electrical activity in 32.8% (n = 21) and asystole in 20.3% (n = 13). A total of 9.4% (n = 6) were thrombolysed and one (1.6%) patient was referred for emergency PCI. 2. Holzer M, et al.: N Engl J Med 2002, 346:549-556. 1. Parnia et al.: Resuscitation 2012, 83:982-985. 2. MacLeod et al.: J Cardiothorac Vasc Anesth 2012, 26:1007-1014. P308 P308 Comparison of 1-year neurological outcome between intra-hospital and extra-hospital cardiac arrest survivors submitted to mild therapeutic hypothermia in a community-based setting in Brazil CA Abreu Filho1, A Andrade2, A Neto2, S Santos2, M Bracco2, E Silva1, A Baruzzi1, J Teixeira2 1Hospital Israelita Albert Einstein, São Paulo, Brazil; 2Hospital Municipal Dr. Moysés Deutsch, São Paulo, Brazil Critical Care 2013, 17(Suppl 2):P308 (doi: 10.1186/cc12246) 2 Results Of the 18 patients, nine patients had ROSC (survivors). The initial rhythm was the same in both groups, six patients in each group had asystole as initial rhythm. In the group of survivors were six female patients, in the nonsurvivors were two female patients. The mean age in ROSC and no-ROSC groups is respectively 75.8 years (SD ±12.8) and 69.4 years (SD ±22.9, P = 0.48). The mean rSO2 at arrival of the emergency medical team was 31.56% (SD ±29.4) and 12.78% (SD ±12.7) respectively in the ROSC group and no-ROSC group (P = 0.1). The mean time between collapse and start of CPR (basic life support of bystanders) was 6.9 minutes (SD ±8.2) in the no-ROSC group and 8.2 minutes (SD ±7.08, P = 0.69) in the ROSC group. Introduction Mild therapeutic hypothermia (MTH) is the most powerful therapy to improve survival and neurologic outcome after out-of- hospital cardiac arrest. Such benefi t may also occur for unconscious patients after in-hospital cardiac arrest. The aim is to compare 1-year evolution of neurological outcomes of patients treated with MTH after in-hospital versus out-of-hospital cardiac arrest. Conclusion Initial rSO2 values in out-of hospital CA patients with ROSC showed a tendency towards higher values compared with nonsurvivors, but no signifi cant diff erence could be demonstrated, probably related to the small number of patients included in this preliminary report. References Methods A prospective study of patients treated with MTH after cardiac arrest in a community hospital in São Paulo, Brazil. After return of spontaneous circulation, unconscious survivors received MTH using topical ice and cold saline infusions in order to achieve a 32 to 34°C goal temperature, within 6 hours of cardiac arrest, and maintained P310 P310 Infrared pupillometry for outcome prediction after cardiac arrest and therapeutic hypothermia T Suys, N Sala, AO Rossetti, M Oddo Lausanne University Hospital, Lausanne, Switzerland Critical Care 2013, 17(Suppl 2):P310 (doi: 10.1186/cc12248) Conclusion Midterm neurological outcome of MTH after in hospital cardiac arrest seems to be not as good as after out-of-hospital cardiac arrest. Delay in hypothermia initiation, older age and associated comorbidities could explain the worse evolution of this group of patients. Introduction Sedation and therapeutic hypothermia (TH) modify neurological examination and alter prognostic prediction of coma after cardiac arrest (CA). Additional tools, such as EEG and evoked potentials, improve prediction of outcome in this setting, but are not widely available and require signifi cant implementation. i Methods Using a new device for infrared pupillometry, we examined the value of quantitative pupillary light reactivity (PLR) to predict outcome in comatose post-CA patients treated with TH. Twenty-four comatose CA patients treated with TH (33°C, 24 hours) were prospectively studied. The percentage variation in PLR was measured during TH (12 hours from CA), using the NeuroLight Algiscan® (IDMED, Marseille, France). For each patient, three consecutive measures were performed and the best value was retained for analysis. The relationship of PLR P309 Primary percutaneous coronary angioplasty and therapeutic hypothermia in out-of-hospital cardiac arrest R Hunt, M Holl, A Bailey, P Macnaughton Derriford Hospital, Plymouth, UK Critical Care 2013, 17(Suppl 2):P309 (doi: 10.1186/cc12247) Introduction The benefi t of primary percutaneous intervention (PCI) in the management of out-of-hospital cardiac arrest (OHCA) is not clear cut [1]. It has historically been used in patients with ST elevation on post-resuscitation electrocardiogram (ECG) although this is a poor predictor of acute coronary occlusion after cardiac arrest [2]. This study investigates the benefi t of PCI regardless of post-resuscitation ECG. Benefi t is widely claimed for therapeutic hypothermia, so cooling parameters were included. Table 1 (abstract P310). False-positive and false-negative rates for outcome (% of patients) p Methods We analysed all 41 consecutive adults admitted post OHCA to a university hospital ICU between January 2010 and December 2011. Patients received PCI regardless of ECG changes. A Cox proportional hazards model was used to determine the relationship between PCI, cooling and survival to discharge. Routinely collected data such as demographics and details of resuscitation (OHCA Utstein data) were also included. Results Survival to hospital discharge was 41% with 29% of survivors discharged to a neurological rehabilitation centre. References 1. Kern KB: Optimal treatment of patients surviving out-of-hospital cardiac arrest. JACC Cardiovasc Interv 2012, 5:597-605. 2. Spaulding CM, et al.: Immediate coronary angiography in survivors of out- of-hospital cardiac arrest. N Engl J Med 1997, 336:1629-1633. 2. Spaulding CM, et al.: Immediate coronary angiography in survivors of out- of-hospital cardiac arrest. N Engl J Med 1997, 336:1629-1633. 3. Dumas F, et al.: Immediate percutaneous coronary intervention is associated with better survival after out-of-hospital cardiac arrest. Circ Cardiovasc Interv 2010, 3:200-207. 3. Dumas F, et al.: Immediate percutaneous coronary intervention is associated with better survival after out-of-hospital cardiac arrest. Circ Cardiovasc Interv 2010, 3:200-207. References S117 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 with other studies suggesting benefi t for primary PCI regardless of the post-resuscitation ECG [3]. Cooling was not found to improve survival to discharge but further analysis is required to determine impact on neurological function. for 24 hours. Esophageal temperature was monitored; continuous intravenous sedation-analgesia was maintained for 48 hours after initiation of MTH. The Glasgow Outcome Scale (GOS) was used to analyze the neurological outcomes after hospital discharge. for 24 hours. Esophageal temperature was monitored; continuous intravenous sedation-analgesia was maintained for 48 hours after initiation of MTH. The Glasgow Outcome Scale (GOS) was used to analyze the neurological outcomes after hospital discharge. Results From January 2009 to April 2012, 84 patients submitted to MTH were divided into two groups: Group 1, 54 patients presented out- of-hospital cardiac arrest; and Group 2, 30 patients had intra-hospital cardiac arrest. Both groups were similar regarding to gender; Group 2 tended to be older (mean age 44.3 years vs. 33.5 years, P = 0.07), and had more frequently asystole as the cardiac arrest rhythm (45% vs. 15 %, P = 0.10). Group 2 had shorter duration of resuscitation (12.3 minutes vs. 33.7 minutes, P = 0.03), longer time to hypothermia initiation (309.3 minutes vs. 212.8 minutes, P = 0.04), longer hospital stay after cardiac arrest (50.7 days vs. 32.4 days, P = 0.01) and worse neurological outcome, characterized by the presence of GOS ≤3 at 30 days (68.4% vs. 35.1%, P = 0.03). Hospital mortality was 5.5% in Group 1 and 13.3% in Group 2 (P = 0.21). The 1-year survival rate was 85.1% in Group 1 and 83.3% in Group 2 (P = 0.43); after 1 year follow-up, GOS ≤3 was present in 30.4% of Group 2 patients and in 11.1% of Group 1 patients (P = 0.04). Eff ect of moderate hyperventilation and induced hypertension on cerebral tissue oxygen saturation in comatose post-cardiac arrest patients treated with hypothermia T Suys, N Sala, M Oddo Lausanne University Hospital, Lausanne, Switzerland Critical Care 2013, 17(Suppl 2):P311 (doi: 10.1186/cc12249) l Methods We performed an observational study in 10 comatose patients after cardiac arrest. Patients were treated with hypothermia for 24 hours. Blood viscosity was measured ex vivo using a Contraves LS300 viscometer. Mean fl ow velocity in the middle cerebral artery (MFVMCA) was measured by transcranial Doppler (TCD) at the same time points. T Suys, N Sala, M Oddo T Suys, N Sala, M Oddo Lausanne University Hospital, Lausanne, Switzerland Critical Care 2013, 17(Suppl 2):P311 (doi: 10.1186/cc12249) Lausanne University Hospital, Lausanne, Switzerland y p Critical Care 2013, 17(Suppl 2):P311 (doi: 10.1186/cc12249) Introduction Maintenance of adequate brain perfusion is an essential component of post-resuscitation care. Mean arterial pressure (MAP) and PaCO2 are important determinants of brain perfusion; however, no precise guidelines exist for optimal MAP and PaCO2 targets in comatose post-cardiac arrest (CA) patients. an essential ssure (MAP) however, no n comatose ive cerebral erventilation ents treated dy including hospital CA face cooling NIRS system erventilation PaCO2 from zation, MAP infusion of SctO2 were 19 minutes) Results are nt reduction 5), P <0.001). and IH tests (n = 10 patients). Figure 1 (abstract P312). Figure 1 (abstract P312). p p Methods Using NIRS, we examined changes in non-invasive cerebral tissue oxygen saturation (SctO2) following moderate hyperventilation (HV) and induced hypertension (IH) in comatose CA patients treated with therapeutic hypothermia (TH). A prospective pilot study including comatose patients successfully resuscitated from out-of-hospital CA treated with TH (33°C for 24 hours, using cold saline and surface cooling device), monitored for continuous SctO2 with the Foresight NIRS system (CAS Medical Systems, Branford, CT, USA). Moderate hyperventilation was induced for approximately 30 minutes by decreasing PaCO2 from ~40 to ~30 mmHg, at stable MAP. After PaCO2 normalization, MAP was increased from ~70 to ~90 mmHg by intravenous infusion of norepinephrine, at stable PaCO2. Eff ects of MV and IH on SctO2 were analyzed with a paired t test. Figure 1 (abstract P312). Figure 1 (abstract P312). y p Results Ten patients (mean age, 69.5; mean time to ROSC, 19 minutes) were studied during the stable TH maintenance phase. Results are summarized in Figure 1. MV was associated with a signifi cant reduction of SctO2 from baseline (75% (73 to 76) to 69% (67.5 to 71.5), P <0.001). 0 patients). Figure 1 (abstract P311). Changes in SctO2 after moderate HV and IH tests (n = 10 patients). Figure 1 (abstract P311). P312f Eff ects of viscosity on cerebral blood fl ow after cardiac arrest L Bisschops, G Pop, S Teerenstra, J Van der Hoeven, C Hoedemaekers Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands Critical Care 2013, 17(Suppl 2):P312 (doi: 10.1186/cc12250) p g Conclusion Quantitative PLR appears highly accurate and superior to standard neurological examination to predict outcome in patients with post-CA coma. Further study is warranted to confi rm these promising fi ndings. Acknowledgements Supported by Grants from the Swiss National Science Foundation (FN 320030_138191) and the European Critical Care Research Network (ECCRN). Introduction After cardiac arrest, microcirculatory reperfusion dis orders develop despite adequate cerebral perfusion pressure. Increased blood viscosity strongly hampers the microcirculation, resulting in plugging of the capillary bed, arteriovenous shunting and diminished tissue perfusion. The aim of the present study was to assess blood viscosity in relation to cerebral blood fl ow in patients after cardiac arrest. P310 Multivariate analysis using a Cox proportional hazards model showed PCI to be an independent predictive factor of survival, unrelated to ECG (hazards ratio, 0.0583; 95% CI, 0.0076 to 0.4485). Cooling had no signifi cant impact on patient survival. See Figure 1. p p g Conclusion In this small retrospective study primary PCI appears to be an independent predictor of survival after OHCA. This is consistent Figure 1 (abstract P309). Comparison of hazards ratios post Cox analysis. Figure 1 (abstract P309). Comparison of hazards ratios post Cox analysis. S118 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 No signifi cant changes in SctO2 were found after IH (74 (72 to 76) vs. 75 (73 to 75.5), P = 0.24). with survival and neurological outcome (CPC scores) at 3 months was analyzed, and the predictive value of PLR was compared with that of standard clinical examination (motor response and brainstem refl exes) performed at 48 hours from CA. Conclusion Moderate HV was associated with signifi cant reduction in cerebral saturation, whilst IH may be detrimental after CA and TH, whilst increasing MAP to supranormal levels with vasopressors does not improve cerebral oxygenation. These data stress the importance of strict control of PaCO2 following CA and TH to avoid secondary cerebral ischemic insults. Results Quantitative PLR was strongly associated with survival (median left-eye PLR 14% (11 to 16%) variation in survivors vs. 5.5% (4 to 8.5%) in nonsurvivors, P < 0.0001) and 3-month neurological outcome (14% (11 to 21%) in patients with CPC 1 to 2 vs. 5.5% (4 to 8.5%) in those with CPC 3 to 5, P <0.0001). Comparable fi ndings were obtained using right-eye PLR. A PLR >10% was 100% predictive of patient prognosis, with false-positive and false-negative rates of 0% for outcome. Clinical examination was signifi cantly associated with outcome; however, motor response (MR) and brainstem refl exes (BRS) yielded higher false- positive and false-negative rates than PLR (Table 1). Acknowledgements MO is supported by Grants from the Swiss National Science Foundation (FN 320030_138191) and The European Critical Care Research Network (ECCRN). P312f Eff ect of moderate hyperventilation and induced hypertension on cerebral tissue oxygen saturation in comatose post-cardiac arrest patients treated with hypothermia Eff ect of moderate hyperventilation and induced hypertension on cerebral tissue oxygen saturation in comatose post-cardiac arrest patients treated with hypothermia T Suys, N Sala, M Oddo Lausanne University Hospital, Lausanne, Switzerland Critical Care 2013, 17(Suppl 2):P311 (doi: 10.1186/cc12249) Association between oxygenation and 6-month mortality during post-cardiac arrest care p M Skrifvars1, J Vaahersalo1, M Reinikainen2, S Bendel3, J Kurola3, M Tiainen1, R Raj1, V Pettilä1, T Varpula1, FinnResusci Study Group1 1Helsinki University Hospital, HUS, Finland; 2North Karelia Central Hosital, Joensuu, Finland; 3Kuopio University Hospital, Kuopio, Finland Critical Care 2013, 17(Suppl 2):P315 (doi: 10.1186/cc12253) M Skrifvars1, J Vaahersalo1, M Reinikainen2, S Bendel3, J Kurola3, M Tiainen1 R Raj1, V Pettilä1, T Varpula1, FinnResusci Study Group1 1Helsinki University Hospital, HUS, Finland; 2North Karelia Central Hosital, Joensuu, Finland; 3Kuopio University Hospital, Kuopio, Finland Critical Care 2013, 17(Suppl 2):P315 (doi: 10.1186/cc12253) g g y g g y Results We had 297 CA patients (168 SR, 129 NR) in whom CPR was done. Return of spontaneous circulation was achieved in 90 patients. TMH was induced in 57 patients (33 SR, 24 NR). Survival to hospital discharge was observed in 27 patients (18/33 (54.5%) SR, 9/24 (37.5%) NR), out of which 18 patients (10/33 (30%) SR, 8/24 (33%) NR) had good neurological outcome. On analysis, TMH was found to be associated with increased odds of survival to hospital discharge (although statistically not signifi cant) in SR patients compared with NR patients (odds ratio (OR) = 2.00; 95% CI = 0.68 to 5.85; P = 0.2837), but it was not associated with any better neurological outcome in terms of CPC score in patients presenting with SR rather than NR (OR = 0.87; 95% CI = 0.28 to 2.68; P = 1.0000). Rather, the odds for good neurological outcome were more in favour of NR (pulseless electrical activity/asystole). Introduction Optimal oxygenation level during post-cardiac arrest (PCA) care is currently undefi ned, and studies have suggested harm from hyperoxia exposure [1]. We aimed to assess the optimal oxygenation level and possible associations of time-weighted exposure to hyperoxia on outcome in patients during PCA care. Introduction Optimal oxygenation level during post-cardiac arrest (PCA) care is currently undefi ned, and studies have suggested harm from hyperoxia exposure [1]. We aimed to assess the optimal oxygenation level and possible associations of time-weighted exposure to hyperoxia on outcome in patients during PCA care. Methods We conducted a prospective observational cohort study in 21 ICUs in Finland between 2009 and 2010. The Utstein Guidelines were used for collecting resuscitation and PCA care data, such as initial rhythm and delay to return of spontaneous circulation (ROSC). P313 p Results Agreement between the diff erent sources of temperature measurement was generally high, with less than 10% outliers beyond ±1.96 SD. The best agreement was seen between intravascular tempera ture measurement and bladder temperature measurement (SD = 0.14°C), with vaginal measurements showing less agreement (SD = 0.23°C), and rectal measurements showing the least (SD = 0.31°C) with intravascular. Eff ect of moderate hyperventilation and induced hypertension on cerebral tissue oxygen saturation in comatose post-cardiac arrest patients treated with hypothermia T Suys, N Sala, M Oddo Lausanne University Hospital, Lausanne, Switzerland Critical Care 2013, 17(Suppl 2):P311 (doi: 10.1186/cc12249) Changes in SctO2 after moderate HV and IH tests (n = 10 patients). S119 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Results The median viscosity on admission was 9.12 (8.19 to 11.19) mPa. second, and remained stable at 3 and 6 hours after admission. From 6 hours after admission, viscosity decreased signifi cantly to 3.66 (3.12 to 4.04) mPa.second (P <0.001). Median MFVMCA was low (27.0 (23.8 to 30.5) cm/second) on admission, and signifi cantly increased to 63.0 (51.0 to 80.0) cm/second at 72 hours (P <0.001). There was a signifi cant association between viscosity and the MFVMCA (P = 0.0019). See Figure 1. where temperature is measured. Measurements of temperature can be made rectally, intravascularly, vaginally, or from the bladder, but agreement between these sites is often uncertain. We measured temperature at multiple sites during experimental induction and reversal of hypothermia in swine with a novel esophageal cooling device, hypothesizing that agreement between sites would fall within standard acceptance criteria (average diff erence ± 1.96 standard deviation of the diff erence) in Bland–Altman analyses. g Conclusion Viscosity decreases in the fi rst 3 days after cardiac arrest and is strongly associated with an increase in cerebral blood fl ow. Since viscosity is a major determinant of cerebral blood fl ow, repeated measurements may guide therapy to help restore cerebral oxygenation after cardiac arrest. f y Methods Five female Yorkshire swine (mean weight 65 kg) were anesthetized and cooled for 24 hours, then gradually rewarmed, with a novel esophageal heat transfer device powered by an external chiller (Gaymar MediTherm III). Swine temperature was measured intra- vascularly, rectally, and either vaginally or in the bladder. Comparisons between temperature readings were then made via Bland–Altman plots. Reference 1. Arrich J, Holzer M, Herkner H, Mullner M: Hypothermia for neuroprotection in adults after cardiopulmonary resuscitation. Cochrane Database Syst Rev 2009, 4:CD004128. Methods We reviewed medical records of all CA patients (in-hospital or out-of-hospital arrest) in whom cardiopulmonary resuscitation (CPR) was performed at our hospital from 1 February 2011 to 31 January 2012 (12 months). The following information was collected: fi rst documented rhythm, whether TMH done or not, and two outcome measures including survival to hospital discharge and neurological outcome at the time of hospital discharge. A measure of good neurological outcome was Cerebral Performance Category score 1 or 2 (CPC, fi ve- point scale; 1 = good cerebral performance to 5 = brain death). Then we quantifi ed the association of TMH with SR as well as NR for both the parameters of outcome – that is, survival to hospital discharge and good neurological outcome – by logistic regression analysis. Association between oxygenation and 6-month mortality during post-cardiac arrest care Measured arterial blood oxygen values during the fi rst 24 hours from admission to the ICU were divided into the following predefi ned oxygenation categories: low (<10 kPa), normal (10 to 19 kPa), intermediate (20 to 29 kPa), and high (>30 kPa). Exposure to hyperoxia was defi ned as paO2 levels higher than 40 kPa [1]. Time spent in diff erent oxygenation categories and the highest, lowest and median oxygen values during the fi rst 24 hours were calculated and included in separate multivariate regression models along with age, delay to ROSC, initial rhythm and the use of therapeutic hypothermia for the prediction of 6-month mortality. Conclusion Although TMH might be associated with better survival chances in patients presenting with SR, neurological outcome was no better (rather worse) in this group of patients when compared with patients with NR as the fi rst documented rhythm. Therapeutic mild hypothermia after cardiac arrest in shockable and nonshockable rhythms: does it improve both survival and neurological outcome? A Gupta Fortis Escorts Heart Institute, New Delhi, India Critical Care 2013, 17(Suppl 2):P313 (doi: 10.1186/cc12251) Introduction Although therapeutic mild hypothermia (TMH) after resuscitation from cardiac arrest (CA) has been postulated and studied to be associated with good outcome of the patients, there is no dearth of data that does not favour TMH. Our aim was to fi nd out whether TMH is associated with good outcome after CA in shockable rhythm (SR) compared with nonshockable rhythm (NR), in terms of survival as well as neurological outcome. Conclusion Bladder temperature measurements demonstrated the best agreement with intravascular temperature measurements during cooling with an esophageal heat transfer device; however, reasonable agreement was demonstrated with rectal and vaginal temperature measurements, suggesting that these sites are also acceptable for use. Reference Diff erences of calcium binding protein immunoreactivities in the young hippocampal CA1 region from the adult following transient ischemic damage Results NSE >45 occurred in 24/68 patients (35.3%) and invariably correlates with bad outcome. The positive predictive value (PPV) NSE >45 for bad outcome is 100%. No patient in this group ever had a GCS ≥12. NSE >33 and <45 occurred in 16/68 patients (23.5%). Thirteen out of 16 patients (81.2%) had bad outcome. However, 7/16 patients (43.8%) woke up at some time (GCS ≥12). NSE <33 occurred in 28/68 patients (41.2%), 17/28 patients (60.7%) had good outcome and 23/28 patients (88.4%) had GCS ≥12 at some time. The PPV NSE <33 for good outcome is 60.7%. The BIS and SR were measured in only 28 patients. Initial BIS ≤10 occurred in 13/28 patients (46.4%) and correlates with bad outcome in 12/13 patients (92.3%). BIS >30 occurred in nine patients, 6/9 (66.7%) had good outcome. Initial SR ≥75 occurred in 11/28 patients (39.3%) and invariably correlates with bad outcome. NSE >25 and SR >60 occurred in 15/28 patients (53.6%) and invariably correlates with bad outcome. g CW Park, HY Lee, JH Cho, MH Won Introduction It has been reported that the young are much more resistant to transient cerebral ischemia than the adult. Introduction It has been reported that the young are much more resistant to transient cerebral ischemia than the adult. Methods In the present study, we compared the chronological changes of calcium binding proteins (CBPs) (calbindin 28k (CB-D 28k), calretinin (CR) and parvalbumin (PV)) immunoreactivities and levels in the hippocampal CA1 region of the young gerbil with those in the adult following 5 minutes of transient cerebral ischemia induced by the occlusion of both the common carotid arteries. Results In the present study, we examined that about 90% of CA1 pyramidal cells in the adult gerbil hippocampus died at 4 days post ischemia; however, in the young hippocampus, about 56% of them died at 7 days post ischemia. We compared immunoreactivities and levels of CBPs, such as CB-D 28k, CR and PV. The immunoreactivities and protein levels of all the CBPs in the young sham were higher than those in the adult sham. In the adult, the immunoreactivities and protein levels of all the CBPs were markedly decreased at 4 days post ischemia; however, in the young, they were apparently maintained. At 7 days post ischemia, they were decreased in the young; however, they were much higher than those in the adult. p References Introduction Neuron-specifi c enolase (NSE) values >33 μg/ml [1] and low bispectral index (BIS) [2] values correlate with bad outcome after cardiac arrest (CA). 1. Wijdicks et al.: Neurology 2006, 67:203-210. 2. Al Thenayan et al.: Neurology 2008, 71:1535-1537. 3. Rossetti et al.: Ann Neurol 2010, 67:301-307. 4. Bisschops et al.: Resuscitation 2011, 82:696-701. 5. Cronberg et al.: Neurology 2011, 77:623-630. 6. Samaniego et al.: Neurocrit Care 2011, 15:113-119. 7. Fugate et al.: Ann Neurol 2010, 68:907-914. 1. Wijdicks et al.: Neurology 2006, 67:203-210. 2. Al Thenayan et al.: Neurology 2008, 71:1535-1537. 3. Rossetti et al.: Ann Neurol 2010, 67:301-307. 4. Bisschops et al.: Resuscitation 2011, 82:696-701. 5. Cronberg et al.: Neurology 2011, 77:623-630. 6. Samaniego et al.: Neurocrit Care 2011, 15:113-119. 7. Fugate et al.: Ann Neurol 2010, 68:907-914. Methods In this nonblinded prospective study, we observed all CA patients from February 2011 until September 2012 surviving at least 24 hours. NSE was measured between 24 and 72 hours after CA. From October 2011 onward, we recorded BIS and suppression ratio (SR) values as soon as possible after arrival in the ICU. Patients treated with therapeutic hypothermia (TH) (33°C for 24 hours) received cisatracurium. Cerebral Performance Category (CPC) [3] 1 and 2 were considered good outcome, CPC 3 to 5 bad outcome and were recorded after 3 months. Statistical analysis was performed using SPSS statistics 19. Diff erences of calcium binding protein immunoreactivities in the young hippocampal CA1 region from the adult following transient ischemic damage Conclusion NSE >45 uniformly correlates with bad outcome after CA. However, we urge caution for the use of intermediate values (33 to 45). In preliminary data, we report that SR >75 might correlate with bad outcome and that combining NSE and SR might improve the predictive value. Also, low NSE and good initial BIS values correlate with preserved cerebral potential and should encourage the clinician. 1. Zandbergen et al.: Neurology 2006, 66:62-68. 1. Zandbergen et al.: Neurology 2006, 66:62-68. 2. Leary et al.: Resuscitation 2010, 81:1133-1137. Conclusion In brief, the immunoreactivities and levels of CBPs were not decreased in the ischemic CA1 region of the young 4 days after transient cerebral ischemia. This fi nding indicates that the longer maintenance of CBPs may contribute to a less and more delayed neuronal death/ damage in the young. 3. Ajam et al.: Scand J Trauma Resusc Emerg Med 2011, 19:38. Neuron-specifi c enolase and bispectral index/suppression ratio for prognostication after cardiac arrest i Conclusion Motor scores at day 3 post cardiac arrest of extension or worse do not reliably predict poor neurological outcome when therapeutic hypothermia has been used. Clinical neurological fi ndings may not be valid predictors of poor neurological outcome after therapeutic hypothermia. g Van Laer1, K Deschilder2, P Lormans2, J Gillet1, W Stockman2 , , , , 1UZ Leuven, Belgium; 2HHRM, Roeselare, Belgium 1UZ Leuven, Belgium; 2HHRM, Roeselare, Belgium g g Critical Care 2013, 17(Suppl 2):P316 (doi: 10.1186/cc12254) g g Critical Care 2013, 17(Suppl 2):P316 (doi: 10.1186/cc12254) Introduction Neuron-specifi c enolase (NSE) values >33 μg/ml [1] and low bispectral index (BIS) [2] values correlate with bad outcome after cardiac arrest (CA). Agreement between temperature measurements during cooling and warming through the esophagus Conclusion Motor scores at day 3 post cardiac arrest of extension or worse do not reliably predict poor neurological outcome when therapeutic hypothermia has been used. Clinical neurological fi ndings may not be valid predictors of poor neurological outcome after therapeutic hypothermia. 2 Conclusion In this multicentre observational study we were unable to defi ne an optimal oxygenation level during PCA care, but hypoxia seemed to be more harmful than hyperoxia. Exposure to hyperoxia was less common than in previous trials, and we were unable to confi rm previous fi ndings indicating an association with mortality. Reference Methods A review of existing literature was undertaken to examine whether the utility of motor scores to predict poor neurological outcome is infl uenced by the use of therapeutic hypothermia.i l Results Six studies were identifi ed [2-7] that investigated the use of motor scores on day 3 post cardiac arrest in patients who had received therapeutic hypothermia. False positive rates (defi ned as 1 – specifi city) for predicting poor neurological outcome were calculable in fi ve of the six studies [2-6] and were 14%, 24%, 11%, 25% and 12% respectively. In all studies the FPR for motor scores of extension or worse were signifi cantly higher than the 0% (0 to 3% 95% CIs) in the AAN guidelines. 1. Kilgannon JH, et al.: JAMA 2010, 303:2165-2171. Motor scores, therapeutic hypothermia and neurological outcome after cardiac arrest M Davidson Agreement between temperature measurements during cooling and warming through the esophagus False positive rates (defi ned as 1 – specifi city) for predicting poor neurological outcome were calculable in fi ve of the six studies [2-6] and were 14%, 24%, 11%, 25% and 12% respectively. In all studies the FPR for motor scores of extension or worse were signifi cantly higher than the 0% (0 to 3% 95% CIs) in the AAN guidelines. Conclusion Motor scores at day 3 post cardiac arrest of extension or worse do not reliably predict poor neurological outcome when therapeutic hypothermia has been used. Clinical neurological fi ndings may not be valid predictors of poor neurological outcome after therapeutic hypothermia. (22 kPa vs. 20 kPa, P = 0.054). In separate multivariate models neither time spent in the low or the intermediate categories, or the median, highest or lowest paO2 was found to correlate with mortality. widespread use of therapeutic hypothermia. The American Association of Neurology guidelines [1] currently recommend that absent or extensor motor scores on day 3 post arrest are reliable indicators or poor neurological outcome with a false positive rate of 0 to 3%. widespread use of therapeutic hypothermia. The American Association of Neurology guidelines [1] currently recommend that absent or extensor motor scores on day 3 post arrest are reliable indicators or poor neurological outcome with a false positive rate of 0 to 3%. desp ead use o t e apeut c ypot e a. e e ca ssoc at o of Neurology guidelines [1] currently recommend that absent or extensor motor scores on day 3 post arrest are reliable indicators or poor neurological outcome with a false positive rate of 0 to 3%. Methods A review of existing literature was undertaken to examine whether the utility of motor scores to predict poor neurological outcome is infl uenced by the use of therapeutic hypothermia. Results Six studies were identifi ed [2-7] that investigated the use of motor scores on day 3 post cardiac arrest in patients who had received therapeutic hypothermia. False positive rates (defi ned as 1 – specifi city) for predicting poor neurological outcome were calculable in fi ve of the six studies [2-6] and were 14%, 24%, 11%, 25% and 12% respectively. In all studies the FPR for motor scores of extension or worse were signifi cantly higher than the 0% (0 to 3% 95% CIs) in the AAN guidelines. Agreement between temperature measurements during cooling and warming through the esophagus p y Results A total of 489 patients were included. The average number of paO2 measurements during the fi rst 24 hours was eight per patient. A total of 6% of patients experienced paO2 values higher than 40 kPa at any time during the fi rst 24 hours. Average times spent in each time oxygenation category during the fi rst 24 hours were as follows: low 14%, normal 69%, intermediate 14%, and high 2% of the time. Survivors spent less time in the low band (P = 0.029) and more time in the intermediate band (P = 0.029) compared with nonsurvivors. The median paO2 during the fi rst 24 hours was higher in survivors than in nonsurvivors (15 kPa vs. 14 kPa, P = 0.016) but there was no diff erence in lowest (11 kPa vs. 10 kPa, P = 0.162) or the highest paO2 values and warming through the esophagus EB Kulstad1, DM Courtney2, P Shanley3, A Metzger3, T Matsuura3, J Rees4, K Lurie3, S McKnite5 1Advocate Christ Medical Center, Oak Lawn, IL, USA; 2Northwestern University, Chicago, IL, USA; 3University of Minnesota Medical Center, Minneapolis, MN, USA; 4Advanced Circulatory Systems, Minneapolis, MN, USA; 5Minneapolis Medical Research Foundation, Minneapolis, MN, USA Critical Care 2013, 17(Suppl 2):P314 (doi: 10.1186/cc12252) Introduction Measurement of temperature during treatment with therapeutic hypothermia can yield variable values depending on Introduction Measurement of temperature during treatment with therapeutic hypothermia can yield variable values depending on S120 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 (22 kPa vs. 20 kPa, P = 0.054). In separate multivariate models neither time spent in the low or the intermediate categories, or the median, highest or lowest paO2 was found to correlate with mortality. widespread use of therapeutic hypothermia. The American Association of Neurology guidelines [1] currently recommend that absent or extensor motor scores on day 3 post arrest are reliable indicators or poor neurological outcome with a false positive rate of 0 to 3%. Methods A review of existing literature was undertaken to examine whether the utility of motor scores to predict poor neurological outcome is infl uenced by the use of therapeutic hypothermia. Results Six studies were identifi ed [2-7] that investigated the use of motor scores on day 3 post cardiac arrest in patients who had received therapeutic hypothermia. P319 Plymouth Hospitals NHS Trust, Plymouth, UK Plymouth Hospitals NHS Trust, Plymouth, UK P319 Early hypothermia improves survival and reduces the rise of serum biomarkers after traumatic brain injury in swine M Kumar, AD Goldberg, M Kashiouris, L Keenan, A Rabinstein Mayo Clinic, Rochester, MN, USA Critical Care 2013, 17(Suppl 2):P319 (doi: 10.1186/cc12257) y p y Critical Care 2013, 17(Suppl 2):P317 (doi: 10.1186/cc12255) Critical Care 2013, 17(Suppl 2):P317 (doi: 10.1186/cc12255) Introduction Accurate prediction of neurological outcome after cardiac arrest is desirable to prevent inappropriate withdrawal of life- sustaining therapy in patients who could have a good neurological outcome, and to limit active treatment in patients whose ultimate neurological outcomes are poor. Established guidelines to predict neurological outcome after cardiac arrest were developed before the y , , , Critical Care 2013, 17(Suppl 2):P319 (doi: 10.1186/cc12257) Introduction Poor outcomes in clinical trials on the use of therapeutic hypothermia following traumatic brain injury (TBI) may be due to the S121 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 delay in reaching target temperature [1]. We hypothesize that early and rapid induction of hypothermia will mitigate neuronal injury and improve survival in a swine model of TBI. Results Table 1 shows the magnitude of the problem, highlighting the leading causes of TBI in Egypt in 2010. Male sex was predominantly aff ected, 79% of cases. Moderate and severe injuries account for 17.2% of all TBI presented cases. delay in reaching target temperature [1]. We hypothesize that early and rapid induction of hypothermia will mitigate neuronal injury and improve survival in a swine model of TBI. Methods Twenty domestic cross-bred pigs (34 to 35 kg) were subjected to a 5 ATM (100 ms) lateral fl uid percussion TBI. The brain temperature and ICP were measured using Camino®. Serum biomarkers for neuronal injury – S-100β, neuron-specifi c enolase, glial fi brillary acid protein (GFAP), and neurofi laments heavy chain – were measured daily using enzyme-linked immunosorbent assay. Twelve of the injured animals were rapidly cooled to 32°C within 90 minutes of the injury using a transpulmonary hypothermia technique [2]. Hypothermia was maintained for 48 hours. Eight injured control animals were maintained at 37°C. In both groups, anesthesia (isofl urane 1%) was discontinued and the animals were weaned off the ventilator after 48 hours. Five days post injury, the surviving animals were euthanized and necropsied. Alcohol intoxication impedes the recognition of traumatic brain injury in the prehospital setting and may worsen 6-month outcome R Raj, J Siironen, R Kivisaari, M Kuisma, M Skrifvars Helsinki University Central Hospital, Helsinki, Finland Critical Care 2013, 17(Suppl 2):P321 (doi: 10.1186/cc12259) Alcohol intoxication impedes the recognition of traumatic brain injury in the prehospital setting and may worsen 6-month outcome R Raj, J Siironen, R Kivisaari, M Kuisma, M Skrifvars Helsinki University Central Hospital, Helsinki, Finland Critical Care 2013, 17(Suppl 2):P321 (doi: 10.1186/cc12259) y g g Results Ten of the 12 hypothermia and four of the eight normothermia animals survived to the end of the 5-day study (χ2 = 2.597, df = 1, P = 0.1071). Although the probability of type I error between survival curves was 11%, the study was clinically signifi cant and showed a clear trend toward improved survival with hypothermia. The intracranial pressures were signifi cantly (P <0.05) lower in the hypothermia group. Both interventions – that is, general anesthesia and hypothermia – mitigated the rise of serum biomarkers following TBI. However, the suppression of biomarkers was sustained during the recovery period only in the hypothermia group. With the exception of the GFAP levels, the curves of all biomarkers were signifi cantly diff erent between the groups.i Introduction Transport directly to a neurosurgical trauma center has shown to reduce mortality in patients with traumatic brain injury (TBI). We hypothesized that alcohol intoxication may impede prehospital recognition of TBI, resulting in transportation to a non-neurosurgical hospital. Methods A retrospective analysis of TBI patients admitted to a designated neurosurgical trauma center’s ICU in 2009/10 and primarily treated by the Emergency Medical Service (EMS). Patients were divided into two categories based either direct or indirect trauma transfer by the EMS. Directly transferred patients are directly transported to the neurosurgical trauma center from the injury scene while indirectly transported patients are initially transported to another non- neurosurgical hospital before re-transfer to the trauma center. Data from patient journals and EMS forms were extracted. The blood alcohol level (BAL) was measured by the EMS using an alcohol breath-test. Logistic regression modeling was used to identify variables present at scene associated with transport destination. Conclusion Our preliminary fi ndings show early initiation, rapid induction, and prolonged maintenance (48 hours) of cerebral hypothermia to lower intracranial pressure, blunt the rise in serum biomarkers, and improve survival following TBI. 1. Clifton GL, et al.: Lancet Neurol 2011, 10:131-139. 2. Kumar M., et al.: Anesth Analg 2012, 114:S-160. 1. Clifton GL, et al.: Lancet Neurol 2011, 10:131-139. 2. Kumar M., et al.: Anesth Analg 2012, 114:S-160. P319 The data were analyzed using a log-rank (Mantel–Cox) test, and ANOVA. Conclusion TBI is a serious public health problem in Egypt. Further data interpretation over wider periods of time should be conducted for better understanding of TBI prevalence is highly recommended to develop eff ective injury prevention programs. 1. Clifton GL, et al.: Lancet Neurol 2011, 10:131-139. 2. Kumar M., et al.: Anesth Analg 2012, 114:S-160. Alcohol intoxication impedes the recognition of traumatic brain injury in the prehospital setting and may worsen 6-month outcome R Raj, J Siironen, R Kivisaari, M Kuisma, M Skrifvars Helsinki University Central Hospital, Helsinki, Finland Critical Care 2013, 17(Suppl 2):P321 (doi: 10.1186/cc12259) Results Totally 470 patients met the inclusion criteria; 60% were transported directly and 40% indirectly. In the direct group 15% of patients had a positive BAL, compared with 26% for those indirectly transported. In the logistic regression model, factors associated with direct transport were: BAL ≥2.3‰ (OR: 0.06, CI: 0.01 to 0.36), male gender (OR: 0.35; CI: 0.16 to 0.76), GCS 13 to 15 (OR: 0.28; CI 0.10 to 0.74), high-energy trauma (OR: 9.42; CI 2.15 to 41.20), major extracranial injury (OR: 7.92; CI 2.57 to 24.41), EMS physician telephone consultation (OR: 6.02; CI 2.51 to 14.11) or presence on scene (OR: 8.63; CI 3.50 to 21.26) and incident at a public place outside (OR: 3.05; CI: 1.34 to 6.4) and inside (OR: 2.92; CI: 1.07 to 8.01). Median time delay to trauma center admission was 1:07 hours (IQR: 0.52 to 1:28) for directly transported patients and 4:06 (IQR: 2.54 to 5:43) for those indirectly transported (P <0.001). There was a clear trend towards poorer neurological outcome for patients with delayed trauma center admission in univariate analysis (P = 0.001). P320 P320 Epidemiology of moderate and severe traumatic brain injury in Cairo University Hospital in 2010 T Montaser1, A Hassan2 1Shobra General Hospital, Cairo, Egypt; 2King Khaled University Hospital, Riyadh, Saudi Arabia Critical Care 2013, 17(Suppl 2):P320 (doi: 10.1186/cc12258) P320 Epidemiology of moderate and severe traumatic brain injury in Cairo University Hospital in 2010 T Montaser1, A Hassan2 1Shobra General Hospital, Cairo, Egypt; 2King Khaled University Hospital, Riyadh, Saudi Arabia Critical Care 2013, 17(Suppl 2):P320 (doi: 10.1186/cc12258) P320 Epidemiology of moderate and severe traumatic brain injury in Cairo University Hospital in 2010 T Montaser1, A Hassan2 1Shobra General Hospital, Cairo, Egypt; 2King Khaled University Hospital, Riyadh, Saudi Arabia Critical Care 2013, 17(Suppl 2):P320 (doi: 10.1186/cc12258) P322 Predictive ability of the IMPACT-TBI prognostic model is improved by adding markers of coagulation R Raj, J Siironen, R Kivisaari, L Handolin, J Hernesniemi, M Skrifvars Helsinki University Central Hospital, Helsinki, Finland Critical Care 2013, 17(Suppl 2):P322 (doi: 10.1186/cc12260) Predictive ability of the IMPACT-TBI prognostic model is improved by adding markers of coagulation Methods A prospective cohort of STBI patients monitored with cerebral microdialysis (CMD) and brain tissue oxygen (PbtO2) was studied. Brain lactate metabolism was assessed by quantifi cation of elevated CMD lactate samples (>4 mmol/l). These were matched to pyruvate and PbtO2, and dichotomized as hyperglycolytic (CMD pyruvate >119 μmol/l) versus nonhyperglycolytic or as hypoxic (PbtO2 <20 mmHg) versus nonhypoxic. Data were expressed as percentages per patient. Global brain perfusion (categorized as oligemic, normal or hyperemic) was assessed with CT perfusion (CTP). Introduction One of the most used prognostic models for traumatic brain injury is the IMPACT-TBI model, which predicts 6-month mortality and unfavorable outcome. Our aim was to study whether adding markers of coagulation improves the model’s predictive power when accounting for extracranial injury. yp p Results Twenty-four patients (total 1,782 CMD samples) were studied. Samples with elevated CMD lactate were frequently observed (41 ± 8% SEM of individual samples). Brain lactate elevations were predominantly hyperglycolytic (73 ± 8.2%), whilst only 14 ± 6.3% of them were hypoxic. Trends over time of both lactate patterns are shown in Figure 1. On CTP (n = 17; average 48 hours from TBI) hyperglycolytic lactate was always associated with normal or hyperemic CTP, whilst hypoxic lactate was associated with oligemic CTP (Table 1). Methods Patients with a TBI admitted to a designated trauma center in 2009/10 were screened retrospectively and included according to the IMPACT study criteria. The predictive outcome was calculated for included patients using the full IMPACT-TBI model. To assess coagulopathy and extracranial injury we used the prothrombin time percentage (PT), platelet count (109), and injury severity score (ISS). PT, platelets and ISS were added to the IMPACT model, creating new models. The predictive power of the new models was tested by comparing their area under the receiver operating characteristic curve (AUC) with the original model. Figure 1 (abstract P323). g Results Totally 342 were included. Six-month mortality was 32% and unfavorable outcome 57%. The IMPACT model’s predictive power for mortality and neurological outcome; AUC: 0.85, CI: 0.81 to 0.89; and AUC: 0.81, CI: 0.76 to 0.86. P320 Epidemiology of moderate and severe traumatic brain injury in Cairo University Hospital in 2010 T Montaser1, A Hassan2 1Shobra General Hospital, Cairo, Egypt; 2King Khaled University Hospital, Riyadh, Saudi Arabia Critical Care 2013, 17(Suppl 2):P320 (doi: 10.1186/cc12258) Nonsurvivors and patients with an unfavorable outcome had lower platelets (159; 165 vs. 191; 198), PT (76; 77 vs. 86; 90) and higher ISS (50; 44% ISS >25 vs. 34; 32% ISS >25) compared with survivors and favorable outcome (P <0.05). Addition of PT but not ISS nor platelets resulted in a signifi cant improvement of the IMPACT model’s predictive power for 6-month mortality; AUC: 0.87, CI: 0.81 to 0.89, P = 0.017 (Figure 1). In multivariate analysis PT remained an independent signifi cant predictor of outcome when adjusting for IMPACT prognosis and ISS (OR: 0.985, CI: 0.975 to 0.996). p g Conclusion Addition of PT to the IMPACT model improves the predictive power for 6-month mortality but not neurological outcome when adjusting for ISS. p p y g when adjusting for ISS. References 1. Steyerberg EW, et al.: PLoS Med 2008, 5:e165. 2. Marmarou A, et al.: J Neurotrauma 2007, 24:239-250. P323 Nonischemic endogenous lactate production in humans with severe traumatic brain injury N Sala1, T Suys1, JB Zerlauth1, J Bloch1, P Magistretti2, R Meuli1, M Oddo1 1Lausanne University Hospital, Lausanne, Switzerland; 2EPFL, Lausanne, Switzerland Critical Care 2013, 17(Suppl 2):P323 (doi: 10.1186/cc12261) Introduction Evidence suggest that endogenous lactate, produced by aerobic glycolysis, is an important substrate for neurons, particularly in Figure 1 (abstract P322). P320 Epidemiology of moderate and severe traumatic brain injury in Cairo University Hospital in 2010 T Montaser1, A Hassan2 1Shobra General Hospital, Cairo, Egypt; 2King Khaled University Hospital, Riyadh, Saudi Arabia Critical Care 2013, 17(Suppl 2):P320 (doi: 10.1186/cc12258) Epidemiology of moderate and severe traumatic brain injury in Cairo University Hospital in 2010 T Montaser1, A Hassan2 1Shobra General Hospital, Cairo, Egypt; 2King Khaled University Hospital, Riyadh, Saudi Arabia Critical Care 2013, 17(Suppl 2):P320 (doi: 10.1186/cc12258) T Montaser1, A Hassan2 y Critical Care 2013, 17(Suppl 2):P320 (doi: 10.1186/cc12258) Introduction Traumatic brain injury (TBI) is a contributing factor to approximately one-third of all injury-related deaths in the USA annually. Updated statistical records for TBI in Egypt are lacking. The current research is aiming to estimate the prevalence of TBI in Egypt in order to develop a comprehensive TBI prevention program. p p p p g Methods A 1-year period (one calendar month every quarter of 2010) descriptive epidemiological study of moderate and severe TBI cases admitted to the emergency department, Cairo main university hospital. The data collection sheet included personal data (age, sex and residency), incident-related data (cause, nature and time of injury) and both clinical and radiological fi ndings. y Conclusion Heavily alcohol intoxicated TBI patients are commonly initially transported to a non-neurosurgical trauma center and this may worsen 6-month neurological outcome. References 1. Hartl R, et al.: J Trauma 2006, 60:1250-1256. 1. Hartl R, et al.: J Trauma 2006, 60:1250-1256. 2. Shahin H, et al.: J Trauma 2010, 69:1176-1181. 2. Shahin H, et al.: J Trauma 2010, 69:1176-1181. Table 1 (abstract P320). Causes of TBI in diff erent age groups in Egypt NO = 844/ Abuse/ Violence/ Struck by/ Other causes/ Age (years) 4 months FFH MVC neglect assaults against not known 1 to 5 54 (6%) 20 (37%) 10 (19%) 8 (15%) – 8 (15%) 8 (14%) 6 to 18 91 (11%) 28 (31%) 20 (22%) 5 (6%) 6 (5 %) 22 (25%) 10 (11%) 19 to 29 150 (18%) 49 (33%) 45 (30%) – 14 (9%) 18 (12%) 24 (16%) 30 to 55 381 (45%) 114 (30%) 134 (35%) – 19 (5%) 50 (13%) 64 (17%) Above 55 168 (20%) 47 (28%) 41 (24%) 11 (7%) 5 (3%) 25 (15%) 39 (23%) Table 1 (abstract P320). Causes of TBI in diff erent age groups in Egypt S122 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 P322 conditions of increased energy demand. This study aimed to examine brain lactate metabolism in patients with severe traumatic brain injury (STBI). j References 1. Steyerberg EW, et al.: PLoS Med 2008, 5:e165. 1. Steyerberg EW, et al.: PLoS Med 2008, 5:e165. 2. Marmarou A, et al.: J Neurotrauma 2007, 24:239-250. 2. Marmarou A, et al.: J Neurotrauma 2007, 24:239-250. Figure 1 (abstract P322). Table 1 (abstract P323). Lactate elevations and brain perfusion ( ) p Oligemia (%) Normal (%) Hyperemia (%) HG lactate 0 58 42 HX lactate 100 0 0 HG, hyperglycolytic; HX, hypoxic. Conclusion Our fi ndings suggest predominant nonischemic lactate release after TBI and identify, for the fi rst time, an association between cerebral hyperglycolytic lactate production and normal to supranormal brain perfusion. Our data support the concept that lactate may be used as energy substrate by the injured human brain. gy y j Acknowledgement Supported by the Swiss National Science Foundation. Figure 1 (abstract P322). P323 P323 Nonischemic endogenous lactate production in humans with severe traumatic brain injury N Sala1, T Suys1, JB Zerlauth1, J Bloch1, P Magistretti2, R Meuli1, M Oddo1 1Lausanne University Hospital, Lausanne, Switzerland; 2EPFL, Lausanne, Switzerland Critical Care 2013, 17(Suppl 2):P323 (doi: 10.1186/cc12261) Nonischemic endogenous lactate production in humans with severe traumatic brain injury Yagi, N Saito, Y Hara, H Hisashi Matumoto, K Mashiko Introduction In the prehospital setting, it is diffi cult to use the Glasgow Coma Scale (GCS) to evaluate the consciousness state using in pediatric patients with severe trauma. The Japan Coma Scale (JCS) is a consciousness scale used widely in Japan and, with its four grades, is simpler and quicker to use than the GCS. This study examined whether Introduction In the prehospital setting, it is diffi cult to use the Glasgow Coma Scale (GCS) to evaluate the consciousness state using in pediatric patients with severe trauma. The Japan Coma Scale (JCS) is a consciousness scale used widely in Japan and, with its four grades, is simpler and quicker to use than the GCS. This study examined whether Introduction Evidence suggest that endogenous lactate, produced by aerobic glycolysis, is an important substrate for neurons, particularly in S123 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Methods In this prospective observational study, measurements of OSND were carried out in 21 patients of a polyvalent 10-bed adult ICU before and 2 minutes after changes of PEEP from a baseline of 5 with 1 cmH2O increments, to a maximum of 12. Two measurements were performed with a 7.5 MHz probe (MicroMaxx; Fujifi lm Sonosite Inc., USA) in each eye before and after PEEP changes. Demographic data (APACHE II score, age, sex, diagnosis) Ppeak and Pm were also recorded. Exclusion criteria were intracranial pathology, ocular neuritis and trauma. Normality with P–P plots and the Kolmogorov–Smirnov test were calculated followed by bivariate and single linear regression analysis (IBM SPPS v17, signifi cance P <0.05). the JCS can predict clinical traumatic brain injury (TBI) and outcome in pediatric patients aged 3 to 15 years in the prehospital setting. Methods This retrospective study analyzed data from the Japan Trauma Data Bank. P323 Registered pediatric patients aged 3 to 15 years with severe trauma (maximum Abbreviated Injury Scale score ≥3 or Injury Severity Score ≥9) were divided into four groups according to JCS score in the prehospital setting (Grade 0: alert, Grade 1: possible eye-opening, not lucid, Grade 2: possible eye-opening upon stimulation, Grade 3: no eye-opening and coma). TBI was defi ned as maximum head AIS ≥3. y gi Results A total of 1,562 patients were included (Grade 0: 673, Grade 1: 410, Grade 2: 230, Grade 3: 249). Victims of blunt trauma accounted for 98.1%. Median age was 9 (interquartile range: 7 to 12) years, median ISS was 16 (9 to 21). There was strong agreement between the JCS in prehospital setting and GCS scores on arrival at hospital (r = –0.745, P <0.001). Multivariate analysis adjusted for age and ISS revealed that the JCS was independently associated with TBI (odds ratio (OR): 2.5; 95% CI: 2.1 to 2.8, P <0.001) and hospital mortality (OR: 3.8; 95% CI 2.4 to 6.0, P <0.001). See Table 1. y gi Results Seventy-two measurements were included for further analysis. Mean ± SD values were: PEEP (7.9 ± 2.3), Ppeak (24.35 ± 5), Pm (12.03 ± 3.16), OSNDright (4.03 ± 0.79), OSNDleft (4.1 ± 0.79), ΔOSND (0.24 ± 0.2), while values for age and APACHE II score were 62 ± 4 years and 14.4 ± 1.4 respectively. PEEP and OSND seem to have a moderate relation (tendency equation: OSND = 2.454 + 0.581×PEEP) compared with the weaker eff ect of Ppeak and Pm (Table 1). Table 1 (abstract P325). Correlations (r2) between airway pressures and OSND OSNDright OSNDleft PEEP 0.581* 0.571* Ppeak 0.467* 0.448* Pm 0.491 0.467 P <0.01, P <0.05. Table 1 (abstract P324) TBI, n (%) Mortality (%) JCS grade 0 213 (31.6) 0.1 JCS grade 1 225 (62.2) 0.7 JCS grade 2 184 (80.0) 2.6 JCS grade 3 230 (92.4) 15.7 Table 1 (abstract P325). Correlations (r2) between airway pressures and OSND Conclusion Our study identifi ed a moderate relation between PEEP and OSND and a weaker one between Ppeak, Pm and OSND. Thus, in selected cases OSND could serve as a bedside marker of eff ect of airway pressure to ICP. Yet, larger studies are needed to come to a safer conclusion. Conclusion There was strong association between JCS score and clinical outcome in pediatric patients with severe trauma. P325f 1. Geeraerts T, et al.: Int Care Med 2008, 34:2062-2067. P325 Eff ect of positive end-expiratory pressure on ultrasound measurement of optical sheath nerve diameter: preliminary study T Aslanidis, E Anastasiou, E Geka, E Efthimiou, A Myrou, A Kontos, D Papadopoulos, E Boultoukas, M Giannakou-Peftoulidou A.H.E.P.A. University Hospital, Thessaloniki, Greece Critical Care 2013, 17(Suppl 2):P325 (doi: 10.1186/cc12263) P325 Eff ect of positive end-expiratory pressure on ultrasound measurement of optical sheath nerve diameter: preliminary study T Aslanidis, E Anastasiou, E Geka, E Efthimiou, A Myrou, A Kontos, D Papadopoulos, E Boultoukas, M Giannakou-Peftoulidou A.H.E.P.A. University Hospital, Thessaloniki, Greece Critical Care 2013, 17(Suppl 2):P325 (doi: 10.1186/cc12263) P326 P326 National survey of current protocols and management of the traumatic brain injury patients in UK ICUs B Lewinsohn, S Panchatsharam, S Wijayatilake, A Billini, G Delacedra, R Jain, J Khan, R Shetty, A Lewinsohn Queens Hospital, Romford, UK Critical Care 2013, 17(Suppl 2):P326 (doi: 10.1186/cc12264) P323 The results support the use of the JCS in the prehospital transport destination criteria for children. Reference Reference National survey of current protocols and management of the traumatic brain injury patients in UK ICUs j y p B Lewinsohn, S Panchatsharam, S Wijayatilake, A Billini, G Delaced R Jain, J Khan, R Shetty, A Lewinsohn Queens Hospital, Romford, UK Critical Care 2013, 17(Suppl 2):P326 (doi: 10.1186/cc12264) Introduction Non-invasive ocular ultrasonography has recently been proposed to detect elevated intracranial pressure (ICP). On the other hand, the eff ect of positive end-expiratory pressure (PEEP) on ICP is well documented. The aim of the present ongoing study is to record the eff ect of changes of airway pressures on optical sheath nerve diameter (OSND). Introduction Following primary neurological insult, initial manage ment of traumatic brain-injured (TBI) patients has a clearly defi ned pathway [1]. However, after arrival at tertiary centers, further manage ment is not Figure 1 (abstract P326). Figure 1 (abstract P326). S124 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 computed for longer periods. Signifi cantly better outcome (Table 1) was observed in patients for whom optimal CPP derived from LAx was maintained. Table 1 (abstract P327) Nonsurvivors Survivors P value Time within CPPopt-% 18.3 (15.5; 24.4) 24.8 (19.7; 28.7) 0.0004 \|CPP-CPPopt\| (mmHg) 7.05 (4.98; 9.73) 5.37 (4.49; 6.82) 0.0024 Table 1 (abstract P327) standardized. Intracranial hypertension (ICH), systemic hypotension, hypoxia, hyperpyrexia and hypocapnia have all been shown to independently increase mortality [2]. Despite numerous studies, there is currently no level 1 evidence to support any specifi c management [3]. Our objective was to provide an overview of the current clinical management protocols in the UK. g p Methods Thirty-one ICUs managing patients with severe TBI were identifi ed from the RAIN (Risk Adjustment In Neurocritical care) study, and a telephone survey was conducted. computed for longer periods. Signifi cantly better outcome (Table 1) was observed in patients for whom optimal CPP derived from LAx was maintained. Results A total 97% of units used a cerebral perfusion pressure protocol for the initial management, with 83% targeting pressures of 60 to 70 mmHg and 17% aimed for >70 mmHg. Ninety-one percent of units monitored CO2 routinely with 61% targeting CO2 of 4.5 to 5 kPa (Figure 1). Regarding osmotherapy, mannitol was still the preferred agent, with 48% of units using it as fi rst line; 32% used hypertonic saline, while 20% of units used either depending on clinicians’ preference. g References 1. NICE: Head Injury; Triage, Assessment, Investigation and Early Management of Head Injury. London: National Collaborating Centre for Acute Care; September 2007. g Methods A retrospective analysis of rcSO2 utility in AMS. rcSO2 data were collected every 30 seconds for AMS patients who had a head CT. Patients with a negative head CT were compared with those with an abnormal head CT. ROC analysis was performed to fi nd the AUC for each summary statistic and performance characteristics. Subgroup analysis was done to determine whether rcSO2 predicted injury and location. 2. Wijayatilake et al.: Updates in the management of intracranial pressure in traumatic brain injury. Curr Opin Anaesthesiol 2012, 25:540-547. 3. Guidelines for the Management of Severe Traumatic Brain Injury. 3rd edition. New York: Brain Trauma Foundation; 2007. 3. Guidelines for the Management of Severe Traumatic Brain Injury. 3rd edition. New York: Brain Trauma Foundation; 2007. c 2 p j y Results rcSO2 readings across 5, 15, 20, and 30 minutes were stable (Figure 1). rcSO2 readings with one or both sides <50% or a wide diff erence between L and R cerebrum was predictive of an abnormal CT scan. A mean diff erence of 4.2 was 82% sensitive for detecting a CT lesion with 62% specifi city, 88% PPV, and 52% NPV; a mean diff erence of 12.2 was 100% specifi c for an abnormal head CT. Lower mean rcSO2 readings localized to the CT pathology side, and higher rcSO2 readings trend toward the EDH group. 4. Barochia et al.: Bundled care for septic shock. Crit Care Med 2010, 38:668-678. 4. Barochia et al.: Bundled care for septic shock. Crit Care Med 2010, 38:668-678. 4. Barochia et al.: Bundled care for septic shock. Crit Care Med 2010, 38:668-678. National survey of current protocols and management of the traumatic brain injury patients in UK ICUs Sixteen percent questioned were currently enrolled on the Eurotherm hypothermia trial, while 16% never used hypothermia and one unit used prophylactic hypothermia routinely. The remaining 65% of units used hypothermia only to manage refractory ICH. P327 f Low-frequency autoregulation index for calculation of optimal cerebral perfusion pressure in severe traumatic brain injury F Guiza1, G Meyfroidt1, M Schuhmann2, G Van den Berghe1, I Piper3, B Depreitere1 1University Hospitals Leuven, Belgium; 2University Hospital Tübingen, Germany; 3Southern General Hospital, Glasgow, UK Critical Care 2013, 17(Suppl 2):P327 (doi: 10.1186/cc12265) Cerebral rSO2 monitoring in pediatric altered mental status patients T Abramo, I Kane Vanderbilt School of Medicine, Nashville, TN, USA Critical Care 2013, 17(Suppl 2):P328 (doi: 10.1186/cc12266) Vanderbilt School of Medicine, Nashville, TN, USA Critical Care 2013, 17(Suppl 2):P328 (doi: 10.1186/cc12266) Conclusion There is no clear consensus on the initial targets used. The surviving sepsis campaign showed that protocol-led care can reduce mortality [4]. Perhaps it is time for a similar approach to be adopted, with specialists coming to together to review the evidence and formulate guidelines that can then be tested. Introduction Pediatric patients with altered mental status (AMS) present with poor histories resulting in delayed testing and potential poor outcomes. Non-invasive detection for altered cerebral physiology related to TBI would improve resuscitation and outcome. Cerebral rSO2 (rcSO2) studies demonstrate its utility in certain neurological emergencies. References References 1. Aries M, et al.: Crit Care Med 2012, 40:2456-2463. 2. Piper I, et al.: Acta Neurochirurgica 2003, 145:615-628. 1. Aries M, et al.: Crit Care Med 2012, 40:2456-2463. 2. Piper I, et al.: Acta Neurochirurgica 2003, 145:615-628. 1. Aries M, et al.: Crit Care Med 2012, 40:2456-2463. 1. Aries M, et al.: Crit Care Med 2012, 40:2456 2463. 2. Piper I, et al.: Acta Neurochirurgica 2003, 145:615-628. Low-frequency autoregulation index for calculation of optimal cerebral perfusion pressure in severe traumatic brain injury F Guiza1, G Meyfroidt1, M Schuhmann2, G Van den Berghe1, I Piper3, B Depreitere1 Conclusion Cerebral rcSO2 monitoring can non-invasively detect altered cerebral physiology and pathology related to TBI as the cause for pediatric altered mental status. The utility of rcSO2 monitoring has shown its potential for localizing and characterizing intracranial lesions among these altered children. Further studies utilizing rcSO2 monitoring as an adjunct tool in pediatric altered mental status evaluation and management are ongoing. Introduction Traumatic brain injury (TBI) is a major cause of permanent disability and death in young patients. Controversy exists regarding the optimal cerebral perfusion pressure (CPP) required in TBI management. A tool for monitoring autoregulation and determining an optimal CPP is the pressure reactivity index (PRx), defi ned as a moving correlation coeffi cient between the mean arterial blood pressure (MAP) and intracranial pressure (ICP) at a frequency of at least 60 Hz. This requirement of high frequency has constrained its use to a few academic centers. An association was shown between outcome and continuous optimal CPP based on 4 hours of PRx [1]. We present a novel low-frequency autoregulation index (LAx), based on correlations between ICP and MAP at a standard minute-by-minute time resolution. Methods A total of 182 patients from the Brain-IT [2] multicentre European database had registered outcome and ICP and MAP for the fi rst 48 ICU hours. Twenty-one TBI patients admitted to the university hospitals of Leuven, Belgium and Tubingen, Germany were continuously monitored using ICM+ software (Cambridge Enterprise) allowing for continuous PRx calculation. Autoregulation indices versus CPP plots for PRx and LAx were computed to determine optimal CPP every minute during the fi rst 48 ICU hours [1]. Intravenous paracetamol for fever control in acute brain-injured patients: cerebral and hemodynamic eff ects E Picetti1, I Rossi1, P Ceccarelli1, S Risolo1, P Schiavi2, V Donelli1, A Crocamo1, M Antonini1, M Caspani1 1I Servizio Anestesia e Rianimazione, Azienda Ospedaliero-Universitaria di Parma, Italy; 2Neurochirurgia e Neurotraumatologia, Azienda Ospedaliero- Universitaria di Parma, Italy Critical Care 2013, 17(Suppl 2):P329 (doi: 10.1186/cc12267) Introduction Fever is a dangerous secondary insult for the injured brain [1]. We investigated the cerebral and hemodynamic eff ects of intravenous (i.v.) paracetamol administration for the control of fever in neurointensive care unit (NICU) patients. Methods The i.v. paracetamol (1 g in 15 minutes) was administered to NICU patients with a body temperature (Temp.) >37.5°C. Its eff ects on mean arterial pressure (MAP), heart rate (HR), intracranial pressure (ICP), cerebral perfusion pressure (CPP), jugular venous oxygen saturation (SjVO2) and Temp. were recorded at the start of paracetamol infusion (T0) and after 30 (T30), 60 (T60) and 120 (T120) minutes. Interventions for the maintenance of CPP >60 mmHg or ICP <20 mmHg were recorded. i Results On the Brain-IT database, LAx resulted in an optimal CPP for 90% of the fi rst 48 hours. Table 1 shows recommendations with respect to outcome. In the Leuven–Tübingen database, PRx and LAx resulted in 44% and 92% recommendations respectively. The average diff erence between methods was 5.26 mmHg.f Conclusion The diff erences in optimal CPPs derived from PRx and LAx were not clinically signifi cant. LAx allowed for recommendations to be Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S125 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Results Fifteen NICU patients (nine subarachnoid hemorrhage, fi ve traumatic brain injury, mean age 54.9 ± 16.8, seven (50%) males, median GCS 7) were prospectively studied. We analyzed the administration of one dose of paracetamol for each patient (total 14 cases). After infusion of paracetamol we found a decrease of Temp. (from 37.8 ± 0.3 to 37.4 ± 0.4°C, P <0.001), MAP (from 94.7 ± 9.9 to 86.1 ± 6.7 mmHg, P = 0.008), CPP (from 79.6 ± 13.1 to 70.8 ± 7.6 mmHg, P = 0.011) and HR (from 71.5 ± 14.9 to 63.8 ± 16.3 bpm, P <0.001) with respect to the starting value (ANOVA for repeated measures), whereas ICP and SjVO2 remained unchanged (Figure 1). In fi ve cases norepinephrine infusion was started for CPP <60 mmHg. In another two cases, for the same reason, the norepinephrine dosage was augmented. 1. Greer DM, Funk SE, Reaven NL, Ouzounelli M, Uman GC: Impact of fever on outcome in patients with stroke and neurologic injury. Stroke 2008, 39:3029-3035. Intravenous paracetamol for fever control in acute brain-injured patients: cerebral and hemodynamic eff ects We evaluated sociodemographic and clinical factors associated with readmissions using a logistic regression model.i P330 Retrospective analysis of the hemodynamic eff ects of induction of barbiturate coma in patients with refractory elevated intracranial pressure S D’Hollander1, M Vander Laenen1, JB Gillet2, C De Deyne1, R Heylen1, W Boer1, F Jans1 1ZOL Genk, Belgium; 2UZ Leuven, Belgium Critical Care 2013, 17(Suppl 2):P330 (doi: 10.1186/cc12268) g g g Results Among 18,342 adult patients with TBI identifi ed in the registry, 14,777 patients were included among which 2,363 had severe, 1,106 moderate and 11,308 mild traumatic brain injury. Most patients were young (mean age: 52 ± 23 years) and had no comorbidity (73.6%). Overall, 1,032 patients (7.0%) were readmitted within 30 days, 12.7% within 3 months and 17.6% within 6 months. At 30 days post discharge, 311 (30.1%) were readmitted for a complication. The median length of stay was 8 days (Q1 to Q3: 3 to 20). More than 10% of patients aged ≥75 years with ≥1 comorbidity or with ≥1 admission prior to index hospitalisation were readmitted. The severity of the TBI was not an independent predictor of readmission. Age, highest AIS, number of comorbidities, number of admissions prior to index hospitalization, level of index trauma center and discharge destination were associated with readmissions on multivariate analysis. Introduction Standard clinical practice in patients with elevated intracranial pressure (ICP) is to keep cerebral perfusion pressure (CPP) at (or above) 60 mmHg: we performed this retrospective study to analyze whether induction of barbiturate coma impedes compliance to this target CPP value. It has been repetitively shown that, although proven as an effi cient therapy for refractory elevated ICP, barbiturate coma may cause a decrease in mean arterial pressure (MAP) and CPP [1]. Methods All patients that had received sodium thiopental in our ICU during 2011 were identifi ed and their medical and nursing records were retrospectively analyzed. The eff ect of administration of sodium thiopental (loading dose 3 to 5 mg/kg, followed by a continuous infusion adjusted to obtain an EEG burst suppression ratio between 40 and 80%) on MAP, ICP and CPP was evaluated. Values are reported as mean ± SD. The changes over time in ICP, MAP and CPP are reported as means with their 95% CI. y Conclusion Readmissions in the months following TBI are frequent, but were not found to be associated with the TBI severity. Intravenous paracetamol for fever control in acute brain-injured patients: cerebral and hemodynamic eff ects The proportion of patients who had infusion of norepinephrine increased from 42.8% at T0 to 78.6% at T120 (P = 0.02, chi-square for trends). Conclusion Use of i.v. paracetamol is eff ective in the maintenance of normothermia in acute brain-injured patients. However, adverse hemodynamic eff ects, which could represent a secondary insult for the injured brain, must be rapidly recognized and treated. Reference 1. Greer DM, Funk SE, Reaven NL, Ouzounelli M, Uman GC: Impact of fever on outcome in patients with stroke and neurologic injury. Stroke 2008, 39:3029-3035. Figure 1 (abstract P328). Figure 1 (abstract P329). P <0.05 versus T0. Figure 1 (abstract P328). Figure 1 (abstract P329). P <0.05 versus T0. reason, the norepinephrine dosage was augmented. The proportion of patients who had infusion of norepinephrine increased from 42.8% at T0 to 78.6% at T120 (P = 0.02, chi-square for trends).f Results Fifteen NICU patients (nine subarachnoid hemorrhage, fi ve traumatic brain injury, mean age 54.9 ± 16.8, seven (50%) males, median GCS 7) were prospectively studied. We analyzed the administration of one dose of paracetamol for each patient (total 14 cases). After infusion of paracetamol we found a decrease of Temp. (from 37.8 ± 0.3 to 37.4 ± 0.4°C, P <0.001), MAP (from 94.7 ± 9.9 to 86.1 ± 6.7 mmHg, P = 0.008), CPP (from 79.6 ± 13.1 to 70.8 ± 7.6 mmHg, P = 0.011) and HR (from 71.5 ± 14.9 to 63.8 ± 16.3 bpm, P <0.001) with respect to the starting value (ANOVA for repeated measures), whereas ICP and SjVO2 remained unchanged (Figure 1). In fi ve cases norepinephrine infusion was started for CPP <60 mmHg. In another two cases, for the same q Conclusion Use of i.v. paracetamol is eff ective in the maintenance of normothermia in acute brain-injured patients. However, adverse hemodynamic eff ects, which could represent a secondary insult for the injured brain, must be rapidly recognized and treated. Reference S126 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 P330 admission, and readmissions in the 12 following months. Primary outcome was unplanned readmission 30 days, 3 months and 6 months post discharge. Intravenous paracetamol for fever control in acute brain-injured patients: cerebral and hemodynamic eff ects Further studies evaluating reasons for readmission are warranted in order to develop strategies to prevent such events. Results In 2011, a total of 20 patients were treated in our ICU with barbiturate coma for refractory elevated ICP. In fi ve patients, systemic hypothermia was simultaneously induced. These patients were excluded from further analysis. Six patients died during their stay in the ICU and nine patients could be discharged to the neurosurgical ward. The mean peak ICP value before induction of barbiturate coma was 26 ± 3 mmHg, and the ICP value 6 hours later was 20 ± 6 mmHg. The MAP at those same time points was 91 ± 12 mmHg and 81 ± 11 mmHg, respectively. The CPP, calculated from both previous values, was 66 ± 13 mmHg and 61 ± 11 mmHg, respectively. At both time points, the dose of noradrenaline was comparable (10.1 ± 9.6 μg/minute and 11.5 ± 13.2 μg/minute, respectively). Analysis of the change in ICP, MAP and CPP over the 6-hour period showed a signifi cant decrease in ICP of –5.8 mmHg (–9.3; –2.3) (mean, 95% CI). The MAP signifi cantly decreased by –10.4 mmHg (–19.1; –1.7). The decrease in CPP was not signifi cant (–4.6 mmHg (–14.2; 5.0)). P332 Risk factors of pituitary disorders following traumatic brain injury F Lauzier1, O Lachance1, I Cote1, B Senay1, P Archambault1, F Lamontagne2 A Boutin1, M Shemilt1, L Moore1, F Bernard3, C Gagnon1, D Cook4, AF Turgeon1 1Universite Laval, Quebec, Canada; 2Universite de Sherbrooke, Canada; 3Universite de Montreal, Canada; 4McMaster University, Hamilton, Canada Critical Care 2013, 17(Suppl 2):P332 (doi: 10.1186/cc12270) Risk factors of pituitary disorders following traumatic brain injury F Lauzier1, O Lachance1, I Cote1, B Senay1, P Archambault1, F Lamontagne2 A Boutin1, M Shemilt1, L Moore1, F Bernard3, C Gagnon1, D Cook4, AF Turgeon1 g 1Universite Laval, Quebec, Canada; 2Universite de Sherbrooke, Canada; 3Universite de Montreal, Canada; 4McMaster University, Hamilton, Canada Critical Care 2013, 17(Suppl 2):P332 (doi: 10.1186/cc12270) g 1Universite Laval, Quebec, Canada; 2Universite de Sherbrooke, Canada; 3Universite de Montreal, Canada; 4McMaster University, Hamilton, Canada Critical Care 2013, 17(Suppl 2):P332 (doi: 10.1186/cc12270) Introduction Pituitary disorders following traumatic brain injury (TBI) are frequent, but their determinants are poorly understood. We performed a systematic review to assess the risk factors of TBI- associated pituitary disorders. p y Methods We searched MEDLINE, Embase, Scopus, The Cochrane Library, BIOSIS, and Trip Database, and references of narrative reviews for cohort, cross-sectional and case–control studies enrolling at least fi ve adults with TBI in whom ≥1 pituitary axis was tested and one potential predictor reported. Two independent investigators selected citations, extracted data and assessed the risk of bias. We pooled the data from all studies assessing a specifi c predictor, regardless of the pituitary axis being evaluated. When more than one pituitary axis was assessed, we used the data related to hypopituitarism or the data from the most defective axis. When a pituitary axis was evaluated several times, we used assessment farthest from the injury. A meta-analysis was performed using random eff ect models and I2 was used to evaluate heterogeneity. Studies were considered at low risk of bias if the authors defi ned inclusion/exclusion criteria, did not use voluntary sampling, and tested > 90% of patients with proper detailed diagnostic criteria. i Conclusion This retrospective study indicates that inducing a thiopental coma in patients with refractory elevated ICP eff ectively reduces ICP. Although the concomitant reduction in MAP was signifi cant, the resulting decrease in CPP was small and nonsignifi cant. Moreover, the target CPP of 60 mmHg was maintained after induction of barbiturate coma. Reference 1. 1. Haddad et al.: Scand J Trauma Resusc Emerg Med 2012, 20:12-27. P332 Haddad et al.: Scand J Trauma Resusc Emerg Med 2012, 20:12-27. P331 H i S127 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 P333 P333 Vasopressin reduces the cumulative epinephrine dose in hypothermic swine with traumatic brain injury AD Goldberg, MG Kashiouris, L Keenan, A Rabinstein, M Kumar Mayo Clinic, Rochester, MN, USA Critical Care 2013, 17(Suppl 2):P333 (doi: 10.1186/cc12271) Introduction Severe traumatic brain injury (TBI) is a major cause of death and of severe neurologic sequelae. Long-term functional outcome of TBI and its best timing of assessment are not well understood, and may be evaluated too prematurely in clinical studies because of resources required to do so without too much missing data. Hence, we conducted a systematic review of studies in severe TBI patients to evaluate the long-term functional outcome. We hypothesized that functional impact measured by the Glasgow Outcome Scale (GOS), or the extended version (GOSe), may plateau after several months in patients with severe TBI. Introduction Severe traumatic brain injury (TBI) is a major cause of death and of severe neurologic sequelae. Long-term functional outcome of TBI and its best timing of assessment are not well understood, and may be evaluated too prematurely in clinical studies because of resources required to do so without too much missing data. Hence, we conducted a systematic review of studies in severe TBI patients to evaluate the long-term functional outcome. We hypothesized that functional impact measured by the Glasgow Outcome Scale (GOS), or the extended version (GOSe), may plateau after several months in patients with severe TBI. Introduction Prevention of secondary neurologic injuries is paramount for improved neurologic outcomes after traumatic brain injury (TBI). Evidence suggests that although therapeutic hypothermia (TH) lowers intracranial pressure and attenuates secondary cerebral insults after TBI [1], it also induces hypotension. Brief episodes of mild hypotension in brain-injured patients can trigger secondary injuries, which have been associated with increased mortality in patients with TBI [2]. Vasopressin mitigates hypotension in septic shock and improves coronary perfusion in hypothermic cardiac arrest models [3]. We hypothesized that a low- dose vasopressin infusion may reduce the cumulative epinephrine dose in hypothermic, brain-injured swine. Methods We performed a systematic review of randomized controlled trials and cohort studies (prospective and retrospective) in patients with severe TBI. We searched MEDLINE, Embase, Cochrane Central, BIOSIS, CINAHL and Trip Database from their inception to December 2011. References of included studies were searched for additional studies. P331 H i P331 Hospital readmissions following traumatic brain injury A Boutin, K Francisque, L Moore, F Lauzier, X Neveu, A Turgeon Université Laval, Québec, Canada Critical Care 2013, 17(Suppl 2):P331 (doi: 10.1186/cc12269) p p p g Results Among 13,559 citations, we included 26 studies (1,708 patients). Increased age was associated with pituitary disorders (weighted mean diff erence = 3.2, 95% CI = 0.3 to 6.1, 19 studies, 1,057 patients, I2 = 59%). This fi nding was no longer signifi cant when only considering studies with low risk of bias. TBI severity was associated with an increased risk of developing pituitary disorders (RR = 1.49, 1.24 to 1.77, I2 = 17% for moderate/severe vs. mild TBI; RR = 1.78, 1.09 to 2.91, I2 = 80% for severe vs. mild/moderate TBI), while sex was not (RR for male = 1.05, 0.98 to 1.13, 15 studies, 870 patients, I2 = 0%). Among CT scan fi ndings, only skull fractures were associated with pituitary disorders (RR = 1.75, 1.13 to 2.70, six studies, 345 patients, I2 = 47%). An insuffi cient number of studies with low risk of bias assessing the association between GCS, CT scan fi ndings and pituitary disorders was retrieved to perform meta-analysis. Introduction Evaluating resource utilization is paramount in critically ill patients with traumatic brain injury (TBI), but little is known on readmissions after hospital discharge. We evaluated rates and determinants of unplanned readmission following TBI. Methods We conducted a multicenter retrospective cohort study from April 1998 to March 2009. Data were obtained from a Canadian provincial trauma system, based on mandatory contribution from 59 trauma centres, and a hospital discharge database. Patients aged ≥16 years with TBI (ICD-9 or ICD-10 codes of 850-854 and S06, respectively) were included. Patients who died during the index hospitalization, who lived outside the province, who could not be linked with the hospital discharge database were excluded. We collected baseline and trauma characteristics, hospital admissions in the 12 months preceding index Conclusion Age, TBI severity and skull fractures are associated with an increased risk of pituitary disorders. Further studies are necessary to identify additional factors that will help developing targeted screening strategies. References 1. Polderman KH, et al.: Intensive Care Med 2002, 28:1563-1573. 2. Fearnside MR, et al.: Br J Neurosurg 1993, 7:267-279. 2. Fearnside MR, et al.: Br J Neurosurg 199 Results All 31 (100%) units used ICP monitoring and parenchymal ICP bolt was the most widely used method (77%). Thirty (96.7%) units used a cerebral perfusion pressure protocol to guide therapy. Twenty-eight (90.3%) units used continuous capnography. Twelve (38.7%) units used transcranial Doppler. Eight (25.8%) units used partial pressure of oxygen in brain tissue. Four (12.9%) units used microdialysis. Only one unit used jugular bulb oximetry. None of the units used near-infrared spectrometry or optic nerve sheath diameter. Eight units (25.8%) used a cerebral function monitor and seven (22.5%) units used the bispectral index for guiding depth of sedation. 3. Voelckel WG, et al.: Anesth Analg 2000, 91:627-634. Monitoring of severe traumatic brain injury patients in UK ICUs: a national survey Monitoring of severe traumatic brain injury patients in UK ICUs: a national survey S Panchatsharam, B Lewinsohn, G De La Cerda, D Wijayatilake Queen’s Hospital, Romford, UK Critical Care 2013, 17(Suppl 2):P335 (doi: 10.1186/cc12273) S Panchatsharam, B Lewinsohn, G De La Cerda, D Wijayatilake Queen’s Hospital, Romford, UK Introduction Prevention of secondary brain injury is the cornerstone in the management of patients with severe traumatic brain injury (TBI) and raised intracranial pressure (ICP). Although a variety of monitoring methods are available, due to lack of strong evidence their use varies considerably [1]. The objective of this survey was to provide an overview of the current practice in monitoring of patients with severe TBI in all neuro-ICUs across the UK. Results The median cumulative epinephrine dose in the animals that received the vasopressin infusion was 715 mg with a 25th to 75th interquartile range (IQR) of 320 to 930 mg. The median cumulative epinephrine dose in the control group was 2,044 mg (IQR 1,640 to 2,344 mg). This was statistically signifi cant (P = 0.003), based on the Wilcoxon rank-sum test. Conclusion A low-dose infusion of vasopressin can signifi cantly reduce vasopressor requirements and improves hemodynamics in hypothermic, brain-injured swine. This hemodynamic stability may improve neurological outcomes. Methods The ICUs managing adult patients with severe TBI were identifi ed from the RAIN (Risk Adjustment In Neurocritical care) study sites, and the Society of British Neurosurgeons. Thirty-one centers were identifi ed, and a telephonic survey was conducted by the investigators. Data were collected using a single-page questionnaire containing 18 questions. P331 H i Two reviewers independently determined study eligibility and collected data. The primary outcome measure was the proportion of unfavourable functional outcome (GOS 1 to 3 or GOSe 1 to 4) at 6 to 12 months, 12 to 18 months, 18 to 24 months and more than 24 months after severe TBI. We calculated Freeman Tukey-type arcsine square- root transformations and pooled data using random-eff ect models. Heterogeneity was assessed with the I2 test and sensitivity analyses were based on a priori hypotheses. Methods Six domestic cross-bred pigs were subjected to epinephrine infusion after general anesthesia, standardized TBI and transpulmonary hypothermia (32°C for 48 hours). All animals received the same care, aiming for a mean arterial pressure >60 mmHg. At hour 24, animals received additional vasopressin infusion at 0.04 units/minute. We measured the cumulative epinephrine dose for each animal pre and post vasopressin infusion (Figure 1) and performed a two- sample Wilcoxon rank-sum test, comparing the median cumulative epinephrine doses in the two groups. Results In total, 4,432 studies were assessed for eligibility; 209 studies (n = 31,540) were included. In the 188 studies using the GOS, a poor functional outcome was observed in 56.6% (95% CI = 54.0 to 59.1%, I2 = 91%), 51.9% (95% CI = 38.0 to 59.0%, I2 = 84%), 57.0% (95% CI = 48.2 to 55.5%, I2 = 93%) and 56.9% (95% CI = 48.2 to 65.1%, I2 = 93%) of patients at 6 to 12 months, 12 to 18 months, 18 to 24 months and beyond 24 months, respectively. In the 18 studies using GOSe, a poor functional outcome was observed in 62.9% of patients at 6 to 12 months (95% CI = 55.9 to 69.2%, I2 = 90%) and 54.6% at 12 to 18 months (95% CI = 43.2 to 65.8%, I2 = 90%). Heterogeneity was present in most analyses and was not entirely explained by the planned sensitivity analyses. Figure 1 (abstract P333). Eff ect of vasopressin on cumulative amount of epinephrine administered. Conclusion Considering that the incidence of patients with an unfavourable outcome remained constant at diff erent assessments, a follow-up of severe TBI patients longer than 12 months does not provide incremental information. Functional outcomes measured longer than 12 months after the injury may not be warranted in clinical studies. Survival analysis after head injury: is a normal INR reassuring? K Leclerc-Gagne Hôpital Sacré-Coeur de Montréal, Canada Critical Care 2013, 17(Suppl 2):P336 (doi: 10.1186/cc12274) Critical Care 2013, 17(Suppl 2):P337 (doi: 10.1186/cc12275) Introduction Intracranial bleeding after head injury is an important issue in the emergency department (ED), and elevated INR increases this risk [1]. A retrospective study showed an association between diff erent levels of normal INRs (<1.6) and elevated risk of intracranial bleeding, with a signifi cant elevation of this risk over an INR threshold of 1.3 [2]. The objective of the study was to evaluate clinical impact of an INR within the normal range in patients with head trauma. We compared mortality between patients with INR <1.3 and those with INR ≥1.3 to <1.6. Introduction Traumatic brain injury (TBI) is a leading cause of death and disability worldwide. Because mortality seriously underestimates the impact of TBI, an outcome tool including vegetative state and severe disability should be used. Our purpose was to study the long- term outcome of a small cohort of severe TBI patients in Brazil. Methods This was a retrospective analysis of a prospectively collected database. We included 34 consecutive adult patients admitted with severe TBI to a tertiary private hospital in Rio de Janeiro, Brazil between 2009 and 2011. We analyzed data on demographics, admission, clinical scores, imaging and complications, as well as hospital mortality and 1-year extended Glasgow Outcome Scale (eGOS) of all patients. Methods A post hoc analysis of prospective data collected from 3,356 patients seen in a tertiary-care ED from March 2008 to February 2011. We included patients aged 16 years old and over with an INR <1.6 and a head CT performed within 24 hours of the trauma. We followed these patients until December 2012, performed a chi-square test between mortality of the two groups and calculated the hazard ratio (HR) from survival analysis using Cox regression. y Results We analyzed 33 patients with severe TBI. Mean age was 36 (± 20) years and 79% were male. All patients were mechanically ventilated and 70% underwent ICP monitoring. Overall hospital mortality was 36% and 60% had unfavorable outcome (eGOS <4) after 1 year. In univariate analysis, higher APACHE II scores, the presence of midriasis and intracranial hypertension were more frequent in nonsurvivors compared with survivors. g References 1. Lingsma HF, et al.: Early prognosis in traumatic brain injury: from prophecies to predictions. Lancet Neurol 2010, 9:543-554. 1. Lingsma HF, et al.: Early prognosis in traumatic brain injury: from h i di i L N l 2010 9 543 554 1. Lingsma HF, et al.: Early prognosis in traumatic brain injury: from prophecies to predictions Lancet Neurol 2010 9:543-554 1. Wijayatilake et al.: Updates in the management of intracranial pressure in traumatic brain injury. Curr Opin Anaesthesiol 2012, 25:540-547. tilake et al.: Updates in the management of intracranial pressure in Wijayatilake et al.: Updates in the management of intracranial pres 1. Wijayatilake et al.: Updates in the management of intracranial pressure in traumatic brain injury. Curr Opin Anaesthesiol 2012, 25:540-547. 2. Chauny JM, et al.: Non Elevated INR and Post Traumatic Intracranial Bleeding. Day of Research in Neurotraumatology. Montreal: Hôpital du Sacré-Cœur; 22 June 2012. 2. Chesnut et al.: A trial of intracranial-pressure monitoring in traumatic brain injury. N Engl J Med 2012, 367:2471-2481. 2. Chesnut et al.: A trial of intracranial-pressure monitoring in traumatic brain injury. N Engl J Med 2012, 367:2471-2481. P337 One-year extended Glasgow Outcome Scale and hospital mortality predictors in patients with severe traumatic brain injury in Brazil R Turon1, FR Ferreira1, D Prado1, P Kurtz2, M Damasceno1 1Hospital de Clínicas de Niteroi, Brazil; 2Clinica São Vicente, Rio de Janeiro, Brazil Critical Care 2013, 17(Suppl 2):P337 (doi: 10.1186/cc12275) P338 P338 Early respiratory deterioration predicts poor outcome after aneurysmal subarachnoid hemorrhage E Van Lummel, L Vergouw, M Van der Jagt Erasmus MC, Rotterdam, the Netherlands Critical Care 2013, 17(Suppl 2):P338 (doi: 10.1186/cc12276) One-year extended Glasgow Outcome Scale and hospital mortality predictors in patients with severe traumatic brain injury in Brazil R Turon1, FR Ferreira1, D Prado1, P Kurtz2, M Damasceno1 One-year extended Glasgow Outcome Scale and hospital mortality predictors in patients with severe traumatic brain injury in Brazil R Turon1, FR Ferreira1, D Prado1, P Kurtz2, M Damasceno1 Survival analysis after head injury: is a normal INR reassuring? K Leclerc-Gagne Hôpital Sacré-Coeur de Montréal, Canada Critical Care 2013, 17(Suppl 2):P336 (doi: 10.1186/cc12274) Furthermore, older age, higher APACHE II score and worse GCS on admission were associated with unfavorable outcome after 1 year, as measured by eGOS <4.i y g g Results Patient mean age was 55.1 years (SD ±23), 65% were men and mean follow-up duration was 3.1 years (SD ±0.8). A total of 115 patients (15.9%) died during follow-up: 16 (36.4%) in the group with INR ≥1.3 and 99 (14.6%) in the group with INR <1.3 (P <0.001). Results showed a Figure 1 (abstract P336). Survival analysis of patients with INR <1.3 and with INR ≥1.3. y y Conclusion Our descriptive results suggest a signifi cant burden of severe TBI in a small cohort of young patients. Moreover, severity of the primary disease, age and pupil refl exes, as well as intracranial hypertension seemed to be associated with worse neurological outcome. Long-term functional outcome in adults with severe TBI: a meta-analysis y M Asselin1, Y Lachance1, G Lalonde1, A Boutin1, M Shemilt1, L Moore1, F Lauzier1, R Zarychanski2, P Archambault1, F Lamontagne1, D Fergusson3, A Turgeon1 1Université Laval, Québec, Canada; 2University of Manitoba, Winnipeg, Canada; 3University of Ottawa, Canada Critical Care 2013, 17(Suppl 2):P334 (doi: 10.1186/cc12272) M Asselin1, Y Lachance1, G Lalonde1, A Boutin1, M Shemilt1, L Moore1, F Lauzier1, R Zarychanski2, P Archambault1, F Lamontagne1, D Fergusson3, A Turgeon1 1Université Laval, Québec, Canada; 2University of Manitoba, Winnipeg, Canada; 3University of Ottawa, Canada Critical Care 2013, 17(Suppl 2):P334 (doi: 10.1186/cc12272) S128 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 http://ccforum.com/supplements/17/S2 Conclusion This survey shows that there is no clear consensus on what types of monitoring should be used to guide management of patients with TBI. A CPP protocol, based on measurement of ICP, is the most widely used method, although a recent randomized control trial did not show any benefi t in outcome [2]. Other invasive monitoring methods, although they may help in individualized care, are still not yet popular to due lack of strong evidence. Larger multicentre portfolio studies are needed to establish their benefi ts. References signifi cantly higher risk of death in the group with INR ≥1.3: HR = 2.99 (95% CI = 1.8 to 5.1); P <0.001 (Figure 1). g Conclusion In patients with head injury and normal INR (<1.6), there is an association between an INR ≥1.3 and higher risk of death. Therefore, it would be useful to request an INR in patients presenting with a head injury when bleeding is suspected, even in the absence of anticoagulant. f P339 Evaluation of proximal–distal velocity gradient in spastic middle cerebral artery after aneurismal subarachnoid hemorrhage M Gotti1, F Stretti1, S Piff eri1, V Conte1, M Zara1, N Stocchetti2 1Fondazione IRCCS Ca’ Granda-Ospedale Maggiore Policlinico, Milan, Italy; 2Fondazione IRCCS Ca’ Granda-Ospedale Maggiore Policlinico, Milan University, Milan, Italy Critical Care 2013, 17(Suppl 2):P339 (doi: 10.1186/cc12277) Results Twenty-fi ve patients (age 57 ± 12 years, WFNS 1 to 5) were included in the analysis. Overall increased levels of MPs were observed at early phase after aSAH when compared with controls. Contrarily to our hypothesis, in the early phase no signifi cant diff erences of MP level were observed in WFNS 4 to 5 (n = 9) when compared with WFNS 1 to 2 (n = 16) patients. Interestingly, patients with most severe ECI and DCI showed the highest values of MPs, but this trend did not reach statistical signifi cance, probably because of the small number of patients included in the study. Introduction Delayed cerebral ischemia (DCI) worsens neurological outcome in patients with aneurysmal subarachnoid hemorrhage (aSAH). DCI pathogenesis is multifactorial and not completely understood, but vasospasm plays a central role. Transcranial color- coded duplex sonography (TCCDS) is a non-invasive bedside tool to explore cerebral vessels, but specifi city is still suboptimal. The aim of this study was to evaluate the proximal–distal gradient of mean cerebral blood fl ow velocity (CBF-V) in middle cerebral artery (MCA) as a possible indicator of critical vasospasm. Conclusion Microparticles and microthrombosis may increase the severity of early and delayed ischemic damage after aSAH. p p Methods Consecutive aSAH patients (WFNS 1 to 5, age 18 to 80 years) admitted to NeuroICU between November 2011 and September 2012 were included in this study. TCCDS was used to assess CBF-V in MCA of both sides: we defi ned TCCDS vasospasm as CBF-V >120 cm/second. Magnetic resonance angiography (MRA, 3D TOF HR) was performed to evaluate vasospasm at early (<3 days) and delayed (7 to 10 days) time points. When patients underwent MRA, in the same clinical conditions, we recorded CBF-V with TCCDS at two levels: at the origin (proximal CBF-V) and at the end (distal CBF-V) of the MCA. Then we calculated the absolute value of proximal–distal CBF-V gradient for MCA of both sides. We defi ned clinical vasospasm as the appearance of a new neurological defi cit confi rmed by imaging. P338 Early respiratory deterioration predicts poor outcome after aneurysmal subarachnoid hemorrhage E Van Lummel, L Vergouw, M Van der Jagt Erasmus MC, Rotterdam, the Netherlands Critical Care 2013, 17(Suppl 2):P338 (doi: 10.1186/cc12276) Introduction Pulmonary complications are frequently occurring medical complications after aneurysmal subarachnoid hemorrhage (aSAH) [1]. Early respiratory deterioration (ERD) may be associated with delayed cerebral ischemia (DCI) or outcome and would then be a potential target for therapeutic interventions. We investigated whether respiratory deterioration within the fi rst 72 hours after admission predicted DCI or poor outcome. Methods We conducted a retrospective study in 137 consecutively admitted patients with aSAH, admitted between October 2007 and October 2011 to the ICU of a university hospital. ERD was defi ned as increased need for ventilatory support the second or third day after admission (Table 1). Elective intubation for a surgical procedure was not included as ERD. Inclusion criteria were availability of detailed information on respiratory status and level of support, admission within 48 hours after hemorrhage and age ≥18 years. Multivariable Figure 1 (abstract P336). Survival analysis of patients with INR <1.3 and with INR ≥1.3. Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S129 Table 1 (abstract P338). Categories of respiratory support Level of respiratory support Category (compared with fi rst admission day) 1 Up to 5 l O2 2 >5 l O2 by nasal canulla or Venturi mask 3 Nonrebreathing mask or non-invasive positive-pressure ventilation 4 Invasive mechanical ventilation When mechanically Decreased PaO2/FiO2 ratio terminated Results We included 26 consecutive aSAH patients (WFNS 1 to 5, age 57 ± 12 years). The absolute value of MCA CBF-V gradient was higher in: (1) vessels aff ected by TCCDS vasospasm when compared with vessels not aff ected (63 cm/second, IR 23 to 85 and 15 cm/second IR 7 to 23 respectively, P <0.001); (2) vessels aff ected by MRA vasospasm when compared with vessels not aff ected (43 cm/second, IR 18 to 78 and 13 cm/second IR 6 to 22, P <0.001); and (3) in patients who developed clinical vasospasm than in patients who did not (56 cm/second IR 11 to 124 and 16 cm/second IR 8 to 30 respectively, P <0.01). Considering only the subset of MCAs aff ected by MRA vasospasm, the CBF-V gradient was higher when clinical vasospasm was also present (75 cm/ second IR 56 to 124 and 23 cm/second IR 17 to 30 respectively, P <0.05). Conclusion Proximal–distal CBF-V gradient of MCA might be a reliable indicator of critical vasospasm but further studies are warranted to defi ne threshold values and specifi city in aSAH patients. P338 Early respiratory deterioration predicts poor outcome after aneurysmal subarachnoid hemorrhage E Van Lummel, L Vergouw, M Van der Jagt Erasmus MC, Rotterdam, the Netherlands Critical Care 2013, 17(Suppl 2):P338 (doi: 10.1186/cc12276) survival analysis was used to investigate associations of DCI, death and Glasgow Outcome Scale (GOS) with ERD adjusted for condition on admission, Hijdra score, treatment of ruptured aneurysm and pulmonary comorbidity. GOS was assessed at 3 to 6 months after the bleed. DCI was defi ned as described recently [2]. P340 P340 Microparticles in aneurismal subarachnoid hemorrhage: role in acute and delayed cerebral ischemia V Conte1, A Artoni1, S Magnoni1, M Leoni1, L Capuano1, V Civelli1, N Stocchetti2 1Fondazione IRCCS Ca’ Granda-Ospedale Maggiore Policlinico, Milan, Italy; 2Fondazione IRCCS Ca’ Granda-Ospedale Maggiore Policlinico, Milan University, Milan, Italy Critical Care 2013, 17(Suppl 2):P340 (doi: 10.1186/cc12278) i y Results Mean age of the patients was 55.9 (± 12.7) and 63.5% was female. A total 46.7% of the patients developed DCI. Mortality was 25.6%. Forty percent of the patients were classifi ed as having ERD. ERD was not associated with DCI (adjusted HR = 1.48; 95% CI = 0.88 to 2.49; P = 0.14). ERD showed a trend towards an association with mortality (adjusted HR = 2.22; 95% CI = 0.96 to 5.14; P = 0.06; additionally adjusted for age, and rebleed). A clear association was found between absence of ERD and functional outcome with ordinal logistic regression analysis (0.98 point increase in GOS score at 3 to 6 months; 95% CI = 0.14 to 1.82; P = 0.02; additionally adjusted for age and rebleed). Introduction Microthrombosis has been demonstrated in early and delayed cerebral ischemia after aneurysmal subarachnoid hemorrhage (aSAH). Markers of coagulation activation as microparticles (MPs) are an established risk factor for thrombosis. Our hypothesis was that levels of microparticles might correlate with aSAH severity, early cerebral ischemia (ECI) and delayed cerebral ischemia (DCI). y j g Conclusion ERD within 72 hours after admission is associated with increased risk of poor functional outcome after aSAH, but not DCI. Further investigations are required to assess whether prevention of ERD may improve outcome. Methods Consecutive aSAH patients (age 18 to 80) admitted to our department between November 2011 and September 2012 were enrolled in the study. Total MPs (anxV+), platelet MPs (PLT-MPs, anx+/ CD41+), tissue factor MPs (TF-MPs, anx+/CD142+), and endothelial MPs (E-MPs, anx+/CD144+) were assessed in venous blood samples. Measurement of MPs levels were obtained at early (<3 days) and delayed phase (7 to 10 days). Multiparameter MRI (T2; FLAIR; T1; AngioRM 3D TOF HR; DWI) was performed to evaluate ischemic damage and vasospasm at the same time points. Qualitative evaluation of severity of ischemic damage was performed on DWI sequences. Levels of MPs were evaluated comparing diff erent groups in terms of SAH severity (World Federation of Neurological Surgeons score or WFNS), ECI severity and occurrence of delayed complications (vasospasm and DCI).i References References 1. Friedman JA, et al.: Neurosurgery 2003, 52:1025-1031. 2. Vergouwen MD, et al.: Stroke 2010, 41:2391-2395. P339 The relationships between the absolute value of TCCDS CBF-V gradient and (1) TCCDS vasospasm, (2) MRA vasospasm and (3) clinical vasospasm were explored. P341 P341 Intracranial pressure after subarachnoid hemorrhage b d 1 l 1 2 h 1 S h 1 Intracranial pressure after subarachnoid hemorrhage A Lombardo1, T Zoerle1, ER Zanier2, L Longhi1, N Stocchetti1 1Fondazione IRCCS Ca’ Granda-Ospedale Maggiore Policlinico, University of Milan, Italy; 2Mario Negri Institute, Milan, Italy Critical Care 2013, 17(Suppl 2):P341 (doi: 10.1186/cc12279) Introduction Elevated intracranial pressure (ICP) may have deleterious eff ects on cerebral metabolism and mortality after aneurysmal subarachnoid hemorrhage (SAH) [1,2], but its relevance has not yet been well explored. Aims of this study are to track ICP changes after SAH, to identify clinical factors associated with it and to explore the relationship between ICP and outcome. Introduction Elevated intracranial pressure (ICP) may have deleterious eff ects on cerebral metabolism and mortality after aneurysmal subarachnoid hemorrhage (SAH) [1,2], but its relevance has not yet been well explored. Aims of this study are to track ICP changes after SAH, to identify clinical factors associated with it and to explore the relationship between ICP and outcome. S130 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Methods A total of 116 consecutive SAH patients with ICP monitoring were enrolled. Episodes of ICP >20 mmHg for at least 5 minutes and the mean ICP value for every 12-hour interval were analyzed. The highest mean ICP collected in every patient was identifi ed. ICP values were analyzed in relation to clinical and CT fi ndings; 6-month outcome and ICU mortality were also introduced in multivariable logistic models. with aSAH. Studies including more than 50% of non-aneurismal SAH were excluded. Studies were considered at low risk of bias if the authors defi ned inclusion/exclusion criteria, avoided voluntary sampling, and tested >90% of included patients with proper detailed diagnostic criteria. Studies testing all pituitary axes were considered as evaluating hypopituitarism, which was defi ned as the dysfunction of ≥1 axis. We used a Freeman Tukey-type arcsine square-root transformation and pooled prevalences using the DerSimonian–Laird random-eff ect method. We determined the degree of heterogeneity with I2 values. Results Among 12,363 citations, we included 28 studies (1,628 patients). Patients were mostly female (64%) aged 50.5 ± 5.4. Sixteen studies reported the severity of aSAH, 14 reported the procedure for securing the aneurysm and 13 reported the location of aneurysm. Overall, hypopituitarism was observed in 53.4% of patients at short- term (<3 months), 36.5% at mid-term (3 to 12 months) and 34.2% at long-term (>12 months) (Table 1). Correlation between the timing of endovascular coiling or microsurgical clipping and long-term outcomes of patients after aneurysmal subarachnoid haemorrhage N Ojukwu, R Jain, S Wijayatilake, A Bellini, R Shetty, J Khan, G De la Cerda Queen’s Hospital, London, UK Critical Care 2013, 17(Suppl 2):P343 (doi: 10.1186/cc12281) Correlation between the timing of endovascular coiling or microsurgical clipping and long-term outcomes of patients after aneurysmal subarachnoid haemorrhage N Ojukwu, R Jain, S Wijayatilake, A Bellini, R Shetty, J Khan, G De la Cerda Queen’s Hospital, London, UK Critical Care 2013, 17(Suppl 2):P343 (doi: 10.1186/cc12281) References 1. Heuer GG, et al.: Relationship between intracranial pressure and other clinical variables in patients with aneurismal subarachnoid hemorrhage. J Neurosurg 2004, 101:408-416. 1. Heuer GG, et al.: Relationship between intracranial pressure and other clinical variables in patients with aneurismal subarachnoid hemorrhage. J Neurosurg 2004, 101:408-416. 2. Nagel A, et al.: Relevance of intracranial hypertension for cerebral metabolism in aneurismal subarachnoid hemorrhage. Clinical article. J Neurosurg 2009, 111:94-101. 2. Nagel A, et al.: Relevance of intracranial hypertension for cerebral metabolism in aneurismal subarachnoid hemorrhage. Clinical article. J Neurosurg 2009, 111:94-101. P341 Intracranial pressure after subarachnoid hemorrhage b d 1 l 1 2 h 1 S h 1 There was an insuffi cient number of studies with low risk of bias to perform sensitivity analyses according to study quality. y Results Eighty-one percent of patients had at least one episode of elevated ICP and 36% had a highest mean ICP >20 mmHg. The number of patients with highest mean ICP >20 mmHg or with episodes of HICP was maximum at day 3 after SAH and decreased only after day 7. Neurological status, aneurysmal rebleeding, amount of blood on CT and CT ischemic lesion occurred within 72 hours from SAH were signifi cantly related to highest mean ICP >20 mmHg in a multivariable model. Patients with highest mean ICP >20 mmHg showed signifi cantly higher mortality in ICU. However, ICP is not an independent predictor of 6 months unfavorable outcome. Conclusion Elevated intracranial pressure is a common complication in the fi rst week after SAH. It is associated with early brain injury severity and ICU mortality. Conclusion The exact prevalence of pituitary disorders following aSAH remains uncertain, mainly due to high heterogeneity and the small number of studies with low risk of bias. However, the prevalence seems to decrease during the recovery phase. The prevalence, risk factors and clinical signifi cance of pituitary disorders in aSAH will require further rigorous evaluation. y References Prevalence of pituitary disorders associated with aneurysmal subarachnoid hemorrhage g F Lauzier, I Cote, A Bureau, M Shemilt, A Boutin, L Moore, C Gagnon, AF Turgeon Universite Laval, Quebec, Canada Critical Care 2013, 17(Suppl 2):P342 (doi: 10.1186/cc12280) Introduction Aneurysmal subarachnoid haemorrhage (aSAH) is associated with a high morbidity and mortality. Although UK Anaesthesia Guidelines advocate early coiling or clipping of the aneurysm within the fi rst 72 hours of admission for all grades of aSAH, the optimal timing of treatment and whether this is linked with better neurological long-term outcome are a subject of debate [1]. We aimed to investigate whether the timing of the occlusion of the aneurysm translates into better outcome. Introduction Pituitary disorders are an often-neglected consequence of aneurysmal subarachnoid hemorrhage (aSAH). We systematically reviewed their prevalence, aiming particularly at studies with low risk of bias. Methods We searched Embase, MEDLINE, The Cochrane Library, Trip Database, references of included studies and narrative reviews. We included cohort studies, cross-sectional studies and RCTs published in any language that tested the integrity of ≥1 pituitary axis in adults Methods A retrospective analysis of prospective collected data in a tertiary neuroscience centre from January to September 2012. Neuroprotective eff ect of a new synthetic aspirin–decursinol adduct in a rat model of ischemic stroke Neuroprotective eff ect of a new synthetic aspirin–decursino adduct in a rat model of ischemic stroke YH Lee, CW Park, JH Cho, MH Won Kangwon National University, Chuncheonsi, South Korea Critical Care 2013, 17(Suppl 2):P345 (doi: 10.1186/cc12283) patients were managed according to the local Guidelines for the management of aSAH. Outcome was assessed at 3 months using the extended Glasgow Outcome Scale (GOSE) defi ning good recovery as a GOSE ≥7 and poor outcome as GOSE ≤6. Introduction Stroke is a major cause of death. This study investigated the preventative eff ect of a new synthetic drug on brain function in experimentally induced ischemic stroke. Introduction Stroke is a major cause of death. This study investigated the preventative eff ect of a new synthetic drug on brain function in experimentally induced ischemic stroke. Results A total of 28 patients were included within the study period. Three patients were not expected to survive the fi rst 24 hours and were not included in the study. Seventeen patients were classifi ed as good grade aSAH (WFNS I to III) and eight as poor grade (IV to V). Twenty-two patients underwent successful coiling while the other three required clipping due to unsuccessful coiling. We did not fi nd any correlation between the timing of coiling/clipping and the 3-month GOSE (Figure 1). A total 44% of the patients had a poor 3-month GOSE while 56% had a good long-term functional outcome. The overall mortality rate was 21%. Methods Male Sprague–Dawley rats were administered aspirin (ASA), decursinol (DA) or ASA-DA before and after ischemic insults. Brain and neuronal damage were examined by TTC staining, PEP-CT, NeuN immunohistochemistry and F-J B histofl uorescence. Gliosis was also observed by GFAP and iba-1 immunohistochemistry. y y Results Pretreatment with 20 mg/kg, but not 10 mg/kg, of ASA-DA protected against ischemic neuronal death and damage, and its neuroprotective eff ect was much more pronounced than that of ASA or DA alone. In addition, treatment with 20 mg/kg ASA-DA reduced the ischemia-induced activation of astrocytes and microglia.i Conclusion Overall mortality in patients with aSAH is low when aneurysm is treated early post rupture of aneurysm. We did not fi nd any correlation between the timing of occlusion of aneurysm and the 3-month functional outcome. Prevalence of pituitary disorders associated with aneurysmal subarachnoid hemorrhage All Table 1 (abstract P342) Hypopituitarism GH ACTH TSH Gonadal ADH Short term All studies 53.4% (20.5 to 84.7) 30.4% (11.5 to 53.2) 13.2% (4.4 to 25.1) 49.2% (0.1 to 17.7) 26.1% (8.8 to 48.0) 6.3% (2.7 to 11.0) n (patients) 5 (235) 8 (346) 13 (537) 7 (327) 8 (351) 3 (481) I2 96.0% 93.5% 90.2% 89.1% 93.0% 61.1% Studies of low risk of bias (patients) 2(56) – – 2(56) 1(26) – Mid-term All studies 36.5% (13.6 to 62.8) 7.3% (0.6 to 18.3) 8.9% (1.3 to 21.0) 8.8% (2.4 to 18.0) 3.9% (0.0 to 11.9) 3.7% (0.6 to 13.5) n (patients) 4 (109) 6 (158) 7 (208) 5 (176) 6 (178) 2(56) I2 85.7% 70.9% 79.4% 59.4% 66.8% 29.0% Studies with low risk of bias (patients) 1(40) – – 1(40) – – Long-term All studies 34.2% (18.7 to 51.4) 17.1% (9.4 to 26.4) 11.2% (4.8 to 19.4) 2.3% (0.4 to 5.3) 3.2% (0.7 to 6.8) 0% (0 to 1.5) n (patients) 12 (486) 13 (531) 13 (531) 12 (486) 13(351) 3 (142) I2 92.5% 82.9% 82.9% 49.0% 62.9% 0% Studies with low risk of bias (patients) 2(40) 2(40) 2(40) 2(40) 1(10) – Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S131 Results All WFNSpt 1 to 2 patients completed neuropsychological tests at each time point. WFNSpt 3 and WFNS 4 patients were testable in 80% and 50% of the cases respectively at early time points. WFNS 5 patients were not testable at any time point. In all testable patients, cognitive functions were severely impaired at early time points. At 3 months in WFNS 1 to 3 a good recovery of language defi cits while only a partial recovery of attention, memory and executive functions were observed; at the same time point 70% of WFNS 4 patients became testable, but they had a worse recovery of all cognitive functions. At 1 month after SAH less than 30% of patients return to work, at 3 months approximately 50%. Despite a good recovery of everyday life activities at 3 months, for all patients quality of life was lower than a normal population.f Figure 1 (abstract P343). Time of coiling/clipping versus GOSE boxplot. Prevalence of acute critical neurological disease in children: a global epidemiological assessment (PANGEA) Prevalence of acute critical neurological disease in children: a global epidemiological assessment (PANGEA) E Fink1, R Tasker2, J Beca3, MJ Bell1, RS Clark1, J Hutchison4, M Vavilala5, RS Watson1, L Weissfeld6, PM Kochanek1, DC Angus7, PANGEA Investigators1 1Children’s Hospital of Pittsburgh, PA, USA; 2Children’s Hospital Boston, MA, USA; 3Starship Children’s Hospital, Auckland, New Zealand; 4The Hospital for Sick Children, Toronto, Canada; 5Seattle Children’s Hospital, Seattle, WA, USA; 6University of Pittsburgh, PA, USA; 7University of Pittsburgh Medical Center, Pittsburgh, PA, USA Critical Care 2013, 17(Suppl 2):P346 (doi: 10.1186/cc12284) Prevalence of pituitary disorders associated with aneurysmal subarachnoid hemorrhage p q y p p Conclusion Cognitive dysfunction has diff erent time courses after aSAH: signifi cant defi cits in diff erent cognitive domains, worse quality of life and diffi culties in return to work persist in more than 50% of patients at 3 months following SAH. P344 Time course of neuropsychological functioning after aneurysmal subarachnoid hemorrhage B Zarino1, G Bertani1, V Conte1, S Magnoni1, A Di Cristofori1, N Stocchetti2 1Fondazione IRCCS Ca’ Granda-Ospedale Maggiore Policlinico, Milan, Italy; 2Fondazione IRCCS Ca’ Granda-Ospedale Maggiore Policlinico, Milan University, Milan, Italy Critical Care 2013, 17(Suppl 2):P344 (doi: 10.1186/cc12282) Reference 1. van der Schaaf I, et al.: Stroke 2006, 37:572-573. 1. van der Schaaf I, et al.: Stroke 2006, 37:572-573. Neuroprotective eff ect of a new synthetic aspirin–decursinol adduct in a rat model of ischemic stroke Conclusion Our fi ndings indicate that ASA-DA, a new synthetic drug, prevents against transient focal cerebral ischemia, which provides a resource for the development of its clinical application for stroke. Time course of neuropsychological functioning after aneurysmal subarachnoid hemorrhage An aff ordable and reliable bedside test could change practice to an individual tailored dosing of LMWH. The aim of our study was to evaluate whether a prophylactic dose regimen of 40 mg enoxaparine in cardiac surgical patients increases the anti-Xa activity to the level necessary for effi cient prevention of a thromboembolic event [1]. Secondarily we tested whether there was a reliable correlation between a bedside anti-Xa measurement compared with a two-stage chromogenic assay at the laboratory [2]. assay remain controversial in daily clinical practice. One of the major obstacles is the cost of such a test. An aff ordable and reliable bedside test could change practice to an individual tailored dosing of LMWH. The aim of our study was to evaluate whether a prophylactic dose regimen of 40 mg enoxaparine in cardiac surgical patients increases the anti-Xa activity to the level necessary for effi cient prevention of a thromboembolic event [1]. Secondarily we tested whether there was a reliable correlation between a bedside anti-Xa measurement compared with a two-stage chromogenic assay at the laboratory [2]. Results Of 3,113 subjects screened, 1,009 (32%) met enrollment criteria. The mean number of subjects enrolled per site for each study day was 2.4. Most sites were dedicated pediatric ICUs with a mean number of 22 ICU beds (range 3 to 72). ICUs had resources to invasively monitor intracranial pressure (93%), continuous electroencephalography (14%), invasive and non-invasive brain tissue oxygenation (14% and 57%), and somatosensory evoked potentials (39%). There were on average 11 ICU faculty and six fellows per site, and nearly one-half reported a neurocritical care ICU team. Subjects were 58% male and 52% white, and 60% had normal pre-admission PCPC scores (85%). Status epilepticus and cardiac arrest (both 21%) had the highest prevalence. Sixty-one per cent of subjects were mechanically ventilated during ICU admission. ICU length of stay was a mean 29 days (median 43 days) and hospital LOS was a mean 43 days (median 61 days). Survival at hospital discharge was 87% with 58% of subjects discharged home and 17% to inpatient rehabilitation.i y y Methods This was an open, single-centre, prospective, nonrandomized clinical trial at a university hospital. All patients that needed prophylactic dosing of enoxaparine after cardiac surgery were duly informed and after giving written consent we included 44 patients with a mean Euroscore of 1.66. Eff ective dosage of enoxaparin for intensive care patients S Robinson, A Zincuk, U Larsen, P Toft Odense University Hospital, Odense, Denmark Critical Care 2013, 17(Suppl 2):P349 (doi: 10.1186/cc12287) Eff ective dosage of enoxaparin for intensive care patients S Robinson, A Zincuk, U Larsen, P Toft Odense University Hospital, Odense, Denmark Critical Care 2013, 17(Suppl 2):P349 (doi: 10.1186/cc12287) gi Results In total, 943 patients met the inclusion criteria with a mean follow-up period of 1 year. The mean age was 74.3 years and 50.4% were men. A total of 108 deaths (11.5%) were recorded within the follow-up period; eight (25%) for dabigatran compared with 44 (12.6%) for warfarin and 56 (9.9%) for aspirin. The mortality risk for patients on dabigatran was signifi cantly higher than for patients on warfarin: HR = 2.1 (95% CI: 1.0 to 4.5), P = 0.05 after controlling for age. Aspirin had a lower (but not statistically signifi cant) mortality risk compared with warfarin; HR = 0.75 (95% CI: 0.50 to 1.14), P = 0.18 after controlling for age. y p Critical Care 2013, 17(Suppl 2):P349 (doi: 10.1186/cc12287) Introduction We hypothesized that higher doses of enoxaparin would improve thromboprophylaxis without increasing the risk of bleeding. Critically ill patients are predisposed to venous thromboembolism, leading to increased risk of adverse outcome [1]. Peak anti-factor Xa (anti-Xa) levels of 0.1 to 0.4 IU/ml, 4 hours post administration of enoxaparin, refl ect adequate thromboprophylaxis for medico-surgical patients. Conclusion The results showed higher overall mortality in patients who presented to the ED with a bleeding complication and were taking dabigatran compared with warfarin or aspirin. Physicians should be aware of the potential higher mortality with dabigatran over warfarin when treating a bleeding patient. However, this was a single- centre retrospective analysis with a small number of patients taking dabigatran (n = 32), and further studies are needed to corroborate the results. p Methods The sample population consisted of 78 patients, randomized to receive subcutaneous (s.c.) enoxaparin: 40 mg ×1 (control group), versus 30 mg ×2, 40 mg ×2 or 1 mg/kg ×1 (test groups) for a period of 3 days. Anti-Xa activity was measured at baseline, and at 4, 12, 16 and 24 hours post administration on each day. Patients did not diff er signifi cantly between groups. i Results On day 1 of administration, doses of 40 mg ×1 and 40 mg ×2 yielded similar mean peak anti-Xa of 0.20 IU/ml and 0.17 IU/ml respectively, while a dose of 30 mg ×2 resulted in subtherapeutic levels of anti-Xa (0.08 IU/ml). Time course of neuropsychological functioning after aneurysmal subarachnoid hemorrhage B Zarino1, G Bertani1, V Conte1, S Magnoni1, A Di Cristofori1, N Stocchetti2 1Fondazione IRCCS Ca’ Granda-Ospedale Maggiore Policlinico, Milan, Italy; 2Fondazione IRCCS Ca’ Granda-Ospedale Maggiore Policlinico, Milan University, Milan, Italy Critical Care 2013, 17(Suppl 2):P344 (doi: 10.1186/cc12282) Introduction Early and delayed cognitive dysfunctions are an under- studied issue after aneurysmal subarachnoid hemorrhage (aSAH), irrespective of neurological outcome. The aim of this study was to describe early and delayed changes of cognitive functions, activities of everyday life, and quality of life in aSAH patients. Introduction Acute neurological injury is a leading cause of morbidity and mortality in children. Global prevalence and regional disparities of etiology, interventions, and outcomes are unknown. The aim of this point-prevalence study was to measure the burden of pediatric neurological injury and to describe variations in interventions and outcomes in ICUs. Methods Consecutive aSAH patients admitted to our ICU between November 2011 and September 2012 were prospectively studied. Patients underwent neuropsychological evaluation at early (<3 days, 10 days) and delayed time points (1 month, 3 months). Patients were tested for language, verbal fl uency, short-term and long-term memory, attention, executive functions, praxis, and neglect. Impairments in activities of everyday life were assessed using the Activities of Daily Living scale and the Instrumental Activities of Daily Living scale. The SF-36 was used to assess the quality of life at 3 months. Since complications of aneurysm treatment in addition to aSAH severity may signifi cantly aff ect cognitive status, patients were evaluated according to the World Federation of Neurological Surgeons score after treatment (WFNSpt). Methods One hundred and three ICUs on six continents enrolled subjects on 4 specifi c days in a 1-year period. Included subjects were between ages 7 days and 17 years who were diagnosed with acute traumatic brain injury, stroke, cardiac arrest, central nervous system infection or infl ammation, status epilepticus, spinal cord lesion, hydrocephalus, or brain mass. Sites completed a secure web-based case report form that included subject and hospital demographics, details about the neurological disease, interventions, length of stay, and Pediatric Cerebral Performance Category (PCPC) score (good outcome = PCPC 1 to 3) and mortality at hospital discharge. S132 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 assay remain controversial in daily clinical practice. One of the major obstacles is the cost of such a test. P347 Survival analysis of patients taking dabigatran who consulted an emergency department for acute bleeding: a fi rst alarm OA Arlet, EN Notebaert, JP Paquet, RD Daoust, MV Vincent, JC Chauny Hôpital du Sacré-Coeur de Montréal, Université de Montréal, Canada Critical Care 2013, 17(Suppl 2):P347 (doi: 10.1186/cc12285) P347 Survival analysis of patients taking dabigatran who consulted an emergency department for acute bleeding: a fi rst alarm OA Arlet, EN Notebaert, JP Paquet, RD Daoust, MV Vincent, JC Chauny Hôpital du Sacré-Coeur de Montréal, Université de Montréal, Canada Critical Care 2013, 17(Suppl 2):P347 (doi: 10.1186/cc12285) Survival analysis of patients taking dabigatran who consulted an emergency department for acute bleeding: a fi rst alarm OA Arlet, EN Notebaert, JP Paquet, RD Daoust, MV Vincent, JC Chauny Hôpital du Sacré-Coeur de Montréal, Université de Montréal, Canada Critical Care 2013, 17(Suppl 2):P347 (doi: 10.1186/cc12285) Introduction The newly approved oral anticoagulant dabigatran has no eff ective antidote. We therefore suspected an overall increase in mortality in patients presenting to the emergency department (ED) with a bleeding complication on dabigatran compared with warfarin or aspirin. Conclusion The bedside anti-Xa activity assay with a Hemochron device tends to show some correlation with the two-stage chromogenic assay, but insuffi cient to be used as an alternative, in this small but uniform patient population. Use of a standard dosing protocol for enoxaparine administration is prone for underdosage in post-cardiac surgery patients and may increase postoperative morbidity. R f p Methods We conducted a post hoc analysis on a database of all patients admitted to a tertiary-care ED with any kind of bleeding or suspicion of one from March 2011 to August 2012 who were taking dabigatran, warfarin, or aspirin. The primary endpoint was long-term survival. Patients were censored at death or at the end of the study period (7 December 2012). We performed a Cox proportional hazard model, controlled for age, to calculate the hazard ratio (HR) for dabigatran versus warfarin and one for warfarin versus aspirin. Statistical signifi cance was set at α = 0.05 and results are presented with 95% CI. Results In total, 943 patients met the inclusion criteria with a mean follow-up period of 1 year. The mean age was 74.3 years and 50.4% were men. A total of 108 deaths (11.5%) were recorded within the follow-up period; eight (25%) for dabigatran compared with 44 (12.6%) for warfarin and 56 (9.9%) for aspirin. The mortality risk for patients on dabigatran was signifi cantly higher than for patients on warfarin: HR = 2.1 (95% CI: 1.0 to 4.5), P = 0.05 after controlling for age. Aspirin had a lower (but not statistically signifi cant) mortality risk compared with warfarin; HR = 0.75 (95% CI: 0.50 to 1.14), P = 0.18 after controlling for age. Methods We conducted a post hoc analysis on a database of all patients admitted to a tertiary-care ED with any kind of bleeding or suspicion of one from March 2011 to August 2012 who were taking dabigatran, warfarin, or aspirin. The primary endpoint was long-term survival. Eff ective dosage of enoxaparin for intensive care patients S Robinson, A Zincuk, U Larsen, P Toft Odense University Hospital, Odense, Denmark Critical Care 2013, 17(Suppl 2):P349 (doi: 10.1186/cc12287) Patients receiving 1 mg/kg enoxaparin achieved near-steady-state levels from day 1 with mean peak anti-Xa levels of 0.34 IU/ml. Steady-state anti-Xa was achieved for all doses of enoxaparin at day 3. At steady state, mean peak anti-Xa levels of 0.13 IU/ ml and 0.15 IU/ml were achieved with doses of 40 mg ×1 and 30 mg ×2 respectively. This increased signifi cantly to 0.33 IU/ml and 0.40 IU/ml for doses of 40 mg ×2 and 1 mg/kg enoxaparin respectively (P = 0.0000) (Figure 1). A dose of 1 mg/kg enoxaparin yielded therapeutic anti-Xa levels for over 80% of the study period. There were no adverse eff ects. Survival analysis of patients taking dabigatran who consulted an emergency department for acute bleeding: a fi rst alarm Patients were censored at death or at the end of the study period (7 December 2012). We performed a Cox proportional hazard model, controlled for age, to calculate the hazard ratio (HR) for dabigatran versus warfarin and one for warfarin versus aspirin. Statistical signifi cance was set at α = 0.05 and results are presented with 95% CI. 1. Ribic C, et al.: J Crit Care 2009, 24:197-205. 1. Ribic C, et al.: J Crit Care 2009, 24:197-205. 1. Ribic C, et al.: J Crit Care 2009, 24:197-205. 2. Gehrie E, et al.: Am J Hematol 2012, 87:194-196. Time course of neuropsychological functioning after aneurysmal subarachnoid hemorrhage The demographic specifi cations, medical and surgical history of all patients were collected. Anti-Xa activity was measured at three diff erent points in time. We determined baseline, peak and trough anti-Xa activity: preoperatively, and respectively 4 hours after the third dose of enoxaparine and 30 minutes before the fourth dose. Each measurement was done with both techniques, the two-stage chromogenic assay at the laboratory (Biophen®) and the bedside assay (Hemochron® Jr). Conclusion Acute neurological disease is a signifi cant pediatric health issue. These data suggest a vital need for increased research and healthcare resources to assist in the challenge of improving outcomes for these children. Results Our dose regimen of enoxaparine achieved in one-half of the included patients a suffi cient anti-Xa activity for prevention of thromboembolic events. One-half of the patients with insuffi cient anti- Xa activity had a body mass index over 30 kg/m2. Comparison of the bedside assay with the two-stage chromogenic assay by means of the Pearson’s correlation coeffi cient showed correlation of the two tests if no variables were taken into account. In the Bland–Altman analysis we could not confi rm this correlation. P348 P348 Bedside anti-Xa measurement for therapeutic assessment of a prophylactic anticoagulation regimen B Dewulf1, I Herck2, F De Somer2, K Francois2, J Decruyenaere2 1AZ Sint-Jan, Brugge, Belgium; 2University Hospital, Gent, Belgium Critical Care 2013, 17(Suppl 2):P348 (doi: 10.1186/cc12286) P351 The fi rst-in-class hepcidin antagonist NOX-H94, a PEGylated anti-hepcidin L-RNA oligonucleotide, is in development for targeted treatment of anemia of infl ammation. We investigated whether NOX-H94 prevents the infl ammation-induced serum iron decrease during experimental human endotoxemia. Methods A randomized, double-blind, placebo-controlled trial in 24 healthy young men. At T = 0 hours, 2 ng/kg E. coli endotoxin was administered intravenously (i.v.), followed by 1.2 mg/kg NOX-H94 or placebo i.v. at T = 0.5 hours. Blood was drawn serially after endotoxin administration for measurements of infl ammatory parameters, Introduction Anemia is very frequently encountered on the ICU. Increased hepcidin production is one of the cornerstones of the pathophysiology of anemia of infl ammation. The fi rst-in-class hepcidin antagonist NOX-H94, a PEGylated anti-hepcidin L-RNA oligonucleotide, is in development for targeted treatment of anemia of infl ammation. We investigated whether NOX-H94 prevents the infl ammation-induced serum iron decrease during experimental human endotoxemia. Introduction Unfractionated heparin is preferred over LMWH in ICU patients but LMWH is used more frequently in many European ICUs. Thromboprophylaxis with standard doses of nadroparin and enoxaparin has been shown to result in signifi cantly lower anti-Xa in ICU patients when compared with medical patients [1,2]. p p p Methods ICU patients (SAPS 44 ± 16, MV, n = 44; pressors n = 32) received 7,500 IU (Group 1, n = 25) or 5,000 IU dalteparin s.c. (Group 2, n = 29). Twenty-nine medical patients receiving 5,000 IU dalteparin served as controls (Group 3). g p Methods A randomized, double-blind, placebo-controlled trial in 24 healthy young men. At T = 0 hours, 2 ng/kg E. coli endotoxin was administered intravenously (i.v.), followed by 1.2 mg/kg NOX-H94 or placebo i.v. at T = 0.5 hours. Blood was drawn serially after endotoxin administration for measurements of infl ammatory parameters, p Results Group 2 had signifi cantly lower areas under the Xa curve (AUC) compared with Groups 1 and 3 (Table 1). Diff erences were not signifi cant between Groups 1 and 3. Peak anti-Xa activities (Cmax-anti-Xa) Table 1 (abstract P351). g Referencesfl 1. Dörffl er-Melly J, deJonge E, dePont AC, et al.: Bioavailability of subcutaneous low-molecular-weight heparin to patients on vasopressors. Lancet 2002, 359:849-850. 1. Cook D, et al.: N Engl J Med 2011, 364:1305-1314. 2. Priglinger U, Delle Karth G, Geppert A, et al.: Prophylactic anticoagulation with enoxaparin: is the subcoutaneous route appropriate in the critically ill? Crit Care Med 2003, 31:1405-1409. 2. Priglinger U, Delle Karth G, Geppert A, et al.: Prophylactic anticoagulation with enoxaparin: is the subcoutaneous route appropriate in the critically ill? Crit Care Med 2003, 31:1405-1409. P350 Abstract withdrawn P350 Abstract withdrawn P351 Anti-Xa activities following 5,000 IU and 7,500 IU of s.c. dalteparin in critically ill patients and 5,000 IU in medical patients: a prospective randomized study GH Heinz1, U Priglinger2, I Pabinger3 1University Clinic of Internal Medicine II, Vienna, Austria; 2University Clinic of Emergency Medicine, Vienna, Austria; 3University Clinic of Internal Medicine I, Vienna, Austria Critical Care 2013, 17(Suppl 2):P351 (doi: 10.1186/cc12289) Randomized double-blind placebo-controlled PK/PD study on the eff ects of a single intravenous dose of the anti-hepcidin Spiegelmer NOX-H94 on serum iron during experimental human endotoxemia L Van Eijk1, DW Swinkels1, A John1, F Schwoebel2, F Fliegert2, L Summo2, S Vauleon2, JM Laarakkers1, K Riecke2, P Pickkers1 1Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands; 2NOXXON Pharma AG, Berlin, Germany Critical Care 2013, 17(Suppl 2):P352 (doi: 10.1186/cc12290) Anti-Xa activities following 5,000 IU and 7,500 IU of s.c. dalteparin in critically ill patients and 5,000 IU in medical patients: a prospective randomized study Randomized double-blind placebo-controlled PK/PD study on the eff ects of a single intravenous dose of the anti-hepcidin Spiegelmer NOX-H94 on serum iron during experimental human endotoxemia L Van Eijk1, DW Swinkels1, A John1, F Schwoebel2, F Fliegert2, L Summo2, S Vauleon2, JM Laarakkers1, K Riecke2, P Pickkers1 1Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands; 2NOXXON Pharma AG, Berlin, Germany Critical Care 2013, 17(Suppl 2):P352 (doi: 10.1186/cc12290) Randomized double-blind placebo-controlled PK/PD study on the eff ects of a single intravenous dose of the anti-hepcidin Spiegelmer NOX-H94 on serum iron during experimental human endotoxemia L Van Eijk1, DW Swinkels1, A John1, F Schwoebel2, F Fliegert2, L Summo2, S Vauleon2, JM Laarakkers1, K Riecke2, P Pickkers1 1Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands; 2NOXXON Pharma AG, Berlin, Germany Critical Care 2013, 17(Suppl 2):P352 (doi: 10.1186/cc12290) GH Heinz1, U Priglinger2, I Pabinger3 1University Clinic of Internal Medicine II, Vienna, Austria; 2University Clinic of Emergency Medicine, Vienna, Austria; 3University Clinic of Internal Medicine I, Vienna, Austria Critical Care 2013, 17(Suppl 2):P351 (doi: 10.1186/cc12289) GH Heinz1, U Priglinger2, I Pabinger3 1University Clinic of Internal Medicine II, Vienna, Austria; 2University Clinic of Emergency Medicine, Vienna, Austria; 3University Clinic of Internal Medicine I, Vienna, Austria Critical Care 2013, 17(Suppl 2):P351 (doi: 10.1186/cc12289) Introduction Anemia is very frequently encountered on the ICU. Increased hepcidin production is one of the cornerstones of the pathophysiology of anemia of infl ammation. Bedside anti-Xa measurement for therapeutic assessment of a prophylactic anticoagulation regimen Introduction Dose adjustments of low molecular weight heparin (LMWH) based on daily anti-Xa measurement by chromogenic S133 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum com/supplements/17/S2 http://ccforum.com/supplements/17/S2 Figure 1 (abstract P349). Peak anti-Xa over 3 days with varying doses of enoxaparin. Figure 1 (abstract P349). Peak anti-Xa over 3 days with varying doses of enoxaparin. Conclusion Current standard s.c. doses of 40 mg ×1 enoxaparin (Europe) or 30 mg ×2 (North America) yield levels of anti-Xa thought to be subtherapeutic for critically ill patients. A weight-based dose yielded the best anti-Xa levels, allowed the establishment of near-steady-state levels from the fi rst day of enoxaparin administration, and may thus be more appropriate, convenient, and eff ective. A new study using 1 mg/ kg enoxaparin s.c. with clinical endpoints has recently been approved by the Danish Ministry of Health. R f were delayed (tmax-anti-Xa) in Group 2 compared with Groups 1 and 3 (Table 1). Conclusion In ICU patients a s.c. dose of 5,000 IU dalteparin results in signifi cantly lower Xa activities when compared with normal ward patients. A s.c. dose of 7,500 IU dalteparin in ICU patients resulted in kinetics and peak anti-Xa activities comparable with medical patients receiving 5,000 IU dalteparin. P351 Pharmacokinetics Group 1 (n = 25) Group 2 (n = 29) Group 3 (n = 29) ICU, 7,500 IU ICU, 5,000 IU Normal ward, 5,000 IU P overall AUC-anti-Xa0–12 hours (U/lhour) 2.51 (1.15 to 4.61) 1.27 (1.15 to 4.4) 2.58 (1.45 to 4.87) <0.001* AUC-anti-Xa12_00 (U/lhour) 1.37 (0.58 to 13) 1.47 (0.65 to 6.3) 0.89 (0.35 to 3.88) 0.003* Cmax-anti-Xa (U/l) 0.29 (0.10 to 0.52) 0.14 (0.1 to 0.43) 0.33 (0.14 to 0.65) <0.001*** tmax-anti-Xa (hours) 3 (3 to 12) 4.5 (1 to 12) 3 (3 to 6) 0.017§ Cmax, peak anti-Xa level; tmax, time of anti-Xa peak. Medians and range. Group 1 versus 2, P = 0.001; Group 1 versus 3, P = 0.51. Group 1 versus 2, P = 0.96; Group 1 versus 3, P = 0.045; Group 2 versus 3, P = 0.13. *Group 1 versus 2, P = 0.001; Group 1 versus 3, P = 0.29. §Group 1 versus 3, P = 0.09; Group 1 versus 2, P = 0.8; Group 2 versus 3, P = 0.018. Table 1 (abstract P351). Pharmacokinetics Cmax, peak anti-Xa level; tmax, time of anti-Xa peak. Medians and range. Group 1 versus 2, P = 0.001; Group 1 versus 3, P = 0.51. Group 1 versus 2, P = 0.96; Group 1 versus 3, P = 0.045; Group 2 versus 3, P = 0.13. Group 1 versus 2, P = 0.001; Group 1 versus 3, P = 0.29. §Group 1 versus 3, P = 0.09; Group 1 versus 2, P = 0.8; Group 2 versus 3, P = 0.018. S134 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 cytokines, NOX-H94 pharmacokinetics, total hepcidin-25, and iron parameters. The diff erence of serum iron change from baseline at T = 9 hours was defi ned as the primary endpoint.ll Introduction According to many authors, acute necrotizing pancreatitis (ANP) still remains one of the diffi cult problems of abdominal surgery. The complexity of the pathogenesis of the disease, features of the pancreas pathomorphology, abdominal hypertension, and high mortality (30 to 70%) necessitate a search for new ways to treat this disease. i y Results Endotoxin administration led to fl u-like symptoms. Infl am- matory parameters (CRP, body temperature, leucocytes, and plasma levels of TNFα, IL-6, IL-10, and IL-1RA) peaked markedly and similarly in both treatment groups. P351 NOX-H94 was well tolerated. Plasma concen- trations peaked at 0.7 ± 0.4 hours after the start of administration, after which they declined according to a two-compartment model, with a T1/2 of 22.5 ± 4.28 hours. In the placebo group, serum iron increased from 19.0 ± 7.6 μg/l at baseline to a peak at T = 3 hours, returned close to baseline at T = 6 hours and decreased under the baseline concentration at T = 9 hours, reaching its lowest point at T = 12 hours. In the NOX-H94 group, serum iron concentrations rose until T = 6 hours and then slowly declined until T = 24 hours. From 6 to 12 hours post LPS, the serum iron concentrations in NOX-H94-treated subjects were signifi cantly higher than in placebo-treated subjects (P <0.0001, ANCOVA). Methods The study was conducted in 44 patients with ANP, who were divided into two groups according to type of analgesia: epidural or opioids. Patients from the fi rst group (n = 23) had epidural analgesia by ropivacaine 6 to 14 mg/hour during 7 to 10 days, and from the second group (n = 21) opioid analgesia by trimeperidine 20 mg three times a day during the same period. We monitored the level of septic and thrombohemorrhagic complications by clinical and instrumental data, during the month after treatment starting. The hemostatic system was evaluated using indicators of hemoviscoelastography (Mednord-01M analyzer). g p y y Results It was found that all patients with ANP initially have hypercoagulation and fi brinolysis inhibition. Levels of hemostatic disorders correlate with the level of septic complications, treatment in the ICU, and mortality. In the fi rst group we noted a deep vein thrombosis, two pneumonia, seven pseudopancreatic cysts and abscesses, two deaths and time of stay in the ICU as 15.4 days. In the second group: three cases of deep vein thrombosis, four pneumonia, 10 pseudopancreatic cysts and abscesses, two episodes of gastroduodenal bleeding, fi ve deaths and time of stay in the ICU as 27.8 days. Conclusion Experimental human endotoxemia induces a robust infl am matory response and a subsequent decrease in serum iron. Treatment with NOX-H94 had no eff ect on innate immunity, but eff ectively prevented the infl ammation-induced drop in serum iron concentrations. These fi ndings demonstrate the clinical potential of the anti-hepcidin drug NOX-H94 for further development to treat patients with anemia of infl ammation. Hemoviscoelastography: a new method of hemostasis monitoring O Tarabrin, S Galich, D Gavrychenko, G Mazurenko, A Suhanov Odessa National Medical University, Odessa, Ukraine Critical Care 2013, 17(Suppl 2):P355 (doi: 10.1186/cc12293) Introduction The association between haemoglobin concentrations and mortality has been studied in patients with various comorbidities [1,2]. This study aims to determine the association between haemo- globin levels on admission to intensive care and patient length of stay and mortality. Introduction Deep vein thrombosis of lower extremities and pulmonary embolism occupy an important place in the structure of postoperative morbidity and mortality. Introduction Deep vein thrombosis of lower extremities and pulmonary embolism occupy an important place in the structure of postoperative morbidity and mortality. y Methods A retrospective collection of data from patient admissions to a single fi ve-bed ICU over a 15-year period identifi ed 5,826 patients between April 1994 and November 2012. Patients were split into groups according to haemoglobin concentration on admission. The data were analysed to determine whether there was any relationship between haemoglobin concentration at ICU admission and any of our outcome measures (unit and hospital mortality, unit and hospital length of stay). Results Patients with haemoglobin concentrations ≤10 g/dl and >10.1 g/dl were used in mortality comparisons. Patients with a haemo globin concentration ≤10 g/dl had an increase in ICU mortality compared with those with haemoglobin levels >10 g/dl (OR = 1.36, 95% CI = 1.30 to 1.71, P <0.0001). A similar diff erence was seen with hospital mortality (≤10 g/dl 37.7% vs. >10 g/dl 27.3%, P <0.0001). Unit length of stay was signifi cantly longer in patients with admission Hb ≤10 g/ dl (5.34 days) compared with an admission Hb >10 g/dl (4.08 days), P <0.0001. Hospital length of stay was also signifi cantly longer in patients with Hb ≤10 g/dl versus Hb >10 g/dl (21.6 days vs. 15.5 days, P <0.0001). There was seen to be an inverse correlation between haemoglobin concentration and patient age (r = –0.174; P <0.0001). Methods After ethics approval and informed consent, we studied the functional state of hemostasis in a group of 57 healthy volunteers, who were not receiving drugs aff ecting coagulation, and 43 patients with postphlebothrombotic syndrome (PPTS). In the PPTS patients we conducted baseline studies of coagulation state and daily monitoring of dynamic changes in the functional state of hemostasis, a comparative evaluation of performance low-frequency piezoelectric vibration hemoviscoelastography (LPVH) and platelet aggregation test (PAT), standard coagulation tests (SCT), and thromboelastogram (TEG). Results We found that LPVH correlated with SCT, PAT and TEG. P355 Hemoviscoelastography: a new method of hemostasis monitoring O Tarabrin, S Galich, D Gavrychenko, G Mazurenko, A Suhanov Odessa National Medical University, Odessa, Ukraine Critical Care 2013, 17(Suppl 2):P355 (doi: 10.1186/cc12293) P353 Haemoglobin concentration on admission to intensive care infl uences hospital mortality rates and length of stay: a retrospective study M Adams, P Dean, P Doherty, S Noble, A Mackay Victoria Infi rmary, Glasgow, UK Critical Care 2013, 17(Suppl 2):P353 (doi: 10.1186/cc12291) P356 P356 Thromboelastographic monitoring of fondaparinux in surgical patients B Martinez1, R Giacomello1, R Paniccia2 1University Hospital of Udine, Italy; 2University of Florence, Italy Critical Care 2013, 17(Suppl 2):P356 (doi: 10.1186/cc12294) P356 Thromboelastographic monitoring of fondaparinux in surgical patients B Martinez1, R Giacomello1, R Paniccia2 1University Hospital of Udine, Italy; 2University of Florence, Italy Critical Care 2013, 17(Suppl 2):P356 (doi: 10.1186/cc12294) Hemoviscoelastography: a new method of hemostasis monitoring O Tarabrin, S Galich, D Gavrychenko, G Mazurenko, A Suhanov Odessa National Medical University, Odessa, Ukraine Critical Care 2013, 17(Suppl 2):P355 (doi: 10.1186/cc12293) However, our proposed method is more voluminous: indexes ICC (the intensity of the contact phase of coagulation), t1 (the time for the contact phase of coagulation), and AO (initial rate of aggregation of blood) are consistent with PAT; indexes ICD (the intensity of coagulation drive), CTA (a constant thrombin activity) and CP (the clot intensity of the polymerization) are consistent with SCT and TEG. In addition, the advantage of this method is to determine the intensity of fi brinolysis – with the indicator IRIS (the intensity of the retraction and clot lysis). Conclusion Haemoglobin concentrations ≤10 g/dl on admission to the ICU are associated with an increase in ICU mortality, hospital mortality, unit length of stay and hospital length of stay when compared with patients admitted with haemoglobin concentrations >10 g/dl. References y y Conclusion LPVH allows one to make a total assessment of all parts of hemostasis: from initial viscosity and platelet aggregation to coagulation and lysis of clots, as well as their interaction. These fi gures are objective and informative, as evidenced by close correlation with the performance of traditional coagulation methods. 1. Zhao Y, et al.: Crit Care 2007, 11:P234. 2. Sekhon MS, et al.: Crit Care 2012, 16:R128. P351 Conclusion Using epidural anesthesia in patients with ANP reduced the number of septic complications on 36.6%, and reduced the mortality rate from 23.8% (second group) to 8.7% (fi rst group). We think that violations of blood coagulation and microcirculation are the basis for ischemia, necrosis in tissues and septic complications. Epidural analgesia is an eff ective method to decrease the level of septic and thrombohemorrhagic complications and mortality in ANP patients. Infl uence of epidural anesthesia on the hemocoagulation disorders and quantity of septic complications in patients with acute necrotizing pancreatitis O Tarabrin, S Shcherbakov, A Tkachenko, O Kushnir, I Grychushenko Odessa National Medical University, Odessa, Ukraine Critical Care 2013, 17(Suppl 2):P354 (doi: 10.1186/cc12292) O Tarabrin, S Shcherbakov, A Tkachenko, O Kushnir, I Grychushenko Odessa National Medical University, Odessa, Ukraine Critical Care 2013, 17(Suppl 2):P354 (doi: 10.1186/cc12292) Introduction Fondaparinux is currently the fi rst therapeutic choice in several clinical settings such as deep venous thrombosis (DVT) S135 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 8.7 ± 3.4 minutes vs. 12.2 ± 1.9 minutes, P <0.001; α-angle 59.7 ± 9.4 vs. 47.2 ± 11.8, P <0.01). This reduction of fi brinolysis was more evident at a urokinase concentration of 160 UI/ml (Figure 1). Conclusion UKIF-TEG could be a feasible point-of-care method to evaluate fi brinolysis in critically ill patients. Reference 1 Gallimore MJ et al : J Thromb Haemost 2005 3:2506-2513 Figure 1 (abstract P357). prophylaxis in orthopedics or in cases of acute coronary syndromes. The main drawback of FOND is that routine monitoring is not currently available. This could be a problem during the management of critical and surgical patients, especially in cases of old patients and renal failure. The aim of this study is to evaluate the ability of thromboelastography (TEG) to determine the level of anticoagulation due to FOND in a surgical population. prophylaxis in orthopedics or in cases of acute coronary syndromes. The main drawback of FOND is that routine monitoring is not currently available. This could be a problem during the management of critical and surgical patients, especially in cases of old patients and renal failure. The aim of this study is to evaluate the ability of thromboelastography (TEG) to determine the level of anticoagulation due to FOND in a surgical population. Figure 1 (abstract P357). Methods We prospectively analyzed all patients to whom elective major orthopedic surgery was consecutively performed in a 2-month period. All the patients received FOND 2.5 mg in the postoperative period according to ACCP 2012 Guidelines. Native and heparinase (hep) TEG (Haemoscope Corporation, Niles, IL, USA) tests activated with kaolin were performed using whole blood citrated samples at four times: T0, before FOND administration; T1, 2 hours after administration; T2, 17 hours after administration (half-life); T3, 24 hours after administration. The following native and hep TEG parameters were analyzed: reaction time (R), α angle, maximum amplitude (MA) and coagulation index (CI). Infl uence of epidural anesthesia on the hemocoagulation disorders and quantity of septic complications in patients with acute necrotizing pancreatitis These parameters were compared with levels of anti-Xa. Unvariate analysis and Spearman’s test were applied to our data. 8.7 ± 3.4 minutes vs. 12.2 ± 1.9 minutes, P <0.001; α-angle 59.7 ± 9.4 vs. 47.2 ± 11.8, P <0.01). This reduction of fi brinolysis was more evident at a urokinase concentration of 160 UI/ml (Figure 1). Conclusion UKIF-TEG could be a feasible point-of-care method to evaluate fi brinolysis in critically ill patients. Reference 1. Gallimore MJ, et al.: J Thromb Haemost 2005, 3:2506-2513. Results Eighteen patients were analyzed. Ten patients met the inclusion criteria. The mean R value increased from T1 to T3. The mean R parameter was in the normal range at any phase of the study and there was no signifi cant diff erences between the R mean value at the diff erent phases. The lowest value of R was at T1, which coincides with plasmatic peak concentration of FOND. This value did not correlate with anti-Xa mean value at T1, which showed the highest value at that time. There was signifi cant diff erence between the mean native and hep R value only at T2 (P <0.05), native and hep α angle at T3, MA and MA hep at T2 (P <0.01) and CI and CI hep at T3 (P <0.02). Only the parameter MA had signifi cant variation over time (P <0.02). 1. Gallimore MJ, et al.: J Thromb Haemost 2005, 3:2506-2513. P358 p References References 1. Falck-Ytter Y, et al.: Chest 2012, 141:2. 1. Falck-Ytter Y, et al.: Chest 2012, 141:2. 2. FUTURA/OASIS Trial Group, Steg PG, Jolly SS, Mehta SR, et al.: Low Methods In this retrospective study, we investigated 1,737 consecutive insertions of central venous catheters on 1,444 patients in a large university hospital during 2009 to 2010. Pre-procedural coagulation status, blood component use, type of catheter, insertion site, and complications during insertion were recorded and compared with bleeding complications documented in electronic charts. 2. FUTURA/OASIS Trial Group, Steg PG, Jolly SS, Mehta SR, et al.: Low-dose vs standard dose unfractioned heparin for percutaneous coronary intervention in acute coronary syndromes treated with fondaparinux: the FUTURA/OASIS randomized trial. JAMA 2010, 304:1339-1349. py TK Kander, US Schött y TK Kander, US Schött TK Kander, US Schött Lund University, Lund, Sweden y Critical Care 2013, 17(Suppl 2):P358 (doi: 10.1186/cc122 Conclusion R represents the time necessary to thrombin formation and in the presence of FOND we hypothesized a prolonged R time. In our population, TEG performed with citrated kaolin-activated whole blood was not able to detect prophylatic doses of FOND in every phase. On the contrary, levels of anti-Xa were able to reveal the exact pharmacokinetics of the drug. Further studies including a large number of patients are necessary. Introduction Bleeding complications in association with insertion of central venous catheters are reported in 0.5 to 1.6% of cases, are rarely fatal, but sometimes require intervention. The aim of this study was to map pre-procedural variables at the insertion of a central venous catheter and to investigate whether any independent variable could be identifi ed as an independent risk factor for bleeding complications associated with insertion of central venous catheters. Urokinase-induced fi brinolysis in thromboelastography (UKIF-TEG) to assess fi brinolysis in critically ill patients Urokinase-induced fi brinolysis in thromboelastography (UKIF-TEG) to assess fi brinolysis in critically ill patients L Zacchetti, M Panigada, M Cressoni, T Marchesi, L Gattinoni Fondazione IRCCS Ca’ Granda Ospedale Policlinico Milano, Milan, Italy Critical Care 2013, 17(Suppl 2):P357 (doi: 10.1186/cc12295) Results No serious bleeding complications were recorded in connection with the insertions of central venous catheters. Sixteen out of 1,769 (0.9%) insertions caused grade 2 bleeding, being defi ned as a bleeding requiring prolonged compression at the insertion site. Insertion of a large-bore central dialysis catheter was found to be an independent risk factor for bleeding complications. Neither conventional coagulation tests nor accidental arterial puncture or the number of needle passes could predict bleeding complications in this study. i y y L Zacchetti, M Panigada, M Cressoni, T Marchesi, L Gattinoni L Zacchetti, M Panigada, M Cressoni, T Marchesi, L Gattinoni Fondazione IRCCS Ca’ Granda Ospedale Policlinico Milano, Milan, Italy Critical Care 2013, 17(Suppl 2):P357 (doi: 10.1186/cc12295) Fondazione IRCCS Ca’ Granda Ospedale Policlinico Milano, Milan, Italy Critical Care 2013, 17(Suppl 2):P357 (doi: 10.1186/cc12295) p itical Care 2013, 17(Suppl 2):P357 (doi: 10.1186/cc12295) Introduction Coagulopathy, particularly a trend toward hyper- coagula bility and hypofi brinolysis, is common in critically ill patients and correlates with worse outcome. Available laboratory coagulation tests to assess fi brinolysis are expensive and time demanding. We investigated whether a modifi ed thromboelastography with the plasminogen activator urokinase (UKIF-TEG) [1] may be able to evaluate fi brinolysis in a population of critically ill patients.i Conclusion This retrospective study shows that serious bleeding complications in association with central line insertion are uncommon and that insertion of dialysis catheter is an independent risk factor for mild bleeding complications. Coagulopathy, measured with conventional coagulation tests, is not an independent risk factor for bleeding complication at insertion of central venous catheters. i y y Methods UKIF-TEG was performed as follows: fi rst urokinase was added to citrate blood to give fi nal concentrations of 160 UI/ml, then thromboelastography (TEG) analysis was started after kaolin activation and recalcifi cation with calcium chloride. Basal TEG (no addition of urokinase) was also performed. Fibrinolysis was determined by the loss of clot strength after the maximal amplitude (MA), and recorded as Ly30 (percentage lysis at 30 minutes after MA) and as Ly60 (percentage lysis at 60 minutes after MA). P359 Effi cacy of tranexamic acid in decreasing blood loss during cesarean section V Zaporozhan, O Tarabrin, D Gavrychenko, G Mazurenko, O Saleh, I Lyoshenko Results UKIF-TEG was performed on 17 healthy volunteers and 18 critically ill patients. Ly60 was predicted by Ly30 according to an exponential function, so we used Ly30 as an indicator of clot lysis. Basal TEG showed increased coagulability and a trend toward less fi brinolysis in critically ill patients compared with healthy volunteers (reaction time Introduction Despite signifi cant progress in obstetric care, the problem of bleeding during labor remains unsolved. Annually in the world, 125,000 women die from obstetric hemorrhage. Introduction Despite signifi cant progress in obstetric care, the problem of bleeding during labor remains unsolved. Annually in the world, 125,000 women die from obstetric hemorrhage. S136 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Methods We performed a randomized, double-blind study in 37 patients who underwent cesarean section. Patients were divided into two groups: the fi rst group (n = 19) received preoperative (30 minutes before operation) tranexamic acid 10 mg/kg; the second group (n = 18) received preoperative placebo. The condition of hemostasis was monitored by haemoviscoelastography. Methods High and low concentrations of both fi brinogen and FXIII were used to reverse coagulopathy induced by 1:1 dilution in vitro with 5% albumin of blood samples from healthy volunteers, monitored by rotational thromboelastometry (ROTEM).i y ( ) Results Haemodilution with albumin signifi cantly attenuated EXTEM maximum clot fi rmness (MCF), α angle (AA), clotting time (CT) and clot formation time (CFT), and FIBTEM MCF (P <0.001). Following haemodilution, both doses of fi brinogen signifi cantly corrected all ROTEM parameters (P ≤0.02), except the lower dose did not correct AA. Compared with the lower dose, the higher dose of fi brinogen signifi cantly improved FIBTEM MCF and EXTEM MCF, AA and CFT (P <0.001). The lower dose of FXIII did not signifi cantly correct any of the ROTEM parameters, and the high dose only improved EXTEM CT (P = 0.004). All combinations of high/low concentrations of fi brinogen/ FXIII signifi cantly improved all ROTEM parameters examined (P ≤0.001). Fibrinogen concentration generally had a greater eff ect on each parameter than did FXIII concentration; the best correction of ROTEM parameters was achieved with high-dose fi brinogen concentrate and either low-dose or high-dose FXIII. 1Hôpital Jeanne de Flandre, Lille, France; 2Centre hospitalier regional universitaire Lille, France Critical Care 2013, 17(Suppl 2):P362 (doi: 10.1186/cc12300) Critical Care 2013, 17(Suppl 2):P362 (doi: 10.1186/cc12300) Introduction Postpartum hemorrhage (PPH) remains the leading cause of maternal mortality. After IOCS introduction in our north of France tertiary-care obstetric unit, the consecutive cohort of severe PPH was collected prospectively and compared regarding the use of IOCS or not. y Methods Citrated venous blood from 10 healthy volunteers was diluted by 33% using 130/0.42 hydroxyethyl starch or Ringer’s acetate. ROTEM was used to evaluate the eff ect of addition of either fi brinogen concentrate corresponding to 4 g/70 kg, or this fi brinogen dose combined with factor XIII equivalent to 20 IU/kg. Blood was analyzed at 33 or 37°C with ROTEM ExTEM and FibTEM reagents.i Methods A case–control prospective open-label study comparing the management and outcome of a consecutive cohort of 324 severe PPHs in elective and emergency hemorrhagic caesarean sections (CS) over 3 years. Results A signifi cant dilutive response was shown in both groups: hypocoagulation was greater in the starch group. Hypothermia lengthened the following: ExTEM clotting time (CT), clot formation time and α angle; FibTEM maximal clot formation (MCF). Irrespective of temperature, fi brinogen overcorrected Ringer’s acetate’s eff ects on all ROTEM parameters and partially reversed starch’s eff ects on ExTEM CT and FibTEM MCF. FibTEM demonstrated that factor XIII provided an additional procoagulative eff ect in the Ringer’s acetate group at both temperatures but not the starch group. The only ExTEM parameter to be improved by addition of factor XIII was MCF at 33°C.i Results IOCS was used in 70 severe PPHs and 254 severe PPH controls were managed without IOCS. Placenta accreta can be selected as the best indication for RBC restitution. In the 1,500 to 3,000 ml PPH, allogeneic transfusion was decreased in the IOCS group: 17.6 versus 56.3% (P = 0.006); PRBC: 0 (0 to 3) versus 3 (0 to 6) (P = 0.045). IOCS spared 87 blood bank PRBC (17,374 ml); that is, 24.2% of the total transfusion need. No amniotic fl uid embolism has been observed in the group with IOCS whereas one case appeared in the control group without IOCS. Conclusion Regarding the literature [1-4] and our study, IOCS could be used safely in PPH during CS. A leukocyte fi lter for retransfusion has been recommended and Rhesus isoimmunization must be precluded and monitored by repeated fetal RBC testing. In vitro correction of hypothermic and dilutive crystalloid and colloid rotational thromboelastography-monitored coagulopathy with fi brinogen and factor XIII i g D Winstedt, O Thomas, US Schött Intraoperative cell savage for life-threatening haemorrhagic caesarean section Lund University, Lund, Sweden AS Ducloy1, C Barre-Drouard2, E Jaillette2, S Depret-Mosser2, B Wibaut2, B Vallet2, S Susen2 1Hôpital Jeanne de Flandre, Lille, France; 2Centre hospitalier regional universitaire Lille, France References y Conclusion ROTEM shows that fi brinogen concentrate can reverse dilutive coagulation defects induced by colloid and crystalloid at both 33 and 37°C. Some additional reversal was provided by factor XIII: higher doses of both fi brinogen and factor XIII may counteract starch’s eff ects on clot structure. 1. Rainaldi et al.: Br J Anaesth 1998, 80:195-198. P361 2. Bernstein et al.: Anesth Analg 1997, 85:831-833. Dose–response of fi brinogen and factor XIII concentrate for correcting albumin-induced coagulopathy US Schött, DW Winstedt, JH Hanna Lund University, Lund, Sweden Critical Care 2013, 17(Suppl 2):P361 (doi: 10.1186/cc12299) Dose–response of fi brinogen and factor XIII concentrate for correcting albumin-induced coagulopathy US Schött, DW Winstedt, JH Hanna Lund University, Lund, Sweden Critical Care 2013, 17(Suppl 2):P361 (doi: 10.1186/cc12299) 3. Catling et al.: Int J Obstet Anesth 1999, 8:79-84. g 4. Waters et al.: Anesthesiology 2000, 92:1531-1536. P359 Effi cacy of tranexamic acid in decreasing blood loss during cesarean section y g p y Results All patients included in the study before surgery had moderate hypercoagulation and normal fi brinolysis: increasing the intensity of clot formation (ICF) to 11.4% compared with normal rates; the intensity of the retraction and clot lysis (IRCL) was 16.45 ± 1.40 in both groups. At the start of the operation in patients (Group 1), ICF decreased by 9.7% (P <0.05), and IRCL decreased by 27.6% (P <0.05) compared with preoperatively. In Group 2, there was ICF decrease by 8.8% (P <0.05), and IRCL increase by 11.4% (P <0.05) compared with preoperatively. At the end of the operation, the condition of hemostasis in both groups came almost to the same value – moderate hypocoagulation, depressed fi brinolysis. In both groups there were no thrombotic complications. Intraoperative blood loss in the fi rst group was 300 ± 40.5 and in the second was 500 ± 60.6.i Conclusion Using of tranexamic acid before surgery signifi cantly reduces intraoperative blood loss by 60%, without thrombotic complications. Conclusion Fibrinogen concentrate successfully corrected initiation, propagation and clot fi rmness defi cits induced by haemodilution with albumin, and FXIII synergistically improved fi brin-based clot strength. Reference AS Ducloy1, C Barre-Drouard2, E Jaillette2, S Depret-Mosser2, B Wibaut2, B Vallet2, S Susen2 Introduction Rotational thromboelastography (ROTEM) can detect dilutive and hypothermic eff ects on coagulation and evaluate corrective treatments. The aim of this in vitro study was to study whether fi brinogen concentrate alone or combined with factor XIII could reverse colloid-induced and crystalloid-induced coagulopathies in the presence and absence of hypothermia. 1Hôpital Jeanne de Flandre, Lille, France; 2Centre hospitalier regional universitaire Lille, France In vitro correction of hypothermic and dilutive crystalloid and colloid rotational thromboelastography-monitored coagulopathy with fi brinogen and factor XIII D Winstedt, O Thomas, US Schött Lund University, Lund, Sweden Critical Care 2013, 17(Suppl 2):P360 (doi: 10.1186/cc12298) 1. Anesth Analg 2008, 106:1360-1365. P363 primary composite endpoint – all-cause mortality, cardiovascular complications, acute respiratory distress syndrome, acute kidney injury requiring renal replacement therapy, septic shock or reoperation at 30 days – occurred in 19.6% of the patients in the liberal strategy group and in 35.6% in the restrictive group (P = 0.012). The liberal strategy group had a signifi cantly lower 30-day mortality rate as compared with the restrictive group (8.2% (95% CI, 4.2 to 15.4%) vs. 22.8% (95% CI, 15.7 to 31.9%), respectively, P = 0.005). The occurrence of cardiovascular complications was lower in the liberal group than in the restrictive group (5.2% (95% CI, 2.2 to 11.5%) vs. 13.9% (95% CI, 8.4 to 21.9%), respectively, P = 0.038). The restrictive strategy group had a higher 60- day mortality rate as compared with the liberal group (23.8% (95% CI, 16.5 to 32.9%) vs. 11.3% (95% CI, 6.5 to 9.2%), respectively, P = 0.022). Conclusion The liberal RBC transfusion strategy with a hemoglobin trigger of 9 g/dl was associated with fewer major postoperative complications in patients undergoing major cancer surgery compared with the restrictive strategy. Methods Data from 240 cardiac surgery patients were retrospectively analysed. Of these, 157 had routine management with a bolus of protamine to correct the activated clotting time and then expectant management of subsequent bleeding, and 47 had the same but also a protamine infusion of 10 to 80 mg/hour for between 3 and 8 hours postoperatively. Blood loss was measured at 1, 6, 12 and 24 hours. In all, excessive bleeding was investigated using thromboelastography (TEG). Rebound heparinisation was determined by a ratio of R-times (heparinase/plain) <0.8. The Mann–Whitney U test and the chi-squared test were used to assess statistical signifi cance.if p y Conclusion The liberal RBC transfusion strategy with a hemoglobin trigger of 9 g/dl was associated with fewer major postoperative complications in patients undergoing major cancer surgery compared with the restrictive strategy. gi Results There was no signifi cant diff erence in blood loss between the two groups. Blood loss at 1 hour in the infusion and non-infusion group was 145 and 88 ml, respectively (P = 0.06); at 6 hours: 450 and 392 ml (P = 0.5); at 12 hours: 620 and 595 ml (P = 0.62); and at 24 hours: 971 and 872 ml (P = 0.12). P364 P364 Transfusion Requirements in Surgical Oncology Patients (TRISOP): a randomized, controlled trial J Almeida1, F Galas1, E Osawa1, J Fukushima1, S Moulin1, C Park1, E Almeida1, S Vieira1, J Vincent2, A Rhodes3, M Balzan1, J Inacio1, H Palomba1, R Nakamura1, F Bergamin1, A Sandrini1, U Ribeiro Jr1, J Auler Jr1, L Hajjar1 1Instituto do Cancer do Estado de São Paulo, Brazil; 2Erasme Hospital, Université libre de Bruxelles, Brussels, Belgium; 3St George’s Healthcare NHS Trust and St George’s University of London, UK Critical Care 2013, 17(Suppl 2):P364 (doi: 10.1186/cc12302) p Results One hundred and thirty-six patients (66.3%) were exposed to RBC transfusion. In the intraoperative room, 63.4% of patients received at least one RBC unit, and in the ICU, 11.2% of children were transfused. From all patients, 66 (32.1%) presented the combined endpoint. Patients with complications had higher RACHS-1 score, were younger (69 months (0 to 137) vs. 73 months (37 to 138), P <0.001), had a lower weight (13 kg (3 to 23) vs. 20 kg (12 to 36), P <0.001), a longer time of surgery (475 minutes (410 to 540) vs. 353 (275 to 433), P <0.001), a longer duration of cardiopulmonary bypass (205 minutes (175 to 235) vs. 106 minutes (73 to 123), P = 0.003), a lower SVO2 at the end of surgery (59% (IQR 39 to 80) vs. 78% (71 to 83), P <0.001), a higher arterial lactate at the end of surgery (6.9 mmol/l (4.3 to 9.2) vs. 2.7 mmol/l (73 to 123), P = 0.003), a lower intraoperative hematocrit (26.2 ± 5.6% vs. 29.5 ± 6% (P <0.001)) and a lower hematocrit at the end of surgery (33.4 ± 6.7% vs. 36.9 ± 6.9% (P <0.001)) as compared with patients without complications. Patients with complications were more exposed to RBC transfusion in the intraoperative room (75% vs. 57%, P = 0.011) and in the ICU (21% vs. 6.4%, P = 0.002). In an adjusted model of logistic regression, RBC transfusion is an independent risk factor of combined endpoint (OR 4.25 (95% CI, 1.359 to 13.328), P = 0.013). Conclusion Blood transfusion after pediatric cardiac surgery is a risk factor for worse outcome including 30-day mortality. P363 Does protamine infusion reduce postoperative blood loss and heparin rebound in cardiac surgery patients? A retrospective analysis of 240 patients on the cardiac ICU p p p heparin rebound in cardiac surgery patients? A retrospectiv Introduction Natural colloid albumin induces a lesser degree of dilutional coagulopathy than synthetic colloids. Fibrinogen concentrate has emerged as a promising strategy to treat coagulopathy, and factor XIII (FXIII) works synergistically with fi brinogen to correct coagulopathy following haemodilution with crystalloids. Objectives were to examine the ability of fi brinogen and FXIII concentrates to reverse albumin- induced dilutional coagulopathy. analysis of 240 patients on the cardiac ICU E Helme, H Meeran, N Al-Subaie St George’s Hospital, London, UK g p Critical Care 2013, 17(Suppl 2):P363 (doi: 10.1186/cc12301) Introduction Cardiac surgery is associated with signifi cant blood loss. Teoh and colleagues demonstrated a reduction in postoperative S137 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 primary composite endpoint – all-cause mortality, cardiovascular complications, acute respiratory distress syndrome, acute kidney injury requiring renal replacement therapy, septic shock or reoperation at 30 days – occurred in 19.6% of the patients in the liberal strategy group and in 35.6% in the restrictive group (P = 0.012). The liberal strategy group had a signifi cantly lower 30-day mortality rate as compared with the restrictive group (8.2% (95% CI, 4.2 to 15.4%) vs. 22.8% (95% CI, 15.7 to 31.9%), respectively, P = 0.005). The occurrence of cardiovascular complications was lower in the liberal group than in the restrictive group (5.2% (95% CI, 2.2 to 11.5%) vs. 13.9% (95% CI, 8.4 to 21.9%), respectively, P = 0.038). The restrictive strategy group had a higher 60- day mortality rate as compared with the liberal group (23.8% (95% CI, 16.5 to 32.9%) vs. 11.3% (95% CI, 6.5 to 9.2%), respectively, P = 0.022). Conclusion The liberal RBC transfusion strategy with a hemoglobin trigger of 9 g/dl was associated with fewer major postoperative complications in patients undergoing major cancer surgery compared with the restrictive strategy. bleeding with the use of a protamine infusion and an abolishment of heparin rebound [1]. The aim of this study was to see whether the use of postoperative protamine infusions in our cardiac ITU was associated with a reduction in heparin rebound and blood loss. 1. Teoh K, et al.: J Thorac Cardiovasc Surg 2004, 128:211-219. 2. Teoh K, et al.: Circulation 1993, 88:420-425. References 1. Teoh K, et al.: J Thorac Cardiovasc Surg 2004, 128:211-219. 2. Teoh K, et al.: Circulation 1993, 88:420-425. y Methods We performed a prospective cohort study of 205 patients undergoing cardiac surgery for congenital heart disease. We recorded baseline characteristics, RACHS-1 score, intraoperative data, transfusion requirement and severe postoperative complications as need for reoperation, acute kidney injury, arrhythmia, severe sepsis, septic shock, bleeding, stroke, and death during 30 days. We performed univariate analysis using baseline, intraoperative and postoperative variables. Selected variables (P <0.10) were included in a forward stepwise multiple logistic regression model to identify predictive factors of a combined endpoint including 30-day mortality and severe complications. Conclusion Blood transfusion after pediatric cardiac surgery is a risk factor for worse outcome including 30-day mortality. Avoiding blood transfusion must be a goal of best postoperative care. P365 Red blood cell transfusion is a predictor of mortality and morbidity in children undergoing cardiac surgery C Colognesi, R Maia, L Hajjar, F Bergamin, J Fukushima, E Osawa, J Almeida, L Camara, S Zeferino, V Guimaraes, F Galas Heart Institute, São Paulo, Brazil Critical Care 2013, 17(Suppl 2):P365 (doi: 10.1186/cc12303) Introduction Red blood cell (RBC) transfusion is associated with morbidity and mortality in critically ill patients. Congenital cardiac surgeries are associated with high rates of bleeding and consequently with high rates of allogeneic transfusion. We aimed to evaluate the association of transfusion with worse outcomes in children undergoing cardiac surgery. P363 There was also no signifi cant diff erence in those getting heparin rebound with 40% in the infusion group and 47% in the non-infusion group (P = 0.54). gy Acknowledgement ClinicalTrials.gov NCT 01560494. Acknowledgement ClinicalTrials.gov NCT 01560494. g Conclusion Unlike Teoh and colleagues [1], we did not fi nd an advantage in using protamine infusions. That there were still cases of heparin rebound in the infusion group suggests that the infusion was not as eff ective as expected and/or the dose was inadequate. However, previous studies assessed heparin rebound using isolated clotting parameters [1,2]. Here, we used TEG. As TEG measures the thrombodynamic properties of whole blood coagulation, perhaps it is a more reliable indicator of heparin activity? As a retrospective study, there are limitations; namely, the nonstandardised management of the patients and the potential bias in the anaesthetists’ selection of patients for an infusion. This group may be inherently higher risk for bleeding. However, heparin rebound is common and protamine is a simple, relatively safe and low-cost intervention compared with transfusion and so further study is needed. 1. Teoh K, et al.: J Thorac Cardiovasc Surg 2004, 128:211-219. 2 Teoh K et al : Circulation 1993 88:420-425 P364 Avoiding blood transfusion must be a goal of best postoperative care Introduction The purpose of this study was to evaluate whether a restrictive strategy of red blood cell (RBC) transfusion was superior to a liberal one for reducing mortality and severe clinical complications among patients undergoing major cancer surgery. Introduction The purpose of this study was to evaluate whether a restrictive strategy of red blood cell (RBC) transfusion was superior to a liberal one for reducing mortality and severe clinical complications among patients undergoing major cancer surgery. Methods The trial was designed as a phase III, randomized, controlled, parallel-group, superiority trial. The inclusion criteria were adult patients with cancer who were undergoing major abdominal surgery requiring postoperative care in an ICU. The patients were randomly allocated to treatment with either a liberal RBC transfusion strategy (transfusion when hemoglobin levels decreased below 9 g/dl) or a restrictive RBC transfusion strategy (transfusion when hemoglobin levels decreased below 7 g/dl). The primary outcome was a composite endpoint of death or severe complications. The patients were monitored for 30 days. Results A total of 1,521 patients were screened for eligibility and 248 met the inclusion criteria. After exclusions for medical reasons or a lack of consent, 198 patients were included in fi nal analysis, with 101 allocated to the restrictive group and 97 to the liberal group. The Conclusion Blood transfusion after pediatric cardiac surgery is a risk factor for worse outcome including 30-day mortality. Avoiding blood transfusion must be a goal of best postoperative care. S138 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 patients in the MT group (23.7%; 27/114) and 87 in the non-MT group. The MT group was signifi cantly higher in the ratio of females (P <0.001), ISS (P <0.01), PT-INR (P <0.001), APTT (P <0.05), ABCs (P <0.001) and TASHs (P <0.001) than in the on-MT group. On the other hand, the MT group was signifi cantly lower in Ps (P <0.05) and fi brinogen level (P <0.001) than the non-MT group. In the receiver operating characteristics (ROC) analysis, the area under the curve (AUC) to distinguish a MT was the highest for TASHs (0.831, P <0.001), followed by fi brinogen (0.758, P <0.001), and ABCs (0.732, P <0.001). P367i i Results During the study period, 913 admissions were documented, 843 of which were included in the study. Among them, 144 (17%) were transfused. When comparing TCs with nontransfused cases (NTCs), the odds ratio (OR) of new or progressive multiple organ dysfunction syndrome (NPMODS) was 2.39 (95% CI = 1.58 to 3.62, P <0.001). This association remained statistically signifi cant in the multivariate analysis for children with admission PRISM score ≤5 (OR = 2.41, 95% CI = 1.08 to 5.37, P = 0.032). TCs were ventilated longer than NTCs (14.1 ± 32.6 days vs. 4.3 ± 9.6 days, P <0.001). This diff erence was still signifi cant after adjustment using a Cox model. Moreover, we observed an adjusted dose–eff ect relationship between RBC transfusions and length of mechanical ventilation. The PICU length of stay was signifi cantly increased for TCs (12.4 ± 26.2 days vs. 4.9 ± 10.2 days, P <0.001), even after multivariate adjustment (hazard ratio of PICU discharge for TCs: 0.61, 95% CI = 0.5 to 0.74, P <0.001). We also observed an adjusted dose–eff ect relationship between RBC transfusions and PICU length of stay. The paired analysis for comparison of pre-transfusion and post- transfusion values showed that the arterial partial pressure in oxygen was signifi cantly reduced after the fi rst transfusion (mean diff erence 42.8 mmHg, 95% CI = 27.2 to 58.3, P <0.001). The paired analysis also showed an increased proportion of renal replacement therapy, while the proportions of sepsis, severe sepsis and septic shock did not diff er. Conclusion RBC transfusions were associated with prolonged mechanical ventilation and prolonged PICU stay. The risk of NPMODS was increased in some transfused children. Moreover, our study questions the ability of stored RBCs to improve oxygenation in critically ill children. These results should help to improve transfusion practice in the PICU. P364 Fibrinogen was only a predictor of a MT without a scoring system such as ABCs and TASHs, and the optimal cutoff value was 205 mg/dl. P366 P366 Red blood cell transfusion results in worse outcomes in patients with left ventricular dysfunction undergoing on-pump cardiac surgery R Miadda, C Colognesi, L Hajjar, M Sundin, L Camara, S Zeferino, F Bergamin, A Leme, V Guimaraes, F Galas Heart Institute, São Paulo, Brazil Critical Care 2013, 17(Suppl 2):P366 (doi: 10.1186/cc12304) Introduction Cardiac surgeries are associated with high rates of bleeding and consequently with high rates of allogeneic transfusion. Patients with left ventricular dysfunction usually present a higher incidence of complications after cardiac surgery, including low output syndrome and organ dysfunction. It is a matter of controversy whether this subgroup of patients need higher hemoglobin levels to optimize oxygen delivery. We aimed to evaluate the association of transfusion with worse outcomes in patients with left ventricular dysfunction. pf g Conclusion We found that the level of fi brinogen was the most valuable predictor of a MT in the coagulation or fi brinolysis markers. It is certain that the level of fi brinogen at admission was not as useful as the TASHs about predicting a MT in this study. Whereas the scoring systems require the assessment of several factors, the measurement of fi brinogen is simple, easy and quick. We strongly suggest that the level of fi brinogen will be a useful predictor of a MT at in severe blunt traumatic patients. p y Methods A prospective cohort study was conducted in 215 patients undergoing cardiac surgery, presenting ejection fraction <35%. We recorded baseline characteristics, intraoperative data, transfusion requirement and severe postoperative complications as need for reoperation, acute kidney injury, arrhythmia, severe sepsis, septic shock, bleeding, stroke, and death during 30 days. We performed univariate analysis using baseline, intraoperative and postoperative variables associated with severe postoperative complications. Selected variables (P <0.10) were included in a forward stepwise multiple logistic regression model to identify predictive factors of a combined endpoint including 30-day mortality and severe complications.i p References 1. Timothy C, et al.: J Trauma 2009, 66:346-352. 2. Yücel N, et al.: J Trauma 2006, 60:1228-1236. 3. Sørensen B, et al.: Semin Thromb Hemost 2012, 38:268-273. 1. Timothy C, et al.: J Trauma 2009, 66:346-352. 1. Timothy C, et al.: J Trauma 2009, 66:346-352. 2. Yücel N, et al.: J Trauma 2006, 60:1228-1236. 3. Sørensen B, et al.: Semin Thromb Hemost 2012, 38:268-273. 1. Timothy C, et al.: J Trauma 2009, 66:346 352. 2. Yücel N, et al.: J Trauma 2006, 60:1228-1236. 3. Sørensen B, et al.: Semin Thromb Hemost 2012, 38:268-273. 2. Yücel N, et al.: J Trauma 2006, 60:1228-1236. 3. Sørensen B, et al.: Semin Thromb Hemost 2012, 38:268-273. Association between red blood cell transfusions and clinical outcome in critically ill children y P Demaret1, M Tucci2, T Ducruet2, H Trottier2, J Lacroix2 Results Two hundred and fi fteen patients were included in the study. The mean ejection fraction was 31%. From all patients, 129 (54.4%) presented the combined endpoint. The incidence of RBC transfusion was higher in the group with complications compared with the group without complications both intraoperative and postoperative, P <0.001. Patients with complications were older, had lower initial hemoglobin levels and had higher EuroSCORE. At multivariate analysis, age (OR = 1.07, 95% CI = 1.013 to 1.137, P = 0.016) and perioperative RBC transfusion (OR = 7.78, 95% CI = 2.13 to 28.41, P = 0.002) were independent predictors of severe postoperative complications after cardiac surgery. Introduction Red blood cell (RBC) transfusions are frequent in critically ill children. Their benefi ts are clear in several situations. However, issues surrounding their safety have emerged in the past decades. It is important to identify the potential complications associated with RBC transfusions, in order to evaluate their risk–benefi t ratio better. i Methods A single-center prospective observational study of all children admitted to the pediatric intensive care unit (PICU) over a 1-year period. The variables possibly related to RBC transfusions were identifi ed before the study was initiated, and their presence was assessed daily for each child. In transfused cases (TCs), a variable was considered as a possible outcome related to the transfusion only if it was observed after the fi rst transfusion. g y Conclusion In patients with left ventricular dysfunction, red blood transfusion after cardiac surgery is a risk factor for worse outcome including 30-day mortality. Defi ning the need for a massive transfusion in severe blunt traumatic patients T Umemura, H Ishikura, Y Nakamura, K Hoshino, T Nishida, T Kamitani Fukuoka University, Fukuoka, Japan Critical Care 2013, 17(Suppl 2):P367 (doi: 10.1186/cc12305) Introduction We do not have enough criteria to make a judgment of the need for a massive transfusion (MT) in severe blunt traumatic patients. As a scoring system to predict the need for a MT, we usually use the Assessment Blood Consumption score (ABCs) and/or the Trauma-Associated Severe Hemorrhage score (TASHs). However, for these scoring systems, the procedure is slightly complicated. The aim of this study was to establish a predictor of a MT using coagulation or fi brinolysis markers. i Methods A retrospective analysis of MT was conducted in patients with severe blunt traumatic injury, which was defi ned as Injury Severity Score (ISS) of 16 or more admitted to the ICU between 1 June 2009 and 31 December 2010. Blood samples were collected from patients immediately after arriving at our level I trauma center. We defi ned the patients who received more than 10 unit packed red blood cells (PRBCs) within the fi rst 24 hours as a MT group and who received less than 9 units PRBCs as a non-MT group. After the demographic data, number of units of PRBCs and the need for massive transfusions were recorded and analyzed in each groups, we compared data between two groups. Results There were 114 patients who met the inclusion criteria. Fifty patients received blood transfusions (43.9%; 50/114). There were 27 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S139 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 P369 Microcirculation and blood transfusions during sepsis: leukoreduced (LR) versus non-LR red blood cells A Donati1, E Damiani1, R Domizi1, C Scorcella1, A Carsetti1, S Tondi1, R Castagnani1, N Mininno1, P Pelaia1, C Ince2 1Università Politecnica delle Marche, Ancona, Italy; 2Academic Medical Center, Amsterdam, the Netherlands Critical Care 2013, 17(Suppl 2):P369 (doi: 10.1186/cc12307) P369 density and fl ow were assessed with sidestream dark-fi eld imaging sublingually. Thenar tissue O2 saturation (StO2) was measured using near-infrared spectroscopy and a vascular occlusion test was performed. Results The De Backer score (P = 0.02), total vessel density (P = 0.08), perfused vessel density (P = 0.04), proportion of perfused vessels (P = 0.01), and microvascular fl ow index (P = 0.04, Figure 1) increased only in Group 2. The StO2 upslope (Figure 2) during reperfusion increased in both groups (P <0.05). Massive blood transfusion for obstetric haemorrhage S Simmons1, WE Pollock1, L Phillips2, S McDonald3 p 1Mercy Hospital for Women, Heidelburg, Australia; 2Monash University, Prahran, Australia; 3La Trobe University, Bundoora, Australia Critical Care 2013, 17(Suppl 2):P370 (doi: 10.1186/cc12308) Figure 1 (abstract P369). Blood transfusion and microvascular fl ow. Prahran, Australia; 3La Trobe University, Bundoora, Australia Critical Care 2013, 17(Suppl 2):P370 (doi: 10.1186/cc12308) Introduction Obstetric haemorrhage remains a leading cause of maternal mortality and severe morbidity. Cardiovascular and haemostatic physiology alters in pregnancy and massive transfusion protocols have been implemented for obstetric haemorrhage based on limited evidence. The objective of this study was to examine the pattern and rate of blood products used in massive transfusion for obstetric haemorrhage in a tertiary obstetric hospital. Methods Massive transfusion was defi ned as 5 or more units of red blood cells within 4 hours in accordance with the Australian Massive Transfusion Registry defi nition. Following ethics approval, all cases fi lling this criterion were identifi ed in the hospital’s birthing and blood bank systems. Data were extracted from the medical histories and analysed using SPSS. P <0.05 was considered statistically signifi cant. i Results Twenty-eight women in three years (2009 to 2011) underwent a massive transfusion for obstetric haemorrhage, with nine receiving more than 10 units of RBCs in 24 hours. Eleven (39%) were admitted to the ICU and 11 underwent a hysterectomy, of which six were admitted to the ICU. The median estimated blood loss was 4,335 ml (IQR 3,000 to 5,000). Median blood product delivery was RBC 8 units (IQR 6 to 13); FFP 4 units (IQR 4 to 8); platelets 4 units (IQR 0 to 8) and cryoprecipitate 3 units (IQR 0 to 10). One-half of the women received the fi rst four units of RBCs in less than 34 minutes. Other blood products were started a median of 49 minutes, 44 minutes and 75 minutes after the RBC transfusion commenced, respectively. Eight women had a fi brinogen level <0.8 g/l on the initial coagulation test during the haemorrhage. The remaining 20 women had a median fi brinogen level of 3.7 g/l (IQR 3.15 to 4.9). There was no diff erence in the transfusion of RBCs (P = 0.20), FFPs (P = 0.96) and platelets (P = 0.48) in women who showed an initial low fi brinogen and those who did not, although there was a diff erence in the number of units of cryoprecipitate (P <0.05). Massive blood transfusion for obstetric haemorrhage The median lowest Hb during the haemorrhage was 66 g/l (IQR 51 to 80) and median discharge Hb was 103 g/l (IQR 96 to 113). No blood product reaction was noted and there was one death. Figure 1 (abstract P369). Blood transfusion and microvascular fl ow. Figure 2 (abstract P369). Blood transfusion and microvascular reactivity. Conclusion Massive transfusion for obstetric haemorrhage involved rapid blood product administration with no consistent pattern in the ratio of products administered. Defi ning the need for a massive transfusion in severe blunt traumatic patients In Group 1 the baseline StO2 and StO2 downslope during ischemia increased, probably refl ecting a lower O2 consumption. Introduction Microcirculatory alterations during sepsis impair tissue oxygenation, which may be further worsened by anemia. Blood transfusions proved not to restore O2 delivery during sepsis [1]. The impact of storage lesions and/or leukocyte-derived mediators in red blood cell (RBC) units has not yet been clarifi ed [2]. We compared the eff ects of leukoreduced (LR) versus nonLR packed RBCs on microcirculation and tissue oxygenation during sepsis. 2 p Conclusion Unlike nonLR RBCs, the transfusion of fresh LR RBCs seems to improve microvascular perfusion and might help to restore tissue oxygenation during sepsis. yg References 1. Marik PE, et al.: JAMA 1993, 269:3024-3029. Methods A prospective randomized study. Twenty patients with either sepsis, severe sepsis or septic shock requiring RBC transfusion randomly received nonLR (Group 1, n = 10) or LR (Group 2, n = 10) fresh RBCs (<10 days old). Before and 1 hour after transfusion, microvascular 2. Sparrow RL: Blood Transfus 2010, 8(Suppl 3):s26-s30. 3 Blood transfusion practices in the ICU of a level 1 trauma centre and tertiary cardiac unit P373 P373 Incidence, risk factors and outcome of TRALI after cardiac surgery A Dijkhuizen, R De Bruin, S Arbous Leiden University Medical Center, Leiden, the Netherlands Critical Care 2013, 17(Suppl 2):P373 (doi: 10.1186/cc12311) Introduction Transfusion-related acute lung injury (TRALI) is a syndrome that presents as a sudden onset of respiratory distress 6 hours after transfusion of blood products. The diagnosis is based on clinical and radiographic fi ndings. Particularly at risk for TRALI are cardiac surgery patients. However, specifi c patient risk factors and data on outcome are largely unknown. The aim of this study was to investigate incidence, risk factors and outcome of TRALI in cardiac surgical patients on cardiopulmonary bypass. g p p Results A total 175 units of PRC were transfused in the ICU, over 105 episodes during the 2-month period (excluding immediately postoperative transfusions). Ninety-four units (53.7%) administered in 64 transfusion episodes (61.0%) occurred contrary to the guidelines. In 89.3% of these cases the recorded reason for transfusion was an apparently low Hb level. The median (IQR (range)) Hb level at which patients were transfused: within guidelines was 6.9 g/dl (6.6 to 7.7 (4.8 to 13.9)); within guidelines, excluding cases of acute blood loss, was 6.6 g/dl (6.5 to 6.8 (5.5 to 6.9)); and outside the guidelines was 7.7 g/dl (7.4 to 8.2 (7.0 to 9.7)). One unit of PRC was transfused in 57 episodes (54.3%), 2 units of PRC were transfused in 36 episodes (34.3%), and 3 to 6 units were transfused in 12 episodes (11.4%), with two-thirds of the latter due to acute haemorrhage. g y y Methods All thoracic surgery patients from a university hospital in the Netherlands of 18 years and older admitted to the ICU from January 2009 until December 2011 were screened. Included patients were observed during surgery and the fi rst 24 hours on the ICU for the onset of possible TRALI. The Canadian Consensus Conference TRALI defi nition was used. Two independent physicians blinded to the predictor variables scored the chest radiographs for the onset of bilateral interstitial abnormalities on 2K monitors. When interpretation diff ered, chest radiographs were reviewed by a third physician to achieve consensus. The European System for Cardiac Operative Risk Evaluation (EURO score) and the American Association of Anesthesiology (ASA) were scored before surgery. 3 Blood transfusion practices in the ICU of a level 1 trauma centre and tertiary cardiac unit Y Mustafa, B Pouchet University Hospitals Birmingham NHS Foundation Trust, Birmingham, UK Critical Care 2013, 17(Suppl 2):P371 (doi: 10.1186/cc12309) Introduction Blood transfusions are associated with longer ICU and hospital inpatient durations, and an increase in mortality [1]. This study was undertaken to investigate whether the practice of packed red cell Figure 2 (abstract P369). Blood transfusion and microvascular reactivity. S140 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 4.9 ± 0.5 (P = 0.09). Cetor® also reduced BALF levels of MIP-2 from 214 (54) to 127 (22) pg/ml (P <0.01). KC and IL-6 levels were not aff ected. Conclusion In a model of antibody-mediated TRALI, C1 inhibitor attenuated pulmonary infl ammation. C1 inhibition may be a potential benefi cial intervention in TRALI. 4.9 ± 0.5 (P = 0.09). Cetor® also reduced BALF levels of MIP-2 from 214 (54) to 127 (22) pg/ml (P <0.01). KC and IL-6 levels were not aff ected. Conclusion In a model of antibody-mediated TRALI, C1 inhibitor attenuated pulmonary infl ammation. C1 inhibition may be a potential benefi cial intervention in TRALI. (PRC) transfusions in the ICU was in accordance with the best clinical evidence. A number of studies, most notably the TRICC study [2], have shown that indications for ICU blood transfusions are a haemoglobin (Hb) level of <7 g/dl or evidence of acute haemorrhage [3]. These criteria were therefore employed. (PRC) transfusions in the ICU was in accordance with the best clinical evidence. A number of studies, most notably the TRICC study [2], have shown that indications for ICU blood transfusions are a haemoglobin (Hb) level of <7 g/dl or evidence of acute haemorrhage [3]. These criteria were therefore employed. y Methods This study prospectively examined episodes of PRC unit transfusions over a 2-month period in the ICU of a large level 1 trauma centre and a tertiary cardiac unit. The number of PRC units transfused in each episode was recorded by nurses, along with the proposed indication and concurrent Hb level. The data were analysed to assess the number of transfusions administered contrary to the guidelines, along with the average Hb level at which a PRC unit was transfused and the average number of units administered per episode. P372 P372 C1 inhibitor attenuates pulmonary infl ammation in an in vivo model of transfusion-related acute lung injury M Müller1, PR Tuinman1, G Jongsma1, AM Tuip-de Boer1, L Boon2, SS Zeerleder1, NP Juff ermans1 1Academic Medical Center, Amsterdam, the Netherlands; 2Bioceros, Utrecht, the Netherlands Critical Care 2013, 17(Suppl 2):P372 (doi: 10.1186/cc12310) 3 Blood transfusion practices in the ICU of a level 1 trauma centre and tertiary cardiac unit By calculating the Acute Physiology and Chronic Health Evaluation (APACHE) II and IV scores the severity of illness was determined on arrival in the ICU. Conclusion Our results indicate a liberal transfusion threshold currently exists in the ICU. Patients are frequently receiving excessive PRC transfusions for Hb levels above the recommended concentration. In the 2-month study period, these were associated with a cost of approximately £12,220. We recommend increased staff awareness of the guidelines to reduce the number of unnecessary transfusions. This would decrease exposure of ICU patients to unnecessary risks of blood transfusion, reduce cost of treatment and help to preserve a valuable resource. f 1Academic Medical Center, Amsterdam, the Netherlands; 2Bioceros, Utrecht, the Netherlands 1Academic Medical Center, Amsterdam, the Netherlands; 2Bioceros, Utrecht, the Netherlands 1Academic Medical Center, Amsterdam, the Netherlands; 2Bioceros, Utrecht, the Netherlands Critical Care 2013, 17(Suppl 2):P372 (doi: 10.1186/cc12310) Introduction Transfusion-related acute lung injury (TRALI) has a high incidence in critically ill and surgical patients and contributes to adverse outcome, while specifi c therapy is absent. Recently it was demonstrated that complement activation plays a pivotal role in TRALI. We aimed to determine whether a C1 inhibitor is benefi cial in a two-hit mouse model of antibody-mediated TRALI. 1. Vlaar APJ, et al.: Crit Care Med 2010, 38:771-778. References Results In total, 1,787 cardiac surgical patients were included. A total of 19 (1.1%) patients developed TRALI within 24 hours following surgery. Patients developing TRALI were older compared with patients not developing TRALI, mean age respectively 71 and 65 years (P = 0.035). Furthermore, patients developing TRALI had higher APACHE II, APACHE IV, EURO and ASA score (P = 0.000, P = 0.000, P = 0.000 and P = 0.37). Compared with patients not developing TRALI, patients developing TRALI had a longer cardiopulmonary bypass time. Of the 19 patients developing TRALI, four patients (21.1%) died during ICU stay, in total six patients (31.6%) died during hospital stay. Patients developing TRALI were ventilated longer and had a longer ICU and hospital stay. 1. Corwin HL, et al.: Crit Care Med 2004, 32:39-52. 1. Corwin HL, et al.: Crit Care Med 2004, 32:39-52. 1. Corwin HL, et al.: Crit Care Med 2004, 32:39-52. 2. Hebert PC: Br J Anaesth 1998, 81(Suppl 1):25-33. 3. Napolitano et al.: Crit Care Med 2009, 37:3124-3157. 2. Hebert PC: Br J Anaesth 1998, 81(Suppl 1):25-33. 3. Napolitano et al.: Crit Care Med 2009, 37:3124-3157. g j y M Müller1, PR Tuinman1, G Jongsma1, AM Tuip-de Boer1, L Boon2, SS Zeerleder1, NP Juff ermans1 g g p y Conclusion Older age, higher APACHE II, IV, EURO and ASA scores and a longer time on cardiopulmonary bypass and ventilation time are risk factors for the development of TRALI. The ICU and hospital stay are longer, and the prognosis is bad for patients developing TRALI. Reference Albumin-induced coagulopathy is less severe and more eff ectively reversed with fi brinogen concentrate than is synthetic colloid-induced coagulopathy y Methods BALB/c mice were primed with lipopolysaccharide (LPS, from E. coli 0111:B4) that was administered intraperitoneally in a dose of 0.1 mg/kg, after which TRALI was induced by injecting MHC-I antibody against H2Kd (IgG2a,k) at a dose of 2 mg/kg. Mice infused with PBS or LPS served as controls. Concomitantly, mice infused with the MHC-I antibody were treated with C1 inhibitor (Cetor®; Sanquin, Amsterdam, the Netherlands) in a dose of 400 IU/kg intravenously. After infusion, mice were mechanically ventilated with a lung-protective pressure- controlled mode for 2 hours and then sacrifi ced, after which a bronchoalveolar lavage (BAL) was done. Statistics were analyzed by one-way ANOVA, values expressed as mean and standard deviation. Results Injection of LPS and MHC-I antibodies resulted in TRALI, indicated by increased levels of protein in the BAL fl uid, wet/dry ratios and levels of KC, MIP-2 and IL-6. C1 inhibitor Cetor® signifi cantly reduced total protein in BAL fl uid from 792 (169) to 421 (230) μg/ml (P <0.001) and tended to reduce the wet/dry ratio from 5.3 ± 0.3 to DW Winstedt, JH Hanna, US Schött Lund University, Lund, Sweden Critical Care 2013, 17(Suppl 2):P374 (doi: 10.1186/cc12312) Introduction Volume resuscitation is essential to restore normovolemia during hemorrhagic shock, burns and sepsis. However, synthetic colloids cause dilutional coagulopathy. The aims were to determine whether the natural colloid albumin induces a lesser degree of coagulopathy compared with synthetic colloids, and the comparative eff ectiveness of fi brinogen concentrate to reverse coagulopathy following dilution with these solutions. Methods Rotational thromboelastometry-based tests were used to examine coagulation parameters in samples from 10 healthy volunteers, in undiluted blood and samples diluted 1:1 with saline, S141 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Ringer’s acetate, hydroxyethyl starch (HES) 130/0.4, buff ered HES 130/0.4, 3% dextran 60, 6% dextran 70 or 5% albumin. Samples were analyzed before and after addition of 2 mg/ml fi brinogen concentrate. Results EXTEM maximum clot fi rmness (MCF), clot formation time (CFT) and α angle (AA) decreased signifi cantly upon dilution with all colloid solutions (P <0.001), although a signifi cantly greater coagulopathic eff ect was seen for samples diluted with synthetic colloid solutions versus albumin (P ≤0.001). Albumin-induced coagulopathy is less severe and more eff ectively reversed with fi brinogen concentrate than is synthetic colloid-induced coagulopathy A signifi cant reduction in the platelet component of clot strength (EXTEM MCF – FIBTEM MCF) was seen for samples diluted with synthetic colloids (P <0.001) but not albumin (P = 0.10). Following addition of fi brinogen, FIBTEM MCF, EXTEM MCF and EXTEM AA were signifi cantly higher, and EXTEM CFT was signifi cantly shorter in samples diluted with albumin versus those treated with HES or dextran (P ≤0.001). P375 P375 Network meta-analysis of clinical trials of fl uid treatments in sepsis demonstrates improved survival with albumin compared with crystalloid and hydroxyethyl starch M Bansal1, A Farrugia2, G Martin3 1Plasma Protein Therapeutics Association, Annapolis, MD, USA; 2University of Western Australia, Crawley, Australia; 3Emory University School of Medicine, Atlanta, GA, USA Critical Care 2013, 17(Suppl 2):P375 (doi: 10.1186/cc12313) Critical Care 2013, 17(Suppl 2):P375 (doi: 10.1186/cc12313) Critical Care 2013, 17(Suppl 2):P375 (doi: 10.1186/cc12313) Introduction Fluid resuscitation is widely practiced in treating sepsis. Comparative assessment of the diff erent fl uid modalities is hampered by a paucity of direct trials. We present a network meta-analysis for assessing the relative eff ectiveness of two fl uid treatments in sepsis when they have not been compared directly in a randomized trial but have each been compared with a common treatment. p g Results The economic model shows the potential cost saving by switching from open to closed albumin administration systems. It is measured in occurrence of BSI in the ICU, estimated occurrence of BSI in the albumin population and incidence of overall mortality in the ICU and cost of BSI per albumin patient. Finally, the model describes the potential cost savings by switching to a closed system. Using closed albumin delivery systems may overall provide a cost saving for the hospital and healthcare sector. Assuming 800 patients treated in an ICU per year with all in all 4,800 ml albumin, the total annual cost saving would be £24,000 switching from open to closed albumin infusions. Methods A systematic review of trials sepsis yielded 13 trials for assessment in network meta-analysis. The indirect comparison between albumin, HES and crystalloid was conducted using Bayesian methods for binomial likelihood, fi xed-eff ects network meta-analysis with a Monte Carlo Gibbs sampling method. Studies in septic patients with crystalloid as a reference treatment compared with any formulation of the colloid treatments albumin or HES were included, as were direct head-to-head trials between the two colloids.f g Conclusion Switching from an open to a closed albumin infusion system may result in a reduction in the risk of external contamination, resulting in less infection and may provide a cost saving for the healthcare system. Conclusion Switching from an open to a closed albumin infusion system may result in a reduction in the risk of external contamination, resulting in less infection and may provide a cost saving for the healthcare system. P376 Assessing the economic impact of catheter-related bloodstream infections when switching from open to closed system delivery of human albumin solutions in ICUs F Axelsen1, P Riss2 1Baxter Healthcare, Zurich, Switzerland; 2Medical University of Vienna, Austria Critical Care 2013, 17(Suppl 2):P376 (doi: 10.1186/cc12314) y References Results Odds ratios between the diff erent treatments were obtained (Figure 1). Ranking the interventions [1] demonstrated that albumin ranked highest in lowering mortality at a 96.4% probability compared with 3.6% and 0.01% for crystalloid and HES, respectively.l 1. Maki et al.: Infect Control Hosp Epidemiol 2011, 32:50-58. 2. Tacconelli et al.: J Hosp Infection 2009, 72:97-103. 3. Franzetti et al.: Epidemiol Infect 2009, 137:1041-1048. 1. Maki et al.: Infect Control Hosp Epidemiol 2011, 32:50-58. 1. Maki et al.: Infect Control Hosp Epidemiol 2011, 32:50-58. 2 Tacconelli et al : J Hosp Infection 2009 72:97-103 1. Maki et al.: Infect Control Hosp Epidemiol 2011, 32:50-58. 2. Tacconelli et al.: J Hosp Infection 2009, 72:97-103. 3. Franzetti et al.: Epidemiol Infect 2009, 137:1041-1048. 2. Tacconelli et al.: J Hosp Infection 2009, 72:97-103. Conclusion Albumin as a fl uid therapy in sepsis is associated with the lowest mortality of the three modalities studied. Reference . Salanti G, Ades AE, Ioannidis JP: Graphical methods and numerical summaries for presenting results from multiple-treatment meta-analysis: an overview and tutorial. J Clin Epidemiol 2011, 64:163-171. Assessing the economic impact of catheter-related bloodstream infections when switching from open to closed system delivery of human albumin solutions in ICUs , 1Baxter Healthcare, Zurich, Switzerland; 2Medical University of Vienna, Austria Critical Care 2013, 17(Suppl 2):P376 (doi: 10.1186/cc12314) Introduction Hospital-acquired infections create a signifi cant burden to the healthcare system. To understand the economic impact, a model was developed to assess the cost of diff erences in catheter-related bloodstream infections (BSI) in ICUs based on either closed or open albumin systems. Conclusion Hemodilution using albumin induced a lesser degree of coagulopathy compared with the synthetic colloids HES and dextran. In addition, albumin-induced coagulopathy was more eff ectively reversed following addition of fi brinogen concentrate compared with coagulopathy induced by synthetic colloids. y Methods A model was developed with the aim of showing the economic consequences of diff erences between intravenous (i.v.) infusions of albumin via open or closed infusion containers. The model took a healthcare perspective in the UK. The impact of central venous catheter-related BSI was evaluated. The model took diff erences in BSI rates and the associated cost consequences for the hospital into account. The model bases on the evidence that closed system delivery helps to reduce the risk of external contamination, which decreases the BSI rate and reduces overall mortality [1-3]. We assessed the total costs using public list prices and literature. The model assumes that infection rates for using open and closed albumin delivery systems are similar to any other closed or open nonalbumin i.v. fl uid delivery system used in the ICU. The model looks at reduction in central-line-associated BSI and the potential cost saving of this reduction. P375 Procedural sedation in the emergency department Methods An electronic-dedicated database was created to retro- spectively collect volume, type of fl uids infused and plasmatic acid– base balance variations in postoperative ICU patients from admission to 9:00 am of the day after. SIDin was calculated as the average SID of all fl uids infused during the whole study period (crystalloids, colloids and blood products). Arterial base excess variation (ΔBEa) was computed as the diff erence between values at 9:00 am on the day after and those at entry. We report data from all patients admitted in 2006 and 2007 (650 patients). MS Shah1, FS Shah2, KP Pope3, AS Abbas4 1Luton and Dunstable Hospital, London, UK; 2Birmingham Hospitals NHS Trust, Birmingham, UK; 3Chesterfi eld NHS Trust, Chesterfi eld, UK; 4Colchester Hospital, Colchester, UK Critical Care 2013, 17(Suppl 2):P380 (doi: 10.1186/cc12318) MS Shah , FS Shah , KP Pope , AS Abbas 1Luton and Dunstable Hospital, London, UK; 2Birmingham Hospitals NHS Trust, Birmingham, UK; 3Chesterfi eld NHS Trust, Chesterfi eld, UK; 4Colchester Hospital, Colchester, UK Critical Care 2013 17(Suppl 2):P380 (doi: 10 1186/cc12318) p , , Critical Care 2013, 17(Suppl 2):P380 (doi: 10.1186/cc12318) Introduction Are safety guidelines being followed when administering procedural sedation in the emergency department? Between November 2004 and November 2008, the NPSA received 498 alerts of patients being given the wrong dose of midazolam for procedural sedation [1]. In the fi rst 5 years of midazolam use there were 86 deaths, most related to procedural sedation [2]. Introduction Are safety guidelines being followed when administering procedural sedation in the emergency department? Between November 2004 and November 2008, the NPSA received 498 alerts of patients being given the wrong dose of midazolam for procedural sedation [1]. In the fi rst 5 years of midazolam use there were 86 deaths, most related to procedural sedation [2]. p Results Nine patients not receiving intravenous infusions were excluded. The remaining population was divided into three groups according to SIDin distribution (Group 1, 18 ± 12; Group 2, 47 ± 6; Group 3, 55 ± 0 mEq/l). We observed a progressive increment in ΔBEa between the groups (1.1 ± 2.0 vs. 2.8 ± 2.9 vs. 3.0 ± 2.8 mmol/l, P <0.001). We further subdivided each group by the median value of baseline HCO3-a (24.3 (22.3 to 26.1) mmol/l) and we analyzed the ΔBEa: we observed a greater increase in patients with lower baseline HCO3-a (Group 1, 1.8 ± 2.9 vs. P379 (SAP), SV and CO were recorded directly before the administration of any colloid (T0) and every 5 minutes for the next 1 hour (T1 to T12). Kolmogorov–Smirnov was used to test normal distribution of data and ANOVA was used for the statistical analysis. P <0.05 was considered statistically signifi cant. P379 Microcirculatory response to fl uid challenge: should we prefer balanced colloids to rebalance tissue perfusion? A Donati, E Damiani, R Domizi, C Scorcella, A Carsetti, S Tondi, R Castagnani, N Mininno, V Monaldi, P Pelaia Università Politecnica delle Marche, Ancona, Italy Critical Care 2013, 17(Suppl 2):P379 (doi: 10.1186/cc12317) y gi Results Demographic data and ASA classifi cation did not diff er statistically signifi cant among the six groups of the study. CO, SV, HR and SAP did not show any statistically signifi cant evolution compared with their baseline value during the study period. Moreover, there were no statistically signifi cant diff erences among the six study groups with regard to any of the recorded parameters. Introduction Fluid resuscitation should improve tissue oxygenation in hypovolemia, besides restoring macrohemodynamic stability [1]. We evaluated the microvascular response to fl uid challenge with diff erent colloid solutions and its relation to macrohemodynamics.l y Conclusion According to our results, volume replacement with the six colloids tested in our study did not result in any hemodynamic response. Within comparison of these six colloids did not reveal any statistically signifi cant diff erence in any of the parameters recorded according to our protocol. Methods An observational study of patients receiving a fl uid challenge (500 ml colloids in 30 minutes) according to the attending physician’s decision. Before and after the infusion, sublingual microcirculation was evaluated with sidestream dark-fi eld imaging (Microscan; Microvision Medical, Amsterdam, the Netherlands). Microvascular fl ow and density were assessed for small vessels [2]. The cardiac index (CI), intrathoracic blood volume index (ITBVI) and extravascular lung water index (ELWI) were measured in seven patients with PiCCO2 (Pulsion Medical System, Munich, Germany). Results Ten patients (two sepsis, four trauma, three intracranial bleeding, one post surgery) received either saline-based hydroxyethyl starch (HES) 130/0.4 (Amidolite®; B.BraunSpA; n = 5) or balanced HES 130/0.42 (Tetraspan®; B.BraunSpA; n = 5). The CI (P = 0.02) and ITBVI (P = 0.07) tended to increase, the EVLWI did not change. Microvascular fl ow and density improved in the whole sample. No correlation was found between macro-circulatory and micro-circulatory parameters. P378 Strong ion diff erence and arterial bicarbonate concentration as cornerstones of the impact of fl uid therapy on acid–base balance D Ottolina, M Ferrari, L Zazzeron, E Scotti, M Stanziano, C Rovati, C Marenghi, L Gattinoni, P Caironi Fondazione IRCCS Ca’ Granda – Ospedale Maggiore Policlinico Università degli Studi di Milano, Milan, Italy Critical Care 2013, 17(Suppl 2):P378 (doi: 10.1186/cc12316) g Conclusion Balanced HES may be more effi cacious than saline-based HES in recruiting the microcirculation, thereby improving tissue O2 delivery. Introduction The biochemical characteristics of infused fl uids may be important in regulating acid–base balance, by modifying plasmatic volume and strong ion diff erence. In vitro and animal studies [1,2] have shown that volume and strong ion diff erence of infused fl uids (SIDin) as well as the arterial baseline bicarbonate concentration (HCO3-a) infl uence acid–base variations. Our aim was to verify these changes in critically ill patients after surgery. Procedural sedation in the emergency department 0.2 ± 2.6, mmol/l, P <0.001; Group 2, 4.0 ± 2.7 vs. 1.5 ± 2.6, mmol/l, P <0.001; Group 3, 4.4 ± 2.8 vs. 1.7 ± 2.0 mmol/l, P <0.001), as compared with those with higher baseline levels. When the study population was divided into quartiles of the diff erence between SIDin and HCO3-a, ΔBEa appeared to increase with the rise of such diff erence (P <0.001).f Methods We searched through the controlled drugs book in resuscitation over a 2-month period and found a list of patients who had received midazolam or fentanyl. From this, we could make a search for the relevant A and E notes for these patients. From these notes, we looked for (see shorthand in Table 1): verbal consent documentation (consent), past medical history recorded (pmhx), safe initial dose of midazolam (midaz), pre-procedure monitoring (pre), post-procedure monitoring (post), and monitoring for 1 hour before discharge (1hr). Following introduction of a reminder in the controlled drugs book/ sedation room and staff education, the case notes were analysed over another 2-month period (24 sets of notes) to assess practise against safety guidelines. f Conclusion SIDin aff ects the acid–base status per se and in relation- ship with HCO3-a. We verifi ed this hypothesis in critically ill patients, highlighting the importance of the diff erence between SIDin and HCO3-a, which better describes and predicts the acid–base modifi cations to fl uid therapy. y g Results See Table 1 (key for shorthand in Methods). Conclusion The re-audit notices within the procedural sedation room and controlled drug book front cover served as a reminder of good practise. The visibility of this reminder (within the CD book) helped ensure better adherence to the audit standard. This reminder will now be kept within the CD book. i References il References 1. Carlesso E, et al.: The rule regulating pH changes during crystalloid infusion. Intensive Care Med 2011, 37:461-468. Carlesso E, et al.: The rule regulating pH changes during crystalloid infusion. Intensive Care Med 2011, 37:461-468. P377 Figure 1 (abstract P375). Forest plot of results of Bayesian fi xed-eff ect network meta-analysis of mortality. Comparing the hemodynamic eff ects of six diff erent colloids N Kteniadakis, V Grosomanidis, E Oloktsidou, B Fyntanidou, C Nouris, K Kotzampasi, D Vasilakos Aristotle Medical School, Thessaloniki, Greece Critical Care 2013, 17(Suppl 2):P377 (doi: 10.1186/cc12315) Comparing the hemodynamic eff ects of six diff erent colloids N Kteniadakis, V Grosomanidis, E Oloktsidou, B Fyntanidou, C Nouris, K Kotzampasi, D Vasilakos Aristotle Medical School, Thessaloniki, Greece Critical Care 2013, 17(Suppl 2):P377 (doi: 10.1186/cc12315) Introduction Despite the fact that colloids are widely used, doubt still remains as to which colloid is the best and what is the ideal volume of administration. The aim of our study was to compare the hemodynamic eff ects of six diff erent colloids. Introduction Despite the fact that colloids are widely used, doubt still remains as to which colloid is the best and what is the ideal volume of administration. The aim of our study was to compare the hemodynamic eff ects of six diff erent colloids. ff Methods A total of 180 patients were enrolled in our study undergoing general surgery procedures, who were assigned to six groups (A, B, C, D, E, F). After fasting fl uid replacement and induction of anesthesia, 500 ml Gelofusine, Haemaccel, Voluven, HES 6%, HES 10% and Hemohes were administered to each group respectively. An oesophageal Doppler monitor probe was inserted into the patients for measuring stroke volume (SV) and cardiac output (CO). Standard monitoring included ECG, IBP, ETCO2 and SpO2, and heart rate (HR). Systemic arterial pressure Figure 1 (abstract P375). Forest plot of results of Bayesian fi xed-eff ect network meta-analysis of mortality. S142 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 References 1. Futier et al.: Crit Care 2011, 15:R214. 2. De Backer et al.: Crit Care 2007, 11:R101. P379 Balanced HES led to a greater increase in capillary density than NaCl HES (Figure 1).fi p References 2. Langer T, et al.: In vivo conditioning of acid–base equilibrium by crystalloid solutions: an experimental study on pigs. Intensive Care Med 2012, 38:686-693. [http://www.npsa.nhs.uk/corporate/news/ reducing-risk-of-midazolam-overdose-in-adults] [http://www.npsa.nhs.uk/corporate/news/ reducing-risk-of-midazolam-overdose-in-adults] S143 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http //ccfor m com/s pplements/17/S2 http://ccforum.com/supplements/17/S2 Figure 1 (abstract P379). Microvascular response to fl uid challenge: eff ects of diff erent colloids. Figure 1 (abstract P379). Microvascular response to fl uid challenge: eff ects of diff erent colloids. Figure 1 (abstract P379). Microvascular response to fl uid challenge: eff ects of diff erent colloids. 2. Epstein B: Data Retrieval Unit HFD-737. Department of Health and Human Services, Offi ce of Epidemiology and Biostatistics, Center for Drug Evaluation and Research; 27 June 1989. P381 Impact of an age, kidney and liver function adjusted sedation protocol in critically ill patients S Haddad, H Tamim, C Gonzales, A Rishu, A Deeb, Y Arabi King Abdulaziz Medical City, Riyadh, Saudi Arabia Critical Care 2013, 17(Suppl 2):P381 (doi: 10.1186/cc12319) Introduction Daily sedation interruption and protocol implementation have been recommended to reduce excessive sedation; however, their use has been inconsistent. We hypothesized that the use of an age, kidney and liver function adjusted sedation protocol would be Table 1 (abstract P380). Before and after education programme and reminder in the controlled drugs book Before (%) After (%) Yes No Yes No Consent 35 65 50 50 Pmhx 55 45 67 33 Midaz 58 42 88 12 Pre 25 75 54 46 Post 10 90 71 29 1hr 55 45 54 46 Table 1 (abstract P380). Before and after education programme and reminder in the controlled drugs book associated with reduced doses and improved outcomes compared with a standard protocol. Table 1 (abstract P380). Before and after education programme and reminder in the controlled drugs book Before (%) After (%) Yes No Yes No Consent 35 65 50 50 Pmhx 55 45 67 33 Midaz 58 42 88 12 Pre 25 75 54 46 Post 10 90 71 29 1hr 55 45 54 46 Methods This was a prospective cohort study comparing 3 months of a standard protocol (control group) with 3 months of an adjusted protocol (intervention group). Benzodiazepines induce hyperglycemia in rats by aff ecting peripheral disposal of glucose VS Salice, FV Valenza, MP Pizzocri, LV Valenti, GC Chevallard, MU Umbrello, SG Gatti, SF Fargion, GI Iapichno, LG Gattinoni Università degli Studi Milano, Milan, Italy Critical Care 2013, 17(Suppl 2):P385 (doi: 10.1186/cc12323) Results We retrieved 902 citations. Among them, nine RCTs fulfi lled inclusion criteria. PS was compared with the standard practice in one RCT, DIS with the standard practice in four, PS with DIS in two, and PS with PS plus DIS (PS/DIS) in two. PS or DIS reduced the duration of mechanical ventilation by 28.9% and ICU stay by 27.8% compared with the standard practice. Neither the duration of mechanical ventilation nor ICU stay diff ered among DIS, PS and PS/DIS. Mortality did not diff er among the three strategies. DIS increased the daily dose of sedatives and the workload of nurses in one recent RCT. Introduction In light of the interest in the relationship between glycemia control in critically ill subjects and outcome, we set up a study to investigate whether benzodiazepine, commonly used in anesthesia and ICUs, interferes with glucose metabolism and to explore the mechanism. Introduction In light of the interest in the relationship between glycemia control in critically ill subjects and outcome, we set up a study to investigate whether benzodiazepine, commonly used in anesthesia and ICUs, interferes with glucose metabolism and to explore the mechanism. Methods A total of 40 sedated and paralyzed Sprague–Dawley rats (301 ± 55 g) were investigated in four consecutive studies. (1) To investigate the eff ects of diazepam on blood glucose, 15 rats were randomly assigned to intraperitoneal anesthesia with tiopenthal 80 mg/kg (DZP0), tiopenthal 40 mg/kg + diazepam 5 mg/kg (DPZ5) or tiopenthal 40 mg/kg + diazepam 15 mg/kg (DZP15). Blood levels of glucose (GEM premier 3000; IL) were measured at time intervals over 2 hours. (2) Ten animals randomized to DZP0 or DZP5 underwent an intravenous glucose tolerance test with glucose bolus (0.5 g/kg). Acute insulin response, the mean value of blood insulin (Insulin ELISA kit; Millipore) from 2 to 10 minutes after glucose bolus, was measured as index of insulin secretion. (3) A hyperinsulinemic euglycemic clamp obtained by a continuous intravenous infusion of insulin (130 mUI/ kg/minute) was run in 10 animals randomized to DZP0 or DZP5 and the glucose infusion rate (GIR, mg/kg/minute) was assessed [1]. Benzodiazepines induce hyperglycemia in rats by aff ecting peripheral disposal of glucose (4) The eff ect of midazolam on blood glucose was tested in fi ve additional animals (M5: tiophental 40 mg/kg + midazolam 5 mg/kg). Data are presented as mean ± SEM. Statistical analysis (ANOVA, t test) was conducted with Sigma Stat 3.1 (Systat Software). Conclusion PS or DIS decreased the duration of mechanical ventilation and ICU stay. PS seems to be superior to DIS based on the doses of sedatives and the workload of nurses. P383 Chemical and physical compatibility of continuous intravenous drug infusion combinations used in paediatric intensive care C Cole1, A Fox1, M Van Der Merwe2, L Dickson2, K Ball2, A Bevan1, A Hocking1, J Pappachan1 1University Hospitals Southampton, UK; 2University of Portsmouth, UK Critical Care 2013, 17(Suppl 2):P383 (doi: 10.1186/cc12321) Introduction The aim of this research was to provide clinically relevant evidence for Y-site compatibility of drug infusion combinations used in the PICU. Pharmacists and clinicians regularly have to interpret limited published data, particularly when more than two drugs are Y-sited. The risk of potential incompatibility must be balanced against that of additional line insertion. Results (1) Diazepam was associated with higher levels of blood glucose in a dose–response fashion: DZP0 128 ± 7 mg/dl, DZP5 166 ± 7.3, DZP15 197 ± 7 (P <0.05). (2) The acute insulin response to intravenous glucose tolerance test was similar in DZP0 and DZP5 (DZP0 3.97 ± 0.42 ng/ml, DZP5 3.68 ± 0.44, P = 0.68), while blood glucose levels were diff erent (DZP0 192 ± 5 mg/dl, DZP5 217 ± 5, P <0.05). (3) During hyperinsulinemic euglycemic clamp, blood glucose levels were similar (109 ± 3 mg/dl, P = 0.2), but the DZP5 group showed a trend through lower values of GIR (DZP0 30.8 ± 2 mg/ kg/minute, DZP5 24.7 ± 2, P = 0.08). (4) Infusion of midazolam was associated with higher blood glucose levels (DZP0 128 ± 5 mg/dl, M5 151 ± 6, P <0.05).i Methods A full 28-factorial design (total 256 combinations) was used to investigate chemical and physical compatibility of fi ve drugs (clonidine, morphine, ketamine, midazolam and furosemide). The drugs were studied at their highest commonly infused concentrations and exposed to three variations in environmental conditions (diluent: sodium chloride 0.9% or glucose 10%; temperature 25 or 37°C; and normal room lighting or blue light phototherapy). Chemical stability was assessed using HPLC; >10% reduction in concentration indicated incompatibility. Benzodiazepines induce hyperglycemia in rats by aff ecting peripheral disposal of glucose Physical incompatibility was confi rmed by precipitation, pH or colour change.f Conclusion Both diazepam and midazolam signifi cantly alter plasma glucose levels in rats. Peripheral disposal of glucose rather than altered pancreas secretion of insulin explains the benzodiazepine-associated hyperglycemia. Results Environmental conditions had no eff ect on the drug mixtures. The precipitation observed in incompatible combinations was due to either a change in pH, or with ketamine the presence of benzethonium chloride. Of 31 possible drug combinations, 12 were incompatible. A further three combinations were incompatible at extreme pH, or were of concern and so should be avoided. The incompatible formulations p References We hypothesized that the use of an age, kidney and liver function adjusted sedation protocol would be S144 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 adjusted protocol group (18% vs. 36%, P = 0.004) with no signifi cant diff erences in MVD, ICU and hospital LOS, and hospital mortality. Conclusion The use of an age, kidney and liver function adjusted sedation protocol is associated with lower doses of analgesics and sedatives, lower risk of agitation and lower ICU mortality. all contained furosemide. All combinations of the sedative agents studied were chemically and physically compatible. adjusted protocol group (18% vs. 36%, P = 0.004) with no signifi cant diff erences in MVD, ICU and hospital LOS, and hospital mortality. y y y Conclusion This work provides evidence for Y-site compatibility of morphine, midazolam, clonidine and ketamine in any combination, which will potentially reduce the need for extra intravenous lines. Furosemide is incompatible with any of these sedative drugs and must be infused via a separate line. These results will aid clinical decision- making and help satisfy the requirements of recent UK Department of Health legislation relating to the mixing of medicines. Reference Conclusion The use of an age, kidney and liver function adjusted sedation protocol is associated with lower doses of analgesics and sedatives, lower risk of agitation and lower ICU mortality. P382 Risk and benefi t of protocol sedation and daily interruption of sedation for mechanically ventilated patients: a systematic review K Hosokawa1, M Egi2, M Nishimura3 1Kyoto Prefectural Yosanoumi Hospital, Kyoto, Japan; 2Okayama University Hospital, Okayama, Japan; 3Tokushima University Hospital, Tokushima, Japan Critical Care 2013, 17(Suppl 2):P382 (doi: 10.1186/cc12320) DeFronzo: Am J Physiol 1979, 273:E214-E223. p References In the adjusted protocol, patients were divided into three categories: category 1 (age <60 years, and normal kidney and liver function), category 2 (age = 60 to 70 years, or moderate kidney or liver function impairment), and category 3 (age >70 years, or severe kidney or liver function impairment). The upper limits of analgesics and sedatives doses were determined by age, and kidney and liver function, being lowest in category 3, and lower in category 2 than category 1. All consecutive adults mechanically ventilated patients who required infusion of analgesics and/or sedatives for >24 hours were included in the study. We compared the main outcomes of both groups including average daily doses of analgesics and sedatives; average Sedation–Agitation Scale (SAS), pain and GCS scores; mechanical ventilation duration (MVD); sedation-related complications during ICU stay; ICU and hospital length of stay (LOS), and ICU and hospital mortality. y y Results Two hundred and four patients were included in the study (control group = 105; adjusted protocol group = 99). There was no diff erence in baseline characteristics between the two groups. The adjusted protocol group, compared with the control group, received signifi cantly lower average daily doses of fentanyl (2,162 ± 2,110 μg vs. 3,650 ± 3,253 μg, P = 0.0001), nonsignifi cant lower average daily doses of midazolam and dexmedetomidine, and a trend toward higher average daily doses of propofol. Pain score was higher in the adjusted protocol group (0.98 ± 0.72 vs. 0.16 ± 0.35, P <0.0001) with no diff erence in SAS or GCS scores. Sedation-related complications during ICU stay were not diff erent between the two groups; however, agitation (SAS = 5) was less frequent in the adjusted protocol group (3% vs. 30%, P <0.0001). ICU mortality was signifi cantly lower in the P381 Impact of an age, kidney and liver function adjusted sedation protocol in critically ill patients S Haddad, H Tamim, C Gonzales, A Rishu, A Deeb, Y Arabi King Abdulaziz Medical City, Riyadh, Saudi Arabia Critical Care 2013, 17(Suppl 2):P381 (doi: 10.1186/cc12319) Introduction Daily sedation interruption and protocol implementation have been recommended to reduce excessive sedation; however, their use has been inconsistent. Reference 1. Mixing of Medicines Prior to Administration in Clinical Practice: Medical and Non-medical Prescribing [http://www.dh.gov.uk/en/ Publicationsandstatistics/Publications/PublicationsPolicyAndGuidance/ DH_116361] 1. Mixing of Medicines Prior to Administration in Clinical Practice: Medical and Non-medical Prescribing [http://www.dh.gov.uk/en/ Publicationsandstatistics/Publications/PublicationsPolicyAndGuidance/ DH_116361] 1. Mixing of Medicines Prior to Administration in Clinical Practice: Medical and Non-medical Prescribing [http://www.dh.gov.uk/en/ Publicationsandstatistics/Publications/PublicationsPolicyAndGuidance/ DH_116361] g 1Kyoto Prefectural Yosanoumi Hospital, Kyoto, Japan; 2Okayama University Hospital, Okayama, Japan; 3Tokushima University Hospital, Tokushima, Japan Critical Care 2013, 17(Suppl 2):P382 (doi: 10.1186/cc12320) Introduction Protocol-directed sedation (PS) and daily interruption of sedation (DIS) are two major strategies to optimize sedation depth for mechanically ventilated patients. To clarify the clinical risk and benefi ts of PS and DIS, we performed a systematic review of randomized controlled trials (RCTs) in previous literature. Introduction Protocol-directed sedation (PS) and daily interruption of sedation (DIS) are two major strategies to optimize sedation depth for mechanically ventilated patients. To clarify the clinical risk and benefi ts of PS and DIS, we performed a systematic review of randomized controlled trials (RCTs) in previous literature. P384 Abstract withdrawn P384 Abstract withdrawn P384 P384 Abstract withdrawn P384 Abstract withdrawn Methods We searched the MEDLINE database from January 1990 to October 2012 for studies on PS and DIS in critically ill patients. English-language manuscripts were included when they assessed the impact of PS or DIS on outcomes in critically ill patients requiring mechanical ventilation for >48 hours. PS is defi ned as sedation practices implemented by ICU staff using a sedation scale and a written protocol. DIS is defi ned as strategies with cessation of the continuous sedatives. P387 Involvement of mitochondrial ATP-sensitive K+ channels in fentanyl- induced mitochondrial dysfunction of cultured human hepatocytes S Djafarzadeh, M Vuda, J Takala, SM Jakob Bern University Hospital (Inselspital) and University of Bern, Switzerland Critical Care 2013, 17(Suppl 2):P387 (doi: 10.1186/cc12325) Introduction Pharmacological agents used to treat critically ill patients may alter mitochondrial function. The aim of the present study was to investigate whether fentanyl, a commonly used analgesic drug, interacts with hepatic mitochondrial function. Introduction We report our experience in the use of isofl urane for prolonged sedation in severe ARDS patients. Prolonged sedation in the ICU may be diffi cult because of tolerance, drug dependence and withdrawal, drug interactions and side eff ects. Inhaled anesthetics have been proposed for sedation in ventilator-dependent ICU patients. AnaConDa is a device that allows a safety and easy administration of inhaled anesthetics in the ICU. Methods The human hepatoma cell line HepG2 was exposed to fentanyl at 0.5, 2 or 10 ng/ml for 1 hour, or pretreated with naloxone (an opioid receptor antagonist) at 200 ng/ml or 5-hydroxydecanoate (5-HD; a specifi c inhibitor of mitochondrial ATP-sensitive K+ (KATP) channels) at 50 μM for 30 minutes, followed by incubation with fentanyl at 2 ng/ml for an additional hour. The mitochondrial complex I-dependent, II-dependent and IV-dependent oxygen consumption rates of the permeabilized cells were measured using a high-resolution oxygraph (Oxygraph-2k; Oroboros Instruments, Innsbruck, Austria). The respiratory electron transfer capacity of intact cells was evaluated using FCCP (carbonyl cyanide p-trifl uoromethoxyphenylhydrazone) to obtain the maximum fl ux. Methods From January 2009 to June 2012, 15 patients were sedated with isofl urane by means of the AnaConDa device. We consider administration of isofl urane as a washout period from common sedative drugs in patients with (at least one of): high sedative drug dosage (propofol ≥300 mg/hour or midazolam ≥8 mg/hour) to reach the target Richmond Agitation Sedation Score (RASS) or inadequate paralysis; two or more hypnotic drugs to reach the target RASS (propofol, midazolam, hydroxyzine, haloperidol, diazepam, quetiapine); and hypertriglyceridemia. During isofl urane administration previous hypnotic drugs were interrupted. We retrospectively collected data before, during and after administration of isofl urane: hemodynamic parameters, renal and hepatic function, level of sedation (RASS) and sedative drug dosage. All data are reported as mean ± standard deviation, otherwise as median (minimum to maximum). P387 l Results Incubation of HepG2 cells with fentanyl (1 hour, 2 ng/ml) induced a reduction in complex II-dependent and IV-dependent respiration (Figure 1). Cells pretreated with 5-HD before the addition of fentanyl exhibited no signifi cant changes in complex activities in comparison with controls. Pretreatment with naloxone tended to abolish the fentanyl-induced mitochondrial dysfunction. Treatment with fentanyl led to a reduction in cellular ATP content (0.24 ± 0.06 in controls vs. 0.17 ± 0.14 μmol/mg cellular protein in stimulated cells; P = 0.02). We did not observe any diff erence in basal or FCCP-uncoupled respiration rates of cells treated with fentanyl at 2 ng/ml compared with controls (data not shown). Results Mean age was 43 ± 12 years and SAPS II was 35.7 ± 11; 13 patients were treated with ECMO for severe ARDS and four had a history of drug abuse; median ICU length of stay was 41 (15 to 127) days and they were ventilated for 41 (12 to 114) days. Due to severe critical illness, target RASS was –4 for all patients, most of which were also paralysed. Isofl urane was administered in nine patients because of a high level of common sedative drugs, in fi ve patients due to the use of two or more hypnotic drugs and in one patient because of hypertrigliceridemia. Isofl urane administration lasted 5.6 ± 1.8 days. During isofl urane administration no alteration in renal function or hemodynamic instability was recorded. After the isofl urane washout period we observed a reduction in sedative drug dosage in 10 patients while two patients were quickly weaned from mechanical ventilation and the target RASS raised to 0. In two patients isofl urane was precautionarily interrupted because of concomitant alteration of liver function and suspected seizures respectively. Conclusion Fentanyl reduces cultured human hepatocyte mito- chondrial respiration by a mechanism that is blocked by a KATP channel antagonist. In contrast, antagonism with naloxone does not seem to completely abolish the eff ect of fentanyl. Acknowledgement This study was supported by the Swiss National Science Foundation (grant number 32003B_127619). P388f Reference S145 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 P386 P386 Prolonged sedation in ARDS patients with inhaled anesthetics: our experience S Redaelli, P Mangili, V Ormas, S Sosio, L Peluso, F Ponzoni, N Patroniti, A Pesenti University of Milan-Bicocca, Monza, Italy Critical Care 2013, 17(Suppl 2):P386 (doi: 10.1186/cc12324) P389 Conjunctival administration of S+ketamine in rabbits L Hess1, J Malek2, A Kurzova2, Z Simunkova1, M Votava2 1Institute for Clinical and Experimental Medicine, Prague, Czech Republic; 23rd Medical Faculty of Charles University, Prague, Czech Republic Critical Care 2013, 17(Suppl 2):P389 (doi: 10.1186/cc12327) Results During the study period 75 patients underwent procedural sedation and were discharged from the ED. The median age was 40 years and 57% were male. The commonest procedure performed was shoulder reduction (52%). In the propofol group (n = 20) the mean LOS was 100 minutes compared with 165 minutes in those receiving midazolam (n = 40) and 141 minutes in those receiving a combination (n = 15), P = 0.004. There was no diff erence in adverse events between groups. See Figure 1. Introduction Delivering analgesics via conjunctival application could provide rapid and convenient pain relief in disaster medicine. There are sporadic reports from the USA concerning inhalation administration of aerosol with various drugs producing a wide variety of eff ects from anxiolysis, sedation, and loss of aggressiveness to immobilisation. We attempted to determine in an animal experiment whether conjunctival administration of S+ketamine could produce signifi cant eff ect without side eff ects. g p g Conclusion Propofol is increasingly used in EDs for procedural sedation due to its short duration of action. This study suggests that a shorter duration of action and faster recovery may result in a reduced LOS in the ED. f Methods After ethic committee approval, 10 rabbits were administered conjunctival S+ketamine 2.5 mg/kg. Measured parameters were SpO2, blood pressure (BP) and heart rate (HR) before administration and in 1-minute intervals and immobilisation time (loss of righting refl ex). The measurements were performed for 20 minutes. Conjunctival irritation was measured 1, 10 and 20 minutes after administration according to modifi ed technical standard EN IS O 10993-10. ANOVA test was used for statistical analysis of hemodynamic parameters. Figure 1 (abstract P390). Length of stay (minutes) in the ED. Figure 1 (abstract P390). Length of stay (minutes) in the ED. Results The immobilisation time was 207 ± 60 seconds. P388 Eff ects of clonidine on microcirculatory alterations during endotoxemia K S h idt C Phili b A Zi k i S H f P388 Eff ects of clonidine on microcirculatory alterations during endotoxemia K Schmidt, C Philipsenburg, A Zivkovic, S Hofer Universitätsklinikum Heidelberg, Germany Critical Care 2013, 17(Suppl 2):P388 (doi: 10.1186/cc12326) Conclusion Inhaled anesthetics could be successfully used in the ICU especially in case of an inadequate sedation plan; for example, in patients with a history of drug abuse or young severe ARDS patients that required deep sedation and paralysis for a long period. Introduction Endothelial dysfunction during endotoxemia is responsible for the functional breakdown of microvascular perfusion and microvessel permeability. The cholinergic anti-infl ammatory pathway (CAP) is a neurophysiological mechanism that regulates the infl ammatory response by inhibiting proinfl ammatory cytokine synthesis, thereby preventing tissue damage. Endotoxemia-induced Figure 1 (abstract P387). Cellular oxygen consumption after incubation with fentanyl, naloxone or 5-HD. S146 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 eff ect of ketamine or due to the method of administration. There were no signs of conjunctival irritation in any animal (S+ketamine is a preservative-free solution). microcirculatory dysfunction can be reduced by cholinergic CAP activation. Clonidine improves survival in experimental sepsis [1] by reducing the sympathetic tone, resulting in the parasympathetic- mediated CAP activation. The aim of this study was to determine the eff ects of clonidine on microcirculatory alterations during endotoxemia. Methods Using fl uorescent intravital microscopy, we determined the venular wall shear rate, macromolecular effl ux and leukocyte adhesion in mesenteric postcapillary venules of male Wistar rats. Endotoxemia was induced over 120 minutes by intravenous infusion of lipopolysaccharide (LPS). Control groups received an equivalent volume of saline. Clonidine 10 μg/kg was applied as i.v. bolus in treatment groups. Animals received either (i) saline alone, (ii) clonidine 10 minutes prior to saline administration, (iii) clonidine 45 minutes prior to LPS administration, (iv) clonidine 10 minutes prior to LPS administration, (v) clonidine 30 minutes after LPS administration or (vi) LPS alone. p Conclusion Conjunctival S+ketamine 2.5 mg/kg in rabbits produced rapid onset without changes in cardiorespiratory parameters and without signs of irritation of the eye. The results of our project warrant further research to increase the variety of drugs and methods of their administration for anxiolysis, sedation and analgesia in disaster medicine. Acknowledgement The study was supported by scientifi c grant IGA NT 11284. Reference 1. Malek J, et al.: Conjunctival administration of sufentanil in rabbits. P388 Eff ects of clonidine on microcirculatory alterations during endotoxemia K S h idt C Phili b A Zi k i S H f Eur J Pain 2011, Suppl 5:212. 1. Malek J, et al.: Conjunctival administration of sufentanil in rabbits. Eur J Pain 2011, Suppl 5:212. Results All LPS groups (iii to vi) showed a signifi cantly reduced venular wall shear rate compared with the saline group after 120 minutes. There were no signifi cant diff erences between the numbers of adhering leukocytes in the clonidine/LPS groups (iii, iv, v) and the LPS group after 120 minutes. Macromolecular effl ux signifi cantly increased in all groups over the time period of 120 minutes. After 120 minutes there was no diff erence between the LPS group and the clonidine 10 minutes prior to LPS administration group (iv) whereas all other groups (i, ii, iii, v) showed a signifi cantly reduced macromolecular effl ux compared with the LPS group.f Use of propofol for procedural sedation reduces length of stay in the emergency department J Millar, F Adamson, P O’Connor, R Wilson, E Ferrie, R McLaughlin Royal Victoria Hospital, Belfast, UK Critical Care 2013, 17(Suppl 2):P390 (doi: 10.1186/cc12328) Use of propofol for procedural sedation reduces length of stay in the emergency department J Millar, F Adamson, P O’Connor, R Wilson, E Ferrie, R McLaughlin Royal Victoria Hospital, Belfast, UK Critical Care 2013, 17(Suppl 2):P390 (doi: 10.1186/cc12328) Introduction Procedural sedation is used in the emergency department (ED) to facilitate short but painful interventions. Many patients are suitable for discharge after completion. Ideally, the agent used to achieve sedation should not have a prolonged eff ect, allowing safe discharge in the shortest time frame. We hypothesised that propofol, with its short onset and off set, may reduce length of stay (LOS) in comparison with traditional benzodiazepines. Conclusion Clonidine has no positive eff ect on microhemodynamic alterations and leukocyte–endothelial interaction during endotoxemia. The reduction of capillary leakage in clonidine-treated groups depends on the time interval relative to the initiation of endotoxemia. Endothelial permeability and leukocyte activation are regulated by diff erent pathways when stimulated by clonidine during endotoxemia. We conclude that clonidine might have an important time-dependent anti-infl ammatory and protective eff ect on endothelial activation during infl ammation. Methods Data from a prospective registry were analysed for the period 1 August 2011 to 31 January 2012. Patients who underwent procedural sedation and who were discharged from the ED were identifi ed. Individuals were grouped as having received propofol, midazolam or a combination of the two. All were discharged when fully alert and able to eat and drink. Demographic details and the type of procedure undertaken were extracted. ANOVA was performed to identify diff erences in the length of stay between groups, in addition to descriptive analysis. Reference Reference 1. Hofer S, et al.: Crit Care 2009, 13:R11. 1. Hofer S, et al.: Crit Care 2009, 13:R11. 1. Hofer S, et al.: Crit Care 2009, 13:R11. P393 Use of physical restraint in Dutch ICUs: prevalence and motives RJ Raijmakers1, RL Vroegop1, H Tekatli1, M Van den Boogaard2, AW Van der Kooi1, AJ Slooter1 1University Medical Center Utrecht, the Netherlands; 2Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands Critical Care 2013, 17(Suppl 2):P393 (doi: 10.1186/cc12331) Use of physical restraint in Dutch ICUs: prevalence and motives RJ Raijmakers1, RL Vroegop1, H Tekatli1, M Van den Boogaard2, AW Van der Kooi1, AJ Slooter1 1University Medical Center Utrecht, the Netherlands; 2Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands Critical Care 2013, 17(Suppl 2):P393 (doi: 10.1186/cc12331) Results Data were collected from 54 adults; 50 received an infusion for ≥24 hours. We noted a signifi cant CK rise in 11 (22%) of our patients that could be attributed to propofol alone. They all had raised TG (≥1.7 IU/l); six (12%) were markedly raised (≥3.4 IU/l). No patients became signifi cantly acidotic, developed cardiac arrhythmias or renal failure as a result of propofol. In the 11 patients who met our criteria, highest daily propofol dose varied from 1.5 to 4 mg/kg/hour. Pre-PRIS developed from between 2 and 6 days of infusion. The mean propofol dose in patients displaying Pre-PRIS was 2.2 mg/kg/hour whilst in those not meeting our criteria it was 2.0 mg/kg/hour. No patients developed sequelae of full PRIS. See Table 1. Introduction Physical restraints are used to facilitate essential care and prevent secondary injuries. However, physical restraint may be regarded as humiliating. It may lead to local injury and increase the risk of delirium and post-traumatic stress syndrome. Research on physical restraint is scarce. The aim of this study is to investigate the scope of physical restraint use. Methods Twenty-one ICUs ranging from local hospitals to academic centres were each visited twice and 327 patients were included. We recorded characteristics of restrained patients, motives and awareness of nurses and physicians. Table 1 (abstract P391) Total Trauma Nontrauma ≥24 hours infusion 50 34 16 CK + TG ≥3.4 6 6 0 CK + TG ≥1.7 5 3 2 Results Physical restraint was applied in 74 (23%) patients, ranging from 0 to 54% in diff erent hospitals. Frequent motives for restraint use were ‘possible threat to airway’ (36%) and ‘pulling lines/probes’ (31%). Restrained subjects more often had a positive CAM-ICU (34% vs. 16%, P <0.001), could less frequently verbally communicate (14% vs. 49%, P <0.001), and received more often antipsychotics (49% vs. P394 Factors infl uencing the use of physical restraints in Canadian ICUs B Sneyers1, E Luk2, M Perreault3, D Williamson3, L Rose2, S Mehta4, L Burry4 1Université Catholique de Louvain, Brussels, Belgium; 2Lawrence S. Bloomberg Faculty of Nursing, University of Toronto, Ontario, Canada; 3Université de Montréal, Canada; 4Mount Sinai Hospital, University of Toronto, Ontario, Canada Critical Care 2013, 17(Suppl 2):P394 (doi: 10.1186/cc12332) Factors infl uencing the use of physical restraints in Canadian ICUs B Sneyers1, E Luk2, M Perreault3, D Williamson3, L Rose2, S Mehta4, L Burry4 1Université Catholique de Louvain, Brussels, Belgium; 2Lawrence S. Bloomberg Faculty of Nursing, University of Toronto, Ontario, Canada; 3Université de Montréal, Canada; 4Mount Sinai Hospital, University of Toronto, Ontario, Canada Critical Care 2013, 17(Suppl 2):P394 (doi: 10.1186/cc12332) P389 There were no changes in cardiorespiratory parameters (initial, 10 and 20 minutes after administration: HR 255.5 ± 24.7, 265.8 ± 26.0 and 267.8 ± 20.4, systolic BP 114.3 ± 8.6, 108.2 ± 10.7 and 113.9 ± 11.9 mmHg, diastolic BP 66.8 ± 11.6, 68.4 ± 10.6 and 69.7 ± 9.3 mmHg and SpO2 99.2 ± 1.0, 98.2 ± 0.6 and 98.7 ± 0.5). These results are in contrast to conjunctival administration of sufentanil 5 μg/kg that caused a signifi cant decrease of SpO2 and HR [1]. We can speculate that the reason for stability of cardiorespiratory parameters was due to the sympathoadrenergic S147 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 P391 Pre-PRIS? Prospective monitoring for early markers of propofol infusion syndrome M Stovell, S Smith, M Udberg, W Loh, P Nair Walton Centre Neurology & Neurosurgery, Liverpool, UK Critical Care 2013, 17(Suppl 2):P391 (doi: 10.1186/cc12329) P391 Conclusion The study shows a 1% adverse event rate. This supports use of propofol sedation by emergency physicians but within the limits of a strict governance framework. Our safety analysis using the World SIVA adverse events tool provides a reference point for further studies. R f P391 Pre-PRIS? Prospective monitoring for early markers of propofol infusion syndrome 1. Green SM, Yealy DM: Procedural sedation goes Utstein: the Quebec guidelines. Ann Emerg Med 2009, 53:436-438. 1. Green SM, Yealy DM: Procedural sedation goes Utstein: the Quebec guidelines. Ann Emerg Med 2009, 53:436-438. 1. Green SM, Yealy DM: Procedural sedation goes Utstein: the Quebec guidelines. Ann Emerg Med 2009, 53:436-438. Introduction The use of propofol for sedation in intensive care has been associated with the propofol infusion syndrome (PRIS) characterised by cardiac dysfunction, metabolic acidosis, renal failure, rhabdomyolysis and hyperlipidaemia. We prospectively monitor biochemical markers that we believe demonstrate early signs of this dangerous, often fatal syndrome. When this Pre-PRIS state is identifi ed, propofol is withdrawn whilst the syndrome is still reversible. 2. Green SM, Krauss B: Barriers to propofol use in emergency medicine. Ann Emerg Med 2008, 52:392-398. 3. Mason KP, Green SM, Piacevoli Q; International Sedation Task Force: Adverse event reporting tool to standardize the reporting and tracking of adverse events during procedural sedation: a consensus document from the World SIVA International Sedation Task Force. Br J Anaesth 2012, 108:13-20. 3. Mason KP, Green SM, Piacevoli Q; International Sedation Task Force: Adverse event reporting tool to standardize the reporting and tracking of adverse events during procedural sedation: a consensus document from the World SIVA International Sedation Task Force. Br J Anaesth 2012, 108:13-20. y Methods We prospectively audited our monitoring of these markers over a 4-month period in propofol-sedated patients: propofol infusion rate, creatine kinase (CK), triglycerides (TG), creatinine, lactate, pH and base defi cit. We defi ned the criteria for Pre-PRIS as requiring a CK ≥320 mmol/l that had doubled from its base level and a rise in TG ≥1.7 IU/l; both that followed a trend with propofol dose. P393 28%, P <0.001), or benzodiazepines (55% vs. 36%, P = 0.003). The use of physical restraint was registered in the patient’s fi les in 48% of cases. Of the 310 interviewed nurses, 258 (83%) were familiar with a physical restraint protocol and 89 (29%) used it in any situation. Thirty percent of the 60 interviewed physicians were aware of the physical restraint status of their patients. Conclusion We propose that a paired rise in CK and TG that can be attributed to propofol alone represents a Pre-PRIS state that is at risk of developing into full PRIS. We noted this in 22% of our patients, all on modest doses of propofol. It is unclear what proportion of patients will develop the full syndrome as it is not ethically possible to continue propofol in this situation. We advocate daily monitoring of CK and TG to identify Pre-PRIS so that propofol can be reduced or substituted to avoid the morbidity and mortality of the full syndrome. Conclusion Physical restraint is frequently used in Dutch ICUs, but the frequency diff ers strongly between diff erent ICUs. Attending physicians are often not aware of physical restraint use. P392 Safety profi le of 1,008 cases of propofol sedation in the emergency department Safety profi le of 1,008 cases of propofol sedation in the emergency department S Bradburn, G Lloyd, B Newstead Royal Devon and Exeter Hospital, Exeter, UK Critical Care 2013, 17(Suppl 2):P392 (doi: 10.1186/cc12330) Introduction Until recently there were no guidelines for the reporting of adverse events (AEs) during procedural sedation [1,2]. A consensus document released in 2012 by the world SIVA International Sedation Task Force proposed a benchmark for defi ning AEs [3]. We analysed 1,008 cases of procedural sedation in the emergency department. Introduction Physical restraint (PR) use in critically ill patients has been associated with delirium, unplanned extubation, prolonged ICU length of stay, and post-traumatic stress disorder. Our objectives were to defi ne prevalence of PR use, and to examine patient, treatment, or institutional factors associated with their use in Canadian ICUs. Introduction Physical restraint (PR) use in critically ill patients has been associated with delirium, unplanned extubation, prolonged ICU length of stay, and post-traumatic stress disorder. Our objectives were to defi ne prevalence of PR use, and to examine patient, treatment, or institutional factors associated with their use in Canadian ICUs. Methods The study is based on 1,008 patients who received procedural sedation with propofol in the emergency department between December 2006 and March 2012. Patients were selected and sedated to a strict protocol by ED consultant staff . We applied the AE tool by performing a search through patient records, discussion with consultants performing the sedation and consensus opinion.i Methods We conducted an analysis of a database of sedative, analgesic, antipsychotic and neuromuscular blocker prescribing practices and PR use in 711 mechanically ventilated (MV) adults in 51 Canadian ICUs (3,619 patient-days). Data were collected during a 2-week period (2008 to 2009). Logistic and Poisson regression analysis were used to identify factors associated with PR use and number of days of use respectively. Results PR was used on 1 or more days in 52.6% (374/711) of patients. For patients restrained, the mean number of days restrained was Results From 1,008 cases we identifi ed 11 sentinel (six of hypotension, fi ve cases of hypoxia), 34 moderate, 25 minor and three minimal risk adverse events. S148 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 4.1 ± 4.0 and the mean number of restraint applications per patient was 1.2 ± 0.5. Current perspectives, beliefs and practices concerning delirium in critically ill patients: a multicenter survey among Dutch healthcare professionals Z Trogrlic, M Van der Jagt, P Van der Voort, J Bakker, E Ista, on behalf of iDECePTIvE Study Group Results We assessed 1,576 patients, corresponding with 8,150 ICU treatment days with a median length of stay of 3 days (IQR 2 to 5). The mean age of the patients was 62 years (SD = 16) and 58% were male. Delirium occurred in 23% (356/1,576) of patients with a median duration of 3 days (IQR 2 to 7) and ranged from 11 to 40% for each ICU. Delirium assessment with the CAM-ICU at any point during ICU stay was performed in 38% of all patients. Screening with CAM-ICU was applied in three ICUs, in 29 to 96% of the patients in these centers. Of 3,564 documented screening days with the CAM-ICU, it was positive in 1,459 (41%); in only 120 (8%) of these CAM-ICU-positive days there was a documented action or treatment started for delirium. However, patients still received haloperidol on 52% (n = 766) of all CAM-ICU- positive days. Patients received benzodiazepines in 49% (n = 1,141) of patient sedation days. Delirium preventive interventions were physiotherapy (19% of 8,150 ICU days), mobilization (10%), glasses use (2.6%) and hearing aid use (0.3%). Presence of hearing or visual impairment at admission was not documented in 65% of patients. Erasmus MC University Medical Center, Rotterdam, the Netherlands Critical Care 2013, 17(Suppl 2):P397 (doi: 10.1186/cc12335) Introduction The beliefs, knowledge and practices regarding ICU delirium among ICU professionals may vary. This may interfere with the implementation of the Dutch ICU Delirium guideline. We aimed to get insight into potential barriers and facilitators for delirium guideline implementation that may help to fi nd an eff ective implementation strategy. Methods An online survey was sent to healthcare professionals from the six participating ICUs. Respondents included ICU physicians, nurses and delirium experts (psychiatrists, neurologists, geriatricians, nurse experts). The survey consisted of statements on beliefs, knowledge and practices towards ICU delirium. Agreement with statements by more than 75% of respondents were regarded as facilitating items and agreement lower than 50% as barriers for implementing protocolled care. Conclusion Daily screening for ICU delirium with a validated screening instrument is applied in less than one-half of the time in critically ill patients and management of delirium is often not guided by this screening. Haloperidol was used as the fi rst-choice medication. P395 Current practices in ICU delirium management: a prospective multicenter study in the Netherlands Z Trogrlic, E Ista, A Slooter, J Bakker, M Van der Jagt, on behalf of iDECePTIvE Study Group Erasmus MC University Medical Center, Rotterdam, the Netherlands Critical Care 2013, 17(Suppl 2):P395 (doi: 10.1186/cc12333) Results Of 768 eligible patients, complete data on delirium could be collected in 738 patients. The incidence of delirium was 14.2% in the dexamethasone group and 14.9% in the placebo group (odds ratio = 0.95, 95% CI = 0.63 to 1.43). No signifi cant diff erence was found on the duration of delirium between the intervention (median = 2 days, interquartile range 1 to 3 days) and placebo (median = 2 days, interquartile range 1 to 2 days) group (P = 0.45). The use of restrictive measures and administration of sedatives, haloperidol, benzodiazepine and opiates were comparable between both groups. Introduction As part of a multicenter prospective study on barriers and facilitators for implementation of protocolled care for delirious critically ill patients, we aimed to describe current practices in delirium management in a representative cohort of ICU patients. p p g p Conclusion Intraoperative injection of dexamethasone seems not to aff ect the incidence or duration of delirium in the fi rst 4 days after cardiac surgery, suggesting this regimen is safe to use in the operative setting with respect to psychiatric adverse events. Reference Methods We included consecutively admitted patients during a 4-month period in a prospective multicenter study in six ICUs (one academic, teaching and nonteaching hospitals) in the southwest of the Netherlands. We assessed: percentage of patients screened for delirium with validated screening tools; pharmacological treatment with haloperidol or other antipsychotic drugs; psychohygiene (use of hearing aids and or glasses, preventing sleep disturbances); and access to early mobilization and physiotherapy. Delirium was pragmatically defi ned as administration of haloperidol and/or delirium reported by physicians or ICU nurses in patient records, as assessed by a designated research nurse. Diff erences between centers were tested with non- parametric tests. 1. Dieleman JM, et al.: JAMA 2012, 308:1761-1767. 1. Dieleman JM, et al.: JAMA 2012, 308:1761-1767. Intraoperative dexamethasone and delirium after cardiac surgery: a randomized clinical trial AC Sauër, AJ Slooter, DS Veldhuijzen, MM Van Eijk, D Van Dijk University Medical Center Utrecht, the Netherlands Critical Care 2013, 17(Suppl 2):P396 (doi: 10.1186/cc12334) Introduction The objective of this study is to investigate the eff ect of intraoperative administration of dexamethasone versus placebo on the incidence of delirium in the fi rst four postoperative days after cardiac surgery. Introduction The objective of this study is to investigate the eff ect of intraoperative administration of dexamethasone versus placebo on the incidence of delirium in the fi rst four postoperative days after cardiac surgery. Methods Within the context of the large multicenter Dexamethasone for Cardiac Surgery (DECS) trial [1] for which patients were randomized to 1 mg/kg dexamethasone or placebo at induction of anesthesia, a monocenter substudy was conducted. The primary outcome of this study was the incidence of delirium in the fi rst four postoperative days. Secondary outcomes were duration of delirium, use of restrictive measures and sedative, antipsychotic and analgesic requirements. Delirium was assessed daily by trained research personnel, using the Richmond Agitation Sedation Scale and the Confusion Assessment Method. Medical, nursing and medication charts were evaluated for signs of delirium and use of prespecifi ed medication. Analysis was by intention to treat. Conclusion We identifi ed multiple factors associated with PR use and number of days of PR use. Particularly among those related to treatment, strategies such as daily sedation interruption or use of antipsychotics are associated with PR use and increasing the number of days of use. P392 Safety profi le of 1,008 cases of propofol sedation in the emergency department Treatment characteristics associated with PR use were: daily benzodiazepine dose (OR = 1.01, 95% CI: 1.00 to 1.01), daily opiate dose (OR = 1.00, 95% CI: 1.00 to 1.01), daily sedation interruption (OR = 1.84, 95% CI: 1.24 to 2.72), use of antipsychotics (OR = 3.42, 95% CI: 1.98 to 5.91) and agitation (Sedation–Agitation Scale (SAS) >4, OR = 2.55, 95% CI: 1.20 to 5.44). Additionally, number of days of PR use was increased according to the following: daily sedation interruption (IRR = 4.52, 95% CI: 2.09 to 9.75), use of antipsychotics (IRR = 18.65, 95% CI: 7.95 to 43.77), the presence of a heavily sedated score (SAS <3, IRR = 2.57, 95% CI: 1.06 to 6.24) or the presence of an adverse event, such as self- extubation (IRR = 10.46, 95% CI: 2.55 to 42.92). Hospital characteristics associated with PR use included the proportion of MV patients in the ICU. University hospitals were associated with reduced PR use, compared with community hospitals (OR = 0.272, 95% CI: 0.15 to 0.50). Nonmodifi able patient characteristics (age, sex, APACHE score, patient category, prior substance abuse, prior psychotropic medication, pre- existing psychiatric condition or dementia) were not associated with PR use. mobilization) were carried only in a small minority or were not documented. To implement protocolled delirium care in the region at study, a multifaceted tailored implementation program is needed. P396 Intraoperative dexamethasone and delirium after cardiac surgery: a randomized clinical trial AC Sauër, AJ Slooter, DS Veldhuijzen, MM Van Eijk, D Van Dijk University Medical Center Utrecht, the Netherlands Critical Care 2013, 17(Suppl 2):P396 (doi: 10.1186/cc12334) International validation of the delirium prediction model for ICU patients (PREDELIRIC): a multicenter observati onal study Methods We included patients in whom days with delirium could be compared with days without delirium, based on the Confusion Assessment Method for the ICU and inspection of medical records. Patients with conditions aff ecting thermal regulation, including infectious diseases, and those receiving therapies aff ecting body temperature were excluded. Twenty-four ICU patients were included after screening 334 delirious ICU patients. Daily temperature variability was determined by computing the mean absolute second derivative of the temperature signal. Per patient, temperature variability during delirious days was compared with nondelirium days using a Wilcoxon signed-rank test. With a linear mixed model, diff erences between delirium and nondelirium days with regard to temperature variability were analysed adjusted for daily mean Richmond Agitation and Sedation Scale scores, daily maximum Sequential Organ Failure Assessment score, and within-patient correlation. M van den Boogaard1, L Schoonhoven2, E Maseda3, C Plowright4, C Jones5, A Luetz6, PV Sackey7, P Jorens8, LM Aitken9, F van Haren10, JG van der Hoeven1, P Pickkers1 M van den Boogaard1, L Schoonhoven2, E Maseda3, C Plowright4, C Jones5, A Luetz6, PV Sackey7, P Jorens8, LM Aitken9, F van Haren10, JG van der Hoeven1, P Pickkers1 1Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands; 2Radboud University Nijmegen Medical Centre, Scientifi c Institute for Quality of Healthcare, Nijmegen, the Netherlands; 3Hospital Universitario La Paz, Madrid, Spain; 4Medway Maritime Hospital, Kent, UK; 5Whiston Hospital, Prescot, UK; 6Charité – Universitaetsmedizin Berlin, Germany; 7Karolinska University Hospital, Stockholm, Sweden; 8University of Antwerpen, Belgium; 9Princess Alexandra Hospital, Woolloongabba, Australia; 10Canberra Hospital, Canberra, Australia Critical Care 2013, 17(Suppl 2):P398 (doi: 10.1186/cc12336) Critical Care 2013, 17(Suppl 2):P398 (doi: 10.1186/cc12336) Critical Care 2013, 17(Suppl 2):P398 (doi: 10.1186/cc12336) Introduction Delirium is a serious and frequent disorder in ICU patients. The aim of this study was to internationally validate the existing Prediction of Delirium ICU (PREDELIRIC) model to predict delirium in ICU patients. Results Temperature variability was increased during delirium days compared with days without delirium (mean diff erence = –0.007, 95% CI = –0.004; –0.011, P <0.001). Adjusting for confounders did not alter our fi ndings (adjusted mean diff erence = –0.005, 95% CI = –0.008; –0.002, P <0.001). Methods A prospective multicenter cohort study was performed in eight ICUs in six countries (Table 1). The 10 predictors (age, APACHE II score, urgent and admission category, infection, coma, sedation, morphine use, urea level, metabolic acidosis) were collected within 24 hours after ICU admission. P398 International validation of the delirium prediction model for ICU patients (PREDELIRIC): a multicenter observati onal study M van den Boogaard1, L Schoonhoven2, E Maseda3, C Plowright4, C Jones5, A Luetz6, PV Sackey7, P Jorens8, LM Aitken9, F van Haren10, JG van der Hoeven1, P Pickkers1 Current perspectives, beliefs and practices concerning delirium in critically ill patients: a multicenter survey among Dutch healthcare professionals Measures aimed at delirium prevention (psychohygiene and early Results Of the 565 surveys distributed, 360 were completed (63.7%). The majority of respondents were ICU nurses (79%). Delirium was considered a major problem (83%) that requires adequate treatment (99%) and is underdiagnosed (81%). Respondents considered that S149 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 routine screening of delirium can improve prognosis (95%). However, only a minority (20%) answered that delirium is preventable. Only 39% of the respondents had received any training about delirium in the previous 3 years and 77% of them found training useful. The mean delirium knowledge score was 6.6 out of 10 (SD = 1.54). When all groups were mutually compared, nurses scored lower than delirium experts (ANOVA, P = 0.013). The respondents (58%; n = 210) from three ICUs indicated that CAM-ICU assessment was department policy. However, 50% (n = 106) of these respondents felt unfamiliar with CAM- ICU and only 47% (n = 99) of them indicated that a positive CAM-ICU was used for treatment decisions. Haloperidol was the fi rst-choice pharmacological treatment. Only 21% of all respondents knew that a national ICU delirium guideline existed, but in-depth knowledge was generally low. was 82 ± 16% and inter-rater reliability 0.87 ± 0.17. Overall delirium incidence was 19.9% (range 27.2%). Despite signifi cant diff erences between centres on all 10 predictors, the overall area under the receiver operating characteristic curve (AUROC) of the eight centers was 0.77 (95% CI: 0.74 to 0.79). was 82 ± 16% and inter-rater reliability 0.87 ± 0.17. Overall delirium incidence was 19.9% (range 27.2%). Despite signifi cant diff erences between centres on all 10 predictors, the overall area under the receiver operating characteristic curve (AUROC) of the eight centers was 0.77 (95% CI: 0.74 to 0.79). Conclusion The overall predictive value of the PREDELIRIC model was good, indicating that the prediction model can now be used internationally. P399 Delirium in ICU patients is associated with increased temperature variability AW Van der Kooi, TH Kappen, RJ Raijmakers, IJ Zaal, AJ Slooter University Medical Center Utrecht, the Netherlands Critical Care 2013, 17(Suppl 2):P399 (doi: 10.1186/cc12337) Delirium in ICU patients is associated with increased temperature variability Delirium in ICU patients is associated with increased temperature variability y AW Van der Kooi, TH Kappen, RJ Raijmakers, IJ Zaal, AJ Slooter University Medical Center Utrecht, the Netherlands Critical Care 2013, 17(Suppl 2):P399 (doi: 10.1186/cc12337) y AW Van der Kooi, TH Kappen, RJ Raijmakers, IJ Zaal, AJ Slooter University Medical Center Utrecht, the Netherlands Critical Care 2013, 17(Suppl 2):P399 (doi: 10.1186/cc12337) y Conclusion Our survey showed that healthcare professionals considered delirium an important but underdiagnosed form of organ failure. In contrast, screening tools for delirium are scarcely used, knowledge can be improved and protocolled treatment based on positive screening is often lacking. These results suggest that the focus of implementation of ICU delirium management should not be on motivational aspects, but on knowledge improvements, training in screening tools and implementation of treatment and prevention protocols. Introduction Delirium is an acute disturbance of consciousness and cognition. It is a common disorder in the ICU and associated with impaired long-term outcome [1,2]. Despite its frequency and impact, delirium is poorly recognized by ICU physicians and nurses using delirium screening tools [3]. A completely new approach to detect delirium is to use monitoring of physiological alterations. Temperature variability, a measure for temperature regulation, could be an interesting parameter for monitoring of ICU delirium, but this has never been investigated before. The aim of this study was to investigate whether temperature variability is aff ected during ICU delirium. International validation of the delirium prediction model for ICU patients (PREDELIRIC): a multicenter observati onal study CAM-ICU was used to identify ICU delirium. Conclusion Temperature variability is increased during delirium in ICU patients, which refl ects the encephalopathy that underlies delirium. Opportunities for delirium monitoring using temperature variability should be further explored. Particularly, in combination with electroencephalography it could provide the input for an objective tool to monitor delirium. Results A total of 2,852 adult ICU patients were screened and 1,824 (64%) were included. Main exclusion reasons were length of stay <1 day (19.1%) and sustained coma (4.1%). Mean ± CAM-ICU compliance Gastrointestinal symptoms in the ICU: a comparison of primary and secondary acute gastrointestinal injury A Reintam Blaser, J Starkopf , p University of Tartu, Tartu University Hospital, Tartu, Estonia y y Critical Care 2013, 17(Suppl 2):P401 (doi: 10.1186/cc12339) Introduction We aimed to clarify the diff erences between primary and secondary acute GI injury. Methods A total of 2,690 consecutive adult patients were retro- spectively studied during their fi rst week in the ICU. Pathology in the GI system or laparotomy defi ned the primary GI insult. If GI symptoms developed without primary GI insult it was considered secondary GI injury. Absent bowel sounds (BS), vomiting/regurgitation, diarrhoea, bowel distension, GI bleeding, and high gastric residuals (GRV >1,000 ml/24 hours) were recorded daily. ventilation with relative risk of 9 to 27% and with mortality of 25 to 50% [1,2]. One of the promoting factors of VAP is the increased pH of the gastric acid, which occurs when H2-receptor antagonists (H2RA) or proton pump inhibitors (PPI) are used for stress ulcer prophylaxis. The aim of this study was to investigate eff ects of PPI versus H2RA on the occurrence of VAP and, as second endpoint, the occurrence of gastrointestinal bleeding (GIB). y Results In total, 2,690 patients (60.4% male), median age 59 years (range 16 to 92), were studied. Eighty-four per cent of them were ventilated, 72% received vasopressor/inotrope. Median (IQR) APACHE II score was 14 (9 to 21) and SOFA on the fi rst day was 6 (3 to 10). A total 35.5% had primary GI pathology. During the fi rst week 34% of patients had absent BS, 38% vomiting/regurgitation, 9% diarrhoea, 7% bowel distension, 6% high GRV and 5% GI bleeding. All symptoms except diarrhoea occurred more often (<0.001) in patients with primary GI insult. Eighty-fi ve per cent of patients with primary GI insult versus 46% without developed at least one GI symptom. The incidence of GI symptoms was signifi cantly higher in nonsurvivors. ICU mortality was lower in patients with primary than secondary GI injury (43.6% vs. 61.2%, P = 0.046). Nonsurvivors without primary GI insult developed GI symptoms later (Figure 1).f Methods After ethics committee approval mechanically ventilated (>48 hours) adults were recruited in a prospective randomized controlled, double-blind clinical trial and randomised into PPI or H2RA groups. Patients with admission diagnosis of pneumonia or other acute infl ammatory pulmonary disease, severe sepsis/septic shock, and/or regular PPI/H2RA users were excluded. References 1. Pisani MA, et al.: Am J Respir Crit Care Med 2009, 180:1092-1097. 2. Ely EW, et al.: JAMA 2004, 291:1753-1762. 1. Pisani MA, et al.: Am J Respir Crit Care Med 2009, 180:1092-1097. 2 El EW t l JAMA 2004 291 1753 1762 Table 1 (abstract P398). PREDELIRIC AUROC of diff erent centers Table 1 (abstract P398). PREDELIRIC AUROC of diff erent centers Table 1 (abstract P398). PREDELIRIC AUROC of diff erent centers Delirium (%) AUROC 95% CI Belgium (%) 86 (15.2) 0.79 0.74 to 0.84 Germany (%) 60 (26.9) 0.85 0.80 to 0.91 Spain (%) 23 (18.0) 0.88 0.81 to 0.94 Sweden (%) 30 (39.0) 0.71 0.60 to 0.83 Australia Brisbane (%) 42 (12.8) 0.81 0.75 to 0.88 Australia Canberra (%) 23 (11.8) 0.80 0.72 to 0.89 UK_Prescot (%) 73 (30.8) 0.65 0.57 to 0.73 UK_Kent (%) 26 (36.6) 0.76 0.65 to 0.88 PREDELIRIC overall 363 (19.9) 0.77 0.74 to 0.79 y 3. van Eijk MM, et al.: Am J Respir Crit Care Med 2011, 184:340-344. P400 Long-term outcome of delirium in critically ill patients A Wolters, D Van Dijk, O Cremer, D De Lange, A Slooter UMC Utrecht, the Netherlands Critical Care 2013, 17(Suppl 2):P400 (doi: 10.1186/cc12338) Long-term outcome of delirium in critically ill patients A Wolters, D Van Dijk, O Cremer, D De Lange, A Slooter UMC Utrecht, the Netherlands Critical Care 2013, 17(Suppl 2):P400 (doi: 10.1186/cc12338) Introduction In ICU patients, little research has been performed on the relationship between delirium and long-term outcome, including health-related quality of life (HRQoL), cognitive functioning and mortality. In addition, results seem to be inconsistent. Furthermore, in studies that reported increased mortality in delirious patients, no proper adjustments were made for severity of illness during ICU admission. This study was conducted to investigate the association S150 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Figure 1 (abstract P401). Daily dynamics of the number of GI symptoms. P <0.001 between survivors and nonsurvivors in all time points. between ICU delirium and long-term HRQoL, cognitive functioning and mortality. The hypothesis was that delirious patients have worse long-term outcome in comparison with nondelirious patients. between ICU delirium and long-term HRQoL, cognitive functioning and mortality. The hypothesis was that delirious patients have worse long-term outcome in comparison with nondelirious patients. Methods A prospective observational cohort study was conducted. Gastrointestinal symptoms in the ICU: a comparison of primary and secondary acute gastrointestinal injury The diagnostic criteria for VAP were: leukocytosis, increased PCT level, fever, purulent tracheal secretion, positive microbiological result of tracheal sample, and new/ increased infi ltrate on chest X-ray. Data are presented as median (interquartile range, IQR). For statistical analysis Mann–Whitney U test and chi-square tests were used. q Results Out of 94 patients recruited 14 were excluded due to early weaning/death (n = 9) or protocol violation (n = 6). The remaining 79 patients were analysed (PPI, n = 38; H2RA, n = 41). There was no signifi cant diff erence (P >0.05) between the groups regarding demographics: age 67 (56 to 77) versus 72 (58 to 79) years; male/ female: 23/15 versus 25/16; length of mechanical ventilation: 5 (3 to 9) versus 5 (2 to 8) days; APACHE II score: 28 (22 to 32) versus 26 (21 to 36) (PPI vs. H2RA, respectively). There was no signifi cant diff erence in the number of cases with VAP in the PPI versus H2RA groups: 9 (24%) versus 10 (24%). None of the patients developed GI bleeding during their stay on the ICU. Conclusion Primary and secondary acute GI injury have diff erent incidence, dynamics and outcome. References A median of 12 months after ICU discharge, questionnaires were sent to all survivors. HRQoL and cognitive functioning were measured with the EuroQol-6D. Age, gender and severity of illness were considered relevant covariates. Severity of illness was estimated using the APACHE IV score and the maximal SOFA score during admission. HRQoL was investigated with linear regression analysis, cognitive functioning using logistic regression and mortality with Cox regression analysis. y y Results The patient population consisted of 690 patients admitted to the ICU, subdivided into delirious (n = 257) and nondelirious patients (n = 433). During follow-up, 181 (26%) patients died. The response rate of the questionnaire was 70.6%. After adjusting for the predefi ned covariates, delirium was signifi cantly associated with a lower HRQoL (adjusted β: –0.137; 95% CI = –0.140 to –0.005) and more mild and severe cognitive impairment (adjusted odds ratio: respectively: 2.3; 95% CI = 1.3 to 4.2 and 5.8; 95% CI = 1.3 to 15.2). No signifi cant association between delirium and long-term mortality was found (adjusted hazard ratio: 1.0; 95% CI = 0.7 to1.4). Conclusion Delirium during ICU admission was associated with lower HRQoL and worse cognitive functioning, 1 year after discharge. Furthermore, delirium on the ICU was not associated with long-term mortality after adjusting for relevant covariates, including severity of illness during ICU admission. Early diagnosis of liver failure in septic patients using the maximal liver function capacity test (LiMAx test): comparison with conventional methods M Kaff arnik, M Stockmann, J Lock Charite Berlin Campus Virchow, Berlin, Germany Critical Care 2013, 17(Suppl 2):P403 (doi: 10.1186/cc12341) Introduction Patients with bacterial sepsis often suff er from multiorgan failure. No reliable parameter exists for the exact estimation of the liver function. The indocyaningreen test (ICG test) showed a correlation between liver function and mortality rate. The new maximal liver function capacity test (LiMAx test) is a real-time method to investigate the liver function. g p pp Results HVPG increased in Group 1 from 3.7 to 5.4 mmHg (P <0.05) and decreased in Group 2 (4.8 to 4.3 mmHg, P = NS) (Table 1). Liver function as assessed by ICG-PDR decreased in Group 1 by day 1 (18.5 to 15.3, P <0.05) and remained stable in Group 2 (25.5 to 26.8, P = NS). Renin, aldosterone, ADH, adrenaline, noradrenaline and dopamine increased signifi cantly (P <0.05) in Group 1 during operation. Group 2 showed a signifi cant rise only in ADH and dopamine. Acute kidney injury occurred in fi ve of 13 patients in Group 1 (P <0.05). Methods Thirty septic patients were prospectively included in the study. The LiMAx test was performed on days 0, 2, 5 and 10 after sepsis onset and was compared with ICG test and liver-specifi c laboratory parameters. Primary endpoint was the mortality rate after 90 days. Secondary endpoint was the comparison with the ICG test. Results The LiMAx test showed low results initially with increasing values on days 5 and 10. The 90-day mortality rate in patients with a low LiMAx test on day 2 (<100 μg/kg/hour) was signifi cantly higher than in patients with a LiMAx test >100 μg/kg/hour. The LiMAx test was comparable with the ICG test. Patients with indication for renal replacement therapy during hospital treatment showed signifi cantly lower LiMAx values than patients without dialysis. See Figure 1. Table 1 (abstract P404) Group 1 Group 2 Renin 36.4 7.9 PostOP 189.7 30.5 Day 1 258 31.3 ADH 3.49 2.59 PostOP 16.11* 10.8* Day 1 10.8 2.39 Creatine 0.8 0.83 Day 1 1.25* 0.91 P <0.05 compared with preOP value. p y g Conclusion With the LiMAx test we can detect a liver dysfunction in septic patients early on days 0 to 2. P403 P403 Early diagnosis of liver failure in septic patients using the maximal liver function capacity test (LiMAx test): comparison with conventional methods M Kaff arnik, M Stockmann, J Lock Charite Berlin Campus Virchow, Berlin, Germany Critical Care 2013, 17(Suppl 2):P403 (doi: 10.1186/cc12341) Early diagnosis of liver failure in septic patients using the maximal liver function capacity test (LiMAx test): comparison with conventional methods The LiMAx test is equal with the ICG test and a result of <100 μg/kg/hour on day 2 suggests a low probability of survival in septic patients. 1. Stockmann et al.: New liver function test for prediction of postoperative outcome in liver surgery. HPB 2010, 12:139. 2. Lock et al.: Early diagnosis of primary nonfunction and indication for reoperation after liver transplantation. Liver Transpl 2010, 16:172. 2. Lock et al.: Early diagnosis of primary nonfunction and indication for reoperation after liver transplantation. Liver Transpl 2010, 16:172. Figure 1 (abstract P403). Kaplan–Meier survival curve of patients with high and low LiMAx results. Conclusion Depending on resection size liver resection acutely increases portal venous pressure and induces neurohumoral activation resulting in compromised renal function and increased risk of developing AKI. P402f Eff ects of proton pump inhibitor versus H2-receptor antagonist stress ulcer prophylaxis on ventilator-associated pneumonia: a pilot study JF Fogas, KK Kiss, FG Gyura, ZT Tóbiás, ZM Molnár University of Szeged, Hungary Critical Care 2013, 17(Suppl 2):P402 (doi: 10.1186/cc12340) Eff ects of proton pump inhibitor versus H2-receptor antagonist stress ulcer prophylaxis on ventilator-associated pneumonia: a pilot study JF Fogas, KK Kiss, FG Gyura, ZT Tóbiás, ZM Molnár University of Szeged, Hungary Critical Care 2013, 17(Suppl 2):P402 (doi: 10.1186/cc12340) University of Szeged, Hungary Critical Care 2013, 17(Suppl 2):P402 (doi: 10.1186/cc12340) y Conclusion The results of this pilot study suggest that there may be no diff erence in the incidence of VAP and GI bleeding if stress ulcer prophylaxis is performed by H2RA or PPI. As the latter is more expensive, its use as fi rst choice in critical care should be questioned. Introduction Ventilator-associated pneumonia (VAP) is the most frequent ICU-acquired infection among patients receiving mechanical S151 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 However, the completion of the study on the planned 198 patients is required to come to the fi nal conclusions. References 1. Efrati S, et al.: J Clin Monit Comput 2010, 24:161-168. 2. Torres A, et al.: Intensive Care Med 2009, 35:9-29. However, the completion of the study on the planned 198 patients is required to come to the fi nal conclusions. References P Biesenbach, T Grünberger, B Payer, A Ferlitsch, W Druml, E Fleischmann, F Luf, J Mühlbacher Medical University of Vienna, Austria Critical Care 2013, 17(Suppl 2):P404 (doi: 10.1186/cc12342) Introduction Liver dysfunction – in correlation to the severity of functional impairment – but also any increase in portal pressure per se (hepatorenal refl ex) can induce alterations in renal function and ultimately result in hepatorenal syndrome (HRS). In this prospective investigation we determined the impact of liver resection on portal venous pressure by measuring the hepatic venous pressure gradient (HVPG), on concentrations of vasoactive peptides and on renal function. Methods Twenty patients (mean age 66.3 years) undergoing elective liver resection surgery because of malignant tumor were assessed and grouped according to resection size: (Group 1) hemihepatectomy, n = 13 versus (Group 2) segmentectomy, n = 7. HPVG was measured before and after resection by canulation of a hepatic vein under fl uoroscopic guidance, liver function was assessed by the indocyanine green plasma disappearance rate (ICG-PDR). However, the completion of the study on the planned 198 patients is required to come to the fi nal conclusions. References 1. Efrati S, et al.: J Clin Monit Comput 2010, 24:161-168. 2. Torres A, et al.: Intensive Care Med 2009, 35:9-29. Impact of liver resection on portal venous pressure and renal function p p p function P Biesenbach, T Grünberger, B Payer, A Ferlitsch, W Druml, E Fleischmann, F Luf, J Mühlbacher Medical University of Vienna, Austria Critical Care 2013, 17(Suppl 2):P404 (doi: 10.1186/cc12342) 1. Efrati S, et al.: J Clin Monit Comput 2010, 24:161-168. 2. Torres A, et al.: Intensive Care Med 2009, 35:9-29. Does just fungal evidence increase the mortality of crit patients with liver cirrhosis? A retrospective analysis patients with liver cirrhosis? A retrospective analysis T Lahmer, U Mayr, M Messer, B Saugel, C Schultheiss, R Schmid, W Huber University Hospital Munich, Germany Introduction Patients with liver cirrhosis (LC) are susceptible to a variety of complications. Especially, infection disorders can reduce their life expectancy markedly. The aim of this retrospective analysis was to detect the incidence and prognosis in patients with LC and fungal evidence (FE). Table 1 (abstract P407) Variable AUC Standard error 95% CI APACHE II score 0.934 0.023 0.889 to 0.980 SOFA score 0.908 0.026 0.857 to 0.960 Number OF 0.901 0.026 0.849 to 0.952 AUC, areas under the curves. Methods A 3-year retrospective analysis of ICU patients with LC. Patients with LC with/without FE were compared with a matched control group based on comparable age and APACHE II score. Specimens were collected based on clinical and/or laboratory (infection signs) reasons and were analyzed by microbiological standard methods. Results A total of 101 patients with LC were enrolled in this analysis. Basic data for both groups: Child–Pugh Score: 4A, 18B, 79C; reason for ICU admission: 28hepatorenal syndrome, 18SBP, 20GI bleeding, 19sepsis, 16pneumonia; etiology of LC: 83alcoholic, 8NASH, 8viral (hepatitis B/C), 2PSC. Fifty-fi ve (54%) patients with LC had FE (mean age: 57.6 ± 14 years, sex M/F: 40/15, APACHE II score 27 ± 2). Compared with the 46 patients with LC but without FE (mean age: 59.2 ± 12 years, sex M/F: 35/11, APACHE II score 23 ± 4), these patients have a signifi cantly higher APACHE II score (27 ± 2 vs. 23 ± 4; P <0.001) and signifi cantly higher mortality (43/55 (78%) vs. 15/46 (33%); P <0.001). Multivariate analysis showed that FE is associated with a higher mortality (P <0.001) but is not associated with age, sex or CHILD score. Seventeen (31%) of the 55 patients in the control group had FE (screening patients n = 147, mean age 65.8 ± 12 years, sex M/F: 35/20, APACHE II score 26.5 ± 3, reason for ICU admission: 19pneumonia, 16sepsis, 9intracerebral haemorrhage, 7acute abdomen, 4lymphatic disorder). The mortality rate was signifi cantly lower (27/55 (49%) vs. 43/55 (78%); P = 0.003) as compared with patients with LC and FE. The main localisation of fungal was in both groups the lung (46 vs. 14), followed by urine (18 vs. 2), blood culture (6 vs. 1) and ascites (4 vs. 0). P405 P405 Value of the preoperative Model for End-stage Liver Disease score in predicting postoperative outcome in living donor liver transplantation recipients Y Nassar, K Omar, A Battah, A Mwafy Cairo University, Giza, Egypt Critical Care 2013, 17(Suppl 2):P405 (doi: 10.1186/cc12343) P405 Value of the preoperative Model for End-stage Liver Disease score in predicting postoperative outcome in living donor liver transplantation recipients Y Nassar, K Omar, A Battah, A Mwafy Cairo University, Giza, Egypt Critical Care 2013, 17(Suppl 2):P405 (doi: 10.1186/cc12343) P405 Value of the preoperative Model for End-stage Liver Disease score in predicting postoperative outcome in living donor liver transplantation recipients Cairo University, Giza, Egypt Cairo University, Giza, Egypt y gyp Critical Care 2013, 17(Suppl 2):P405 (doi: 10.1186/cc12343) Introduction Partial liver grafting predisposes recipients to a unique set of potential technical and anatomic complications that are not prevalent in whole deceased donor grafts. We aimed to assess the preoperative Model for End-stage Liver Disease (MELD) score, postoperative 30-day clinical and laboratory follow-up, and detection of the value of MELD score in predicting 30-day postoperative outcome of LDLT recipients. Figure 1 (abstract P403). Kaplan–Meier survival curve of patients with high and low LiMAx results. Methods A prospective multicenter registry involving 142 patients who underwent LDLT from October 2004 to December 2010. Preoperative S152 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 and long lengths of stay [1]. Survival among patients with predicted SAP at admission has been shown to correlate with the duration of organ failure (OF) [2]. The systemic determinant of severity in a new classifi cation of acute pancreatitis (AP) is also based on identifi cation of patients with transient or persistent OF [3]. MELD score was calculated: (0.378×log (bilirubin) (mg/dl) + 1.120×log (INR) + 0.957×log (creatinine) (mg/dl) + 0.643)×10 then rounded to the nearest integer. Postoperative daily recording of all the clinical and laboratory data was done for a period of 30 days. Results The survival rate recorded in our study was 86.62%. The most frequent complications were renal complications (86.6%), pulmonary complications (73.9%), neurological complications (14%), cardiovascular complications (12.6%), infectious complications (13.3%), intra-abdominal infections (10.5%) and immunosuppressant toxicity (7.7%). MELD score proved to be a good predictor of perioperative outcome, patients with MELD score (>12) was associated with less favorable outcome, accuracy was (80.3%). P407 0 Predictors of persistent organ failure in ICU patients with severe acute pancreatitis P408 Increased plasma levels of thrombopoietin in patients with severe acute pancreatitis 1Città della Salute e della Scienza Hospital, Torino, Italy; 2University of Torino, Italy y Critical Care 2013, 17(Suppl 2):P408 (doi: 10.1186/cc12346) Introduction The aim of this study was to evaluate the accuracy of thrombopoietin (TPO) plasma levels as a biomarker of clinical severity in patients with acute pancreatitis (AP). TPO is a humoral growth factor that stimulates megakaryocyte proliferation and diff erentiation [1]. Furthermore, it favors platelet aggregation and polymorphonuclear leukocyte activation [2]. Elevated plasmatic concentrations of TPO have been shown in patients with critical diseases, including ACS, burn injury and sepsis [2]. In particular, clinical severity is the major determinant of elevated TPO levels in patients with sepsis [3]. AP is a relatively common disease whose diagnosis and treatment are often diffi cult, especially in the clinical setting of the emergency department (ED). About 20% of patients with AP develop a severe form of the disease. In order to early identify those patients aff ected by severe AP, several biomarkers have been studied. No data regarding TPO plasma levels in patients with AP are currently available. y p Conclusion ICU patients with LC have a high incidence of FE. The mortality rate is therefore signifi cantly higher as compared with patients with LC without FE and also as compared with a control group without LC. Therefore early antimycotic treatment should be considered. P405 Methods The aim of the study was to retrospectively determine the predictors of early persistent OF in ICU patients with SAP. The analysis involved 152 patients. The median time interval between the onset of AP and admission was 24 (9; 48) hours. The patients were divided into two groups: the fi rst group (n = 46) had transient OF and the second group (n = 106) had persistent OF. The ability of the APACHE II score, total SOFA score and number of organ/system failure to discriminate transient from persistent OF was explored with receiver operating characteristic (ROC) curves.i Conclusion MELD score >12 was associated with less favorable outcome with an accuracy of 80.3%. Results Hospital mortality was signifi cantly higher in the second group as compared with the fi rst group (45% vs. 7%, P = 0.000); while infectious complications were 39% versus 11% (P = 0.001) and median lengths of ICU stay were 8 (5; 18) days for the second group and 6 (4; 7) days for the fi rst group (P = 0.001). Optimum cutoff levels (by ROC curve analysis) were APACHE II score ≥12 (sensitivity 0.790; 1 – specifi city 0.119), total SOFA score ≥4 (sensitivity 0.829; 1 – specifi city 0.190), and failure ≥2 organs/systems (sensitivity 0.819; 1 – specifi city 0.214). See Table 1. Does just fungal evidence increase the mortality of crit patients with liver cirrhosis? A retrospective analysis Predominantly candida species were detected (especially C. albicans/glabrata), fi ve patients in the LC group had aspergillus fumigatus. In LC patients, FE could be detected in many cases in several compartments. Conclusion Persistent OF in ICU patients with SAP is associated with the highest risk of hospital mortality. Early identifi cation of patients with a high likelihood of persistent OF (APACHE II score ≥12, total SOFA score ≥4, failure ≥2 organs/systems) is an important goal in determining optimal management of ICU patients with SAP. p References References 1. Harrison DA, et al.: Crit Care 2007, 11(Suppl 1):S1. 2. Johnson CD, et al.: Gut 2004, 53:1340-1344. 3 D lli EP t l A S 2012 [E b h d f i t] 3. Dellinger EP, et al.: Ann Surg 2012. [Epub ahead of print] P411 Urinary neutrophil gelatinase-associated lipocalin as an early predictor of acute kidney injury in cardiac surgery patients T García Rodríguez San Miguel Hospital Santa Creu i Sant Pau, Barcelona, Spain Critical Care 2013, 17(Suppl 2):P411 (doi: 10.1186/cc12349) Urinary neutrophil gelatinase-associated lipocalin as an early predictor of acute kidney injury in cardiac surgery patients T García Rodríguez San Miguel Hospital Santa Creu i Sant Pau, Barcelona, Spain Critical Care 2013, 17(Suppl 2):P411 (doi: 10.1186/cc12349) Introduction To evaluate whether urinary neutrophil gelatinase- associated lipocalin (uNGAL) detects acute kidney injury (AKI) earlier than the estimated glomerular fi ltration rate (eGFR) in cardiac surgery patients. Introduction The objective was to evaluate the eff ect of the renal dysfunction measured by pRIFLE on clinical outcome in children admitted to the pediatric intensive care unit (PICU). Introduction The objective was to evaluate the eff ect of the renal dysfunction measured by pRIFLE on clinical outcome in children admitted to the pediatric intensive care unit (PICU). p Methods Two-hundred and seventy-four adult patients undergoing cardiac surgery were consecutively included from February to December 2011. Exclusion criteria were absence of diuresis due to end-stage renal disease or chronic renal failure and a previous cardiac catheterism with i.v. contrast use the week before surgery. Four serial blood and urine samples immediately before (PRE) and after (POST) surgery, and 1 day (1d) and 2 days (2d) after surgery were obtained. uNGAL was measured in an Architect 6200 (Abbott Diagnostics). AKIN criteria were used to diagnose AKI. The study was approved by the local ethics committee and all patients gave informed consent. Delta uNGAL was defi ned as the diff erence between the PRE and the POSTs concentrations. Methods A retrospective cohort involving all children admitted between August 2009 and July 2010 to a Brazilian PICU located in a university-affi liated hospital. Patients were classifi ed according to the pRIFLE at admission as well the maximum pRIFLE during the PICU stay. The endpoints were: length of stay (LOS) in the PICU, LOS on mechanical ventilation, mortality rate quoted by predicted mortality of the Pediatric Index of Mortality (PIM2). y Results In total, 375 children were included in the study. A total 169 (45%) of them presented renal dysfunction according to the pRIFLE. Renal injury and renal failure were associated with higher LOS in the PICU and length of mechanical ventilation. The observed and predicted mortality by PIM2 did not show diff erences. However, the more severe renal presentation (Injury and Failure) was associated with higher mortality rate and was not predicted by PIM2 (8% vs. 11%, P <0.01). Thorough evaluation for the new acute kidney injury criteria by Kidney Disease Improving Global Outcomes Thorough evaluation for the new acute kidney injury criteria by Kidney Disease Improving Global Outcomes Thorough evaluation for the new acute kidney injury criteria by Kidney Disease Improving Global Outcomes y J Izawa, S Uchino, T Fujii, T Arii, T Fukushima, S Kawano Jikei University School of Medicine, Tokyo, Japan Critical Care 2013, 17(Suppl 2):P410 (doi: 10.1186/cc12348) J Izawa, S Uchino, T Fujii, T Arii, T Fukushima, S Kawano Jikei University School of Medicine, Tokyo, Japan Critical Care 2013, 17(Suppl 2):P410 (doi: 10.1186/cc12348) Introduction Two previous classifi cations of acute kidney injury (AKI) that are known as RIFLE criteria and AKIN criteria have shown that AKI is associated with increased morbidity and mortality. Diff erences in predicting ability for prognosis, however, have been reported. In 2012, Kidney Disease Improving Global Outcomes (KDIGO) created the new AKI criteria, combining RIFLE and AKIN criteria. However, such a combination might cause inconsistency among each defi nition in the criteria. We have investigated all of the defi nitions in the new KDIGO criteria in detail. Methods This is a retrospective historical cohort study including adult patients admitted to the ICU (Jikei University, Tokyo, Japan) between January 2010 and October 2011. Patients undergoing chronic dialysis were excluded. KDIGO criteria were applied to all patients to diagnose AKI. Hospital mortality of patients with AKI diagnosed by the 10 defi nitions in the criteria was compared. Results We studied 41 patients with AP (17 severe and 24 mild pancreatitis). No diff erences for gender and age were detected between patients with mild and severe disease. TPO plasma levels were signifi cantly higher in patients with severe AP (99.33 ± 23.68 pg/ ml) than in those with mild AP (50.81 ± 6.73 pg/ml) (Figure 1). The ROC curve led us to calculate a cutoff value of 51.55 pg/ml. This value identifi ed correctly 12 out of the 17 patients aff ected by severe AP. i Results A total of 2,399 patients were evaluated. AKI occurred in 26.6% with standard defi nition of KDIGO; 18.0% with creatinine criteria alone; 15.5% with urine output alone. By multivariable analysis, each AKI stage was associated with hospital mortality: 5.6%, odds ratio 2.95, for Stage 1; 10.1%, odds ratio 5.52, for Stage 2; 30.2%, odds ratio 21.30, for Stage 3. Crude hospital mortality stratifi ed by the 10 defi nitions showed increasing trends with stage progression. P411 Urinary neutrophil gelatinase-associated lipocalin as an early predictor of acute kidney injury in cardiac surgery patients T García Rodríguez San Miguel Hospital Santa Creu i Sant Pau, Barcelona, Spain Critical Care 2013, 17(Suppl 2):P411 (doi: 10.1186/cc12349) Results One-hundred and eighty-one patients (66.1%) were men; mean age was 68.2 ± 12.2 years. Valve replacement was performed in 123, coronary artery bypass graft (CABG) in 81, valve surgery + CABG in 48, cardiac transplant in fi ve, aorta aneurism surgery in nine, and other procedures in eight patients. ICU and hospital stays were 6.7 ± 8.1 and 15.7 ± 13.9 days, respectively. Renal replacement therapy (RRT) was required in 16 patients (5.8%) within 48 hours of ICU stay and in 28 patients (10.2%) within 4 weeks. Mortality at 28 days was 2.9%. Eighty-six patients (31.4%) were diagnosed with AKI within 48 hours of surgery. Area under the ROC curve of POST uNGAL for AKI diagnosis was 0.72 (0.66 to 0.79) (P <0.0001) at an optimal cutoff value of 180 μg/l, Conclusion Renal dysfunction measured by pRIFLE is a very common fi nding (45%) among pediatric patients admitted to the PICU. The more severe renal aff ection is associated with higher mortality, length of MV and length of PICU stay. Thorough evaluation for the new acute kidney injury criteria by Kidney Disease Improving Global Outcomes Mortality of the three defi nitions in Stage 1 was from 4.2% to 8.8%. Stage 2 had two defi nitions and their mortality was 12.1% and 12.9%. Stage 3 had fi ve defi nitions and their mortality was from 20.5% to 55.6%.i i yf y Conclusion TPO may be proposed as biomarker of clinical severity in patients with AP at the time of fi rst evaluation in the ED. Further studies, involving larger numbers of patients, are needed in order to validate these preliminary data. References 1. Kaushansky K: J Thromb Haemost 2003, 1:1587-1592. 2. Lupia E, et al.: Mediators Infl amm 2012, in press. 3. Zakynthinos SG, et al.: Crit Care Med 2004, 32:1004-1010. i Conclusion AKI defi ned by the new KDIGO criteria was associated with increased hospital mortality. In addition, defi nitions in the KDIGO criteria seem to be appropriate because of clear relations between mortality and stage progression. P409 P409 Severe renal dysfunction measured by pRIFLE in children is an independent factor associated with death and not predicted by PIM2 J Piva1, P Lago2, G Pedro3, F Cabral3 1UFRGS, Porto Alegre, Brazil; 2Hospital de Clinicas de Porto Alegre, Brazil; 3PUCRS, Porto Alegre, Brazil Critical Care 2013, 17(Suppl 2):P409 (doi: 10.1186/cc12347) Predictors of persistent organ failure in ICU patients with severe acute pancreatitis Predictors of persistent organ failure in ICU patients with severe acute pancreatitis I Aleksandrova, M Ilynskiy, S Rei, V Kiselev, G Berdnikov, L Marchenkova Hospital Research Institute for Emergency Medicine N.V. Sklifosovsky, Moscow, Russia Critical Care 2013, 17(Suppl 2):P407 (doi: 10.1186/cc12345) I Aleksandrova, M Ilynskiy, S Rei, V Kiselev, G Berdnikov, L Marchenkova Hospital Research Institute for Emergency Medicine N.V. Sklifosovsky, Moscow, Russia Critical Care 2013, 17(Suppl 2):P407 (doi: 10.1186/cc12345) Methods We enrolled patients with AP at the moment of the fi rst clinical evaluation in the ED. AP patients were classifi ed as having mild or severe forms of AP on the basis of the APACHE II score (≥8). TPO concentrations were determined by ELISA. Introduction Severe acute pancreatitis (SAP) requiring admission to an ICU is associated with high mortality (hospital mortality reached 42%) S153 Critical Care 2013, Volume 17 Suppl 2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Figure 1 (abstract P408). Plasma TPO levels in patients with AP. 2. Hassinger AB, Becker CL: Predictive power of serum cistatin C. Pediatr Crit Care Med 2012, 13:435-440. 1. Picca S, Picardo S: Acute kidney injury in an infant after cardiopulmonary bypass. Semin Nephrol 2008, 28:470-476. P413 Perioperative sodium bicarbonate to prevent acute kidney injury after cardiac surgery: a multicenter double-blind randomized controlled trial A Haase-Fielitz1, M Haase1, M Plass2, P Murray3, M Bailey4, R Bellomo5, S Bagshaw6 Perioperative sodium bicarbonate to prevent acute kidney injury after cardiac surgery: a multicenter double-blind randomized controlled trial Atrial natriuretic peptide attenuates metabolic acidosis and infl ammation of the kidney, lung and heart in a rat model of renal ischemia–reperfusion injury Reference 1. Vollmar AM: The role of atrial natriuretic peptide in the immune system. Peptide 2005, 26:1086-1094. Introduction Renal ischemia–reperfusion injury (IRI) is a common cause of acute kidney injury and occurs in various clinical conditions including shock and cardiovascular surgery. Renal IRI releases proinfl ammatory cytokines within the kidney. Atrial natriuretic peptide (ANP) has natriuretic, diuretic and anti-infl ammatory eff ects [1] and plays an important role of regulating blood pressure and volume homeostasis. The hypothesis was that renal IRI induces infl ammation not only in the kidney but also in remote organs such as the lung and heart and ANP attenuates renal injury and infl ammation in the kidney, lung and heart. References 1. Picca S, Picardo S: Acute kidney injury in an infant after cardiopulmonary bypass. Semin Nephrol 2008, 28:470-476. 1. Picca S, Picardo S: Acute kidney injury in an infant after cardiopulmonary bypass. Semin Nephrol 2008, 28:470-476. 2. Hassinger AB, Becker CL: Predictive power of serum cistatin C. Pediatr Crit Care Med 2012, 13:435-440. S154 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 with 78.7% specifi city, 64% sensitivity and 74.1% accuracy. uNGAL advanced diagnosis of AKI in 44 patients (51.2%), whereas diagnosis was achieved at the same time as AKI criteria in 11 patients; AKI criteria outperformed uNGAL in only 36% of cases. Accordingly, uNGAL was useful to diagnose postoperative AKI in 63.9% of cases. Median delta uNGAL was 12.5 (from –1.9 to 71.1) and 154.5 (from 16.6 to 484.5) μg/l in non-AKI and AKI patients, respectively (P <0.0001) and its area under the ROC curve for AKI prediction was 0.70 (0.63 to 0.77) (P <0.0001). measured. The kidney, lung and heart were immunostained to examine the localization of IL-6 and NF-κB and assigned an expression score. The wet/dry ratio of the lung was also measured. Results Renal IRI induced metabolic acidosis, pulmonary edema, mRNA expression of IL-6 in the kidney, lung and heart. Renal IRI increased immunohistochemical localization of IL-6 in the proximal convoluted tubule of the left kidney and NF-κB in the bronchial epithelial cells of the lung. ANP attenuated metabolic acidosis, pulmonary edema and expression of IL-6 mRNA in the kidney, heart, and lung. ANP decreased immunohistochemical localization of IL-6 in the left kidney and NF-κB in the lung. p Conclusion Compared with AKIN criteria, a urinary NGAL concentration >180 μg/l anticipates AKI diagnosis in more than 50% of cardiac surgery patients in the fi rst 24 to 48 hours after intervention. Conclusion These fi ndings suggested that infl ammation within the kidney after renal IRI was extended into the lung and heart. ANP attenuated metabolic acidosis and infl ammation in the kidney, lung and heart in a rat model of renal IRI. ANP may attenuate organ cross- talk between the kidney, lung and heart. P412 P412 Atrial natriuretic peptide attenuates metabolic acidosis and infl ammation of the kidney, lung and heart in a rat model of renal ischemia–reperfusion injury M Khin Hnin Si, C Mitaka, M Tulafu, S Abe, S Ikeda Tokyo Medical and Dental University Graduate School, Tokyo, Japan Critical Care 2013, 17(Suppl 2):P412 (doi: 10.1186/cc12350) P414 p Methods We connected the urinary catheter of 39 ICU patients to a quasi-continuous urine analyzer (Kidney INstant monitorinG®), allowing measurement of pH (pHU), sodium, chloride, potassium and ammonium concentrations (Na+U, Cl–U, K+U, NH4+U) every 10 minutes. The study period lasted 3 hours after a single intravenous bolus of furosemide (time 0). In 13 patients receiving two or more administrations over a longer period (46 (26 to 49) hours), according to clinical needs, we reviewed data on fl uid therapy, hemodynamics and acid–base balance from the beginning to the end of the observation. Introduction Here, we compare the value of acute tubular damage markers measured early in the course after cardiac surgery for prediction of postoperative AKI compared with conventional markers used in clinical routine for risk assessment of acute renal function loss. Methods One hundred adult patients undergoing cardiac surgery in the control arm of a RCT (NCT00672334) were analyzed. We quantifi ed the following biomarkers in urine: NGAL, hepcidin, midkine, IL-6, α1- microglobulin; in plasma: NGAL, hepcidin, haptoglobin, CK, CKMB, CRP, leukocytes, lactate, urea and creatinine. Introduction Here, we compare the value of acute tubular damage markers measured early in the course after cardiac surgery for prediction of postoperative AKI compared with conventional markers used in clinical routine for risk assessment of acute renal function loss. Results Ten minutes after furosemide administration, Na+U and Cl–U rose from 65 ± 6 to 140 ± 5 and from 109 ± 6 to 150 ± 5 mEq/l respectively, while K+U fell from 60 ± 5 to 39 ± 4 mEq/l (P <0.001 for all electrolytes vs. time 0) with a consequent increase in urinary anion gap (AGU = Na+U + Cl–U – K+U). Urinary output increased from 10 (5 to 19) to 53 (29 to 71) ml/10 minutes (P <0.05). After the fi rst hour Cl–U remained higher than Na+U, which progressively decreased, leading to a reduction in AGU and pHU over time. In parallel, a progressive increment in NH4+U was observed. In patients receiving more than one administration we observed an increase in arterial base excess (1.8 ± 0.8 vs. 5.0 ± 0.6 mmol/l, P <0.001) and plasmatic strong ion diff erence (SIDpl) (31 (30 to 33) vs. 35 (34 to 36) mEq/l, P = 0.01) during the study period. These changes were due to a decrease in plasmatic Cl– concentration (109.0 ± 1.1 vs. P414 P414 Tubular damage biomarkers linked to infl ammation or iron metabolism predict acute kidney injury A Haase-Fielitz1, S Westphal1, R Bellomo2, P Devarajan3, M Westerman4, P Mertens1, M Haase1 1Otto von-Guericke University, Magdeburg, Germany; 2Austin Hospital, Melbourne, Australia; 3Cincinnati Children’s Hospital, Cincinnati, OH, USA; 4Intrinsic LifeSciences, La Jolla, CA, USA Critical Care 2013, 17(Suppl 2):P414 (doi: 10.1186/cc12352) balance in response to the administration of furosemide. Methods We connected the urinary catheter of 39 ICU patients to a quasi-continuous urine analyzer (Kidney INstant monitorinG®), allowing measurement of pH (pHU), sodium, chloride, potassium and ammonium concentrations (Na+U, Cl–U, K+U, NH4+U) every 10 minutes. The study period lasted 3 hours after a single intravenous bolus of furosemide (time 0). In 13 patients receiving two or more administrations over a longer period (46 (26 to 49) hours), according to clinical needs, we reviewed data on fl uid therapy, hemodynamics and acid–base balance from the beginning to the end of the observation. Results Ten minutes after furosemide administration, Na+U and Cl–U rose from 65 ± 6 to 140 ± 5 and from 109 ± 6 to 150 ± 5 mEq/l respectively, while K+U fell from 60 ± 5 to 39 ± 4 mEq/l (P <0.001 for all electrolytes vs. time 0) with a consequent increase in urinary anion gap (AGU = Na+U + Cl–U – K+U). Urinary output increased from 10 (5 to 19) to 53 (29 to 71) ml/10 minutes (P <0.05). After the fi rst hour Cl–U remained higher than Na+U, which progressively decreased, leading to a reduction in AGU and pHU over time. In parallel, a progressive increment in NH4+U was observed. In patients receiving more than one administration we observed an increase in arterial base excess (1.8 ± 0.8 vs. 5.0 ± 0.6 mmol/l, P <0.001) and plasmatic strong ion diff erence (SIDpl) (31 (30 to 33) vs. 35 (34 to 36) mEq/l, P = 0.01) during the study period. These changes were due to a decrease in plasmatic Cl– concentration (109.0 ± 1.1 vs. 106.6 ± 0.9 mEq/l, P = 0.009). Plasmatic sodium and potassium concentrations did not change. In these patients, considering the total amount of administered fl uids and urine, a negative water and chloride balance was observed (–460 ± 403 ml and –48 ± 48 mEq, respectively). P415 increase in urinary NGAL in patients receiving bicarbonate infusion was observed compared with control (P = 0.011). The incidence of postoperative RRT was similar but hospital mortality was increased in patients treated with bicarbonate compared with chloride (11/174 (6.3%) vs. 3/176 (1.7%), OR 3.89 (1.07 to 14.2), P = 0.031). See Figure 1. Conclusion On this basis of our fi ndings we do not recommend the use of perioperative infusions of sodium bicarbonate to reduce the incidence or severity of AKI in this patient group. Renal response and acid–base balance alterations during furosemide administration Renal response and acid–base balance alterations during furosemide administration L Zazzeron, D Ottolina, E Scotti, M Ferrari, M Stanziano, C Rovati, F Zadek, C Marenghi, L Gattinoni, P Caironi Fondazione IRCCS Ca’ Granda – Ospedale Maggiore Policlinico, Milan, Italy Critical Care 2013, 17(Suppl 2):P415 (doi: 10.1186/cc12353) Renal response and acid–base balance alterations during furosemide administration L Zazzeron, D Ottolina, E Scotti, M Ferrari, M Stanziano, C Rovati, F Zadek, C Marenghi, L Gattinoni, P Caironi Fondazione IRCCS Ca’ Granda – Ospedale Maggiore Policlinico, Milan, Italy Critical Care 2013, 17(Suppl 2):P415 (doi: 10.1186/cc12353) Introduction Furosemide is one of the most employed diuretics in the ICU for its ability to induce negative water balance. However, one common side eff ect is metabolic alkalosis [1]. We aimed to describe the time course of urinary excretion and changes in plasmatic acid–base balance in response to the administration of furosemide. P414 106.6 ± 0.9 mEq/l, P = 0.009). Plasmatic sodium and potassium concentrations did not change. In these patients, considering the total amount of administered fl uids and urine, a negative water and chloride balance was observed (–460 ± 403 ml and –48 ± 48 mEq, respectively). Methods One hundred adult patients undergoing cardiac surgery in the control arm of a RCT (NCT00672334) were analyzed. We quantifi ed the following biomarkers in urine: NGAL, hepcidin, midkine, IL-6, α1- microglobulin; in plasma: NGAL, hepcidin, haptoglobin, CK, CKMB, CRP, leukocytes, lactate, urea and creatinine. y Results Preoperatively, no biomarker predicted AKI. At ICU arrival, four urinary tubular damage markers showed good discriminatory ability (AUC ≥0.80) for subsequent development or absence of AKI (Figure 1). An excellent predictive value was found for uNGAL/uhepcidin ratio (AUC 0.90, Figure 1). This ratio combines an AKI prediction marker (NGAL) and a marker of protection from AKI (hepcidin), potentiating their individual discriminatory values. Contrarily, at ICU admission, none of the plasma biomarkers was a good early AKI predictor with AUC-ROC ≥0.80. Conclusion Several urinary markers of acute tubular damage predict AKI after cardiac surgery and the biologically plausible combination of NGAL and hepcidin provides excellent AKI prediction. Conclusion Furosemide acts immediately after administration, causing a rise in urinary output, Na+U and Cl–U concentrations. Loop-diuretic- induced metabolic alkalosis may be due to an increased urinary chloride loss and the associated increase in SIDpl. Figure 1 (abstract P414). 1. Wilcox CS: Metabolic and adverse eff ects of diuretics. Semin Nephrol 1999, 19:557-568. Perioperative sodium bicarbonate to prevent acute kidney injury after cardiac surgery: a multicenter double-blind randomized controlled trial A Haase-Fielitz1, M Haase1, M Plass2, P Murray3, M Bailey4, R Bellomo5, S Bagshaw6 g 1Otto von-Guericke University, Magdeburg, Germany; 2German Heart Center, Berlin, Germany; 3University College, Dublin, Ireland; 4ANZIC, Melbourne, Australia; 5Austin Hospital, Melbourne, Australia; 6University of Alberta, Canada Critical Care 2013, 17(Suppl 2):P413 (doi: 10.1186/cc12351) g Methods Male Sprague–Dawley rats were anesthetized with pento- barbital. Tracheostomy was performed and rats were ventilated at VT 10 ml/kg with 5 cmH2O PEEP. The right carotid artery was catheterized for blood sampling and continuous blood pressure measurements. The right femoral vein was catheterized for infusion of saline or ANP. Rats were divided into three groups; IRI group (n = 10), left renal pedicle was clamped for 30 minutes; IRI+ANP group (n = 10), left renal pedicle was clamped for 30 minutes, ANP (0.2 μg/kg/minute, for 3 hours 25 minutes) was started 5 minutes after clamp; and Sham group (n = 6), the sham- operated rats. Hemodynamics, arterial blood gas, and plasma lactate levels were measured at baseline and at 1 hour, 2 hours and 3 hours after declamp. The mRNA expression of IL-6 in the kidney, lung, and heart were Introduction Evidence suggests a nephroprotective eff ect of urinary alkalinization in patients at risk of acute kidney injury (AKI). y y Methods In a multicenter, double-blind, RCT we enrolled 350 adult cardiac surgery patients. At induction of anesthesia, patients received either 24 hours of intravenous infusion of sodium bicarbonate (5.1 mmol/kg) or sodium chloride (5.1 mmol/kg). The primary endpoint was the proportion of patients developing AKI. Results Sodium bicarbonate increased urinary pH (from 6.0 to 7.5, P <0.001). More patients in the bicarbonate group (83/174 (47.7%)) developed AKI compared with control (64/176 (36.4%), OR = 1.60 (95% CI, 1.04 to 2.45); unadjusted P = 0.032). A greater postoperative Figure 1 (abstract P413). Figure 1 (abstract P413). Figure 1 (abstract P413). S155 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 P416 Red cell distribution width is not a predictor of mortality in acute kidney injury P Purvis1, J Kinsella1, R Docking2 1University of Glasgow, UK; 2Glasgow Royal Infi rmary, Glasgow, UK Critical Care 2013, 17(Suppl 2):P416 (doi: 10.1186/cc12354) Value of Tc-99m DMSA renal scan in assessing the prognosis and outcome of acute renal failure due to sepsis Methods A retrospective observational study of renal function in all patients admitted to a London teaching hospital ICU for ≥5 days and surviving to hospital discharge in 2011. We excluded cases with current or new diagnosis of end-stage renal disease or renal transplant. We assessed AKI in ICU by KDIGO 1 criteria and hospital discharge eGFR by the CKD-EPI equation. For comparison we assumed a normal GFR in a healthy individual as 120 ml/minute/1.73 m2 at age 20 decreasing by 0.8 per year over age 20. Value of Tc-99m DMSA renal scan in assessing the prognosis and outcome of acute renal failure due to sepsis S Fawzi, K Mashour, A Amin, H Gamal Cairo University, Cairo – Manial, Egypt y gyp Critical Care 2013, 17(Suppl 2):P417 (doi: 10.1186/cc12355) Introduction Acute renal failure (ARF) is a common complication in patients admitted to the ICU. Sepsis is also a well-known risk factor for the development of ARF. The combination of ARF and severe sepsis was reported to carry a mortality up to 70% whereas the mortality of ARF alone is 40 to 45%. The aim of the study is to evaluate the role of renal perfusion scanning in detecting the prognosis and outcome of patients with acute renal failure due to sepsis. Results We identifi ed 282 patients, 180 of whom had AKI. Median age was 50 and 68% were male. Median hospital discharge serum creatinine was 57 μmol/l (range 16 to 654), median eGFR was signifi cantly higher than predicted normal GFR for age at 115 versus predicted 95 (P <0.001, median diff erence 16). In patients who had not had AKI discharge the eGFR was 119 versus normal predicted 98 (P <0.001, median diff erence 19), suggesting that eGFR could be overestimating true GFR in our population by at least a factor of 1.23 (Figure 1). Applying this correction factor to eGFRs of patients who had recovered from AKI resulted in 44% more diagnoses of CKD (eGFR <60) at hospital discharge (36 vs. 25). p Methods Forty patients with acute renal failure due to sepsis, aged between 15 and 74 years, were enrolled in the study. They were monitored for their ICU prognosis and outcome after doing renal perfusion scanning. All patients were subjected to routine ICU and laboratory investigations including APACHE II and SOFA score. Results Thirty patients had normal renal scan and 10 patients had abnormal renal scan. Red cell distribution width is not a predictor of mortality in acute kidney injury Red cell distribution width is not a predictor of mortality in acute kidney injury P Purvis1, J Kinsella1, R Docking2 1University of Glasgow, UK; 2Glasgow Royal Infi rmary, Glasgow, UK Critical Care 2013, 17(Suppl 2):P416 (doi: 10.1186/cc12354) Introduction Given the signifi cant morbidity and mortality associated with acute kidney injury (AKI), there is a need to fi nd factors to help aid decision-making regarding levels of therapeutic support. As a prognostic biomarker, the red cell distribution width (RDW) has attracted interest in the setting of critical care when added to existing scoring systems [1]. By examining RDW in a previously studied AKI cohort, we aimed to evaluate the utility of this routine blood test. Methods A cohort of 209 mixed critical care patients who received renal replacement therapy for AKI had their demographic and biochemical data retrieved from electronic databases. Outcomes were gathered for ICU and hospital mortality. Incomplete datasets were discarded, leading to 153 complete sets. RDW data were taken from the fi rst sample after admission to the ICU, as were all other biochemical values apart from pre-RRT creatinine and potassium. Overall cohort characteristics were gathered, and two groups were created: those with a RDW value within normal range (≤14.5%) and those with a greater than normal value Figure 1 (abstract P414). S156 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 (>14.5%). We then further subgrouped RDW to assess the correlation between rising levels and ICU mortality. Figure 1 (abstract P418). eGFR at hospital discharge versus expected normal GFR for age in 280 ICU survivors. Results A total 77.1% of our cohort had a RDW greater than the normal laboratory range at time of ICU admission. Key baseline characteristics (age, APACHE II score, length of stay, ICU mortality) did not diff er signifi cantly between patients with normal and abnormal RDW. When subgroup analysis was performed, no statistically signifi cant correlation between rising RDW and ICU mortality was found (Spearman correlation = 0.426, P = 0.233). Conclusion In this cohort of critically ill patients with AKI, RDW was not found to be a predictor of mortality. Our results contradict those of recent studies [1,2]. However, both groups of RDW patients in our study suff ered a higher ICU mortality than in other studies. Red cell distribution width is not a predictor of mortality in acute kidney injury To further explain these fi ndings, we intend to perform multivariate logistic regression analysis and assess the eff ect of social deprivation on RDW. R f 1. Hunziker et al.: Red cell distribution width improves the simplifi ed acute physiology score for risk prediction in unselected critically ill patients. Crit Care 2012, 16:R89. 1. Hunziker et al.: Red cell distribution width improves the simplifi ed acute physiology score for risk prediction in unselected critically ill patients. Crit Care 2012, 16:R89. Figure 1 (abstract P418). eGFR at hospital discharge versus expected normal GFR for age in 280 ICU survivors. 2. Oh et al.: Red blood cell distribution width is an independent predictor of mortality in acute kidney injury patients treated with continuous renal replacement therapy. Nephrol Dial Transplant 2012, 27:589-594. 2. Oh et al.: Red blood cell distribution width is an independent predictor of mortality in acute kidney injury patients treated with continuous renal replacement therapy. Nephrol Dial Transplant 2012, 27:589-594. to survivors of critical illness who may have reduced muscle mass. We hypothesized that eGFR may underestimate rates and severity of CKD in ICU survivors. Varying models of intra-abdominal hypertension and their eff ect on renal function in a porcine model Varying models of intra-abdominal hypertension and their eff ect on renal function in a porcine model R Wise1, R Rodseth1, I De Laet2, L Luis3, L Correa-Martin4, M Garcia4, G Castellanos5, M Malbrain2 1University of Kwazulu Natal, Durban, South Africa; 2ZNA Stuivenberg, Antwerp, Belgium; 3Neuron NPh, S.A., Granada, Spain; 4Jesús Usón Minimally Invasive Surgery Centre, Cacères, Spain; 5‘Virgen de la Arrixaca’ University Hospital, Murcia, Spain Critical Care 2013, 17(Suppl 2):P419 (doi: 10.1186/cc12357) , 1University of Kwazulu Natal, Durban, South Africa; 2ZNA Stuivenberg, Antwerp, Belgium; 3Neuron NPh, S.A., Granada, Spain; 4Jesús Usón Minimally Invasive Surgery Centre, Cacères, Spain; 5‘Virgen de la Arrixaca’ University Hospital, Murcia, Spain Critical Care 2013, 17(Suppl 2):P419 (doi: 10.1186/cc12357) 1. Ympa YP, et al.: Am J Med 2005, 118:827-832. Introduction Intra-abdominal hypertension (IAH) is an independent predictor of renal impairment and mortality [1]. Organ dysfunction caused by the pressure eff ect of IAH is well understood, but how this is modifi ed in the presence of bowel obstruction is unclear. The aim of this study was to determine how diff erent IAH models cause renal dysfunction in a pig model. 1. Ympa YP, et al.: Am J Med 2005, 118:827-832. Value of Tc-99m DMSA renal scan in assessing the prognosis and outcome of acute renal failure due to sepsis The mortality percentage was higher among abnormal renal scan cases (three out of 10, 30%) compared with cases with normal renal scan (seven out of 30, 23.3%) with nonsignifi cant P value: 0.6. The median length of stay/day in ICU was longer among nonsurvivors than survivors 15.5 ± 10, 11.5 ± 8, P value: 0.058 (approaching signifi cance). APACHE II score was higher in nonsurvivors than survivors 23.9± 3.2, 19.6± 4.2, P value: 0.0001. The percentage of mortality among cases that needed mechanical ventilation was higher (nine out of 16, 56.3%) compared with mortality cases that did not need mechanical ventilation (one out of 24, 4.2% with P value: 0.0001). Conclusion ARF may exert an independent adverse eff ect on outcome in septic and septic shock patients. It is also a risk factor for mortality. Tc-99m DMSA scanning is useful for detecting renal dysfunction and help to predict the outcome and prognosis. R f Conclusion eGFR may overestimate renal function in survivors of critical illness confounding identifi cation of CKD in this at-risk population. Prospective studies with measurement of actual GFR are required to assess the burden of CKD in survivors of critical illness. Use, type and duration of cardiopulmonary bypass are related to acute kidney injury occurrence and urinary NGAL concentrations T García Rodríguez San Miguel Use, type and duration of cardiopulmonary bypass are related to acute kidney injury occurrence and urinary NGAL concentrations T García Rodríguez San Miguel Use, type and duration of cardiopulmonary bypass are related to acute kidney injury occurrence and urinary NGAL concentrations T García Rodríguez San Miguel Hospital Santa Creu i Sant Pau, Barcelona, Spain Critical Care 2013, 17(Suppl 2):P422 (doi: 10.1186/cc12360) Methods We included medical ICU patients with sepsis and oliguria (urine output <1 ml/kg/hour) who were prescribed i.v. furose mide. Patients with known chronic renal failure, hypovolemia (CVP <10 mmHg) or severe hypotension (MAP <80 mmHg) were excluded. Resistive index (1 − (end diastolic velocity / maximum systolic velocity)×100) was measured in at least three segmental arteries of both kidneys, the average of all measurements was reported as the result. Repeated assessments were viewed as independent if separated by more than 24 hours. Furosemide was given intravenously in the dose of 40 mg after RI measurement. Positive response to furosemide was defi ned as doubling of hourly dieresis or achieving urine output >1.5 ml/kg/hour after drug administration. Introduction To analyze whether variables related to cardiopulmonary bypass (CPB) infl uence acute kidney injury (AKI) occurrence and urinary neutrophil gelatinase-associated lipocalin (uNGAL) in cardiac surgery patients. Introduction To analyze whether variables related to cardiopulmonary bypass (CPB) infl uence acute kidney injury (AKI) occurrence and urinary neutrophil gelatinase-associated lipocalin (uNGAL) in cardiac surgery patients. Methods A total of 274 adult cardiac surgery patients were consecu- tively included from February to December 2011. Exclusion criteria were absence of diuresis due to end-stage renal disease or chronic renal failure and cardiac catheterism with i.v. contrast in the week before surgery. CPB, when performed, was used as standard CPB (SCPB) or MiniCPB. We obtained four serial blood and urine samples, immediately before (PRE) and after (POST) surgery, and 1 day (1d) and 2 days (2d) after surgery. uNGAL was measured by Architect 6200 (Abbott Diagnostics). AKIN criteria were used to diagnose AKI. The study was approved by the local ethics committee and all patients gave informed consent. g g g Results We included 47 patients with a total of 59 measurements. In 28 cases patients had positive response to furosemide. Median RI in responders was 0.67 (range 0.55 to 0.78) and in nonresponders 0.79 (range 0.58 to 0.81); P = 0.027. Estimated glomerular fi ltration rate based on hospital discharge creatinine may signifi cantly overestimate renal function and underestimate chronic kidney disease in survivors of critical illness l d ll l Methods Twenty-four pigs were divided into three groups; a control group (n = 5), a pneumoperitoneum (Pn) (n = 10), and an intestinal occlusion (Oc) model (n = 10). IAP was maintained for 3 hours at 20 mmHg during which time creatinine, urea, urine output, potassium, and glomerular fi ltration pressure (GFP) were measured. Statistical analysis was performed using repeated-measures ANOVA.ii Introduction Acute kidney injury (AKI) complicates over 50% of ICU admissions. Episodes of AKI are a major risk factor for development or progression of chronic kidney disease (CKD); however, methods of estimated glomerular fi ltration rate (eGFR) may be poorly calibrated Results Over the fi rst 3 hours there was a statistically signifi cant diff erence between the control group and both IAH models for S157 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 potassium (P = 0.002), urea (P = 0.045), creatinine (P = 0.012), GFP (P <0.001), and urine output (P = 0.003). Over the full 5-hour period there was a statistically signifi cant diff erence in the potassium measurement between the Pn and the Oc model (P = 0.01). The other parameters did not show a signifi cant diff erence: urea (P = 0.171), creatinine (P = 0.074), GFP (P = 0.141), and urine output (P = 0.242).i potassium (P = 0.002), urea (P = 0.045), creatinine (P = 0.012), GFP (P <0.001), and urine output (P = 0.003). Over the full 5-hour period there was a statistically signifi cant diff erence in the potassium measurement between the Pn and the Oc model (P = 0.01). The other parameters did not show a signifi cant diff erence: urea (P = 0.171), creatinine (P = 0.074), GFP (P = 0.141), and urine output (P = 0.242).i Methods A prospective single-center cohort study started in May 2012 in an academic cardiac surgery ICU. Blood and urine were collected preoperatively (baseline), at ICU admission and at several time points until 48 hours after ICU admission. Patients of age <18 years were excluded, as well as patients with AKI based on the KDIGO sCr and urine output criteria, patients with chronic kidney disease stage 5 and/or with recent renal transplantation (≤3 months before cardiac surgery). AKI was defi ned on KDIGO sCr criteria only. Estimated glomerular fi ltration rate based on hospital discharge creatinine may signifi cantly overestimate renal function and underestimate chronic kidney disease in survivors of critical illness l d ll l Baseline sCr was assessed as the sCr concentration measured before start of cardiac surgery. Urinary CHI3L1 was normalized to urinary creatinine. For both serum and urine CHI3L1, we measured changes from baseline. Data are expressed as median (interquartile range), and number (%). Conclusion As expected the IAH models resulted in signifi cantly worse renal function after 3 hours. This early renal dysfunction may be as a result of an early infl ammatory process that has been associated with the pathophysiology of acute kidney injury. Potassium was signifi cantly elevated in the Pn group as compared with the Oc group. Early changes in potassium levels with IAH may be a marker of early renal dysfunction and the usefulness of other renal biomarkers, such as NGAL, prompts further investigation. Results Data for 17 patients (nine males, eight females) were generated for interim analysis. Five patients (29.4%), of which four were females, developed AKI KDIGO class 1. AKI patients were older compared with non-AKI patients (82.0 years (77.5 to 83.5) vs. 66.5 (60.3 to 75.8); P = 0.002). The baseline sCr did not diff er signifi cantly between the AKI (1.12 mg/dl (0.80 to 1.15)) and non-AKI (0.90 mg/dl (0.86 to 1.01); P = 0.383) group. Baseline serum CHI3L1 was highly variable in both AKI and non-AKI patients. Moreover, no trend towards a higher serum CHI3L1 level in AKI versus non-AKI patients was observed either at baseline or during the study follow-up. In contrast, the increase in the urinary CHI3L1 to urinary creatinine ratio from baseline was higher in the AKI versus non-AKI group at time points 4 hours and 6 hours after admission to the ICU (2.00 ng/mg Cr (1.38 to 3.03) vs. 0.26 (0.05 to 0.88); P = 0.002, resp. 2.47 (1.09 to 7.26) vs. 0.23 (–0.18 to 0.74); P = 0.006) and, more importantly, occurred before the diagnosis of AKI by sCr (>16 hours after ICU admission). Use, type and duration of cardiopulmonary bypass are related to acute kidney injury occurrence and urinary NGAL concentrations T García Rodríguez San Miguel Construction of receiver operating characteristic curve showed 83% sensitivity and 81% specifi city for the cutoff RI 0.73. No other measured patient characteristic was found to be predictive of response to diuretic treatment. Conclusion Our results show that the RI could be used to guide diuretic treatment in nonhypovolemic, nonhypotensive septic patients. Further studies are needed to confi rm those preliminary results. Results One hundred and eighty-one patients (66.1%) were men; mean age was 68.2 ± 12.2 years. ICU and hospital stays were 6.7 ± 8.1 and 15.7 ± 13.9 days, respectively. Twenty-eight-day mortality was 2.9%. Eighty-six patients (31.4%) were diagnosed with AKI within 48 hours after surgery. In total, 219 patients required CPB (195 SCPB, 24 MiniCPB) and 55 did not (no-CPB). Seven no-CPB patients (12.7%) developed AKI and their median uNGAL POST was 330 (42.6 to 489.9) μg/l compared with 13.6 (6.9 to 38.3) μg/l in the 48 patients without AKI (P <0.0001). Of the 195 patients undergoing SCPB, 76 (38.9%) developed AKI and 119 did not; POST uNGAL was 204 (34.8 to 575.7) μg/l and 44.5 (13.2 to 175.8) μg/l (P <0.0001), respectively. In the 24 patients under MiniCPB, POST uNGAL was 113 (58.8 to 211.8) μg/l in those (three patients, 12.5%) developing AKI and 19.1 (9.2 to 41.8) μg/l in those without AKI (P = 0.01). Aortic clamp time (r = 0.31, P <0.0001) and cardiopulmonary bypass time (r = 0.30, P <0.0001) correlated with POST uNGAL concentrations. Reference 1. Malbrain ML, et al.: Incidence and prognosis of intraabdominal hypertension in a mixed population of critically ill patients: a multiple- center epidemiological study. Crit Care Med 2005, 33:315-322. 1. Malbrain ML, et al.: Incidence and prognosis of intraabdominal hypertension in a mixed population of critically ill patients: a multiple- center epidemiological study. Crit Care Med 2005, 33:315-322. Renal resistive index can predict response to furosemide in septic patients with oliguria I Gornik, A Godan, V Gasparovic y p g , Critical Care 2013, 17(Suppl 2):P420 (doi: 10.1186/cc12358) Introduction Oliguria is common in septic patients and is frequently therapeutically addressed with loop diuretics; that is, furosemide. Diuretic treatment in shock and hypovolemia is not rational, but can be tried in oliguric patients with normovolemia or hypervolemia and without hypotension. In such patients it still does not always increase dieresis and can also be harmful. The resistive index is a measure of pulsatile blood fl ow that refl ects the resistance to blood fl ow caused by the microvascular bed distal to the site of measurement. It can refl ect functional status of the tissue distal to the point of measurement. We investigated whether measuring the renal resistive index (RI) could be helpful in determining which patients will respond to furosemide treatment. Conclusion This interim analysis of an ongoing pilot study, with 300 ICU patients in three cohorts (general ICU, pediatric and adult cardiac surgery ICU), shows promising results for urinary CHI3L1 (ng/mg Cr) as an early diagnostic biomarker for AKI in adult ICU patients that undergo elective cardiac surgery. P422 P421 P421 Urinary chitinase 3-like 1, a novel biomarker for acute kidney injury in adult cardiac intensive care patients: a pilot study J De Loor1, E Hoste2, I Herck2, K Francois2, L De Crop2, C Clauwaert2, K Demeyere1, E Meyer1 1Faculteit diergeneeskunde, Merelbeke, Belgium; 2UZ Gent, Belgium Critical Care 2013, 17(Suppl 2):P421 (doi: 10.1186/cc12359) P423 Symmetrical dimethylarginine is a more sensitive biomarker of renal dysfunction than creatinine t e apy ( ). Stat st cs: OC UC. S SS 0. Results There were 91 patients (34 female, 57 male), 65 ± 14 years, APACHE II score 20.6 ± 8.3, SOFA score 9.3 ± 4.2. Etiology: 10 ARDS, 36 sepsis, 22 cirrhosis, six pancreatitis, eight cardiogenic shock, two CNS-aff ections, seven various. Mortality was best predicted by SOFA (ROC-AUC: 0.747; P <0.001) and APACHE II (AUC: 0.705; P <0.001). Among admission parameters of renal function only NGAL signifi cantly predicted mortality (AUC: 0.647; P = 0.023), whereas creatinine (P = 0.290) and BUN (P = 0.067) were not predictive. ROC-AUCs for NGAL further increased after 12 hours (0.659), 24 hours (0.691; P = 0.01) and 48 hours (0.728; P = 0.004). NGAL on admission also predicted requirement of RRT during the ICU stay (AUC: 0.678; P = 0.015), which was also predicted by creatinine (AUC 0.688; P = 0.010) and BUN (AUC: 0.649; P = 0.043). Among baseline TPTD parameters, only the pulmonary vascular permeability index (PVPI) signifi cantly predicted mortality (AUC: 0.700; P = 0.007). The EVLWI (AUC 0.628; P = 0.083) slightly failed signifi cance. Cardiac index, global end-diastolic volume index and heart rate were not predictive. Furthermore, PVPI was the only TPTD-derived parameter to predict requirement of RRT (AUC: 0.693; P = 0.009).i Symmetrical dimethylarginine is a more sensitive biomarker of renal dysfunction than creatinine JJ Dixon1, K Lane1, RN Dalton2, IA MacPhee1, BJ Philips1 1Acute Kidney Injury Research Group, St George’s Hospital and University of London, UK; 2King’s College, University of London, UK Critical Care 2013, 17(Suppl 2):P423 (doi: 10.1186/cc12361) Symmetrical dimethylarginine is a more sensitive biomarker of renal dysfunction than creatinine y JJ Dixon1, K Lane1, RN Dalton2, IA MacPhee1, BJ Philips1 1Acute Kidney Injury Research Group, St George’s Hospital and University of London, UK; 2King’s College, University of London, UK Critical Care 2013, 17(Suppl 2):P423 (doi: 10.1186/cc12361) y JJ Dixon1, K Lane1, RN Dalton2, IA MacPhee1, BJ Philips1 1Acute Kidney Injury Research Group, St George’s Hospital and University of London, UK; 2King’s College, University of London, UK Critical Care 2013, 17(Suppl 2):P423 (doi: 10.1186/cc12361) Introduction Current defi nitions of acute kidney injury (AKI) are based upon changes in serum creatinine (SCr) concentration and urine output: both have limitations in patients with AKI. Diagnosis may be delayed if using these criteria alone. Assessment of neutrophil gelatinase-associated lipocalin as a biomarker of acute kidney injury in acute heart failure patients Results Mean GFR measured by single injection was 78.7 ± 28.5 ml/ minute/1.73 m2, and 78.9 ± 28.6 ml/minute/1.73 m2 when measured by CIVLDI (P = 0.82). Mean SDMA concentration was similar on both occasions (641 ± 38 vs. 623 ± 22 nmol/l; P = 0.68). Tubular reabsorption of SDMA was lower in subjects with GFR <60 ml/minute/1.73 m2 (12 ± 8% vs. 18 ± 8% in subjects with GFR >60 ml/minute/1.73 m2; P = 0.0002), and tubular secretion of SCR was higher in subjects with GFR <60 ml/minute/1.73 m2 (29% vs. 23%; P <0.0001). The third-order polynomial equation (r = 0.93) estimated GFR better than quadratic (r = 0.92) and linear (r = 0.87) equations. Bland–Altman comparison revealed no bias when the third-order equation was used (precision: ±20 ml/minute/1.73 m2). p Critical Care 2013, 17(Suppl 2):P425 (doi: 10.1186/cc12363) Introduction Growing evidence hints that bidirectional interaction between heart failure and kidney disease and renal insuffi ciency is a strong predictor of mortality as well as causally linked to the progression of heart failure. Neutrophil gelatinase-associated lipocalin (NGAL) is an early predictor of acute kidney injury (AKI). We evaluated the impact of NGAL on morbidity and mortality in patients with acute heart failure. Methods Seventy-six patients presenting with symptoms consistent with acute heart failure (median age 72 years, 56% male) were enrolled. Plasma NGAL levels were measured by an ELISA at admission and compared with the glomerular fi ltration rate (eGFR) and B-natriuretic peptide (BNP) levels. The primary outcome was AKI development defi ned by RIFLE criteria (fall in GFR >25% or creatinine rise ≥50% from baseline, or a fall in urine output <0.5 ml/kg/hour) and secondary outcomes were duration of hospital stay and in-hospital mortality. Conclusion SDMA appears to be an accurate and precise estimate of GFR and a more sensitive biomarker of renal dysfunction than SCr. We predict SDMA will perform better than SCr as a biomarker of AKI. This forms the basis of a future study. P425 Assessment of neutrophil gelatinase-associated lipocalin as a biomarker of acute kidney injury in acute heart failure patients H Michalopoulou, H Michalopoulou, P Stamatis, J Xenogiannis, D Stamatis Metaxa Hospital, Athens, Greece Critical Care 2013, 17(Suppl 2):P425 (doi: 10.1186/cc12363) Urinary chitinase 3-like 1, a novel biomarker for acute kidney injury in adult cardiac intensive care patients: a pilot study J D L 1 E H 2 I H k2 K F 2 L D C 2 C Cl 2 Introduction As a proof of concept, the potential added value of chitinase 3-like 1 (CHI3L1) as a more early and specifi c diagnostic parameter for acute kidney injury (AKI) was investigated in adult ICU patients that underwent elective cardiac surgery. Introduction As a proof of concept, the potential added value of chitinase 3-like 1 (CHI3L1) as a more early and specifi c diagnostic parameter for acute kidney injury (AKI) was investigated in adult ICU patients that underwent elective cardiac surgery. S158 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Incidence of AKI in patients without CPB or with MiniCPB was identical and signifi cantly lower (P <0.0001) than in SCPB patients. Conclusion The incidence of AKI was higher in patients undergoing SCPB. Aortic clamp and cardiopulmonary bypass time correlated with the POST uNGAL concentration. The higher values found in patients without AKI undergoing SCPB suggest that a subclinical AKI, only detectable by uNGAL, could exist in this group. with mortality. Regarding a delayed increase in creatinine, other biomarkers of AKI including neutrophil gelatinase-associated lipocalin (NGAL) have been suggested. It was the aim of our study to compare the predictive capabilities of NGAL and transpulmonary thermodilution (TPTD)-derived parameters within 48 hours after ICU admission. Incidence of AKI in patients without CPB or with MiniCPB was identical and signifi cantly lower (P <0.0001) than in SCPB patients. i Conclusion The incidence of AKI was higher in patients undergoing SCPB. Aortic clamp and cardiopulmonary bypass time correlated with the POST uNGAL concentration. The higher values found in patients without AKI undergoing SCPB suggest that a subclinical AKI, only detectable by uNGAL, could exist in this group. p Methods Urinary NGAL, serum creatinine and BUN as well as TPTD- derived parameters were measured 0 hours, 12 hours, 24 hours and 48 hours after ICU admission. Primary endpoint: prediction of ICU mortality. Secondary endpoint: requirement of renal replacement therapy (RRT). Statistics: ROC-AUC. IBM SPSS 20. P423 Symmetrical dimethylarginine is a more sensitive biomarker of renal dysfunction than creatinine We have previously validated a method of measuring the glomerular fi ltration rate (GFR) intended for use in patients with AKI (a continuous infusion of very low dose iohexol; CIVLDI). In this study we compare the performance of symmetrical dimethylarginine (SDMA) against SCr and CIVILDI. SDMA is the structural isomer of the endogenous nitric oxide inhibitor asymmetrical dimethylarginine. SDMA increases in parallel with SCr. Despite this, no formula exists to estimate GFR from SDMA concentration. Tubular secretion of SCr may be as high as 40%. SDMA may be a more sensitive biomarker of renal dysfunction than SCr; however, the tubular reabsorption of SDMA is unknown. The aims were to compare the performance of SDMA against SCr and accurately measured GFR, and to derive a formula to estimate GFR from SDMA concentration. Conclusion Urinary NGAL on admission signifi cantly predicts mortality, whereas creatinine and BUN were not predictive. The predictive capabilities of NGAL further increased after 12 hours, 24 hours and 48 hours, with the ROC-AUC of NGAL 48 hours exceeding the AUC of the APACHE II score. NGAL on admission also predicts requirement of RRT. Among TPTD-derived parameters, PVPI on admission is signifi cantly associated with mortality and requirement of RRT. Methods Seventeen volunteers had GFR measured twice via measuring the clearance of a 5 ml i.v. bolus of iohexol, or CIVLDI. Serum and urine iohexol, Cr and SDMA were measured by high-performance liquid chromatography/tandem mass spectrometry (HPLC-ms/ms) at 10 time points. Fractional excretion of SDMA and SCr were calculated using iohexol as the denominator. SDMA was plotted against measured GFR, and estimated GFR equations were derived from linear, quadratic and third-order polynomial plots. P424 Prognostic value of neutrophil gelatinase-associated lipocalin and transpulmonary thermodilution-derived parameters within 48 hours after admission W Huber, J Rauch, B Saugel, S Mair, M Messer, T Lahmer, C Schultheiss, P Luppa, RM Schmid Klinikum Rechts der Isar, Technical University of Munich, II. Medizinische Klinik, Munich, Germany Critical Care 2013, 17(Suppl 2):P424 (doi: 10.1186/cc12362) Prognostic value of neutrophil gelatinase-associated lipocalin and transpulmonary thermodilution-derived parameters within 48 hours after admission W Huber, J Rauch, B Saugel, S Mair, M Messer, T Lahmer, C Schultheiss, P Luppa, RM Schmid Klinikum Rechts der Isar, Technical University of Munich, II. Medizinische Klinik, Munich, Germany Critical Care 2013, 17(Suppl 2):P424 (doi: 10.1186/cc12362) Results AKI developed in 11 patients (14.4%). The subgroup that during hospitalization developed renal dysfunction had increased NGAL values with respect to patients with preserved renal function (146 ± 42 vs. 84 ± 24 ng/ml, P <0.05). NGAL signifi cantly associated with admission eGFR (r = 0.72, P <0.01) and BNP levels (r = 0.80, P <0.01). For prediction of AKI, NGAL >140 ng/ml has been related to renal insuffi ciency with sensitivity of 83% and specifi city of 74% with area under the receiver operator curve of 0.70 (0.58 to 0.82). In the follow-up setting, NGAL was associated with recurrent hospital admission for heart failure (HR: 3.1, CI: 1.5 to 6.2) and cardiac death (HR: 2.2, CI: 1.8 to 4.5). Neutrophil gelatinase-associated lipocalin predicts postoperative fl uid overload after cardiac surgery Survivors of acute kidney injury requiring renal replacement therapy rarely receive follow-up: identifi cation of an unmet need CJ Kirwan, R Taylor, JR Prowle Survivors of acute kidney injury requiring renal replacement therapy rarely receive follow-up: identifi cation of an unmet need CJ Kirwan, R Taylor, JR Prowle The Royal London Hospital, Barts Health NHS Trust, London, UK Critical Care 2013, 17(Suppl 2):P427 (doi: 10.1186/cc12365) l g y M Haase1, P Devarajan2, P Michael3, R Bellomo4, A Haase-Fielitz1 1Otto von-Guericke University, Magdeburg, Germany; 2Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA; 3German Heart Center, Berlin, Germany; 4Austin Hospital, Melbourne, Australia Critical Care 2013, 17(Suppl 2):P426 (doi: 10.1186/cc12364) Introduction Acute kidney injury (AKI) occurs in more than 50% of ICU admissions, requiring renal replacement therapy (RRT) in around 10% of cases. There is now increasing evidence that AKI is a risk factor for the development and progression of chronic kidney disease (CKD); however, when AKI occurs as a complication of critical illness appropriate follow-up may be neglected. Accordingly, we reviewed the follow-up of renal function in all patients who received RRT on our ICU and survived to hospital discharge. Introduction Neutrophil gelatinase-associated lipocalin (NGAL), measured early after cardiac surgery, has been demonstrated to predict postoperative acute kidney injury (AKI). Fluid overload potentially masks a subsequent acute renal function loss through dilution of serum creatinine and maintenance of urine output just above AKI- defi ning criteria. g Methods A retrospective audit of patients who received RRT in a central London adult critical care unit during 2011. i Methods We investigated the early postoperative value of NGAL versus that of simultaneously measured serum creatinine to predict subsequent fl uid overload. We studied 100 adult cardiac surgery patients in the control arm of a RCT (NCT00672334). Severe postoperative fl uid overload was defi ned as positive fl uid balance >10% of preoperative body weight within 48 hours after surgery.l Results Of 921 patients admitted, 203 received RRT with 109 surviving to hospital discharge. We excluded 52 patients who had end-stage renal disease, renal transplant or known glomerular disease. Of the remaining 57 AKI patients, median age was 60 (range: 18 to 77) and 37 (65%) were male. Median discharge creatinine was 74.5 μmol/l (27 to 662). Forty-two (74%) were off ered follow-up, but in only six cases (11%) was this to nephrology services. Neutrophil gelatinase-associated lipocalin predicts postoperative fl uid overload after cardiac surgery Twenty-eight attended follow-up (fi ve to nephrology) at a median time of 6 weeks; however, creatinine was measured at in only 14 and in six of these it had risen (by median 16.5 μmol/l). In addition, 14 patients had creatinine measured 3 to 6 months post discharge and in eight it had risen (by median 31.5 μmol/l). y y Results Severe postoperative fl uid overload was present in 5% of patients with a mean positive fl uid balance of 15.8 ± 9.5 l. At ICU admission, urine NGAL predicted severe fl uid overload (AUC-ROC 0.82 (95% CI = 0.70 to 0.94)) (Figure 1) and mortality (AUC 0.88 (0.78 to 0.97)). Serum creatinine measured at the same time did not predict severe fl uid overload (AUC 0.52 (0.26 to 0.79)) or mortality (AUC 0.61 (0.16 to 0.99)).l Conclusion Early NGAL-guided adjustments to fl uid management may reduce organ edema after cardiac surgery. Findings should be validated in larger cohorts. Conclusion Follow-up of patients who received RRT for AKI in the ICU was poor and they were rarely referred to nephrologists. Where renal function was measured after discharge, there was evidence of progressive renal dysfunction; however, renal function was often not assessed. We propose an algorithm for clinicians to guide follow-up. See Figure 1. Figure 1 (abstract P426). NGAL at ICU admission predicts fl uid overload of >10% of body weight. Figure 1 (abstract P426). NGAL at ICU admission predicts fl uid overload of >10% of body weight. P427 P426 Neutrophil gelatinase-associated lipocalin predicts postoperative fl uid overload after cardiac surgery M Haase1, P Devarajan2, P Michael3, R Bellomo4, A Haase-Fielitz1 1Otto von-Guericke University, Magdeburg, Germany; 2Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA; 3German Heart Center, Berlin, Germany; 4Austin Hospital, Melbourne, Australia Critical Care 2013, 17(Suppl 2):P426 (doi: 10.1186/cc12364) Prognostic value of neutrophil gelatinase-associated lipocalin and transpulmonary thermodilution-derived parameters within 48 hours after admission W Huber, J Rauch, B Saugel, S Mair, M Messer, T Lahmer, C Schultheiss, P Luppa, RM Schmid Klinikum Rechts der Isar, Technical University of Munich, II. Medizinische Klinik, Munich, Germany Critical Care 2013, 17(Suppl 2):P424 (doi: 10.1186/cc12362) Introduction Outcome of ICU patients is predicted by multifactorial scores such as APACHE II and SOFA. Furthermore, markers of single organ failure such as the extravascular lung water index (EVLWI) and several biomarkers of acute kidney injury (AKI) have been associated Conclusion NGAL is emerging as a promising biomarker of AKI in the setting of acute heart failure and elevated NGAL levels indicate a poor prognosis in this population regarding morbidity and mortality. S159 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 P426 Eff ect of continuous high-volume hemofi ltration on patients with acute respiratory distress syndrome Eff ect of continuous high-volume hemofi ltration on patients with acute respiratory distress syndrome A Kuzovlev1, E Tishkov2, O Bukaev2 1V.A. Negovsky Scientifi c Research Insitute of General Reanimatology RAMS, Moscow, Russia; 2A.I. Evdokimov Moscow State University of Medicine and Dentistry, Moscow, Russia Critical Care 2013, 17(Suppl 2):P431 (doi: 10.1186/cc12369) Eff ect of continuous high-volume hemofi ltration on patients with acute respiratory distress syndrome A Kuzovlev1, E Tishkov2, O Bukaev2 1V.A. Negovsky Scientifi c Research Insitute of General Reanimatology RAMS, Moscow, Russia; 2A.I. Evdokimov Moscow State University of Medicine and Dentistry, Moscow, Russia Critical Care 2013, 17(Suppl 2):P431 (doi: 10.1186/cc12369) A Kuzovlev1, E Tishkov2, O Bukaev2i , , 1V.A. Negovsky Scientifi c Research Insitute of General Reanimatology RAMS, Moscow, Russia; 2A.I. Evdokimov Moscow State University of Medicine and Dentistry, Moscow, Russia , , 1V.A. Negovsky Scientifi c Research Insitute of General Reanimatology RAMS, Moscow, Russia; 2A.I. Evdokimov Moscow State University of Medicine and Dentistry, Moscow, Russia Critical Care 2013, 17(Suppl 2):P431 (doi: 10.1186/cc12369) Introduction The aim of this study was to investigate the eff ects of intermittent venovenous high-volume hemofi ltration (IHVHF) on extravascular lung water, respiratory function and cytokine removal in patients with acute respiratory distress syndrome (ARDS) associated with postoperative multiple organ dysfunction syndrome in severe abdominal infection patients. Results The average quantity of the substitutive renal therapy procedures in the CG was 13.4 ± 0.7, in the IG it was 8 ± 0.6 (P <0.05). The recuperation of the renal excretory functions was on 19 ± 1 day in 12 patients of the CG, and on 11 ± 1.3 day in the IG, from the moment of substitutive renal therapy start (P <0.05). Lethality in the CG was 43% (nine patients), and in the IG it was 29% (six patients, P <0.05). The duration of the MLV in the CG and IG was 21 ± 1.2 days and 16 ± 1.2, respectively (P <0.05). In the IG the duration of the ICU was lower by 23%, hospitality duration was lower by 17% (P <0.05).fi Methods Thirty patients with severe abdominal infection and ARDS were randomized into the IHVHF group (n = 15) and control group (n = 15). All patients were mechanically ventilated. The IHVHF technique with a rate of 100 to 120 ml/kg/hour during 6 to 8 hours was used. Can treatment with the molecular adsorbent recirculation system be the solution for type-1 hepatorenal syndrome? L Lavayssiere University Hospital, Toulouse, France Critical Care 2013, 17(Suppl 2):P430 (doi: 10.1186/cc12368) Can treatment with the molecular adsorbent recirculation system be the solution for type-1 hepatorenal syndrome? L Lavayssiere University Hospital, Toulouse, France Critical Care 2013, 17(Suppl 2):P430 (doi: 10.1186/cc12368) University Hospital, Toulouse, France Introduction The aim of this retrospective study was to determine whether the molecular adsorbent recirculation system (MARS) can improve renal function in HSR1 patients. Methods Thirty-two patients with chronic liver disease and HRS1 were treated by MARS sessions that were performed every other day. The endpoint was renal function improvement by 28 days after diagnosis of HRS1 that was defi ned as a serum creatinine level <133 μmol/l. Partial renal recovery was defi ned as a 10% decrease in baseline serum creatinine level. y Conclusion Individuals who develop dialysis-dependent AKI in the ICU setting in general terms either die or recover. Sepsis is the most common association with death. The need for mechanical ventilation and inotropic therapy are both associated with increased incidence of death. Results The mean number of MARS sessions required by each patient was 3.5 ± 1.5. The median time between admission and the start of MARS therapy was 3 (0 to 15) days. Of the total patients, 13 (40%) had improved renal function. Among these, nine (28%) had complete renal recovery. Among the patients that survived, only 40% (6/15) had improved renal function. When MARS was started, 53% of patients had failed renal failure according to RIFLE criteria. Seven patients received a liver transplant after diagnosis of HRS. Of these, four had complete or partial recovery after transplantation (57%) versus nine of the 25 patients who did not undergo liver transplantation (36%), P = NS. The 28-day survival rate was 47%. P429fi P429 Effi ciency of substitutive renal therapy in the complex intensive care of multiple organ failure in patients with polytrauma S Kravtsov, A Shatalin, V Agadzhanyan, D Skopintsev Federal State Budgetary Medical Prophylactic Institution ‘Scientifi c Clinical Center of the Miners’ Health Protection’, Leninsk-Kuznetsky, Russia Critical Care 2013, 17(Suppl 2):P429 (doi: 10.1186/cc12367) Introduction This study was to evaluate the effi ciency of the early start of intermittent substitutive renal therapy in patients with polytrauma complicated by multiple organ failure syndrome. 1. Nadim MK, Genyk YS, Tokin C, et al.: Impact of etiology of acute kidney injury on outcomes following liver transplantation: acute tubular necrosis versus hepatorenal syndrome. Liver Transpl 2012, 18:539-548. 2. Cholongitas E, Senzolo M, Patch D, Shaw S, O’Beirne J, Burroughs AK: Cirrhotics admitted to intensive care unit: the impact of acute renal failure on mortality. Eur J Gastroenterol Hepatol 2009, 21:744-750. 1. Nadim MK, Genyk YS, Tokin C, et al.: Impact of etiology of acute kidney injury on outcomes following liver transplantation: acute tubular necrosis versus hepatorenal syndrome. Liver Transpl 2012, 18:539-548. p y p g y Methods Forty-two patients with polytrauma complicated by multiple organ failure syndrome were included in the study. The age of the patients was from 20 to 60 years (38.3 ± 1.6 years average). All patients were divided into two equal groups. In the control group (CG) the criteria for the start of the substitutive renal therapy were: hyperkalemia ≥6 mmol/l, plasma creatinine ≥280 μmol/l, diuresis ≤20 ml/hour. In the investigation group (IG) there were subtests to carry out the substitutive renal therapy, allowing one to start it in the earlier period of the multiple organ failure progression. These are increase of Na+ >150 mmol/l, osmolarity >300 mOsm/l, elevation of the plasma toxicity according to the average molecule concentration ≥1.0, diuresis decrease ≤40 ml/hour. These were examined: lethality, quantity of the substitutive renal therapy procedures, mechanical lung ventilation duration (MLV), intensive care and hospital duration. The substitutive renal therapy was carried out by AK-200-Ultra apparatus (Gambro, Sweden). The statistical analysis was realized using Statistica 6.1 and the Mann–Whitney U test. 2. Cholongitas E, Senzolo M, Patch D, Shaw S, O’Beirne J, Burroughs AK: Cirrhotics admitted to intensive care unit: the impact of acute renal failure on mortality. Eur J Gastroenterol Hepatol 2009, 21:744-750. 2. References 1. Metnitz PG, et al.: Eff ect of acute renal failure requiring renal replacement therapy on outcome in critically ill patients. Crit Care Med 2002, 30:2051-2058. 2. Uchino S, et al.: Acute renal failure in critically ill patients: a multinational, multicenter study. JAMA 2005, 294:813-818. 2. Uchino S, et al.: Acute renal failure in critically ill patients: a multinational, multicenter study. JAMA 2005, 294:813-818. y Conclusion MARS therapy improved renal function in only very few patients with HRS1. Even if liver transplantation is the best option, MARS could be used as a bridge until transplantation. Some authors argue that prolonged duration of dialysis (>1 month) before transplantation may result in chronic renal failure probably because there are two etiologies of AKI in end-stage liver disease: acute tubular necrosis (ATN) and real functional renal failure. ATN before liver transplantation has been identifi ed as a risk factor for patients’ mortality and chronic renal failure at 1 and 5 years post liver transplantation. Further prospective controlled studies including large number of patients are required. References P429fi Cholongitas E, Senzolo M, Patch D, Shaw S, O’Beirne J, Burroughs AK: Cirrhotics admitted to intensive care unit: the impact of acute renal failure on mortality. Eur J Gastroenterol Hepatol 2009, 21:744-750. Aetiology and outcomes for dialysis-dependent acute kidney injury patients on the ICU M Hameed1, P Carmichael2 1Salford Royal NHS Foundation Trust, Salford, UK; 2The Royal Wolverhampton Hopsitals NHS Trust, Wolverhampton, UK Critical Care 2013, 17(Suppl 2):P428 (doi: 10.1186/cc12366) Introduction AKI is a common occurrence in sick hospitalized patients, in particular those admitted to intensive care. Published data suggest that 4 to 5% of all critically ill patients develop severe AKI and require initiation of renal replacement therapy (RRT) [1,2]. Such patients have high mortality rates often exceeding 60% [2]. We aimed to review the outcomes of patients admitted to the ICU and required renal replacement therapy for AKI. We examined whether aetiology of AKI, comorbidity burden, hospital length of stay and treatment in ICU had any signifi cant association with survival in the study cohort.i i Methods During 2009, 56 patients were identifi ed to have received RRT with AKI who were admitted to the ICU at the Royal Wolverhampton Hospitals NHS Trust. Computerised and paper-based case records were examined for these patients to collect the data. AKIN classifi cation was used to classify the severity of AKI. Figure 1 (abstract P426). NGAL at ICU admission predicts fl uid overload of >10% of body weight. Figure 1 (abstract P426). NGAL at ICU admission predicts fl uid overload of >10% of body weight. Figure 1 (abstract P427). A suggested follow-up pathway for AKI survivors. S160 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Results Median age at admission was 66 years (27 to 85) with 29 males and 27 females. Thirty-one (55.4%) patients had sepsis and 20 (35.7%) patients had ATN as the main cause of AKI. Thirty-two patients (57%) had three organ failures at the time of commencement of RRT. Forty- six patients (82.1%) received haemofi ltration only. Thirty-two (57%) patients died, with more than 80% of these occurring in the ITU. There was no signifi cant diff erence in survival when compared with duration of haemofi ltration, length of stay, number of organs failed and number of comorbidities. However, signifi cantly more patients that died had AKI due to sepsis (P = 0.003) or if they received mechanical ventilation (P = 0.48) or inotropes (0.04). Of the 27 patients who survived until discharge from hospital, 18 (66.7%) had normal renal function, eight (29.6%) had AKIN stage I and only one patient required maintenance haemodialysis. Dialyzer clearance of myoglobin with middle molecule fi lter and low blood fl ow CVVHD in patients with rhabdomyolysis-associated acute kidney injury y j y I Leppanen1, T Ahonen1, J Tenhunen2 1Tampere University Hospital, Tampere, Finland; 2Uppsala University Hospital, Uppsala, Sweden Critical Care 2013, 17(Suppl 2):P432 (doi: 10.1186/cc12370) y j y I Leppanen1, T Ahonen1, J Tenhunen2 1Tampere University Hospital, Tampere, Finland; 2Uppsala University Hospital, Uppsala, Sweden Critical Care 2013, 17(Suppl 2):P432 (doi: 10.1186/cc12370) Figure 1 (abstract P433). Daily fl uid balance and 95% CIs among survivors and nonsurvivors. Introduction Rhabdomyolysis-associated acute kidney injury can be treated with various modes of renal replacement therapy (RRT) [1,2]. We have used continuous venoveno hemodialysis (CVVHD) with middle molecule fi lter and low extracorporeal blood fl ow (100 to 180 ml/minute) to avoid albumin loss and hemodynamic instability.f y y Methods We treated nine patients suff ering from rhabdomyolysis and acute kidney injury with various causes. We used a CVVHD mode with low (100 to 180 ml/minute) blood fl ow and a middle molecule fi lter (Ultrafl ux EMiC2; 1.8 m2 surface area, polysulfone membrane, cutoff at 40 kDa, blood fi lling volume 130 ml, blood fl ow range 100 to 350 ml/ minute, maximum dialysate fl ow 1,000 ml/minute). Myoglobin and albumin concentrations were measured from prefi lter and postfi lter samples and dialyzer clearances were calculated (CLdial = blood fl ow×Cpre – Cpost/Cpre + UF×Cpre/Cpost). Measurements were taken at 0, 15 minutes, 30 minutes, 4 hours, 12 hours, 24 hours, 36 hours and 48 hours from the start of the CRRT or until CRRT was no longer needed. Results The baseline and prefi lter plasma albumin concentrations varied from 8 to 29 g/l. Postfi lter and post-treatment albumin concentrations remained comparable. Prefi lter concentrations of myoglobin (17.8 kDa) varied from 96,109 to 747 μg/l and the dialyzer clearance of myoglobin from 50.6 to 0 ml/minute. The mean dialyzer clearance was 23.1 ml/ minute and the median clearance 22.2 ml/minute. Maximal changes between corresponding prefi lter and postfi lter samples were: absolute concentration change 27,091 μg/l, percentage 34%, dialyzer clearance 50.6 ml/minute. Clearances were achieved with low extracorporeal blood fl ow between 100 and 180 ml/minute, most commonly 120 ml/ minute. Ultrafi ltration (UF) was used only at fi ve of the 56 time points calculated, because of hemodynamic instability. All patients required either vasopressor or vasopressor and inotrope support. P432 P432 Dialyzer clearance of myoglobin with middle molecule fi lter and low blood fl ow CVVHD in patients with rhabdomyolysis-associated acute kidney injury I Leppanen1, T Ahonen1, J Tenhunen2 1Tampere University Hospital, Tampere, Finland; 2Uppsala University Hospital, Uppsala, Sweden Critical Care 2013, 17(Suppl 2):P432 (doi: 10.1186/cc12370) Dialyzer clearance of myoglobin with middle molecule fi lter and low blood fl ow CVVHD in patients with rhabdomyolysis-associated acute kidney injury The highest dialyzer clearance of myoglobin (50.6 ml/minute) was measured at the prefi lter myoglobin of 29,266 μg/l, blood fl ow 180 ml/ml and no UF. Figure 1 (abstract P433). Daily fl uid balance and 95% CIs among survivors and nonsurvivors. Conclusion Among critically ill patients who are receiving CRRT, daily fl uid balance is an independent predictor of mortality, even after adjusting for the daily severity of illness and comorbidities. References Conclusion Among critically ill patients who are receiving CRRT, daily fl uid balance is an independent predictor of mortality, even after adjusting for the daily severity of illness and comorbidities. References 1. Bagshaw SM, et al.: Crit Care 2008, 12:169. 2. Bellomo R, et al.: N Engl J Med 2009, 361:1627. 3. Bellomo R, et al.: Crit Care Med 2012, 40:1753-1760. Eff ect of continuous high-volume hemofi ltration on patients with acute respiratory distress syndrome The PiCCOplus system was used to monitor cardiac output (CO), extravascular lung water index (EVLWI) and intrathoracic blood volume index (ITBVI). Arterial partial pressure of oxygen (PaO2), arterial partial pressure of carbon dioxide (PaCO2), oxygenation index (PaO2/FiO2) and dynamic lung compliance (Cdyn), oxygen delivery and oxygen consumption were measured. Serum concentrations of TNFα, IL-6, IL-8 Conclusion The effi ciency of the substitutive renal therapy depends directly on the hydroelectrolytic and metabolic changes and toxicosis degrees in the polytrauma complicated by multiple organ failure syndrome. The early start of the dialysis methods treatment allows one to achieve the earlier recuperation of the renal functions and to decrease the lethality level by 14%. S161 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 P433l and lactate were measured by ELISA method. The APACHE II scores before and after IHVHF therapy were detected.i Daily fl uid balance predicts hospital mortality in critically ill patients receiving continuous renal replacement therapy M Kashiouris, A Akhoundi, S Chaudhary, V Velagapudi, A Goldberg, K Kashani Mayo Clinic, Rochester, MN, USA Critical Care 2013, 17(Suppl 2):P433 (doi: 10.1186/cc12371) Results All indexes in the control group did not show any signifi cant improvement before and after treatment (P <0.05). There was a signifi cant decrease of serum concentrations of TNFα, IL-6, IL-8 and lactate, increase of CO and decrease of EVLWI 24 hours after IHVHF (P <0.05). The indexes of oxygen delivery, consumption, PaO2, and Cdyn improved signifi cantly, PaO2/FiO2 increased markedly compared with those before IHVHF and the control group. Signifi cant diff erences were detected in all of the hemodynamics and oxygen indexes (P <0.05). Introduction It has been suggested that fl uid balance is a biomarker in critically ill patients [1]. There is a paucity of randomized trials examining the eff ect of daily fl uid balance on outcomes in patients on continuous renal replacement therapy (CRRT). The RENAL trial did not fi nd mortality diff erence with higher CRRT dose [2], but did not investigate the eff ect of daily fl uid balance on patient outcomes. A post hoc analysis suggested survival benefi t in patients with negative fl uid balance [3]. In this study, we hypothesize that daily fl uid balance is an independent predictor of mortality in critically ill patients. y yg Conclusion IHVHF presents with a signifi cant benefi cial eff ect on respiratory function in patients with ARDS as a result of removal of cytokines, decrease in EVLWI, improvement of hemodynamic parameters, and correction of disturbances in oxygen balance. IHVHF adjuvant treatment for ARDS reduces pulmonary edema, improves PaO2/FiO2 and Cdyn and mechanical ventilation parameters. IHVHF may be a promising treatment for ARDS in postoperative multiple organ dysfunction syndrome in severe abdominal infection patients. Reference Methods We conducted a retrospective cohort study in eight ICUs of a tertiary academic center. We constructed a robust clustered linear regression model of daily fl uid balance and all-cause hospital mortality among 595 critically ill patients receiving CRRT. We adjusted the model for the Charlson comorbidity score, the daily SOFA scores in the fi rst week after initiation of CRRT as well the type of ICU. 1. 1. Honoré P, et al.: Anestesiol Reanimatol 2008, 6:4-11. 2. Sorrentino SA, et al.: Crit Care Med 2011, 39:184-186. 1. Albert C, et al.: Crit Care Resusc 2012, 14:159-162. P433l Honoré P, et al.: Anestesiol Reanimatol 2008, 6:4-11. Results After adjusting for the type of ICU and the daily severity of illness, patients who died had on average 779 ml higher daily fl uid balance compared with patients who survived (P <0.001, 95% CI = 385 to 1,173 ml, Figure 1). Severity of illness predicted daily fl uid accumulation; each additional point of the SOFA score predicted an additional 57 ml of extra daily fl uid (P = 0.002). P432 Fluid balance and renal outcomes in patients requiring renal replacement therapy in the ICU prefi lter myoglobin of 29,266 μg/l, blood fl ow 180 ml/ml and no UF. Conclusion In a rhabdomyolysis-associated kidney injury a middle molecule fi lter and a low blood fl ow CVVHD off er a safe and eff ective treatment choice for patients requiring vasopressor or vasopressor and inotrope for hemodynamic support. Introduction Fluid overload is associated with mortality in critically ill patients with acute kidney injury (AKI) [1-3]. We explored this relationship in patients with AKI who received renal replacement therapy (RRT) in the ICU to investigate the relationship between fl uid 1. Albert C, et al.: Crit Care Resusc 2012, 14:159-162. 2 Sorrentino SA et al : Crit Care Med 2011 39:184-186 Pharmacokinetics of gentamicin and vancomycin during continuous venovenous hemofi ltration in critically ill septic patients with acute kidney injury p Results A total of 522 patients were included (319 male, mean age 64 years); 264 (50.6%) died in hospital. Survivors and those who died were similar with respect to age, weight and incidence of heart failure, liver cirrhosis and primary renal diagnoses. Independent risk factors for hospital mortality were higher Sequential Organ Failure Assessment score at RRT initiation (odds ratio (OR) = 1.18, 95% confi dence interval = 1.11 to 1.25), higher Charlson comorbidity index (OR = 1.22, 1.09 to 1.37), lower baseline creatinine (OR = 0.984 per 10 μmol/l, 0.970 to 0.998), lower minimum mean arterial pressure (MAP) on day 1 (OR = 0.81 per 10 mmHg, 0.66 to 0.95) and more positive fl uid balance for the fi rst 7 days (OR 1.03 per 100 ml, 1.02 to 1.05). Of 172 hospital survivors, 57 (33.1%) were RRT dependent at discharge. Although univariable analysis suggested lower SOFA scores and higher baseline serum creatinine levels in those who remained RRT dependent, no factor was independently associated with RRT dependence at discharge in a multivariate model. y j y N Petejova1, J Duricova1, A Martinek1, J Zahalkova2 1University Hospital, Ostrava, Czech Republic; 2Hospital, Sternberk, Czech Republic Introduction Acute kidney injury (AKI) is a common complication of critical illness and sepsis [1]. Dosing of antibacterial agents in septic patients is complicated by altered pharmacokinetics due to both acute renal failure and critical illness [2]. Current dosing regimens for administration of gentamicin and vancomycin to septic patients with AKI on continuous venovenous hemofi ltration (CVVH) at a fi ltration rate of 45 ml/kg/hour are missing. Methods Seventeen septic patients with AKI treated with vancomycin and seven patients with gentamicin on CVVH were included. In the vancomycin group, patients received the fi rst dose of 1.0 g intravenously followed by 1.0 g/12 hours if not adjusted. In the gentamicin group, patients received a loading dose of 240 mg followed by a maintenance dose every 24 hours. The vancomycin maintenance dose was optimized to achieve AUC0–24/MIC ≥400 (Cmin >10 mg/l), gentamicin target was Cmax/MIC of 8 to 10. Maintenance doses were adjusted according to drug level simulation using a pharmacokinetic programme. g Conclusion In this cohort of patients with AKI requiring RRT, a more positive fl uid balance over 1 week and lower initial minimum MAP were associated with mortality. References 1. Uchino S, et al.: JAMA 2005, 294:813-818. 2. Roberts JA, et al.: Crit Care Med 2009, 37:840-851. Methods Esomeprazole 40 mg was administered intravenously once daily in nine intensive care patients and ranitidine 50 mg was administered intravenously three times daily in nine intensive care patients with acute renal failure undergoing CVVHDF who required stress ulcer prophylaxis. The concentration of esomeprazole and ranitidine in serum and ultradiafi ltrate was determined by high- performance liquid chromatography. The intragastric pH was obtained via intragastric pH-metry.i Pharmacokinetics and pharmacodynamics of esomeprazole and ranitidine during continuous venovenous hemodiafi ltration pi Conclusion CVVH at a fi ltration rate of 45 ml/kg/hour leads to high removal of both antibiotics. Due to rapid change in patient’s clinical status it was impossible to predict a fi xed dosage regimen. We recommend administration of unreduced loading dose and: blood sampling as early as 6 hours after fi rst vancomycin dose; blood sampling 30 to 60 minutes after gentamicin administration and before the next dose; and the maintenance dose should be based on drug- level monitoring. Introduction Esomeprazole, a proton pump inhibitor, and ranitidine, a histamine-2 receptor antagonist are frequently used in intensive care patients requiring stress ulcer prophylaxis. Continuous venovenous hemodiafi ltration (CVVHDF) is an important extracorporeal renal replacement therapy in critically ill patients suff ering from multiple organ failure. This study investigates the pharmacokinetics and pharmacodynamics of esomeprazole and ranitidine in anuric critically ill patients undergoing CVVHDF. p References S162 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 balance and intradialytic hypotension with mortality and recovery of renal function. balance and intradialytic hypotension with mortality and recovery of renal function. intragastric pH >5 and median intragastric pH decreased signifi cantly in the ranitidine group over time (P <0.05) but not in the esomeprazole group. Methods We conducted a retrospective cohort study among patients aged ≥16 years who had RRT initiated and continued for ≥2 days in a level 2 or 3 ICU at two academic centres, and had fl uid balance data available. Patients with end-stage kidney disease, within 1 year of a renal transplant or who had RRT initiated to treat a toxic ingestion were excluded. We used multivariable logistic regression to determine the relationship between mean daily fl uid balance over the fi rst 7 days following RRT initiation and the outcomes of mortality and RRT dependence in survivors. g Conclusion Although pharmacokinetics of esomeprazole and raniti- dine are only little infl uenced by CVVHDF, we found a signifi cant decrease of intragastric pH in patients treated with ranitidine. Esome- prazole may be superior for long-term stress ulcer prophylaxis in critically ill patients undergoing CVVHDF. P436 P436 P436 Pharmacokinetics of gentamicin and vancomycin during continuous venovenous hemofi ltration in critically ill septic patients with acute kidney injury N Petejova1, J Duricova1, A Martinek1, J Zahalkova2 1University Hospital, Ostrava, Czech Republic; 2Hospital, Sternberk, Czech Republic Critical Care 2013, 17(Suppl 2):P436 (doi: 10.1186/cc12374) P435 P435 Pharmacokinetics and pharmacodynamics of esomeprazole and ranitidine during continuous venovenous hemodiafi ltration V Fuhrmann1, P Schenk2, W Jaeger3, M Miksits3, R Kitzberger4, U Holzinger1, C Madl5 1Medical University Vienna, Austria; 2Landesklinikum Thermenregion Hochegg, Grimmenstein, Austria; 3University Vienna, Austria; 4Kaiser Franz Josef Krankenhaus, Vienna, Austria; 5Krankenanstalt Rudolfstiftung Wien, Vienna, Austria Critical Care 2013, 17(Suppl 2):P435 (doi: 10.1186/cc12373) Pharmacokinetics and pharmacodynamics of esomeprazole and ranitidine during continuous venovenous hemodiafi ltration V Fuhrmann1, P Schenk2, W Jaeger3, M Miksits3, R Kitzberger4, U Holzinger1, C Madl5 Pharmacokinetics of gentamicin and vancomycin during continuous venovenous hemofi ltration in critically ill septic patients with acute kidney injury Among survivors, a less positive fl uid balance was not associated with increased risk of RRT dependence at discharge, suggesting that conservative fl uid management does not signifi cantly attenuate renal recovery. gi y References References 1. Bouchard J, et al.: Kidney Int 2009, 76:422-427. 2. Grams ME, et al.: Clin J Am Soc Nephrol 2011, 6:966-973. 3. Payen D, et al.: Crit Care 2008, 12:R74. Results The median vancomycin total clearance (Cltot) was 0.89 and 0.55 ml/minute/kg on the fi rst and second day of the study. CRRT clearance accounted for about 50 to 60% of vancomycin Cltot found in a population with normal renal function (0.97 ml/minute/kg). Vancomycin serum concentrations after the fi rst dose were below the required target of 10 mg/l as early as 6 hours in 10 patients. AUC0–24/ MIC ≥400 ratio was achieved in 67% of patients on the fi rst day. The median gentamicin Cltot was 0.68 and 0.79 ml/minute/kg on the fi rst and second day of the study. CRRT clearance accounted for about 50% of gentamicin Cltot found in a population without renal impairment (0.73 ml/minute/kg). The target Cmax/MIC ratio was achieved in 78% of patients after the fi rst dose. Muscle wasting in the ICU can be reliably monitored using ultrasound H Jørgensen1, B Nedergaard2, T Gilsaa1 1Lillebaelt Hospital, Kolding, Denmark; 2Odense University Hospital, Svendborg, Denmark Critical Care 2013, 17(Suppl 2):P439 (doi: 10.1186/cc12377) g Critical Care 2013, 17(Suppl 2):P439 (doi: 10.1186/cc12377) Introduction The aim of this study was to establish the intraobserver and interobserver variation of ultrasonographic measurements of the rectus femoris muscle cross-section area (RF-CSA). Muscle wasting is frequent in the ICU, aff ecting more than one-half of the patients with severe sepsis [1]. Muscle mass reduces rapidly, and 15 to 20% is lost within the fi rst week [1]. To monitor muscle mass, ultrasound has the benefi ts of being both readily available in the ICU and non-invasive. Ultrasonographic measurement of RF-CSA has an almost perfect correlation with MRI (mean interclass correlation (ICC) = 0.999) [2] and RF-CSA is linearly related to maximum voluntary contraction strength in both healthy subjects and COPD patients (r = 0.78) [3]. Conclusion Adipogenesis and accumulation of M2-macrophages are hallmarks of critical illness, irrespective of nutritional management in humans and mice. Critical illness evokes macrophage polarization to the M2-state not only in adipose tissue but also in liver and lungs, which is further accentuated by fasting. P440 Critical illness induces nutrient-independent adipogenesis and accumulation of alternatively activated tissue macrophages M Marques, S Vander Perre, A Aertgeerts, S Derde, F Guiza, M Casaer, G Hermans, G Van den Berghe, L Langouche KU Leuven, Belgium Critical Care 2013, 17(Suppl 2):P440 (doi: 10.1186/cc12378) P437 ff P437 Eff ects of two diff erent regional citrate anticoagulation CVVH protocols on acid–base status and phosphate supplementation S Morabito1, V Pistolesi1, V Pistolesi1, L Tritapepe2, L Tritapepe2, E Vitaliano3, E Vitaliano3, L Zeppilli1, L Zeppilli1, F Polistena1, F Polistena1, E Fiaccadori4, E Fiaccadori4, A Pierucci1, A Pierucci1 1Umberto I, Policlinico di Roma, ‘Sapienza’ University, Rome, Italy; 2Umberto I, Policlinico di Roma, Rome, Italy; 3Pertini Hospital, Rome, Italy; 4University of Parma Medical School, Parma, Italy Critical Care 2013, 17(Suppl 2):P437 (doi: 10.1186/cc12375) Results The mean peak prefi lter concentration, AUC0–24, volume of distribution, half-life, total clearance and hemodiafi ltration clearance of esomeprazole were 6.9 μmol/l, 30.6 μmol.hour/l, 53.3 l, 2.0 hours, 1.4 l/ hour and 0.2 l/hour, respectively. In the ranitidine group the mean peak prefi lter concentration, AUC0–8, volume of distribution, half-life, total clearance and hemodiafi ltration clearance were 5.5 μmol/l, 12.0 μmol. hour/l, 91.4 l, 14.4 hours, 5.8 l and 1.3 l, respectively. Median time of Introduction Regional citrate anticoagulation (RCA) is increasingly used in high bleeding risk patients undergoing CRRT. Regardless of anticoagulation protocol, hypophosphatemia occurs frequently in Introduction Regional citrate anticoagulation (RCA) is increasingly used in high bleeding risk patients undergoing CRRT. Regardless of anticoagulation protocol, hypophosphatemia occurs frequently in S163 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 distance from the anterior superior iliac spine to the superior patellar border [1]. RF-CSA was calculated by planimetry. At each scan, three measurements were made. For intraobserver variation, the 3×3 scans were analyzed using the interclass correlation coeffi cient. For interobserver variation, the three measurements from each observer were averaged and compared using Bland–Altman statistics. Results Intraobserver variation: 15 healthy adults, age 39.6 ± 2.4 years, weight 66.8 ± 2.3 kg, sex three male/12 female. ICC: 0.996 (95% CI: 0.990 to 0.998). Interobserver variation: 15 ICU patients, age: 77 ± 8.3 years, weight: 71.3 ± 9.1 kg, sex nine male/six female. Bland–Altman: bias: –0.07 cm2, 95% limits of agreement –0.188 to 0.048 cm2. Conclusion Ultrasonographic measurement of RF-CSA is easily distance from the anterior superior iliac spine to the superior patellar border [1]. RF-CSA was calculated by planimetry. At each scan, three measurements were made. For intraobserver variation, the 3×3 scans were analyzed using the interclass correlation coeffi cient. References 1. Langouche L, et al.: Am J Respir Crit Care Med 2010, 182:507-516. 2. Casaer MP, et al.: N Engl J Med 2011, 365:506-517. References 1. Parry SM, et al.: BMJ Open 2012, e001891. doi:10.1136/bmjopen 2. Reeves ND, et al.: Eur J Appl Physiol 2004, 91:116-118. 3. Seymour JM, et al.: Thorax 2009, 64:418-423. g p Results In 30 cardiac surgery patients with AKI, acid–base status and electrolytes have been evaluated comparing protocol A (20 patients, running time 5,283 hours) versus protocol B (10 patients, 1,170 hours) (median (IQR)): pH 7.40 (7.36 to 7.44) versus 7.43 (7.41 to 7.47) (P <0.0001), bicarbonate 22.1 (20.9 to 23.5) versus 24.4 (23.2 to 25.6) mmol/l (P <0.0001), base excess –3.1 (–4.6 to –1.15) versus 0 (–1.5 to 1.1) (P <0.0001), systemic Ca2+ 1.16 (1.1 to 1.23) versus 1.14 (1.08 to 1.19) mmol/l (P <0.0001), phosphate 0.7 (0.5 to 1) versus 1.1 (0.9 to 1.4) mmol/l (P <0.0001). Protocol A required bicarbonate infusion in 90% of patients (6 ± 6.4 mmol/hour) and sodium phosphate supplementation in all cases (4.1 ± 2.4 g/day). A lower amount of phosphate supplementation (0.9 ± 2 g/day) was needed in 30% of patients undergoing protocol B while bicarbonate infusion was never required. Filter life was comparable (51.8 ± 36.5 vs. 46.8 ± 30.3 hours, P = NS).f P437 ff For interobserver variation, the three measurements from each observer were averaged and compared using Bland–Altman statistics. CRRT. The aim was to evaluate the eff ects on electrolyte and acid–base status of a new RCA-CVVH protocol using an 18 mmol/l citrate solution combined with a phosphate-containing replacement fl uid, compared with a previously adopted RCA-CVVH protocol combining a 12 mmol/l citrate solution with a conventional replacement fl uid. CRRT. The aim was to evaluate the eff ects on electrolyte and acid–base status of a new RCA-CVVH protocol using an 18 mmol/l citrate solution combined with a phosphate-containing replacement fl uid, compared with a previously adopted RCA-CVVH protocol combining a 12 mmol/l citrate solution with a conventional replacement fl uid. distance from the anterior superior iliac spine to the superior patellar border [1]. RF-CSA was calculated by planimetry. At each scan, three measurements were made. For intraobserver variation, the 3×3 scans were analyzed using the interclass correlation coeffi cient. For interobserver variation, the three measurements from each observer were averaged and compared using Bland–Altman statistics. pl Methods Until September 2011, RCA-CVVH was routinely performed in our centre with a 12 mmol/l citrate solution and a postdilution replacement fl uid with bicarbonate (HCO3 – 32, Ca2+ 1.75, Mg2+ 0.5, K+ 2 mmol/l) (protocol A). In cases of metabolic acidosis, not related to inappropriate citrate metabolism and persisting after optimization of RCA-CVVH parameter setting, bicarbonate infusion was scheduled. Starting from September 2011, in order to optimize buff er balance and to reduce the need for phosphate supplementation, a new RCA-CVVH protocol has been designed using an 18 mmol/l citrate solution combined with a recently introduced phosphate-containing replacement fl uid with bicarbonate (HCO3 – 30, phosphate 1.2, Ca2+ 1.25, Mg2+ 0.6, K+ 4 mmol/l) (protocol B). g g Results Intraobserver variation: 15 healthy adults, age 39.6 ± 2.4 years, weight 66.8 ± 2.3 kg, sex three male/12 female. ICC: 0.996 (95% CI: 0.990 to 0.998). Interobserver variation: 15 ICU patients, age: 77 ± 8.3 years, weight: 71.3 ± 9.1 kg, sex nine male/six female. Bland–Altman: bias: –0.07 cm2, 95% limits of agreement –0.188 to 0.048 cm2. g Conclusion Ultrasonographic measurement of RF-CSA is easily learned and quickly performed. It has a very low intraobserver and interobserver variation and can be recommended as a reliable method for monitoring muscle wasting in the ICU. R f Critical illness induces nutrient-independent adipogenesis and accumulation of alternatively activated tissue macrophages M Marques, S Vander Perre, A Aertgeerts, S Derde, F Guiza, M Casaer, G Hermans, G Van den Berghe, L Langouche KU Leuven, Belgium Critical Care 2013, 17(Suppl 2):P440 (doi: 10.1186/cc12378) Critical Care 2013, 17(Suppl 2):P440 (doi: 10.1186/cc12378) Introduction In artifi cially fed critically ill patients, adipose tissue reveals an increased number of small adipocytes and accumulation of M2-type macrophages [1]. We hypothesized that nutrient-independent factors of critical illness explain these fi ndings, and also that M2- macrophage accumulation during critical illness may not be limited to adipose tissue. Conclusion Protocol B provided a buff er balance more positive than protocol A and allowed one to adequately control acid–base status without additional bicarbonate infusion and in the absence of alkalosis, despite the use of a standard bicarbonate concentration replacement solution. Furthermore, the combination of a phosphate-containing replacement fl uid appeared eff ective to prevent hypophosphatemia. p Methods We performed a randomized investigation in a septic mouse model of critical illness and a study of ICU patient biopsies. In the critically ill mouse, we compared the eff ect of parenteral nutrition (n = 13) with fasting (n = 11) on body composition, adipocyte cell size, and macrophage accumulation in adipose tissue, liver and lung. Fed healthy control mice (n = 11) were studied for comparison. In vivo adipose tissue was harvested after 1 week of illness from human patients (n = 40) who participated in a RCT on early parenteral nutrition versus tolerating nutrient restriction [2], adipose tissue morphology was characterized and compared with healthy controls (n = 13). P438 Abstract withdrawn P438 Abstract withdrawn P438 Results Irrespective of nutritional intake, critically ill mice lost body weight, total fat and fat-free mass. Part of the fat loss was explained by reduced ectopic fat accumulation. Adipocyte cell number and the adipogenic markers peroxisome proliferator-activated receptor γ and CCAT/enhancer binding-protein β increased with illness, again irrespective of nutritional intake. Macrophage accumulation with predominant M2-phenotype was observed in adipose tissue, liver and lungs of critically ill mice, further accentuated by fasting in visceral tissues. Macrophage M2-markers correlated with chemoattractant factor expression in all studied tissues. In human subcutaneous adipose tissue biopsies of critically ill patients, increased adipogenic markers and M2 macrophage accumulation were present irrespective of nutritional intake. P439 Muscle wasting in the ICU can be reliably monitored using ultrasound H Jørgensen1, B Nedergaard2, T Gilsaa1 1Lillebaelt Hospital, Kolding, Denmark; 2Odense University Hospital, Svendborg, Denmark Critical Care 2013, 17(Suppl 2):P439 (doi: 10.1186/cc12377) P444 Strong ion gap can be accurately estimated with a simple bedside equation g y y Methods Obstetric patients were identifi ed from the ICU admissions database and divided into two groups – those treated with magnesium (for suspected pre-eclampsia) and those admitted for other obstetric indications (postpartum hemorrhage, infection, etc.). The baseline calcium values were compared, as well as the lowest and discharge values. Albumin and magnesium values were also compared. All comparisons used Student’s t test. Introduction The anion gap (AG) is used routinely in the assessment of metabolic acidosis, but can be misleading in patients with hypoalbuminemia and other disorders commonly encountered in intensive care. This approach to acid–base analysis relies on assessment of pH, pCO2, sodium, bicarbonate and chloride, and can lead to underestimation or overestimation of the true electrochemical status of a patient, as it does not include important ions such as lactate, calcium, magnesium, and albumin. The strong ion gap (SIG) is an alternative to the AG and is based upon Stewart’s physical chemistry approach. However, the SIG is cumbersome to calculate. As such, a number of shortcut equations have been developed in an eff ort to approximate the SIG. We sought to compare three such equations, the Kellum corrected anion gap (KellAGc), the Moviat equation, and EZSIG, in an eff ort to evaluate precision and accuracy [1-3]. Results Data were collected on 88 parturients admitted over 2 years including 40 (45%) who received magnesium and 48 (55%) who did not. Magnesium-treated women were younger (age: 31 ± 7 vs. 36 ± 5 years, P = 0.02). The baseline calcium concentrations were similar for the two groups (2.2 ± 0.2 vs. 2.2 ± 0.1 mmol/l, P = 0.85). Patients receiving magnesium had signifi cantly higher magnesium concentrations (2.1 ± 0.4 vs. 0.7 ± 0.2 mmol/l, P <0.001), and signifi cantly lower calcium concentrations during therapy (1.6 ± 0.3 vs. 1.9 ± 0.3 mmol/l, P <0.001). At discharge, the calcium levels were closer (magnesium treated 1.9 ± 0.2 vs. untreated 2.1 ± 0.1 mmol/l, P = 0.02). The albumin concentrations did not diff er between the two groups (magnesium treated 27 ± 13 vs. nontreated 33 ± 23 g/l, P = 0.134). Normal values: calcium 2.15 to 2.55 mmol/l, magnesium 0.7 to 0.95 mmol/l, albumin 35 to 50 g/l. f y Methods We conducted a retrospective chart review of consecutive patients admitted to the ICU of George Washington University Medical Center from September 2010 to March 2011. References 1. Kellum JA: Crit Care 2005, 9:500-507. 2. Moviat M, et al: Crit Care 2003, 7:R41-R45. 3. Busse L, et al.: Chest J 2011, 140(4_MeetingAbstracts):1012A-1012A. 3. Busse L, et al.: Chest J 2011, 140(4_MeetingAbstracts):1012A-1012A. P444 Strong ion gap can be accurately estimated with a simple bedside equation Of the 1,516 patients screened, 200 met inclusion criteria, which included availability of all laboratory components to calculate the SIG, obtained within 1 hour of each other. Demographic data and serum values for pH, pCO2, albumin, lactate, sodium, potassium, chloride, bicarbonate, magnesium, phosphate, and calcium were collected. The AG, SIG, KellAGc, EZSIG, and Moviat equations were subsequently calculated and compared using Pearson correlation and Bland–Altman analysis. Conclusion Magnesium therapy was associated with hypocalcemia. Potential causative mechanisms include a renal excretion interaction and magnesium-induced suppression of parathyroid hormone secretion. Physicians should be aware of the potential for symptomatic hypocalcemia during magnesium therapy. g y Results The mean SIG was 3.25 ± 3.5. Mean values for KellAGc, Moviat, and EZSIG were 4.5 ± 5.0, 1.77 ± 2.2, and 3.6 ± 3.7, respectively. Pearson correlation coeffi cients for KellAGc, Moviat, and EZSIG when compared with the SIG were r = 0.77, P = 0.0001; r = 0.88, P = 0.001; and r = 0.89, P = 0.001, respectively. In Bland–Altman analysis, the mean bias for the test equations versus the SIG were: KellAGc (1.25), Moviat (–1.48), and EZSIG (0.40). References Methods The study had two purposes: to determine the intraobserver variation for RF-CSA by one observer scanning 15 healthy adult volunteers three times each at 2-day intervals; and to determine the interobserver variation for RF-CSA by two observers each scanning 15 adult ICU patients on the same day. Patients were in a supine position, legs in passive extension. The transducer was placed perpendicular to the long axis of the right thigh over the RF, two-thirds of the 1. Langouche L, et al.: Am J Respir Crit Care Med 2010, 182:507-516. 2. Casaer MP, et al.: N Engl J Med 2011, 365:506-517. P441 Abstract withdrawn P441 Abstract withdrawn P441 S164 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 P442 of low serum sodium levels, or if hyponatremia is simply a marker for ‘sicker’ patients, is not known. R f of low serum sodium levels, or if hyponatremia is simply a marker for ‘sicker’ patients, is not known. Reference P442 Hypocalcemia during magnesium therapy for obstetric ICU admissions PD Levin, A Szalat, M Vizana, CL Sprung, R Zaguri, Y Haviv Hadassah Hebrew University Medical Center, Jerusalem, Israel Critical Care 2013, 17(Suppl 2):P442 (doi: 10.1186/cc12380) 1. Asadollahi et al.: Hyponatraemia as a risk factor for hospital mortality. QJM 2006, 99:877-880. Introduction Intravenous magnesium sulfate is commonly used in obstetric patients with pre-eclampsia. Following a case of acute symptomatic hypocalcemia we retrospectively examined a cohort of patients to investigate the frequency of hypocalcemia. P445 Relative adrenal insuffi ciency in burns A Mokline, L Gharsallah, I Rahmani, H Oueslati, B Gasri, S Tlaili, R Hammouda, A Ksontini, A Ghanem, AA Messadi Burn and Trauma Center, Tunis, Tunisia Critical Care 2013, 17(Suppl 2):P445 (doi: 10.1186/cc12383) Relative adrenal insuffi ciency in burns Hyponatremia and intensive care outcome Hyponatremia and intensive care outcome P Dean, R Docking, D Govenden, A Davidson, A Mackay Victoria Infi rmary, Glasgow, UK Critical Care 2013, 17(Suppl 2):P443 (doi: 10.1186/cc12381) i y g Critical Care 2013, 17(Suppl 2):P443 (doi: 10.1186/cc12381) Introduction Disorders of sodium (Na+) and water homeostasis are common in hospitalised patients. Hyponatremia in particular has been associated with worse hospital outcome and length of stay [1]. We aimed to defi ne the incidence of hyponatremia (serum Na+ ≤134 mmol/l) in our intensive care population and to determine whether it was associated with ICU outcome or length of stay. Conclusion While all three equations correlated highly with the SIG, the EZSIG and Moviat outperformed the KellAGc in Pearson and Bland– Altman analysis. The EZSIG had a smaller bias than the Moviat equation and a slightly better correlation (0.89 vs. 0.88). In the assessment of critically ill patients, EZSIG is a candidate scanning equation for the measurement of the SIG when all SIG components are not available. f Methods Demographics, APACHE II score, outcome data and admission sodium were retrieved from the Ward Watcher system in the Victoria Infi rmary ICU for 2,440 consecutive admissions from January 2005 to present. We divided patients into three groups depending on serum Na+ (≤134 mmol/l, 135 to 144 mmol/l, ≥145 mmol/l) and compared APACHE II score, length of stay and ICU outcome between patients with a low versus a normal serum Na+. Data were analysed using the chi-squared test, Student’s t test and the Mann–Whitney test where appropriate. P444 P444 Strong ion gap can be accurately estimated with a simple bedside equation L Busse, L Chawla, R Panchamia, D Choi, E Nobakht, E Brasha-Mitchell, M Seneff George Washington University Medical Center, Washington, DC, USA Critical Care 2013, 17(Suppl 2):P444 (doi: 10.1186/cc12382) Etiology of low testosterone levels in mal sepsis requiring mechanical ventilation h A Bech, H Van Leeuwen, H De Boer A Bech, H Van Leeuwen, H De Boer Rijnstate Hospital, Arnhem, the Netherlands yp Results Data from 1,038 patients were included. Admission iCa was available for 976 (94%) patients. A total of 539 (55%) were hypocalcaemic (iCa <1.1 mmol/l). adjCa was available for 1,031 (99%), with 602 (58%) classifi ed as hypocalcaemic (adjCa <2.2 mmol/l). adjCa <2.2 mmol/l had sensitivity of 78% and specifi city of 63% for detecting iCa <1.1 mmol/l. The best previously published formula had an AUC of 0.81 (95% CI = 0.78 to 0.83), compared with an AUC of 0.78 (0.75 to 0.81) for the locally validated formula [1]. It was hypothesized that albumin, pH or phosphate derangements could aff ect the proportion of albumin- bound calcium and the performance of adjustment formulae. Albumin concentration was not correlated with the diff erence between adjCa and iCa. Weak but statistically signifi cant correlations were found for pH (r = 0.164; P <0.001) and phosphate (r = 0.135; P <0.001). The sensitivity and specifi city of adjCa <2.2 mmol/l for detecting hypocalcaemia did not improve when looking at patients with normal pH (sensitivity 60.4%; specifi city 69%) or phosphate (32.2%; 78.9%). j p Critical Care 2013, 17(Suppl 2):P448 (doi: 10.1186/cc12386) Introduction Low testosterone levels are frequently found in critically ill male patients. The etiology and clinical signifi cance is still poorly understood. In the present study we have investigated the kinetics and pathophysiology of altered gonadal hormone synthesis in male patients with severe sepsis and respiratory failure. Methods All male patients with severe sepsis and respiratory failure who were admitted to the ICU of a large teaching hospital in the Netherlands between September 2011 and June 2012 were included. Steroid hormone levels were measured on days 1, 3 and 7. y Results In total, 18 patients were included. The mean age was 69 ± 2 years, mean weight 76 ± 2 kg, APACHE II score 23 ± 2 and most patients suff ered from pneumosepsis. On the fi rst day of intubation, total and free testosterone levels were extremely low in most patients and remained low during the fi rst week (Figure 1). 17β-Estradiol levels were elevated on day 1 and decreased during the fi rst week. LH and i Conclusion adjCa is a relatively poor predictor of iCa in critically ill patients. Table 1 (abstract P447) Table 1 (abstract P447) Low Intermediate High Tertile (n = 216) (n = 219) (n = 220) P value 25(OH)D (ng/ml) 10.4 (± 3.6) 17.6 (± 5.1) 30.7 (± 11.4) <0.001 SAPS 2 32 (± 15) 28 (± 16) 28 (± 16) 0.007 Hospital mortality 58 (26.9%) 48 (21.2%) 29 (13.2%) 0.002 Lethal sepsis 11 (5.1%) 9 (4.1%) 0 (0.0%) 0.005 Conclusion Low 25(OH)D status is predictive of all-cause and sepsis mortality in the critically ill. Interventional studies are needed to investigate the eff ect of vitamin D on mortality and sepsis incidence and outcomes. Low Intermediate High Tertile (n = 216) (n = 219) (n = 220) P value 25(OH)D (ng/ml) 10.4 (± 3.6) 17.6 (± 5.1) 30.7 (± 11.4) <0.001 SAPS 2 32 (± 15) 28 (± 16) 28 (± 16) 0.007 Hospital mortality 58 (26.9%) 48 (21.2%) 29 (13.2%) 0.002 Lethal sepsis 11 (5.1%) 9 (4.1%) 0 (0.0%) 0.005 T Steele , R Kolamunnage-Dona , C Downey , C Toh , I Welters 1University of Liverpool, UK; 2Royal Liverpool University Hospital, Liverpool, UK Critical Care 2013, 17(Suppl 2):P446 (doi: 10.1186/cc12384) Introduction Hypocalcaemia is common in critical illness and accurate assessment is crucial. Small studies have shown that albumin-adjusted calcium (adjCa) does not accurately predict the ionised calcium (iCa) concentration in critically ill patients, yet adjCa continues to be widely used [1]. We investigated the reliability of using adjCa to identify hypocalcaemia in a large, diverse population requiring intensive care. Introduction Hypocalcaemia is common in critical illness and accurate assessment is crucial. Small studies have shown that albumin-adjusted calcium (adjCa) does not accurately predict the ionised calcium (iCa) concentration in critically ill patients, yet adjCa continues to be widely used [1]. We investigated the reliability of using adjCa to identify hypocalcaemia in a large, diverse population requiring intensive care. Methods In a retrospective study of patients admitted to the ICUs of a tertiary care hospital between January 2008 and 2012, iCa and pH were extracted from routine blood gas results and total calcium, albumin and phosphate from routine biochemistry results. adjCa was calculated using a formula derived from and validated on the local population [2]. Sensitivity, specifi city, positive and negative predictive values (PPV and NPV) and area under the curve (AUC) of adjCa for predicting hypocalcaemia (iCa <1.1 mmol/l) were calculated. Vitamin D status and overall and sepsis mortality in critically ill patients Table 1 (abstract P445). Characteristics of the two groups G1 G2 P value Age 38 ± 13 34 ± 12 NS TBSA 57.8 ± 30 26 ± 22 0.019 UBS 158 ± 100 70 ± 99 NS ABSI 8 ± 3 6 ± 3 NS CS T0 37 ± 11 19 ± 14 0.01 D CS 2.2 ± 1.8 19 ± 6 <0.001 Shock 6 (86%) 4 (36%) 0.04 Table 1 (abstract P445). Characteristics of the two groups y , Critical Care 2013, 17(Suppl 2):P447 (doi: 10.1186/cc123 Introduction Vitamin D status may aff ect hospital mortality and infectious complications in critical illness. Methods A total of 655 mixed critically ill patients with available 25(OH)D levels hospitalized between 2008 and 2010 were included. Cox regression analysis adjusted for SAPS 2, age and gender was performed. 25(OH)D levels were categorized by month-specifi c tertiles (high, intermediate, low) to refl ect seasonal variation. Primary endpoint was the correlation between 25(OH)D and hospital mortality. Secondary endpoints were sepsis mortality and blood culture positivity.i y y Results Hospital mortality was signifi cantly higher in patients in the low (HR = 1.98) and the mid-range tertile (HR = 1.88) compared with the highest tertile. Vitamin D levels were signifi cantly lower in patients with lethal sepsis compared with other causes of death (12.3 ± 5.0 vs. 18.2 ± 11.2, P = 0.02). Blood culture positivity rates did not diff er between the groups (23.0% vs. 26.8% vs. 17.3%, P = 0.361). See Table 1. Conclusion RAI is common in severely burned patients during the acute phase, and is associated with shock. Further prospective controlled studies will be necessary to establish risk factors of RAI in severely burned patients and its impact on their prognosis. Relative adrenal insuffi ciency in burns With direct iCa measurements now readily available on ward-based analysers, adjCa should not be used to determine calcium status in critical illness. phosphate or pH. With direct iCa measurements now readily available on ward-based analysers, adjCa should not be used to determine calcium status in critical illness. university-affi liated teaching hospital in Tunis. Patients admitted within the fi rst 24 hours post burn with greater than 10% total body surface area (TBSA) burned were enrolled in this study from 1 January 2009 to 30 June 2010. Exclusion criteria were pregnancy, history of adrenal insuffi ciency, or steroid therapy within 6 months prior to burns. A short corticotrophin test (250 μg) was performed, and cortisol levels were measured at baseline (CS T0) and 60 minutes post test. Adrenal insuffi ciency was defi ned by a response ≤9 μg/dl. Relative adrenal insuffi ciency was further defi ned by a baseline cortisol >20 μg/dl. Results Patients were assigned into two groups: G1 (RAI, n = 7) and G2 (absence AI, n = 11). Comparative study of the two groups shows the results presented in Table 1. 1. Slomp J, et al.: Albumin-adjusted calcium is not suitable for diagnosis of hyper- and hypocalcemia in the critically ill. Crit Care Med 2003, 31:1389-1393. 1. Slomp J, et al.: Albumin-adjusted calcium is not suitable for diagnosis of hyper- and hypocalcemia in the critically ill. Crit Care Med 2003, 31:1389-1393. 2. James M, et al.: Derivation and internal validation of an equation for albumin-adjusted calcium. BMC Clin Pathol 2008, 8:12. Relative adrenal insuffi ciency in burns Relative adrenal insuffi ciency in burns pp p Results Of the 2,440 patients studied, 1,993 had APACHE II data and serum Na+ recorded and so were included for analysis. In total, 453 patients (22.7%) had a serum Na+ ≤134 mmol/l and 1,388 patients (67.1%) had a serum Na+ of 135 to 144 mmol/l. Patients with a low Na+ had a higher mortality (OR = 1.48, 95% CI = 1.16 to 1.90, P <0.001), a higher APACHE II score (22 vs. 19, P <0.001) and higher mean age (60 years vs. 58 years, P <0.001) than patients with a normal serum Na+. Mean length of stay of patients with low serum Na+ was also longer (5.1 days vs. 4.6 days) although this was not statistically signifi cant (P = 0.09). Introduction Relative adrenal insuffi ciency (RAI) is an uncommon disorder among burn patients, which can often go unrecognized. However, there are no or almost no data about the incidence of this disease in burns. The goal of the current study is to evaluate the incidence of RAI in burn patients during the acute phase. Methods A prospective study, approved by our Institutional Ethics Committee, was conducted in a 20-bed adult burn ICU at a Conclusion In summary, hyponatremia is a useful index of severity of illness in our ICU population. Whether this is a direct adverse eff ect S165 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 university-affi liated teaching hospital in Tunis. Patients admitted within the fi rst 24 hours post burn with greater than 10% total body surface area (TBSA) burned were enrolled in this study from 1 January 2009 to 30 June 2010. Exclusion criteria were pregnancy, history of adrenal insuffi ciency, or steroid therapy within 6 months prior to burns. A short corticotrophin test (250 μg) was performed, and cortisol levels were measured at baseline (CS T0) and 60 minutes post test. Adrenal insuffi ciency was defi ned by a response ≤9 μg/dl. Relative adrenal insuffi ciency was further defi ned by a baseline cortisol >20 μg/dl. Results Patients were assigned into two groups: G1 (RAI, n = 7) and G2 (absence AI, n = 11). Comparative study of the two groups shows the results presented in Table 1. phosphate or pH. Table 1 (abstract P447) Methods In a retrospective study of patients admitted to the ICUs of a tertiary care hospital between January 2008 and 2012, iCa and pH were extracted from routine blood gas results and total calcium, albumin and phosphate from routine biochemistry results. adjCa was calculated using a formula derived from and validated on the local population [2]. Sensitivity, specifi city, positive and negative predictive values (PPV and NPV) and area under the curve (AUC) of adjCa for predicting hypocalcaemia (iCa <1.1 mmol/l) were calculated. Etiology of low testosterone levels in mal sepsis requiring mechanical ventilation h Unpaired t test showed a P value of 0.07. Conclusion Calcium replacement with 40 ml of 10% calcium gluconate is eff ective when the adjusted calcium is <2.0 mmol/l. Replacement with 20 ml of 10% calcium gluconate when the adjusted calcium is between 2.0 and 2.15 mmol/l is not much more eff ective than no replacement. This suggests that either replacement is not needed or there is under-replacement in mild hypocalcaemia. Adjusted calcium may be giving falsely low calcium results in mild hypocalcaemia. The measurement of ionised calcium is a possible solution [2,3]. References 1. Zaloga GP: Crit Care Med 1992, 20:251-262. 2. Slomp J, et al.: Crit Care Med 2003, 31:1389-1393. 3. Byrnes MC, et al.: Am J Surg 2005, 189:310-314. P450 Endogenous melatonin in critically ill patients VS Salice, IG Galluccio, BS Salihovic, IP Piva, FM Marazzo, CV Villa, MT Taverna, MU Umbrello, GM Mistraletti, GI Iapichino Figure 1 (abstract P448). Figure 2 (abstract P448). Figure 1 (abstract P448). the adjusted calcium result and the calcium replacement were documented. Adequate replacement is defi ned as 40 ml of 10% calcium gluconate if the adjusted calcium is <2.0 mmol/l and 20 ml of 10% calcium gluconate if the adjusted calcium result is between 2.0 and 2.15 mmol/l. Figure 2 (abstract P448). Figure 2 (abstract P448). Results Eighty-eight adjusted calcium results were <2.0 mmol/l. In 74 results adequate replacement was given and the average increase in calcium was 0.2 mmol/l. This led to an increase in the adjusted calcium to >2.15 mmol/l in 36% of results and improvement to >2.0 mmol/l in 89% of results. In 14 results no replacement was given and the average increase in calcium was 0.07 mmol/l, which equated to 57% of calcium results remaining <2.0 mmol/l. Unpaired t test showed a P value of 0.0006. A total of 150 adjusted calcium results were between 2.0 and 2.15 mmol/l. In 111 results adequate replacement was given and the average increase was 0.006 mmol/l. The adjusted calcium fell to <2.0 mmol/l in 26% of results and normalised to >2.15 mmol/l in 23%. Where the adjusted calcium fell despite replacement, 51% of results were below 2.05 mmol/l. In 39 results no replacement was given, leading to an average fall in calcium of 0.03 mmol/l. Thirty- three percent of calcium results fell to <2.0 mmol/l and 20% of results normalised. Unpaired t test showed a P value of 0.07. References 1. Zaloga GP: Crit Care Med 1992, 20:251-262. 2. Slomp J, et al.: Crit Care Med 2003, 31:1389-1393. 3. Byrnes MC, et al.: Am J Surg 2005, 189:310-314. Etiology of low testosterone levels in mal sepsis requiring mechanical ventilation h FSH levels were inappropriately low. All lipoprotein fractions and their apo-proteins were reduced as well as 17-OH-progesterone, DHEA and DHEAS. In contrast, androstenedione (adione) levels were elevated. This suggests preferential and stimulated synthesis of androstenedione (Figure 2). The high 17β-estradiol levels indicate that androstenedione is shunted into the estrogen pathway, a process that requires high aromatase activity. The high estradiol/total testosterone ratio supports this conclusion. p Conclusion Calcium replacement with 40 ml of 10% calcium gluconate is eff ective when the adjusted calcium is <2.0 mmol/l. Replacement with 20 ml of 10% calcium gluconate when the adjusted calcium is between 2.0 and 2.15 mmol/l is not much more eff ective than no replacement. This suggests that either replacement is not needed or there is under-replacement in mild hypocalcaemia. Adjusted calcium may be giving falsely low calcium results in mild hypocalcaemia. The measurement of ionised calcium is a possible solution [2,3]. Conclusion Hyperestrogenic hypotestosteronemia is a frequent fi nding in the acute phase of severe sepsis in male patients with respiratory failure. It is suggested to be caused by decreased androgen production and shunting of androgen to estrogen synthesis as a result of increased aromatase activity. The clinical relevance of gonadal hormone substitution needs further study. Effi cacy of calcium replacement in hypocalcaemia H Chatha, K Sim Whiston Hospital, Liverpool, UK Critical Care 2013, 17(Suppl 2):P449 (doi: 10.1186/cc12387 Effi cacy of calcium replacement in hypocalcaemia H Chatha, K Sim Whiston Hospital, Liverpool, UK Critical Care 2013, 17(Suppl 2):P449 (doi: 10.1186/cc12387) Etiology of low testosterone levels in mal sepsis requiring mechanical ventilation h This cannot be explained by abnormalities of albumin, S166 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum com/supplements/17/S2 http://ccforum.com/supplements/17/S2 FSH levels were inappropriately low. All lipoprotein fractions and their apo-proteins were reduced as well as 17-OH-progesterone, DHEA and DHEAS. In contrast, androstenedione (adione) levels were elevated. This suggests preferential and stimulated synthesis of androstenedione (Figure 2). The high 17β-estradiol levels indicate that androstenedione is shunted into the estrogen pathway, a process that requires high aromatase activity. The high estradiol/total testosterone ratio supports this conclusion. Conclusion Hyperestrogenic hypotestosteronemia is a frequent fi nding in the acute phase of severe sepsis in male patients with respiratory failure. It is suggested to be caused by decreased androgen production and shunting of androgen to estrogen synthesis as a result of increased aromatase activity. The clinical relevance of gonadal hormone substitution needs further study. P449 Effi cacy of calcium replacement in hypocalcaemia H Chatha, K Sim Whiston Hospital, Liverpool, UK Critical Care 2013, 17(Suppl 2):P449 (doi: 10.1186/cc12387) the adjusted calcium result and the calcium replacement were documented. Adequate replacement is defi ned as 40 ml of 10% calcium gluconate if the adjusted calcium is <2.0 mmol/l and 20 ml of 10% calcium gluconate if the adjusted calcium result is between 2.0 and 2.15 mmol/l. Results Eighty-eight adjusted calcium results were <2.0 mmol/l. In 74 results adequate replacement was given and the average increase in calcium was 0.2 mmol/l. This led to an increase in the adjusted calcium to >2.15 mmol/l in 36% of results and improvement to >2.0 mmol/l in 89% of results. In 14 results no replacement was given and the average increase in calcium was 0.07 mmol/l, which equated to 57% of calcium results remaining <2.0 mmol/l. Unpaired t test showed a P value of 0.0006. A total of 150 adjusted calcium results were between 2.0 and 2.15 mmol/l. In 111 results adequate replacement was given and the average increase was 0.006 mmol/l. The adjusted calcium fell to <2.0 mmol/l in 26% of results and normalised to >2.15 mmol/l in 23%. Where the adjusted calcium fell despite replacement, 51% of results were below 2.05 mmol/l. In 39 results no replacement was given, leading to an average fall in calcium of 0.03 mmol/l. Thirty- three percent of calcium results fell to <2.0 mmol/l and 20% of results normalised. 50 Endogenous melatonin in critically ill patients The higher melatonin peak in renal failure may be due to an increased distribution volume; greater AUC in patients with liver failure could be due to a less effi cient removal of the hormone from the systemic circulation. The fi nding of increased peak and AUC in nonsurvivor patients could be due to a hormonal response increased by the body stress reaction, potentially similar to cortisol [1], or to a higher production of a physiological antioxidant [2] with a decreased ability to use it. References 1. Venkatesh B, et al.: Best Practice Res Clin End Met 2011, 25:719-733. 2. Flaring UB, et al.: Intensive Care Med 2005, 31:1072-1078. P451 Succinate ameliorates mitochondrial oxygen consumption of metformin-intoxicated human platelets A Protti1, M Monti2, A Lecchi1, A Artoni1, N Greppi1, L Gattinoni1 1Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy; 2Università degli Studi di Milano, Milan, Italy Methods Platelet-rich-plasma was incubated for 72 hours with metformin at a fi nal concentration of 0 mg/l (control), 1.66 mg/l (therapeutic dose) or 166 mg/l (toxic dose). Platelet VO2 was then measured with a Clark-type electrode, in the presence of glutamate plus malate (complex I electron donors) (fi nal concentration: 20 mmol/l for both) or succinate (complex II electron donor) (30 mmol/l), before and after adding cyanide (40 mmol/l). Mitochondrial (cyanide-sensitive) and extra-mitochondrial (cyanide-insensitive) VO2 were corrected for platelet count. Results The main results, from four preliminary experiments, are shown in Figure 1. In the presence of glutamate plus malate, only platelets incubated with a high dose of metformin had a mitochondrial VO significantly lower than controls. In the presence of succinate, Figure 1 (abstract P450). Figure 1 (abstract P451). Mitochondrial oxygen use of human platelets incubated with metformin. Figure 1 (abstract P450). this study is to describe the endogenous blood melatonin values in ICU patients and their correlation with clinical parameters. Figure 1 (abstract P451). Mitochondrial oxygen use of human platelets incubated with metformin. Methods Seventy-three high-risk critically ill patients mechanically ventilated for >48 hours were enrolled. Blood samples for melatonin assay were collected between the 3rd and the 8th day of the ICU stay. Melatonin was determined by radioimmunoassay and ELISA. The peak and the area under the curve (AUC) calculated for each patient were correlated with the clinical parameters using the regression for quantiles test. 50 Endogenous melatonin in critically ill patients Endogenous melatonin in critically ill patients VS Salice, IG Galluccio, BS Salihovic, IP Piva, FM Marazzo, CV Villa, MT Taverna, MU Umbrello, GM Mistraletti, GI Iapichino Università degli Studi Milano, Milan, Italy Critical Care 2013, 17(Suppl 2):P450 (doi: 10.1186/cc12388) Introduction The aim of the study is to examine the effi cacy of calcium replacement in patients with hypocalcaemia in the ICU. Hypocalcaemia aff ects between 15 and 50% of admissions to the ICU [1]. Introduction The aim of the study is to examine the effi cacy of calcium replacement in patients with hypocalcaemia in the ICU. Hypocalcaemia aff ects between 15 and 50% of admissions to the ICU [1]. Introduction Melatonin could have a meaningful role in critically ill patients, because of its immunomodulatory, antioxidant and sleep regulation properties; it is reduced in critical illness. The purpose of Introduction Melatonin could have a meaningful role in critically ill patients, because of its immunomodulatory, antioxidant and sleep regulation properties; it is reduced in critical illness. The purpose of f Methods Adjusted calcium results <2.0 mmol/l were followed up from admission for 5 days or until discharge or death. On each day S167 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 this study is to describe the endogenous blood melatonin values in ICU patients and their correlation with clinical parameters. Methods Seventy-three high-risk critically ill patients mechanically ventilated for >48 hours were enrolled. Blood samples for melatonin assay were collected between the 3rd and the 8th day of the ICU stay. Melatonin was determined by radioimmunoassay and ELISA. The peak and the area under the curve (AUC) calculated for each patient were correlated with the clinical parameters using the regression for quantiles test. Results Endogenous melatonin was found lower in critically ill patients compared with healthy subjects (Figure 1), although it showed a great individual variability and it generally maintained a night-time increase. In the univariate analysis the peak was found related to: blood creatinine (P = 0.034); patients in coma (P = 0.024); hospital mortality (P = 0.016). The AUC was found related to: SAPS II (P = 0.047); creatinine (P <0.001); AST (P <0.001); ALT (P <0.001); hospital mortality (P <0.022). Peak and AUC were found higher in nonsurvivor patients. Conclusion In accord with previous studies, the endogenous blood melatonin was found reduced in ICU patients. References 1. Venkatesh B, et al.: Best Practice Res Clin End Met 2011, 25:719-733. 2. Flaring UB, et al.: Intensive Care Med 2005, 31:1072-1078. 50 Endogenous melatonin in critically ill patients Results Endogenous melatonin was found lower in critically ill patients compared with healthy subjects (Figure 1), although it showed a great individual variability and it generally maintained a night-time increase. In the univariate analysis the peak was found related to: blood creatinine (P = 0.034); patients in coma (P = 0.024); hospital mortality (P = 0.016). The AUC was found related to: SAPS II (P = 0.047); creatinine (P <0.001); AST (P <0.001); ALT (P <0.001); hospital mortality (P <0.022). Peak and AUC were found higher in nonsurvivor patients. g p Conclusion In accord with previous studies, the endogenous blood melatonin was found reduced in ICU patients. The higher melatonin peak in renal failure may be due to an increased distribution volume; greater AUC in patients with liver failure could be due to a less effi cient removal of the hormone from the systemic circulation. The fi nding of increased peak and AUC in nonsurvivor patients could be due to a hormonal response increased by the body stress reaction, potentially similar to cortisol [1], or to a higher production of a physiological antioxidant [2] with a decreased ability to use it. Figure 1 (abstract P451). Mitochondrial oxygen use of human platelets incubated with metformin. Methods Platelet-rich-plasma was incubated for 72 hours with metformin at a fi nal concentration of 0 mg/l (control), 1.66 mg/l (therapeutic dose) or 166 mg/l (toxic dose). Platelet VO2 was then measured with a Clark-type electrode, in the presence of glutamate plus malate (complex I electron donors) (fi nal concentration: 20 mmol/l for both) or succinate (complex II electron donor) (30 mmol/l), before and after adding cyanide (40 mmol/l). Mitochondrial (cyanide-sensitive) and extra-mitochondrial (cyanide-insensitive) VO2 were corrected for platelet count. 1. Venkatesh B, et al.: Best Practice Res Clin End Met 2011, 25:719-733. 2. Flaring UB, et al.: Intensive Care Med 2005, 31:1072-1078. Does chronic metformin therapy off er cardiomyoprotection in coronary artery bypass grafting surgery patients? A case–control study A Delaporte, JP Hacquebard, P Origer, I Pastijn, M Maatouk, O Germay, E Stevens, P Kapessidou, A Roman CHU Saint-Pierre, Brussels, Belgium Critical Care 2013, 17(Suppl 2):P452 (doi: 10.1186/cc12390) g Results Ten patients were selected, fi ve males and fi ve females, with a mean age of 72.2. On admission, nine of 10 patients had a framework of general illness and acute renal failure; one patient appeared with a probable acute abdomen not subsequently confi rmed on CT. Eight patients had a state of dehydration resulting from gastroenteritis, diarrhea, fever; two patients had taken NSAIDs in the days prior to hospitalization. No patient, in these conditions, has discontinued treatment with metformin. All patients showed a critical clinical framework with a SAPS II mean score of 77.5. Cardiovascular and respiratory support was required in all cases. The hemogas analysis showed that patients had a severe metabolic acidosis (mean pH = 6.96) with increased plasma lactate (mean lactate = 15.93 mmol/l). Prothrombin activity was normal in eight of 10 patients. The overall mortality was 70%. Introduction Metformin, widely used as an antidiabetic drug, activates the AMP activated protein kinase, a key regulator of the metabolism providing protection under fuel defi ciency. Chronic metformin therapy has been shown in long-term follow-up clinical studies to reduce cardiovascular mortality [1]. In animal experiments, acute metformin pretreatment has been shown to reduce ischemia–reperfusion injury on cardiomyocytes [2]. We want to evaluate whether outcomes are aff ected in coronary artery bypass grafting (CABG) surgery. f Methods We performed a retrospective case–control study on 341 patients admitted exclusively for CABG surgery with cardiopulmonary bypass between January 2006 and April 2012. Eighty-one patients chronically on metformin were matched 1:3 with nonmetformin- treated ones using gender, age, body mass index and number of arterial bypass grafts. Global mortality, ICU-free 28 postoperative days and creatinine kinase MB (CKMB) at postoperative baseline, 8 and 18 hours post baseline were compared.i Conclusion Patient education on correct use of metformin is essential to prevent MALA, specially in those clinical conditions of increased risk (for example, acute renal failure), as recommended by the AIFA 2011 guidelines [4]. In our study, a higher plasma concentration of lactate represents the main negative prognostic factor, as pointed out by other studies [5]. P452 Does chronic metformin therapy off er cardiomyoprotection in coronary artery bypass grafting surgery patients? A case–control study A Delaporte, JP Hacquebard, P Origer, I Pastijn, M Maatouk, O Germay, E Stevens, P Kapessidou, A Roman CHU Saint-Pierre, Brussels, Belgium Critical Care 2013, 17(Suppl 2):P452 (doi: 10.1186/cc12390) Multiscale modeling of acute insulin resistance in critical care Critical Care 2013, 17(Suppl 2):P454 (doi: 10.1186/cc12392) Critical Care 2013, 17(Suppl 2):P454 (doi: 10.1186/cc12392) Introduction Stress hyperglycemia in the critically ill is a complex process in which insulin signaling is systematically hijacked to provide energy substrate for metabolic priorities such as cell healing or infection containment. Fluctuating levels of plasma glucose are associated with increased mortality in the ICU [1]. We develop a multiscale mathematical model that can characterize the severity of stress hyperglycemia based on a fundamental understanding of the signaling molecules involved. Conclusion These data suggest that chronic preoperative treatment with metformin is associated with lower CKMB levels at postsurgery baseline, 8 and 18 hours post baseline with a shorter ICU stay in patients receiving CABG surgery under extracorporeal circulation. References g g Methods Insulin resistance following insult has been shown to be driven primarily by the immune response via the cytokine IL-6 [2]. We created a multiscale mathematical model that links circulating glucose and insulin concentration dynamics from the extended minimal model [3] to a cellular insulin response model [4] that captures insulin- mediated glucose uptake in an insulin-responsive cell. p References References . UK Prospective Diabetes Study Group: Lancet 1998, 352:854-865. 2. Lalau JD: Drug Saf 2010, 33:727-740. 3. Le Gall JR, et al.: JAMA 1993, 270:2957-2963. 4. Italian Medicines Agency 2011 [http://www.agenziafarmaco.gov.it/it/content/ raccomandazioni-sull%E2%80%99utilizzo-dei-medicinali-base-di-metformina- nella-gestione-del-diabete-m] Table 1 (abstract P452). Case–control analysis Table 1 (abstract P452). Case–control analysis Table 1 (abstract P452). Case–control analysis Table 1 (abstract P452). Case–control analysis Statistics, Metformin Nonmetformin P value Mortality 1/81 8/260 0.24 28 days free ICU (days) 23.4 22.9 <0.04 CKMB T 0 hours (ng/ml) 21 26 <0.003 CKMB T+8 hours (ng/ml) 22 53 <0.004 CKMB T+18 hours (ng/ml) 21.5 27.5 <0.012 5. Dell’Aglio DM, et al.: Ann Emerg Med 2009, 54:818-823. 6. Peters N, et al.: Crit Care 2008, 12:R149. Succinate ameliorates mitochondrial oxygen consumption of metformin-intoxicated human platelets Succinate ameliorates mitochondrial oxygen consumption of metformin-intoxicated human platelets A Protti1, M Monti2, A Lecchi1, A Artoni1, N Greppi1, L Gattinoni1 1Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy; 2Università degli Studi di Milano, Milan, Italy Critical Care 2013, 17(Suppl 2):P451 (doi: 10.1186/cc12389) p Results The main results, from four preliminary experiments, are shown in Figure 1. In the presence of glutamate plus malate, only platelets incubated with a high dose of metformin had a mitochondrial VO2 signifi cantly lower than controls. In the presence of succinate, mitochondrial VO2 of controls did not change signifi cantly whereas that of platelets incubated with metformin did. The eff ect of succinate tended to become larger as the dose of metformin was increased from 0 up to 166 mg/l (0.3 ± 0.2 vs. 0.6 ± 0.3 vs. 1.0 ± 0.3 nmol/minute*106 cells) (P = 0.068). Even so, mitochondrial VO2 of platelets incubated with the highest dose of metformin did not return to the levels of controls. Extra-mitochondrial VO2 was always the same. A Protti1, M Monti2, A Lecchi1, A Artoni1, N Greppi1, L Gattinoni1 1Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy; 2Università degli Studi di Milano, Milan, Italy Critical Care 2013, 17(Suppl 2):P451 (doi: 10.1186/cc12389) Introduction Metformin intoxication inhibits mitochondrial complex I and oxygen consumption (VO2). Succinate bypasses complex I by donating electrons to complex II. The aim of this study was to clarify whether succinate ameliorates mitochondrial VO2 of metformin- intoxicated human platelets. S168 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Conclusion Succinate ameliorates (but does not return to normal) mitochondrial VO2 of human platelets incubated with a toxic dose of metformin. predisposing conditions, the use of metformin may result in metformin- associated lactic acidosis (MALA), a rare adverse event associated with a high mortality rate [2]. The aim of this study is to assess risk factors and prognostic factors in patients with MALA. Methods We conducted a retrospective study of patients with MALA admitted to the ICU of ASL 2 Chieti between 1 January 2008 and 30 September 2012. The eligibility criteria were: diagnosis of diabetes mellitus type 2, treatment with oral hypoglycemic drugs containing metformin, increased anion gap metabolic acidosis (pH <7.35, HCO3 – <22 mmol/l, lactate >5 mmol/l). For each patient we evaluated: sex, age, home care, SAPS II score [3], blood tests. Does chronic metformin therapy off er cardiomyoprotection in coronary artery bypass grafting surgery patients? A case–control study The prothrombin activity, which is considered to be a decisive prognostic factor in the study of Peters and colleagues [6], was not impaired in patients with poor outcome. Results Metformin-treated patient mortality was not signifi cantly lower than in nonmetformin (1.2% vs. 3.1%, P = 0.24, Fischer exact test). ICU-free 28 postoperative days were higher in the metformin group (median 23.4 days; 95% CI = 22.5 to 24.3) than in the nonmetformin group (median 22.9 days; 95% CI 22.3 to 23.5, P <0.04, Wilcoxon test). Median CKMB levels at postoperative baseline were 21 versus 26 ng/ml (P <0.003), at 8 hours were 22 versus 53 ng/ml (P <0.004) and 18 hours post baseline were 21.5 versus 27.5 ng/ml (P <0.012). See Table 1. Impact of a real-time electronic persistent hyperglycemia alert on glucose control in the ICU g K Colpaert, S Oeyen, T van den Driessche, C Danneels, J Decruyenaere Ghent University Hospital, Ghent, Belgium Critical Care 2013, 17(Suppl 2):P455 (doi: 10.1186/cc12393) Introduction Hyperglycemia is frequently encountered in critically ill patients, and associated with adverse outcome. Improvement of glucose protocol adherence may be accomplished using electronic alerts. We confi gured a non-intrusive real-time electronic alert, called a GLYC sniff er, as part of our Intensive Care Information System (ICIS) that continuously evaluates the occurrence of persistent hyperglycemia and hypoglycemia. The GLYC sniff er is confi gured in such a way to give an alert: if two consecutive glucose values are >150 mg/dl, with a minimum interval of 60 minutes; or if a glucose value is <80 mg/dl. We wanted to evaluate whether the GLYC sniff er would improve the glucose control. p p Conclusion The MPC–MHE algorithm achieves targeted glucose control in response to changing patient dynamics and multiple measured disturbances for a pilot population of 10 VPs. Furthermore, the MHE scheme updates patient parameters in real time in response to changing patient dynamics. P457 Methods A single-center, prospective intervention study during a 6-month period in our 22-bed surgical ICU. Two study phases were compared: a 3-month pre-alert phase with no alerting to the nurses, and a 3-month intervention phase where the GLYC sniff er was alerting through the Clinical Notifi cation System of our ICIS.f Hyperglycemia in intensive care identifi es patients at high risk of developing diabetes: preliminary data of a multicentre study E Lukic, I Gornik, M Grgic-Medic, D Milicic, V Vegar, V Ivancan, M Peric, V Gasparovic, I Pavlic Renar University Hospital Centre Zagreb, Croatia Critical Care 2013, 17(Suppl 2):P457 (doi: 10.1186/cc12395) i y Results A total of 652 diff erent patients having a total of 699 admission episodes was recorded during the study period. There were no signifi cant diff erences between the two study groups regarding baseline demographic data, fi rst glucose value upon admission. A total of 2,335 GLYC sniff er alerts were recorded during the whole study period: 84.3% persistent hyperglycemia alerts (1,021 in the pre-alert group, 948 in the alert group), and 15.7% hypoglycemia alerts (139 vs. 227). A signifi cantly lower percentage of glucose values in the alert group were hyperglycemic (19.5% vs. 26.5%, P <0.001). The proportion of persistent hyperglycemic values (i.e. P455 Results MPC controller performance on one VP is shown in Figure 1. Across a population of 10 VPs, the average [Gbl] under MPC is 6.31 mmol/l, the average minimum is 4.62 mmol/l, the population individual minimum is 3.49 mmol/l and the average absolute average residual error is 0.83 mmol/l from a 5.6 mmol/l target. With standard intervention, the 10 VPs have an average [Gbl] of 9.32 mmol/l, an average minimum [Gbl] of 3.77 mmol/l, and a population minimum [Gbl] of 2.78 mmol/l. Algorithm performance deteriorates signifi cantly if the imputed sampling time exceeds 30 minutes, underlining the importance of dynamic variations in insulin sensitivity in this population. y References References 1. Van den Berghe G: N Engl J Med 2001, 345:1359-1367. 2. Thorell A: Clin Nutr 1996, 15:75-79. 3. Roy A: Diabetes Technol Ther 2006, 8:617-626. 4. Sedaghat A: Am J Physiol Endocrinol Metab 2002, 283:E1084-E1101. References 1. Van den Berghe G: N Engl J Med 2001, 345:1359-1367. 2. Thorell A: Clin Nutr 1996, 15:75-79. 3. Roy A: Diabetes Technol Ther 2006, 8:617-626. 4. Sedaghat A: Am J Physiol Endocrinol Metab 2002, 283:E1084-E1101. References 1. Van den Berghe G: N Engl J Med 2001, 345:1359-1367. 2. Thorell A: Clin Nutr 1996, 15:75-79. 3. Roy A: Diabetes Technol Ther 2006, 8:617-626. 4. Sedaghat A: Am J Physiol Endocrinol Metab 2002, 283:E1084-E1101. 3. Roy A: Diabetes Technol Ther 2006, 8:617-626. 4. Sedaghat A: Am J Physiol Endocrinol Metab 2002, 283:E1084-E1101. P453 Metformin-associated lactic acidosis: risk factors and prognostic factors Metformin-associated lactic acidosis: risk factors and prognostic factors V Di Falco, A Milano, M Battilana, F Araosta, A Grosso, D Albanese, F Petrini, L Di Liberato ASL Chieti SS. Annunziata, Chieti, Italy Critical Care 2013, 17(Suppl 2):P453 (doi: 10.1186/cc12391) V Di Falco, A Milano, M Battilana, F Araosta, A Grosso, D Albanese, F Petrini, L Di Liberato ASL Chieti SS. Annunziata, Chieti, Italy Critical Care 2013, 17(Suppl 2):P453 (doi: 10.1186/cc12391) Results Inhibitory dynamics driven by IL-6 were incorporated into the cellular model to attenuate an insulin signaling intermediate (insulin receptor substrate 1) according to the proposed biological mechanisms. The percentage reduction in glucose uptake as a function of IL-6 concentration was fi t to data from patients who underwent elective abdominal surgery [2], shown in Figure 1. The overall multiscale model captures decreased insulin signaling as a result of increased IL-6 levels and the subsequent hyperglycemia that may ensue. y Critical Care 2013, 17(Suppl 2):P453 (doi: 10.1186/cc12391) Introduction Metformin, an oral hypoglycemic drug, belongs to the biguanide class and is now generally accepted as fi rst-line treatment in type 2 diabetes mellitus, especially in overweight patients [1]. In some Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S169 Critical Care 2013, Volume 17 Suppl 2 http://ccforum com/supplements/17/S2 http://ccforum.com/supplements/17/S2 Figure 1 (abstract P454). Reduced glucose uptake driven by increased plasma IL-6 levels. group (9.9% vs. 15.4%, P <0.001). The patients in the alert group spend signifi cantly more time within the set target glucose interval of 80 to 150 mg/dl (82.3% vs. 75.0%, P = 0.009). A signifi cantly lower proportion of patients experienced a new-onset hypoglycemic event (<80 mg/dl) in the alert group (19.3% vs. 26.2%, P = 0.04). g p Conclusion A real-time electronic persistent glycemia sniff er resulted in a signifi cantly higher proportion of normoglycemia, without increasing the variability. Furthermore, hypoglycemic events occurred less frequently, and were resolved more timely. Smart alerting is able to improve quality of care, while diminishing the problem of alert fatigue. P456 P456 Individualized targeted glucose control to avoid hypoglycemia SP Gawel1, G Clermont2, T Ho1, BM Newman3, B Yegneswaran2, RS Parker1 1University of Pittsburgh, PA, USA; 2University of Pittsburgh Medical Center, Pittsburgh, PA, USA; 3Iowa State University, Ames, IA, USA Critical Care 2013, 17(Suppl 2):P456 (doi: 10.1186/cc12394) Introduction Hyperglycemia and hypoglycemia have been linked to worse outcomes in critically ill patients. While there is controversy as to the optimal tightness of glucose control in critically ill patients, there is agreement that an upper limit to safe glucose levels exists and that avoiding hypoglycemic episodes should be prioritized. Our algorithm can assist clinicians in maintaining blood glucose ([Gbl]) within a desired target range while avoiding hypoglycemia. Figure 1 (abstract P454). Reduced glucose uptake driven by increased plasma IL-6 levels. Conclusion A multiscale model has been developed to describe the inhibitory eff ects of IL-6 on insulin-mediated glucose uptake. Cellular inhibitory dynamics were shown to capture reduced insulin sensitivity on the macroscale, which could then be used to characterize insulin sensitivity and to provide insulin treatment advice to reduce glucose variability. Methods Our model predictive control (MPC) algorithm uses insulin and glucose as control inputs and a linearized model of glucose– insulin–fatty acid interactions. To allow the controller model to learn from data, a moving horizon estimation (MHE) technique tailored the tissue sensitivity to insulin to individual responses. Patient data ([Gbl] measurements, insulin and nutritional infusion rates) were from the HIDENIC database at the University of Pittsburgh Medical Center. [Gbl] measurements, typically hourly, were interpolated to impute a measurement every 5 minutes. The model captured patient [Gbl] via nonlinear least squares by adjusting insulin sensitivity (SI) and endogenous glucose production (EGP0). The resulting virtual patient (VP) is used to evaluate the performance of the MPC–MHE algorithm. Impact of a real-time electronic persistent hyperglycemia alert on glucose control in the ICU consecutive glucose values exceeding the limit of 150 mg/dl) was signifi cantly lower in the alert Introduction A recent study showed that hyperglycaemia (blood glucose ≥7.8 mmol/l) in nondiabetic patients hospitalised in a medical ICU is associated with increased risk of diabetes [1]. We investigated a large mixed ICU population to confi rm these results. Methods This study retrospectively included patients with negative history of diabetes admitted to ICUs during the year 2007. We excluded patients receiving steroids, with newly diagnosed diabetes and those with end-stage disease. Patients were followed-up 5 years after index S170 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Figure 1 (abstract P456). Figure 1 (abstract P456). Figure 1 (abstract P456). Figure 1 (abstract P456). admission. Diagnosis of diabetes within 6 months from the index admission was presumed as revealing DM at inclusion, which excluded the patient. Patients who were taking glucocorticoids during the follow- up period were excluded. The rest of the patients, if consented, were assessed for their diabetic status with a single HbA1c measurement. Introduction The objective of the study was to determine the association between average glucose level (AVG) and hospital outcomes in critically ill patients. Methods We performed a chart review of the MIMICII and HIDENIC databases, prospective cohorts of over 32,000 and 46,000 patients admitted to the ICUs at Beth Israel Deaconess Medical Center between 2001 and 2007 and the ICUs at University of Pittsburgh Medical Center between 1991 and 2008, respectively. All admissions of patients ≥18 years without the diagnosis of DKA and NKHS were included. Diabetics were identifi ed from ICD-9 documentation. Propensity score for death (pDead) was computed from either SAP1 (MIMICII) or APACHE III (HIDENIC) to assess the risk of death. Hypoglycemia was defi ned as AVG ≤60 mg/dl. AVG was computed as the area under the glucose curve throughout ICU admission. Mortality was examined within bins (each bin is categorized by a 10 mg/dl increase in AVG) and was compared between adjacent categories using a chi-square test. The same method was repeated among diabetics, nondiabetics, patients with lower (pDead greater than median) and higher (pDead lower than Results A total of 2,696 patients were included in the study: 1,441 medical and 1,255 surgical, hyperglycaemia (≥7.8 mmol/l) was registered in 1,419 (52%). Impact of a real-time electronic persistent hyperglycemia alert on glucose control in the ICU During the 5 years from index admission 409 died and 38 were taking glucocorticoids, and 10 refused participation. Diabetes was diagnosed in 189 patients during the 6 months after index admission. The remaining cohort of 2,320 patients had 1,169 patients who were hyperglycaemic during hospitalisation. Diabetes was already diagnosed in 132 patients, of whom 112 were hyperglycaemic in the ICU; prediabetes was already diagnosed in 121 patients, of whom 83 had ICU hyperglycaemia. Since 125 patients (55 hyperglycaemic) refused HbA1c testing, it was performed on 1,852 patients and revealed diabetes in 136 patients (107 had hyperglycaemia) and prediabetes in 103 patients (75 were hyperglycaemic in ICU). Overall, patients who had hyperglycaemia had 19.8% cumulative 5-year incidence of diabetes and 14.3% of prediabetes. Nonhyperglycaemic ICU patients had cumulative incidences of 4.5% and 6.6%, respectively. Relative risk for developing diabetes during 5 years after ICU admission is 4.2 (95% CI = 3.2 to 5.9) for patients who did have hyperglycaemia.i Figure 1 (abstract P458). Figure 1 (abstract P458). p yp g y Conclusion The results of this study confi rm in a large mixed medical/ surgical ICU population that hyperglycaemia occurring in severe illness or the perioperative period is associated with increased risk of developing diabetes. Suggesting lifestyle changes to reduce the risk and regular follow-up should be implemented at least for these patients. p Reference p Reference 1. Gornik I, et al.: A prospective observational study of the relationship of critical illness associated hyperglycaemia in medical ICU patients and subsequent development of type 2 diabetes. Crit Care 14:R130. Association between average glucose levels and hospital mortality among critically ill patients B Yegneswaran1, R Parket2, S Gawel2, A Pritchard-Bell2, T Ho2, G Clermont2 1UPMC Hamot, Erie, PA, USA; 2University of Pittsburgh, PA, USA Critical Care 2013, 17(Suppl 2):P458 (doi: 10.1186/cc12396) Figure 1 (abstract P458). Figure 1 (abstract P458). S171 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 with increased risk of mortality (M). Heightened attention to identifi ed risk factors (RF) and causes of hypoglycemic events (HE) may lead to improvement in glycemic control. A limited literature describes RF for H but a detailed root cause analysis (RCA) of individual HE has not been published. median) risk of death. Statistical signifi cance was noted following a Bonferroni correction. Results MIMICII database: 21,209 admissions met inclusion criteria. Mean age: 62.5 ± 16.4 years; female: 9,070 (42.8%); diabetes: 5,496 (25.9%); mean admission SAP1 score: 13.8 ± 5.2; mean LOS: 9 days (IQR 6.15); hospital mortality: 10.3%. HIDENIC database: 38,872 admissions met inclusion criteria. Mean age: 59.4 ± 17.3 years; female: 16,675 (42.9%); diabetes: 11,326 (29.1%); mean admission APACHE III score: 80.2 ± 14; mean LOS: 11 days (IQR 7.22); hospital mortality: 14.3%. In both databases, association between average glucose and hospital mortality followed a bathtub shape with mortality nadir between 80 and 130 mg/dl. No mortality diff erence was observed between adjacent AVG glucose bins in the bottom of the bathtub (Figure 1). This relationship was preserved in nondiabetics, and for patients with higher risk of death. p Methods This is a retrospective analysis of prospectively collected data including 835 patients admitted consecutively between 1 February 2012 and 31 October 2012 to the 16-bed medical–surgical ICU of a university-affi liated teaching hospital. The blood glucose (BG, mg/dl) target was 90 to 120 and the monitoring frequency was every 3 hours, or every 1 hour when the patient received i.v. IN infusion. HE data were collected by the bedside nurse, using a cutoff of BG <60, and information was abstracted from the unit’s database. RF included: shock (SH), renal insuffi ciency (RI), hepatic failure (HF), mechanical ventilation (MV), and diabetes mellitus (DM). Attributable causes included: IN treatment, improper monitoring frequency (MF), interruption of feeding (FE), and spontaneous (SP). P461 Continuous arterial and venous glucose monitoring by quenched chemical fl uorescence in ICU patients after cardiac surgery S Bird1, L Macken1, O Flower1, F Bass1, N Hammond1, S Webb1, N Kennedy1, A Baker1, E Yarad1, C Chau2, M Librande2, P Strasma2, S Finfer1 1Royal North Shore Hospital, Sydney, Australia; 2Glumetrics, Irvine, CA, USA Critical Care 2013, 17(Suppl 2):P461 (doi: 10.1186/cc12399) Results Two groups had similar characteristics at baseline. A total of 1,060 CGM/ABG pairs were analyzed and reference glucose ranged from 4.3 to 18.8 mmol/l. Median (IQR) absolute relative deviation was lower in the FC arm (7.0% (3.5, 13.0) vs. 12.8% (6.3, 21.8), FC vs. MC, P <0.001). Similarly, the percentage of points in the Clarke error grid zone A points meeting ISO criteria were higher with FC (87.8% vs. 70.2%). Sensor bias (median (IQR)) was signifi cantly lower in the FC arm (–0.1 (–0.7, 0.4) mmol/l vs. –1.1 (–2.3, –0.1) mmol/l, P <0.001). The median (IQR) interval between calibrations in FC arm was 169 (122, 213) minutes. Introduction Continuous glucose monitoring (CGM) in ICUs has the potential to improve patient safety outcomes. The GluCath Intravascular CGM System uses a novel quenched chemical fl uorescence sensing mechanism to measure glucose concentration in venous or arterial blood (BG). This is the fi rst report of this system deployed for 48 hours in both arteries and veins of ICU patients. Methods This ongoing clinical study evaluates up to two sensors per subject in 20 patients undergoing cardiac surgery. An arterial sensor is deployed via a standard 20 G radial artery catheter inserted for routine care and an optional venous sensor is deployed percutaneously in an upper arm vein. Data are presented from the fi rst fi ve patients. Outcome measures are qualitative (ease of use, workfl ow fi t) and quantitative (accuracy vs. reference analyzer). Sensors were inserted shortly after ICU admission, with ultrasound guidance for venous sensor insertion. In vivo calibration was performed at 1 and 2 hours, then each morning. Glucose values were recorded every 10 seconds by the system. Hourly arterial reference samples were analyzed via Radiometer ABL 800 Flex Blood Gas Analyzer (BGA).i Conclusion CGM accuracy in the MC arm was comparable with accuracy in subjects with type 1 diabetes. Further signifi cant improvements to CGM accuracy in critical care are possible by increasing the frequency of calibrations. References References 1. Weinstein RL, et al.: Diabetes Care 2007, 30:1125-1130. 2. Kovatchev B, et al.: Diabetes Care 2008, 31:1160-1164. Results Arterial sensors were successfully deployed in all fi ve patients and did not interfere with clinical care, blood pressure monitoring or sampling. One arterial catheter failed resulting in sensor removal at 36 hours. The venous sensor was deployed in three patients, but removed from two patients due to thrombosis identifi ed during surveillance ultrasound examinations. A total of 202 reference BG samples ranging from 5.3 to 11.3 mmol/l were collected. Precision between arterial and venous sensors (n = 2) was 9.3% CV. Arterial sensor accuracy compared with BGA was 5.5% MARD. One hundred Association between average glucose levels and hospital mortality among critically ill patients We measured rates of severe (<40) and mild (40 to 69) H, and compared them as well as percentage of values in the optimum range (70 to 139) and hyperglycemic range (>139) with data from the preceding 3 months (PRE). Conclusion Low and high average glucose are associated with mortality. P461 Such accurate CGM may provide valuable information to guide insulin therapy in critically ill subjects. f Continuous glucose monitoring in critically ill adults: comparison of two diff erent calibration protocols p g Results Sixty-three (7.5%) patients sustained a total of 79 HE. They were older, more likely to be diabetic, and had higher APACHE IV predicted (37.3% vs. 15.4%, P <0.0001) and observed (25.4% vs. 8.6%, P <0.0001) M than those without H. RF: SH 24.1%; RI 24.1%; HF 8.9%; MV 46.8%; DM 36.5%. Attributable causes: IN 58.2%; MF 8.9%; FE 6.3%; SP 36.7%. BG <40, 40 to 69 and >139 decreased 78.6%, 39.0% and 9.3% (P = 0.0006, P <0.0001, P <0.0001). BG 70 to 139 increased 5.5% (P <0.0001). In total, 4.2% and 22.4% of PRE patients sustained severe and mild H. In total, 0.7% and 14.1% of RCA patients sustained severe and mild H (P = 0.0029, P = 0.0003). two diff erent calibration protocols L Leelarathna1, S English2, H Thabit1, K Caldwell1, J Allen1, K Kumareswaran1, M Wilinska1, M Nodale1, J Mangat1, M Evans1, R Burnstein2, R Hovorka1 1University of Cambridge, UK; 2Addenbrookes Hospital, Cambridge, UK Critical Care 2013, 17(Suppl 2):P459 (doi: 10.1186/cc12397) f p L Leelarathna1, S English2, H Thabit1, K Caldwell1, J Allen1, Introduction We evaluated the clinical and numerical accuracy of the Freestyle Navigator continuous glucose monitoring (CGM) system, in critically ill adults using two diff erent methods of calibration. Studies looking at intensive glucose control have yielded confl icting results with increased rates of iatrogenic hypoglycemia. Availability of accurate real-time glucose information may improve safety and effi cacy of glucose control in the critical care unit. Conclusion IN treatment was associated with barely more than one-half of HE in this population of intensively monitored patients. Implementation of an initiative mandating RCA of every HE, including identifi cation of RF and attributable causes, was associated with a signifi cant improvement in glycemic control. g Methods In a randomized prospective trial, paired CGM and reference glucose (hourly arterial blood gas (ABG)) were collected from 24 adults with critical illness (age 60 ± 14 years, BMI 29.6 ± 9.3 kg/m2, APACHE score range 6 to 19) and hyperglycemia (glucose ≥10 mmol/l or treated with intravenous insulin), over 48 hours. In 12 subjects, CGM was force- calibrated at variable 1 to 6 hourly intervals using ABG glucose (FC arm). In the other 12 subjects, the sensor was calibrated according to the manufacturer’s instructions (1, 2, 10, 24 hours after insertion) using arterial blood and built-in glucometer (MC arm). P461 Performance of the Medtronic Sentrino® continuous glucose management system in the cardiac ICU Performance of the Medtronic Sentrino® continuous glucose management system in the cardiac ICU M Kosiborod1, R Gottlieb2, J Sekella2, D Peterman1, A Grodzinsky1, P Kennedy1, M Borkon1 1Saint Luke’s Mid America Heart Institute, Kansas City, MO, USA; 2Medtronic Minimed, Northridge, CA, USA Critical Care 2013, 17(Suppl 2):P462 (doi: 10.1186/cc12400) 1. Lazar HL, et al.: Ann Thorac Surg 2009, 87:663-669. P460 P460 Root cause analysis of hypoglycemic events in critically ill patients A McDonald, J Krinsley Stamford Hospital, Stamford, CT, USA Critical Care 2013, 17(Suppl 2):P460 (doi: 10.1186/cc12398) Introduction Hypoglycemia (H) is a complication of intensive insulin (IN) therapy of critically ill patients and is independently associated S172 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 htt // f / l t /17/S2 http://ccforum.com/supplements/17/S2 Figure 1 (abstract P461). Arterial sensor ISO-modifi ed Bland–Altman plot. Figure 1 (abstract P461). Arterial sensor ISO-modifi ed Bland–Altman plot. Figure 1 (abstract P461). Arterial sensor ISO-modifi ed Bland–Altman plot. percent (202/202) of arterial sensor measurements met ISO 15197 criteria (within =/–20% of reference measurements if BG ≥4.2 mmol/l; Figure 1). was 12.2%. No diff erences in CGM system accuracy were seen within subgroups of low versus high Society of Thoracic Surgeons (STS) score (MARD 12.1% and 10.6% for STS >8% vs. ≤8%, respectively) or hemodynamic status (MARD 12.0% and 12.4% for compromised vs. stable hemodynamics). Consensus grid analysis showed >99% of SG values within A/B zones, and 0% in D/E zones. No device or study-related adverse events were reported. In total, 80% and 100% of clinicians found Sentrino® easy to use after one and two patients, respectively. Conclusion The GluCath System measured glucose concentration continuously in cardiac surgery ICU patients. Arterial catheter deployment did not appear to compromise line function or patient care. Percutaneous venous deployment was feasible, but may be associated with risk of local venous thrombosis. p y Conclusion The Sentrino® CGM system demonstrated good analytic and clinically relevant accuracy, excellent reliability and safety in critically ill cardiac patients; and was easy to use and integrate in the cardiac ICU. Future studies are needed to determine whether CGM can improve BG control and reduce hypoglycemia in this patient group. Reference P463 Intensive insulin therapy in critically ill children: impact on blood glucose dynamics and its relation with mortality M Van Tornout, M Gielen, T Van Herpe, D Vlasselaers, L Desmet, I Vanhorebeek, G Van den Berghe, D Mesotten University Hospitals Leuven, Belgium Critical Care 2013, 17(Suppl 2):P463 (doi: 10.1186/cc12401) Introduction Blood glucose (BG) control reduces morbidity and length of stay, and is standard practice in patients undergoing cardiac surgery [1]. However, maintaining BG in the target range, while avoiding hypoglycemia, is challenging. Continuous glucose monitoring (CGM) is a promising technology that may help address these challenges. We investigated the performance and safety of Medtronic Sentrino®, a newly developed CGM for critically ill adults, in the cardiac ICU. P464 P464 Implementation of evidence-based protocols improves survival: a 15-year surgical ICU experience in 10,172 patients SA Nasraway Tufts Medical Center, Boston, MA, USA Critical Care 2013, 17(Suppl 2):P464 (doi: 10.1186/cc12402) Tufts Medical Center, Boston, MA, USA Table 1 (abstract P465) Admitted Admitted with without decubitus decubitus P Variable (n = 180) (n = 2,543) value Age 65 or older 106 (58.9%) 1,062 (41.8%) <0.001 APACHE IV score on admission 80.2 (28.2%) 58.7 (30.7) <0.001 Diabetes 53 (29.4%) 420 (16.5%) <0.001 Dialysis 16 (8.9%) 58 (2.3%) <0.001 Hepatic failure 3 (1.7%) 56 (2.2%) 1.000 Mechanically ventilated during 123 (68.3%) 1,457 (57.3%) hospitalization Admission source to the ICU Direct admit 10 (5.6%) 154 (6.1%) ER 84 (46.7%) 1,206 (47.4%) Floor 25 (13.9%) 214 (8.4%) 0.017 OR 19 (10.6%) 459 (18.1%) Other location 42 (23.3%) 510 (20.1%) Outcomes ICU LOS (days) 4.9 (4.9) 3.3 (4.7) <0.001 Hospital LOS (days) 15.6 (20.7) 10.5 (11.1) <0.001 ICU mortality 30 (16.7%) 308 (12.1%) 0.073 Hospital mortality 58 (32.2%) 464 (18.3%) <0.001 Readmission rate for 20 out of 112 173 out of 2,079 0.002 survivors (16.4%) (8.3%) Conclusion The presence of decubiti on admission to the ICU is associated with longer hospitalizations even after adjusting for age, acuity, and organ supportive therapies. DU on admission to ICU provide a unique, unambiguous marker of increased resource utilization. Introduction There has been enormous interest in measuring ICU performance in terms of mortality and resource use, owing to increased public and health insurer scrutiny. We elected to describe the performance of our surgical ICU, using a standardized mortality ratio (SMR = observed / predicted mortality). y Methods The primary cohort was all patients admitted to the surgical ICU from March 2010 through February 2012 and related outcomes. The change in SMR was longitudinally determined from the latest 15- year study period (1997 to 2011) comprised of 10,172 patients. y y Results There were 1,799 ICU admissions in the primary cohort. Hospital mortality, observed and predicted by APACHE IV, was measured. Crude hospital mortality was 8.4%. The hospital SMR (observed / predicted mortality) was 0.58 (95% CI: 0.49 to 0.65). The SMR decreased by 20% from 0.73 to 0.58 over the 15-year study period, an absolute 1% per year decrease (P = 0.039; 95% CI: –0.02 to –0.002), as shown in Figure 1. ( ; ), g Conclusion Mortality was less than predicted and steadily declined during the previous 15 years. Intensive insulin therapy in critically ill children: impact on blood glucose dynamics and its relation with mortality Introduction A large RCT showed that tight glucose control (TGC), targeting age-adjusted normal fasting blood glucose levels with insulin infusion, decreased morbidity and mortality in critically children [1]. However, the incidence of hypoglycemia increased substantially in the TGC group. We aimed to assess the eff ect of TGC on the three domains of blood glucose dynamics (hyperglycemia, hypoglycemia and blood glucose variability) and their independent association with mortality in the pediatric ICU. Methods Adult patients with actual or planned cardiac ICU admission at a single tertiary center were approached for participation and signed consent. Other inclusion criteria were treatment with i.v. insulin (target BG <140 mg/dl) and life expectancy >96 hours. After initiation of i.v. insulin, Sentrino® subcutaneous glucose sensors were inserted into patients’ anterior thighs with planned study participation of 72 to 96 hours. Reference BG was collected according to ICU protocol, obtained from central venous catheter and analyzed with bedside blood gas analyzer (BGA; i-STAT®, Abbott, USA). Sensor glucose (SG) results were displayed, and its predictive alerts and alarms fully enabled. Additional reference BGs were obtained during alarms and calibration. All treatment decisions were based on BGA data, not on SG values. p Methods This is a preplanned substudy of a published RCT in one 10-bed pediatric ICU. Seven hundred patients (age 1 to 16 years), admitted to the PICU between October 2004 and December 2007, were randomized to either TGC (50 to 80 mg/dl in infants, 70 to 100 mg/dl in children) or to the usual care tolerating hyperglycemia up to 215 mg/ dl (UC). Patients with at least two arterial blood glucose measurements were included (UC n = 349; TGC n = 348). Results A total of 21 patients were enrolled; all successfully completed the study. Mean age was 65 years, 38% were women, 67% had diabetes. Types of surgery were CABG (38%), valve replacement (29%), combined CABG and valve (19%) and cardiac transplant (14%). SG was displayed 95% of the time during the study, and 864 paired BG–SG points were used for analysis. Overall mean absolute relative diff erence (MARD) Results Mean blood glucose levels were lowered from 143 ± 34 mg/dl in the UC group to 104 ± 24 mg/dl (P <0.0001). The median number of samples per patient did not diff er between UC (21 (13 to 48)) and TGC (22 (15 to 49)). Decubitus ulcers are associated with prolonged length of stay in critically ill patients Decubitus ulcers are associated with prolonged length of stay in critically ill patients Decubitus ulcers are associated with prolonged length of sta critically ill patients WT McGee1, BH Nathanson2, E Lederman1, TL Higgins1 1Baystate Medical Center, Springfi eld, MA, USA; 2OptiStatim LLC, Longmeadow, MA, USA Critical Care 2013, 17(Suppl 2):P465 (doi: 10.1186/cc12403) y WT McGee1, BH Nathanson2, E Lederman1, TL Higgins1 1Baystate Medical Center, Springfi eld, MA, USA; 2OptiStatim LLC, Longmeadow, MA, USA Critical Care 2013, 17(Suppl 2):P465 (doi: 10.1186/cc12403) WT McGee1, BH Nathanson2, E Lederman1, TL Higgins1 1Baystate Medical Center, Springfi eld, MA, USA; 2OptiStatim LLC, Longmeadow, MA, USA Introduction How decubitus ulcers (DU) diagnosed on admission to the ICU aff ect resource use is unknown. We hypothesize that this easily identifi able clinical fi nding would be associated with longer hospitalizations. Methods Clinical assessment was used to identify patients with DU on admission to the ICU. The association of DU on hospital and ICU length of stay (LOS) was assessed with generalized linear models that adjusted for ICU admission source, age, APACHE IV score, diabetes, sepsis, hepatic failure, dialysis and mechanical ventilation. i Conclusion TGC substantially reduced the hyperglycemic index, at the expense of increased hypoglycemia incidence, but without aff ecting blood glucose variability. Statistical analysis suggested strictly avoiding hyperglycemia in order to bring about outcome benefi t with insulin in critically ill children. p y Results DU was present on admission in 180 of 2,723 (6.6%) during a 19-month period in 2011 to 2012 at a single teaching hospital in New England. Patients with decubiti were sicker on admission (mean (SD) APACHE IV 80.2 (28.2) vs. 58.7 (30.7), P <0.001), more likely to be age 65 or older (58.9% vs. 41.8%, P <0.001) and require more organ supportive therapies (Table 1). DU patients had a higher in-hospital mortality rate (32.2% vs. 18.3%, P <0.001) and longer mean ICU and hospital LOS (4.9 vs. 3.3 days and 15.6 vs. 10.5 days, respectively (P <0.001)). After multivariate adjustment, DU patients stayed 3.2 days, 95% CI (1.6, 4.8) longer in the hospital than non-DU patients and stayed 2.5 days longer, 95% CI (0.7, 0.43) among survivors. DU patients had longer ICU stays (1.3 days, 95% CI (0.3, 1.3)) in general but were statistically similar among survivors. Reference 1. Vlasselaers D, et al.: Lancet 2009, 373:547-556. 1. Vlasselaers D, et al.: Lancet 2009, 373:547-556. Intensive insulin therapy in critically ill children: impact on blood glucose dynamics and its relation with mortality TGC lowered the hyperglycemic index, a marker of S173 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 hyperglycemia over time, from median 44 (IQR 29 to 66) mg/dl in UC to 12 (7 to 23) mg/dl, as well as the glycemic penalty index, an aggregate measure of dysglycemia, from median 44 (32 to 58) in UC to 22 (16 to 30) (both P <0.0001). Despite frequent hypoglycemia <40 mg/dl (1.4% in UC, 25.0% in TGC, P <0.0001), TGC did not result in increased blood glucose variability, as refl ected by the standard deviation of the blood glucose time series (TGC: 36 (24 to 50) mg/dl, UC 34 (26 to 48) mg/dl, P = 0.82). In multivariable logistic regression analysis, adjusting for baseline risk factors (age, severity of illness, malignancy, type of admission), hypoglycemia (OR = 1.6, 95% CI = 0.19 to 2.22) and blood glucose variability (OR = 1.0, 95% CI = 0.97 to 1.02, per mg/dl) were not associated with PICU mortality. In contrast, hyperglycemic index (OR = 1.02, 95% CI = 1.01 to 1.04, per mg/dl) was independently associated with mortality and its lowering statistically explained the outcome benefi t of the intervention. P464 Mortality predictors in septic HIV patients in the ICU P Vidal-Cortés1, P Lameiro-Flores1, A Aller-Fernández2, M Mourelo-Fariña2, P Fernández-Ugidos1, R Gómez-López1, M Alves-Pérez1, E Rodríguez-García1 1CHU Ourense, Spain; 2CHU A Coruña, Spain Critical Care 2013, 17(Suppl 2):P468 (doi: 10.1186/cc12406) Introduction Sepsis in HIV patients carries a high mortality. Our purpose is to identify mortality predictors in this population. Introduction Sepsis in HIV patients carries a high mortality. Our purpose is to identify mortality predictors in this population. Methods A retrospective study of HIV patients admitted to our ICU between January 2005 and December 2009. We identify septic patients and analyzed demographic factors, etiology, organ failure and outcome. We used logistic regression to calculate the relative mortality risk for each qualitative variable and the beta (β) coeffi cient for quantitative variables. Conclusion Depending on the patient and the cause of admission, ICU admission may represent an excellent opportunity as a screening method to determine HIV status. Given the greater effi cacy of HAART at present, most patients with medical or surgical conditions unrelated to HIV infection will be eligible to join the ICU. People with HIV can and should benefi t from using reasonable and individualized care in an ICU. References q Results Sixty-two septic patients were admitted to our ICU during the study period (71% men, mean age: 40.59 ± 8.12). A total of 56.5% came from the ED, mean hospital stay before ICU admission: 6.14 ± 10.96 days. A total of 66.1% had a history of intravenous drug use, other comorbidities: COPD (9.7%), cirrhosis (8.1%), solid or hematologic malignancy (12.9%). A total of 40.3% were under HAART. Mean CD4 count at admission: 219.62 ± 353.93 cells/mm3. Mean viral load: 4.57 ± 3.25 log. Mean albumin levels: 2.3 ± 0.53 g/dl. APACHE II: 21.98 ± 7.97. The lung was the most frequent focus of infection (65.6%) followed by the CNS. The most common pathogen was S. pneumoniae (28.8%), followed by P. jirovecii (13.6%). In total, 62.9% needed vasopressors, 79% mechanical ventilation and 19.4% renal replacement. Mean ICU and hospital length of stay: 10.43 ± 10.52 and 34.76 ± 29.64 days. ICU mortality: 33.9%; hospital mortality: 41.9%. P464 The SMR can be used to track success when new quality measures are introduced or changes in the delivery of care are made. ICUs should report their standardized mortality ratios to evaluate performance. Figure 1 (abstract P464). Tufts surgical ICU, 15-year outcome standardized hospital mortality ratio. Conclusion The presence of decubiti on admission to the ICU is associated with longer hospitalizations even after adjusting for age, acuity, and organ supportive therapies. DU on admission to ICU provide a unique, unambiguous marker of increased resource utilization. Figure 1 (abstract P464). Tufts surgical ICU, 15-year outcome standardized hospital mortality ratio. Figure 1 (abstract P464). Tufts surgical ICU, 15-year outcome standardized hospital mortality ratio. S174 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 P466 HIV patients admitted to an ICU of a university hospital – experience of 15 years: 1995 to 2009 A Agrifoglio, M Arce, M Hernández, P Millan, C Guallar, I Pozuelo, M Jiménez Hospital Universitario La Paz, Madrid, Spain Critical Care 2013, 17(Suppl 2):P466 (doi: 10.1186/cc12404) Results We included 139 patients with HIV/AIDS admitted to the ICU. Median age was 40 (31 to 48) years and 71% were male. Severe malnutrition was common (34%). The CD4 cell count was 84 (25 to 274) cells/mm3 and viral load was 17,733 (67 to 174,214) copies/ml; 57% had at least one opportunistic infection; 55% had used antiretroviral therapy previous to ICU admission. Mechanical ventilation was used by 46% of patients and hospital mortality was 42%. Total SOFA score was 5 (2 to 9) points. Cardiovascular dysfunction was the most common on the fi rst day of stay (51%), followed by respiratory (42%), neurological (40%), renal (35%), hematological (27%) and hepatic (17%). Cardiovascular and renal dysfunctions presented with higher rate of severe dysfunction (30% and 15%, respectively). Rates of neurological (P = 0.002), renal (P = 0.009) and hematological (P = 0.003) dysfunctions were higher in nonsurvivors. Age, CD4 cell count, malnutrition, and opportunistic infections were included in the multivariate analysis. Neurological dysfunction was the independent risk factor for hospital mortality (odds 3.2 (1.4 to 7.2)). The presence of neurological dysfunction was dichotomized: associated or not with primary neurological diagnosis; survival was lower in the patients with neurological dysfunction and without primary neurological diagnosis (log-rank test 0.001 in the 7-day and 0.02 in the 28-day analysis). P464 Sixty-day survival was similar in primary and secondary neurological dysfunction, but it remained lower than in patients without neurological impairment. P466 Introduction HIV infection is a major public health problem in the world. The use of prophylaxis against opportunist infection and the introduction of HAART in 1996 increased life expectancies. The therapeutic use of ICU resources for HIV patients has been controversial, questioning the admission of these patients especially in advanced stages of the disease, given the poor prognosis. The aim of this study was to determine the experience of the past 15 years in relation to the income of these patients in an ICU. Methods A retrospective case series consisting of patients with diagnosis of HIV infection (known or unknown) admitted between January 1995 and December 2009. We collected demographic and epidemiological data, process of acquisition of the disease, infection status: known or unknown patient infected, whether or not receiving antiretroviral therapy and whether it was eff ective (undetectable viral load at the time of admission), APACHE II, cause of admission, need for mechanical ventilation (MV), pathology related or unrelated to HIV infection and ICU mortality. Conclusion Neurological dysfunction was independently associated with hospital survival, mainly in those AIDS critically patients without primary neuropathy. y Results During this period 12,607 patients were admitted to the ICU, 188 (1.5%) HIV-positive. Mean APACHE II score 17.6, median age 39 years, 73% men and 90% Spanish nationality. Principal risk behavior: addiction drugs injection (67%). Seventeen percent of patients did not know who was infected with HIV at the time of admission to the ICU. Fifty-three percent were not receiving HAART. Of the 88 patients treated, 93% were receiving HAART (eff ective in 88% of cases). Sixty percent of the patients came from the emergency department of the hospital. Main admission diagnoses: acute respiratory failure caused by infection (Streptococcus pneumoniae and Pneumocystis jirovecii), neurological disorders (coma for illicit drugs and psychotropic) and septic shock. Seventy percent required MV. Of patients whose HIV infection was not known, 93.5% were admitted for related pathology. In patients of known infection, the pathology associated with HIV was 30%. Average length of stay 9 was days. ICU mortality was 35%. Most frequent causes of death: septic shock and multiple organ failure. P464 ICU mortality increases for each day of hospital stay before ICU admission (β = 1.062, 95% CI = 1.009 to 1.118, P = 0.022), each point of: cardiac frequency at admission (β = 1.059, 95% CI = 1.024 to 1.096, P = 0.001), FiO2 (β = 1.034, 95% CI = 1.011 to 1.057, P = 0.003), pCO2 (per mmHg) (β = 1.052, 95% CI = 1.007 to 1.098, P = 0.023); and decreases for each point of: albumin (g/dl) (β = –0.307, 95% CI = 0.095 to 0.986, P = 0.047), mean arterial pressure (mmHg) (β = 0.951, 95% CI = 0.904 to 0.999, P = 0.048), pH (β = 0.002, 95% CI = 0.0 to 0.172, P = 0.006), fi rst ICU 24-hour diuresis (ml) (β = 0.999, 95% CI = 0.998 to 1.000, P = 0.004) and base excess (β = 0.912, 95% CI = 0.836 to 0.995, P = 0.039). Mortality increases threefold (β = 3.134, 95% CI = 1.051 to 9.345, P = 0.04) if patients do not come from the ED, almost 10-fold if the patients need vasopressors for more than 24 hours (β = 9.975, 95% CI = 2.054 to 48.451, P = 0.004) and fi vefold if patients need renal replacement (β = 5.692, 95% CI = 1.466 to 22.099, P = 0.012). 1. Huang L, et al.: N Engl J Med 2006, 355:173-181. 1. Huang L, et al.: N Engl J Med 2006, 355:173-181. g g 2. Akgun K, et al.: J Intensive Care Med 2011, 26:151-164. 2. Akgun K, et al.: J Intensive Care Med 2011, 26:151-164. 2. Akgun K, et al.: J Intensive Care Med 2011, 26:151-164. 2. Akgun K, et al.: J Intensive Care Med 2011, 26:151-164. Prevalence and prognosis of organ dysfunctions of AIDS critically ill patients AJ Japiassu, RA Amâncio, D Medeiros, EM Mesquita, EM Motta, FA Bozza Oswaldo Cruz Foundation, Rio de Janeiro, Brazil Critical Care 2013, 17(Suppl 2):P467 (doi: 10.1186/cc12405) AJ Japiassu, RA Amâncio, D Medeiros, EM Mesquita, EM Motta, FA Bozza Oswaldo Cruz Foundation, Rio de Janeiro, Brazil Critical Care 2013, 17(Suppl 2):P467 (doi: 10.1186/cc12405) Introduction The aim was to analyze the prognosis of AIDS patients with organ dysfunctions at ICU admission. Introduction The aim was to analyze the prognosis of AIDS patients with organ dysfunctions at ICU admission. y Methods A prospective cohort study, including all patients with HIV/ AIDS diagnosis, who were admitted to a specialized ICU from November 2009 until May 2012. Patients with less than 24 hours of ICU stay were excluded. Demographics and nutritional status were collected. The organ dysfunctions were classifi ed according to the SOFA score, and categorized as absent (0 SOFA point), mild (1 to 2 points) and severe (3 to 4 points). We expressed numeric variables as median and interquartile interval (25% to 75%). We performed a multivariate analysis of possible variables associated with hospital mortality (P <0.2), and we explored the 7-day, 28-day and 60-day survival of patients with and without independent risk factors. S175 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Conclusion Neither immune status-related variables nor comorbidity or infection focus are mortality predictors. Poor nutritional status, delayed ICU admission, shock or renal failure increase the ICU relative mortality risk. Tachycardia, hypotension, hypercapnia, acidosis, and oliguria in the fi rst ICU 24 hours increase signifi cantly ICU mortality. Mechanical ventilation is not a mortality predictor. Methods A retrospective study was conducted in consecutive medical and surgical older patients with cancer admitted to a university hospital ICU in São Paulo, between 2007 and 2012. Univariate and multivariate analysis were performed to identify associated and independent factors related to hospital mortality. p y Results From 8,976 patients with cancer requiring ICU at the period, 600 patients were 80 years old or higher. Most patients were male (53%), had solid neoplasm (86%), were from medical admission (54%) and 37% had metastatic disease. The mean age was 84 years (± 3). The ICU and hospital mortality rates were 30% and 44%, respectively. Identifying risk factors for readmission to the ICU: a qualitative approach Results From 597 patients included in the study, 477 were medical admissions (79.8%) and 120 were surgical admissions (20.2%). Four hundred and twenty (70%) patients had metastasis, 222 patients (37%) required the ICU because of respiratory failure and 121 (20%) because of septic shock. The ICU and hospital mortality rates were 53.9% and 64%, respectively. In the univariate analysis, variables associated with hospital mortality were diagnosis of nonsmall-cell lung cancer, higher Charlson morbidity index, medical admission, active neoplasm, vasopressor need at admission to and at 24 hours of ICU, acute renal failure at admission, non-invasive ventilation or mechanical ventilation need at admission to and at 24 hours of ICU and a higher admission arterial lactate. By multivariate analysis, risk factors of hospital mortality were diagnosis of nonsmall-cell lung cancer (OR = 3.32; 95% CI, 2.08 to 5.29, P <0.001), medical admission (OR = 2.89; 95% CI, 1.75 to 4.77, P <0.001), acute renal failure at admission (OR = 4.09; 95% CI, 1.49 to 11.21, P <0.001), non-invasive ventilation at 24 hours of ICU (OR = 2.50; 95% CI, 1.48 to 4.23, P = 0.001) and mechanical ventilation at 24 hours of ICU (OR = 3.68; 95% CI, 1.87 to 7.26, P <0.001). DG Strange, K Tjelle, A Lindhardt, K Jensen Introduction Readmission to the ICU within 48 hours is an indicator of quality of intensive care and is associated with an increase in mortality. During the last years several groups have published data based on multivariate logistic regression analysis to describe characteristics of patients who needed readmission to the ICU. Older age, comorbid conditions and severity of illness (APACHE score) have been among the strongest predictors for readmission. In our ICU most patients are in the groups formerly identifi ed as risk groups, which means that stratifi cation and prediction of readmission is diffi cult. Because of the unusual high severity of acute and pre-existing illnesses we could not fi nd a data match on comparable patient groups. To investigate whether we could reduce our rate of readmission we therefore decided to perform a qualitative investigation to identify risk factors related to readmission. After identifi cation of the risk factors we will take actions to optimize care and perform ongoing control of the implemented actions to secure that they decreases the rate of readmission. Conclusion Hospital survival in patients with lung cancer requiring ICU admission was 36%. P469 Outcome of intensive care treatment for lung cancer patients L Hajjar1, F Galas2, D Goto2, A Tavares2, M Santos2, A Freitas2, T Oliveira2, P Tonini2, L Cavichio2, M Bazan2, J Almeida2, M Cavalcante2, J Fukushima2, S Vieira2, E Osawa2 1Heart Institute, São Paulo, Brazil; 2Cancer Institute of University of São Paulo, Brazil Critical Care 2013, 17(Suppl 2):P469 (doi: 10.1186/cc12407) Introduction Patients with lung cancer commonly require the ICU for a variety of acute illnesses related to the underlying malignancy, treatment, or comorbid conditions. ICU admission of patients with nonresectable lung cancer has been criticized based on the high mortality rate in this population. However, recent advances in critical care may have changed this scenario. The aim of this study was to identify factors associated with hospital mortality in this group of patients. Conclusion Hospital survival in older patients with cancer requiring ICU admission is acceptable. Our results provide evidence that ICU management may be appropriate in older patients with cancer and appoint risk factors for mortality, helping to better triage cancer patients who will benefi t from ICU care. Methods A retrospective study was conducted in consecutive medical and surgical patients with lung cancer admitted to a university hospital ICU in São Paulo, between 2007 and 2012. A univariate analysis was performed to identify associated variables with hospital mortality. Selected variables were included in the multivariate model. Prevalence and prognosis of organ dysfunctions of AIDS critically ill patients In the univariate analysis, variables associated with hospital mortality were diagnosis of lung cancer, medical admission, active neoplasm, vasopressor need at 24 hours of ICU, acute renal failure at admission, mechanical ventilation need at admission to and at 24 hours of ICU and a higher admission arterial lactate. By multivariate analysis, risk factors of hospital mortality were diagnosis of lung cancer (OR = 2.32; 95% CI, 1.08 to 3.29, P <0.001), medical admission (OR = 6.473; 95% CI, 2.76 to 15.15, P <0.001), acute renal failure at admission (OR = 3.09; 95% CI, 1.49 to 9.21, P <0.001), mechanical ventilation at 24 hours of ICU (OR = 5.68; 95% CI, 2.87 to 7.26, P <0.001) and lactate levels at admission (OR = 1.02; 95% CI, 1.006 to 1.051, P <0.001). Identifying risk factors for readmission to the ICU: a qualitative approach Our results provide evidence that ICU management may be appropriate in patients with nonresectable lung cancer and appoint risk factors for mortality, helping to better triage cancer patients who will benefi t from ICU care. Methods Retrospective data on patients readmitted to the ICU within 48 hours during an 8-month period (November 2011 to June 2012) were drawn from the Critical Information System (CIS) at ICU ZIT, Bispebjerg Hospital, Denmark. ZIT is a multidisciplinary unit with 10 beds and 500 to 600 admissions/year and a median SAPS II score of 47. A group of consultants, junior doctors and nurses from the ICU and the ward each read the patient fi les with focus on pattern recognition and suggested trigger points to focus on. Data on trigger points were then drawn from the CIS system and re-evaluated. Finally, fi ve key points were identifi ed and serves as basis for future actions. Complementarity of prognosis models SAPS 3 and EuroSCORE in cardiac surgery Discrimination was assessed using the area under the ROC curve. With the standardized mortality ratio (SMR) we evaluated the agreement between predicted and observed mortality. We used multiple logistic regression for multivariate analysis Introduction The aim of this study is to evaluate the performance of SAPS 3 and EuroSCORE and whether there is complementarity between them in ICU patients admitted after cardiac surgery. Methods An observational, prospective and multicenter study of patients included in the ARIAM registry of adult cardiac surgery. We analyzed clinical variables, surgery data and postoperative compli- cations, outcomes and scores (SAPS 3 and EuroSCORE). Discrimination was assessed using the area under the ROC curve. With the standardized mortality ratio (SMR) we evaluated the agreement between predicted and observed mortality. We used multiple logistic regression for multivariate analysis. y Results A total of 5,642 patients were included, admitted to any fi ve hospitals, consecutively between 2008 and 2012. Mean age was 63.62 ± 12.80 years. Surgery room mortality was 1.4%, ICU mortality was 7.9% and in-hospital mortality was 11.2%. SAPS 3 was calculated in 5,383 patients. SAPS 3 was 40.91 ± 10.46, and the expected rate of mortality was 11.09% (with the southwestern Europe equation), with an observed hospital mortality of 9.6%, SMR = 0.87 (0.79 to 0.94). With the general equation, the expected mortality was 10.47%, SMR = 0.917 (0.843 to 0.996). Discrimination of SAPS 3 was evaluated using the area under the ROC curve, it was 0.77 (0.75 to 0.79). The 30-day mortality was 9.6%, with an expected 30-day mortality predicted by EuroSCORE of 7.86% (SMR = 1.22; CI = 1.12 to 1.32). The area under the ROC curve was 0.734 for EuroSCORE (0.712 to 0.755). Multivariate analysis with logistic regression showed complementarity between SAPS 3 and logistic EuroSCORE (vers. I). There is a relationship between hospital mortality and the probability of dying predicted by SAPS 3 (general equation), OR 1.05 (1.04 to 1.06), and logistic EuroSCORE (vers. I), OR 1.024 (1.015 to 1.032). The model with both variables has a similar discrimination to SAPS 3 alone. The area under the ROC curve of the combined model was 0.778 (0.758 to 0.799) and 0.77 (0.75 to 0.79) using SAPS 3 only. Reference 1. Breslow MJ, Badawi O: Severity scoring in the critically ill: part 1 – interpretation and accuracy of outcome prediction scoring systems. Chest 2012, 141:245-252. P474 g Methods A prospective registry involving patients who underwent LDLT in the period from October 2004 to December 2010. Postoperative 30- day clinical and laboratory data included: clinical BP, HR, temperature, ICU stay, liver functions AST, ALT, GGT, ALP, TotPtn, Alb, TBil, DBil, INR, PTT, CRP, kidney functions Crea, Na, K, blood picture Hb, Hct, TLC and Plt count. Complementarity of prognosis models SAPS 3 and EuroSCORE in cardiac surgery MJ Delgado-Amaya1, E Curiel-Balsera1, MD Arias-Verdu1, E Castillo-Lorente2, FJ Carrero-Gómez3, E Aguayo-DeHoyos4, A Herruzo-Avilés5 1Hospital Regional Carlos Haya, Málaga, Spain; 2Complejo Hospitalario de Jaén, Spain 3Puerta del Mar Hospital, Cádiz, Spain; 4Virgen de las Nieves Hospital, Granada, Spain; 5Virgen del Rocio Hospital, Seville, Spain Critical Care 2013, 17(Suppl 2):P474 (doi: 10.1186/cc12412) Are APACHE II scores better predictors of mortality than routine laboratory values? y Z Baykara, H Özocak, A Kuş, Z Arslan, B Yüksel, C Aksu, M Ertagın, M Solak, K Toker University of Kocaeli, Turkey Critical Care 2013, 17(Suppl 2):P473 (doi: 10.1186/cc12411) Introduction Even though more accurate and updated versions such as APACHE III and IV have been developed, APACHE II has remained the most widely used severity scoring system in the ICU [1]. In the present study, we aimed to develop a model mainly based on the laboratory data available upon admission to predict mortality and to compare its performance with that of APACHE II in a mixed ICU in Turkey. g y Conclusion There is complementarity between the SAPS 3 model and EuroSCORE. However, a joint model with both prognosis scores has no appreciable improvement in discrimination, compared with the SAPS 3 model. In our study, SAPS 3 shows very useful to rule out that the superior observed mortality (compared with the mortality predicted by EuroSCORE) is not attributable to inadequate care of patients in the ICU, the observed mortality being even slightly below the predicted mortality for patients undergoing cardiac surgery at the reference hospitals in which the SAPS 3 system was developed. Methods A total of 645 adult patients who were over 18 years old and who stayed in the ICU for more than 24 hours were included in this study. Using stepwise multivariate logistic regression analysis, a mortality prediction model was developed based on the laboratory data, diagnostic category and age. The adjusted probability of death, according to the diagnostic category of the APACHE II score (adj-APACHE II), was calculated. The ability of the laboratory model and the adj- APACHE II model to discriminate between survivors and nonsurvivors was assessed using receiver operating characteristic (ROC) curves. The calibration of the laboratory model and the adj-APACHE II model was assessed by the Hosmer and Lemeshow goodness-of-fi t test. Complementarity of prognosis models SAPS 3 and EuroSCORE in cardiac surgery p Conclusion Predictors of mortality were INR >3.06 (accuracy 87.5%) followed by PTT >53.3 (79.2%), HR >94 (76.8%), ICU stay >8 days (76.2%), Na >142.9 (72.9%), TBil >15 (71.3%), ALT >379 (67.5%), TProt <4.6 (63.5%), AST >145 (57.4%), Hct <24.4 (54.2%), CRP >12 (Acc 51.6%) and GGT >22 (51.4%). Complementarity of prognosis models SAPS 3 and EuroSCORE in cardiac surgery g y MJ Delgado-Amaya1, E Curiel-Balsera1, MD Arias-Verdu1, MJ Delgado-Amaya1, E Curiel-Balsera1, MD Arias-Verdu1, E Castillo-Lorente2, FJ Carrero-Gómez3, E Aguayo-DeHoyos4, A Herruzo-Avilés5 y Results Our study involved 142 recipients, 124 (87%) males and 18 (13%) females, who underwent living donor liver transplantation; mean age was 53 ± 14.8 years, ranging from 24 to 66 years; 29 cases (20.43%) at Kasr Al Aini University Hospital and 113 cases (79.57%) at the International Medical Center. The 30-day mortality rate was 13.38%. Clinical and laboratory parameters showed gradual resolution towards normal values in survivors or deterioration in nonsurvivors, allowing one to predict mortality at various cutoff points. Clinical parameters: HR >94 cutoff (Acc 76.8%, Sens 89.5%, Spec 62.6%, PPV 27, NPV 97) and ICU stay >8 days (Acc 76.2%, Sens 75.3%, Spec 95.7%, PPV 95.7, NPV 35.3). Laboratory parameters: INR >3.06 cutoff (Acc 87.5%, Sens 92.9%, Spec 78.6%, PPV 96.3, NPV 64.7), followed by PTT >53.3 (Acc 79.2%, Sens 78.6%, Spec 75%, PPV 93.6, NPV 42.9), Na >142.9 (Acc 72.9%, Sens 80%, Spec 72.3%, PPV 26.7, NPV 96.6), TBil >15 (Acc 71.3%, Sens 95.5%, Spec 50%, PPV 92.4, NPV 63.6), ALT >379 (Acc 67.5%, Sens 72.2%, Spec 73.8%, PPV 28.9, NPV 94.7), TProt <4.6 (Acc 63.5%, Sens 55.6%, Spec 73.3%, PPV 24.4, NPV 91.4), AST >145 (Acc 57.4%, Sens 50%, Spec 84.4%, PPV 32.1, NPV 92), Hct <24.4 (Acc 54.2%, Sens 47.4%, Spec 81.5%, PPV 34.6, NPV 88.2), CRP >12 (Acc 51.6%, Sens 43.7%, Spec 73.3%, PPV 90.5, NPV 17.7), GGT >22 (Acc 51.4%, Sens 83.3%, Spec 36.9%, PPV 16.3, NPV 93.7). 1Hospital Regional Carlos Haya, Málaga, Spain; 2Complejo Hospitalario de Jaén, Spain 3Puerta del Mar Hospital, Cádiz, Spain; 4Virgen de las Nieves Hospital, Granada, Spain; 5Virgen del Rocio Hospital, Seville, Spain Critical Care 2013, 17(Suppl 2):P474 (doi: 10.1186/cc12412) 1Hospital Regional Carlos Haya, Málaga, Spain; 2Complejo Hospitalario de Jaén, Spain 3Puerta del Mar Hospital, Cádiz, Spain; 4Virgen de las Nieves Hospital, Granada, Spain; 5Virgen del Rocio Hospital, Seville, Spain Critical Care 2013, 17(Suppl 2):P474 (doi: 10.1186/cc12412) Introduction The aim of this study is to evaluate the performance of SAPS 3 and EuroSCORE and whether there is complementarity between them in ICU patients admitted after cardiac surgery. Methods An observational, prospective and multicenter study of patients included in the ARIAM registry of adult cardiac surgery. We analyzed clinical variables, surgery data and postoperative compli- cations, outcomes and scores (SAPS 3 and EuroSCORE). P470 P470 Outcome of older patients with cancer requiring ICU admission L Hajjar1, M Franca Cavalcante2, F Galas2, J T Fukushima2, M R Sundin2, F Bergamin2, C Park2, H Palomba2, D M Goto2, A Tavares2, M Santos2, A Freitas2, P Tonini2, L Cavichio2, J P Almeida2, M Bazan2 1Heart Institute, São Paulo, Brazil; 2Cancer Institute of University of São Paulo, Brazil Critical Care 2013, 17(Suppl 2):P470 (doi: 10.1186/cc12408) Results In a qualitative analysis, readmissions to the ICU are related to the following fi ve key points – discharge outside day hours, lack of infection control, stay in ICU <2 days, lung physiotherapy ordinated but not eff ectuated, and several minor organ dysfunctions (atrial fi brillation and acute kidney injury). Age, diagnosis, SAPS II score or ventilator treatment during intensive care was not diff erent in patients with successful discharge and patients readmitted in this group of patients. Conclusion It is possible and suitable to identify key points for future eff orts in a given subgroup of patients using a systematic qualitative approach. Critical Care 2013, 17(Suppl 2):P470 (doi: 10.1186/cc12408) Introduction Because the prognosis of older patients with cancer may be poor compared with younger patients, it remains controversial whether they benefi t from ICU treatment. The objective of this study was to identify factors associated with hospital mortality in older patients with cancer requiring the ICU. S176 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 P472 P472 Clinical and laboratory postoperative impact on outcome of living donor liver transplantation recipients Y Nassar, K Omar, A Battah, A Mwafy Cairo University, Giza, Egypt Critical Care 2013, 17(Suppl 2):P472 (doi: 10.1186/cc12410) 2.01 (P = 0.98) for the laboratory model and 13.2 (P = 0.10) for the adj- APACHE II model. Conclusion If the aim is to predict mortality as accurately as APACHE II, the mortality prediction model based mainly on routine admission laboratory tests can achieve this using computer technology, without labor-related costs, as soon as the patient is admitted to the ICU. However, it is necessary to perform multicenter validation studies. Reference Introduction Adult recipients of living donor liver transplantation have a wide range of postoperative medical comorbidities altering various clinical and laboratory parameters. We aimed to assess 30- day postoperative clinical and laboratory changes and their value in predicting outcome of LDLT recipients. P477 Eldicus scores for admission to our ICU: a retrospective study G Zani, B Gemelli, S Vitali, C Villani, R Ragazzi, S Spadaro, CA Volta, R Alvisi University of Ferrara, Italy Critical Care 2013, 17(Suppl 2):P477 (doi: 10.1186/cc12415) Results A total of 273 charts were reviewed. In total, 242 were categorized into A (n = 126), B (n = 79) or C (n = 37). D (n = 31) consisted mainly of patients with hematological malignancies (n = 12) and patients with chemotherapy or immunosuppressive treatment (n = 14). The groups diff ered in length of stay with A< B< C. During the fi rst 3 days the SOFA score was higher in A compared with C and in B compared with C. The duration of antibiotic therapy was longer in both B and C compared with A. There were no diff erences in 28-day mortality (A: 34/126 = 27%, B: 30/79 = 38%, C: 15/37 = 40%); however, the proportions of patients dying between days 8 and 28 were higher in B (14/63 = 22%) and C (7/29 = 24%) compared with A (5/95 = 5%). Introduction The indication for ICU admission is poorly defi ned and identifying patients who may benefi t from it is extremely diffi cult. As reported in literature, the Eldicus scores require further validation in centers in diff erent countries and with diff erent patient populations [1]. Previous studies have demonstrated that patients with intermediate severities of illness are those that benefi t from ICU treatment; instead, patients who are too well or too sick should not be admitted. There are many unmeasurable factors that infl uence the triage decision and can infl uence the subsequent outcome. The Eldicus scores could be the fi rst triage decision rule. The aim of this study was to confi rm the ability of the Eldicus scores to discriminate the survival status in our general ICU. Methods We retrospectively enrolled all patients >18 years admitted to the Ferrara ICU from January to June 2012. Patients’ data were recorded using Prosafe international data collection software and consulting the individual medical records. Data were chosen based on variables used in the Eldicus scores. As indicated in the Eldicus algorithm, for each patient the initial refusal score was calculated, to evaluate the too severe patients. Reference Results A total of 481 patients aged 80 or over were admitted between 1 January 2001 and 31 December 2010, representing 9.6% of the admissions during this time with minimal annual change. Admission age ranged from 80 to 96 years (median 83) with a median APACHE II score of 20 (6 to 48, IQR 16 to 25). In total, 318 (66.1%) survived to critical care discharge and 256 (53.2%) survived to hospital discharge; 228 (89.0%) went home rather than to a care facility. Those under 80 years demonstrated 80.6% critical care survival, 65.5% hospital survival and a median APACHE II score of 17 (0 to 51, IQR 13 to 23). Eighty patients died within the 22-month follow-up period with an average life expectancy of 7.4 months. None reached life expectancy (average curtailment of 5.6 years compared with age-matched and sex-matched population statistics). Neither of these fi gures changed signifi cantly through the 1. Sprung CL, et al.: Crit Care Med 2012, 40:125-131. 1. Sprung CL, et al.: Crit Care Med 2012, 40:125-131. Comparing intensive care treated primary, secondary and tertiary sepsis with respect to length of stay, organ failure and mortality: a pilot study Conclusion Older people represent a growing proportion of the population although their representation in the critical care population remained constant in this 10-year study. These patients had a slightly higher median APACHE II score and 14.5% greater critical care mortality than the younger patients. The majority of survivors were able to go home; however, 31% died within 22 months with signifi cant life expectancy curtailment, surviving on average only 7.4 months after discharge; this has not changed in the last 10 years. Those who survived this initial period (69%) had a much better outlook. This information may be vital to patients and physicians when discussing admission to critical care. p p y Methods The Swedish ICU registry was used to identify all adult patients diagnosed with severe sepsis or septic shock in a single Swedish ICU during 2006 to 2011. The charts were reviewed and patients categorized into four groups based on whether the patient had suff ered systemic infl ammatory-inducing insults previous to the septic episode. The patients were categorized as: (A) primary sepsis, no recent (<30 days) trauma/infection, ongoing systemic infl ammatory or immunosuppressive disease or malnutrition; (B) secondary sepsis, recent (<7 days) trauma/infection or systemic infl ammatory disease, but no immunosuppressive disease or malnutrition; (C) tertiary sepsis, >1 fulfi lled criteria of secondary sepsis during the last 10 days or immunosuppressive disease or malnutrition; and (D) patients not attributable to A to C. After the categorization, physiological and laboratory parameters during the ICU stay as well as therapy and outcome were extracted from the charts. Only signifi cant diff erences (P <0.05) are presented; Mann–Whitney and chi-squared tests were used. P477 Then we counted the fi nal triage score, to discover whether some patients were too healthy to be admitted to the ICU. Correlation between the severity of the scores and patients’ outcome was estimated using the Wilcoxon’s rank-sum test. Conclusion In this retrospective material it was possible to categorize 88.6% of all patients as having primary, secondary or tertiary sepsis. The categories diff ered in clinical picture at presentation as well as in outcome. A prospective study is warranted to validate the results of this study. P476 Comparing intensive care treated primary, secondary and tertiary sepsis with respect to length of stay, organ failure and mortality: a pilot study yi Results The areas under the ROC curves of the laboratory models and the adj-APACHE II scores for the prediction of mortality were 0.80 (95% CI: 0.76 to 0.85) and 0.78 (95% CI: 0.74 to 0.83), respectively (P >0.05, z statistic). The Hosmer–Lemeshow statistic had a chi-squared value of y S Castegren1, M Jonasson1, J Sjölin2, M Castegren1 1Centre for Clinical Research Sörmland, Uppsala University, Uppsala, Sweden; 2Medical Sciences, Uppsala University, Uppsala, Sweden Critical Care 2013, 17(Suppl 2):P475 (doi: 10.1186/cc12413) S Castegren1, M Jonasson1, J Sjölin2, M Castegren1 1Centre for Clinical Research Sörmland, Uppsala University, Uppsala, Sweden; 2Medical Sciences, Uppsala University, Uppsala, Sweden Critical Care 2013, 17(Suppl 2):P475 (doi: 10.1186/cc12413) S177 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 decade. However, 10-year follow-up on 2001/2002 admissions showed that, despite this early mortality eff ect, the average post-discharge lifespan was 5.1 years with 2.2 years shortfall in life expectancy. Conclusion Older people represent a growing proportion of the population although their representation in the critical care population remained constant in this 10-year study. These patients had a slightly higher median APACHE II score and 14.5% greater critical care mortality than the younger patients. The majority of survivors were able to go home; however, 31% died within 22 months with signifi cant life expectancy curtailment, surviving on average only 7.4 months after discharge; this has not changed in the last 10 years. Those who survived this initial period (69%) had a much better outlook. This information may be vital to patients and physicians when discussing admission to critical care. decade. However, 10-year follow-up on 2001/2002 admissions showed that, despite this early mortality eff ect, the average post-discharge lifespan was 5.1 years with 2.2 years shortfall in life expectancy. Introduction It may be hypothesized that a patient’s infl ammatory status might aff ect the clinical picture and outcome of a subsequent septic insult. To test this hypothesis, patients were categorized according to previous infl ammatory activity and analyzed with respect to clinical picture and outcome in a retrospective study. Introduction It may be hypothesized that a patient’s infl ammatory status might aff ect the clinical picture and outcome of a subsequent septic insult. To test this hypothesis, patients were categorized according to previous infl ammatory activity and analyzed with respect to clinical picture and outcome in a retrospective study. Reference 1. Offi ce of National Statistics Population Data [http://www.ons.gov.uk/ons/ taxonomy/index.html?Nscl=Population] 1. Offi ce of National Statistics Population Data [http://www.ons.gov.uk/ons/ taxonomy/index.html?Nscl=Population] P476 Survival of older patients after critical care D Ashton-Cleary, R Sinclair Royal Cornwall Hospital, Truro, UK Critical Care 2013, 17(Suppl 2):P476 (doi: 10.1186/cc12414) Survival of older patients after critical care D Ashton-Cleary, R Sinclair Royal Cornwall Hospital, Truro, UK Critical Care 2013, 17(Suppl 2):P476 (doi: 10.1186/cc12414) g Results Ninety-eight patients were fi nally enrolled. Data are presented as median ± SD. The SAPS II score was 33 ± 13 and was similar to those reported in the literature [1]. The Karnofsky performance status scale was 69 ± 20. The Eldicus initial score was 56 ± 30. The fi nal score was 70 ± 48. Seventy-eight percent of the analyzed patients were alive at hospital discharge. A signifi cant correlation between the severity scores and in-hospital outcome was found (P <0.001). Limitations of our study were the low sample size and the fact that patients not admitted to the ICU were not considered. Introduction We aimed to assess the survival of older patients after admission to critical care compared with the population in general. Introduction We aimed to assess the survival of older patients after admission to critical care compared with the population in general. p admission to critical care compared with the population in general. Methods We reviewed admission data for a 12-bed critical care unit in a district general hospital in the UK between 2001 and 2010. We calculated critical care and hospital discharge survival rates and studied the trend in these through the decade. We searched hospital records to establish whether these patients were still alive at November 2012 to determine life-expectancy (minimum 22-month follow-up on all patients). This was compared with UK Offi ce of National Statistics data for the local population [1]. Discharge destination (care facility or home address) was recorded as a surrogate marker of change in quality of life post discharge. Conclusion The Eldicus scores appear to be useful in identifying patients that may benefi t more from ICU admission. Further studies should confi rm the ability of these scores in helping identify those patients who really benefi t from ICU admission. Reference Prediction of sequences of organ dysfunction in critical patients studied with Bayesian analysis M Sandri1, P Berchialla2, D Gregori3, L Gottin1, V Sweiger1, E Polati1, RA De Blasi4 1University of Verona, Italy; 2University of Torino, Italy; 3University of Padova, Italy; 4University of Rome ‘La Sapienza’, Rome, Italy Critical Care 2013, 17(Suppl 2):P479 (doi: 10.1186/cc12417) p p g p p Results Lower age, NT-Pro-BNP and TnI were signifi cantly associated with 1-year survival. We built a neural network model using the training database. We used the test database to compare our model with the IGS 2 score. The diff erence between the AUC of the biomarker score and the IGS 2 score was statistically signifi cant (Figure 1). Introduction The multiorgan dysfunction syndrome (MODS) is a dynamic process involving simultaneously or consecutively two or more organ systems [1]. The organ dysfunction’s degree can be assessed by three severity scores (SOFA [2], MODS [3], LODS [4]), but they have some limitations: they do not allow the evaluation of the clinical course of a patient, they are not reliable in populations diff erent from the reference one, and they do not support clinicians’ decisions. Because MODS implies a systemic infl ammatory reaction leading to microcirculatory dysfunction, our hypothesis was that organ failures follow a predictable sequence of appearance. Our aims were to verify the presence of more likely organ failure sequences and to assess an online method to predict the evolution of MODS in a patient. The high mortality and morbidity rate of MODS in ICUs can in fact be reduced only by a prompt and well-timed treatment [5]. y gi g Conclusion Our new neural network model built using age, NT-PRO- BNP and TnI succeeded the internal-validation processes and predicts 1-year mortality better than IGS 2. P481 Signifi cance of preadmission quality of life for mortality in the ICU: a prospective cohort study RB Bukan1, A Moeller1, M Henning1, K Mortensen2, T Klausen1, T Waldau1 1Herlev University Hospital, Herlev, Denmark; 2University of Copenhagen, Denmark Critical Care 2013, 17(Suppl 2):P481 (doi: 10.1186/cc12419) Figure 1 (abstract P480). Comparison of area under ROC curves (AUC) of the biomarker score and IGS 2. Figure 1 (abstract P480). Comparison of area under ROC curves (AUC) of the biomarker score and IGS 2. Methods We selected 73 patients consecutively admitted to the ICU of Sant’Andrea Hospital from January to June 2012. The inclusion criteria were at least two organ systems with SOFA ≥2, ICU length of stay >48 hours. Developing a screening instrument for predicting psychological morbidity after critical illness Ö A Schandl1, M Bottai2, E Hellgren1, Ö Sundin3, P Sackey1 1Karolinska University Hospital Solna, Stockholm, Sweden; 2Karolinska Institutet, Stockholm, Sweden; 3Mid Sweden University, Östersund, Sweden Critical Care 2013, 17(Suppl 2):P478 (doi: 10.1186/cc12416) Introduction The purpose was to develop a predictive screening instrument for use at ICU discharge to identify patients at risk for post- traumatic stress, anxiety or depression. Introduction The purpose was to develop a predictive screening instrument for use at ICU discharge to identify patients at risk for post- traumatic stress, anxiety or depression. S178 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Methods Potential risk factors for psychological problems were prospectively collected at ICU discharge. Two months after ICU discharge 252 ICU survivors received the questionnaires Post- Traumatic Stress Symptom scale-10 (PTSS-10) and Hospital Anxiety and Depression Scale (HADS) to estimate the degree of post-traumatic stress, anxiety and depression. Sepsis-Related Problems of the European Society of Intensive Care Medicine. Intensive Care Med 1996, 22:707-710. Marshall JC, et al.: Multiple organ dysfunction score: a reliable descriptor of a complex clinical outcome. Crit Care Med 1995, 23:1638-1652. Le Gall JR, et al.: The logistic organ dysfunction system: a new way to assess organ dysfunction in the intensive care unit. JAMA 1996, 276:802-810. Kilica YA, et al.: Visualizing multiple organ failure: a method for analyzing temporal and dynamic relations between failing systems and interventions. Crit Care 2007, 11:417. y Results Of the 150 responders, 31% had adverse psychological outcome, defi ned as PTSS-10 >35 and/or HADS subscales ≥8. After analysis, six predictors with weighted risk scores were included in the screening instrument: major pre-existing disease, being a parent to children younger than 18 years of age, previous psychological problems, in-ICU agitation, being unemployed or sick-listed at ICU admission and appearing depressed in the ICU. Each predictor corresponded to a given risk score. The total risk score, the sum of individual risk scores, was related to the probability for adverse psychological outcome in the individual patient. The predictive accuracy of the screening instrument, as assessed with area under the receiver operating curve, was 0.77. When categorizing patients in three risk probability groups – low (0 to 29%), moderate (30 to 59%) and high (60 to 100%) risk – the actual prevalence of adverse psychological outcome in respective groups was 12%, 50% and 63%. 6. Prediction of sequences of organ dysfunction in critical patients studied with Bayesian analysis For each patient we calculated the SOFA since the beginning of the inclusion criteria and daily for 8 days. For the statistical analysis we used Dynamic Bayesian Networks (DBNs) [6]. DBNs were applied to model SOFA changes in order to identify the most probable sequences of organs failures in a patient who experienced a fi rst known failure. gi Results We created a DBN for the analysis of MODS studying the relations between organ failures at diff erent times. The DBN was made so that each organ failure is dependent on the previous one. We also considered a corrective factor to take account that not all patients completed the observation. Using software (GeNie) we obtained the probabilities of the organ failure sequences. Figure 1 (abstract P480). Comparison of area under ROC curves (AUC) of the biomarker score and IGS 2. Figure 1 (abstract P480). Comparison of area under ROC curves (AUC) of the biomarker score and IGS 2. g Conclusion The use of DBNs, although with our limited set of data, allowed us to identify the most likely organ dysfunction sequences associated with a fi rst known one. Capability to predict these sequences in a patient makes DBNs a promising prognostic tool for physicians in order to treat patients in a timely manner, or to test a treatment effi cacy. References Developing a screening instrument for predicting psychological morbidity after critical illness Ö Murphy K, et al.: Modelling Gene Expression Data using Dynamic Bayesian Networks. Technical report. University of California; 1999. Comparison of IGS 2 score and a new neural network biomarker score performance in predicting 1-year mortality Comparison of IGS 2 score and a new neural network biomarker score performance in predicting 1-year mortality C Romdhani, I Labbene, M Belhadj Amor, Z Hajjej, A Massoudi, M Ferjani Military Hospital of Tunis, Tunisia Critical Care 2013, 17(Suppl 2):P480 (doi: 10.1186/cc12418) C Romdhani, I Labbene, M Belhadj Amor, Z Hajjej, A Massoudi, M Ferjani Military Hospital of Tunis, Tunisia Critical Care 2013, 17(Suppl 2):P480 (doi: 10.1186/cc12418) Introduction The aim of this study was to compare the IGS 2 score with a predictive model built using clinical characteristics and biomarkers associated with 1-year mortality from procalcitonin, C-reactive protein, troponin I (TnI) and NT-Pro-BNP. Conclusion The preliminary screening instrument may aid ICU clinicians in identifying patients at risk for adverse psychological outcome after critical illness. Prior to wider clinical use, external validation is needed. Methods We conducted a prospective observational cohort study in a polyvalent ICU. We calculated the IGS 2 severity score in the fi rst 24 hours. We dosed NT-Pro-BNP, TnI, procalcitonin and C-reactive protein at admission to the ICU. To test the biomarker model, the patient database was divided into training (70%) and test (30%) databases. A neural network model was trained from variables associated with 1-year mortality. All statistics were done using R v.2.15.1. We used the pROC package to plot and compare ROC curves.i P481 Signifi cance of preadmission quality of life for mortality in the ICU: a prospective cohort study RB Bukan1, A Moeller1, M Henning1, K Mortensen2, T Klausen1, T Waldau1 1Herlev University Hospital, Herlev, Denmark; 2University of Copenhagen, Denmark Critical Care 2013, 17(Suppl 2):P481 (doi: 10.1186/cc12419) Predicting physical function and mental health in trauma intensive care patients 2 years after hospitalisation Predicting physical function and mental health in trauma intensive care patients 2 years after hospitalisation L Aitken1, B Macfarlane1, W Chaboyer2, M Schuetz1, C Joyce1, AG Barnett3 1Princess Alexandra Hospital, Brisbane, Australia; 2Griffi th University, GoldCoast, Australia; 3Queensland University of Technology, Brisbane, Australia Critical Care 2013, 17(Suppl 2):P482 (doi: 10.1186/cc12420) gi Results We present the results of an 18-month period, 9 months prior to and 9 months after the introduction of the SwissDRG. Data of 1,491 and 1,492 patients were analyzed, respectively. When all patients were included, we found prior to and after the introduction of the DRGs a comparable length of stay on the ICU (mean ± SD of 52.1 ± 2.2 hours vs. 50.82 ± 2.2 hours), no diff erence in the severity of illness at discharge detected by the SAPS II (mean ± SD of 27.9 ± 0.3 vs. 28.4 ± 0.3) and the number of readmissions (91 vs. 92). There was also no signifi cant diff erence when only percentiles 6 to 94 were included or when the three subgroups were analyzed. p y p L Aitken1, B Macfarlane1, W Chaboyer2, M Schuetz1, C Joyce1, AG Barnett3 1Princess Alexandra Hospital, Brisbane, Australia; 2Griffi th University, GoldCoast, Australia; 3Queensland University of Technology, Brisbane, Australia Critical Care 2013, 17(Suppl 2):P482 (doi: 10.1186/cc12420) Introduction Long-term compromise after traumatic injury is signifi cant; however, few modifi able factors that infl uence outcome have been identifi ed. The aim of this study was to identify acute and early post-acute predictors of long-term recovery amenable to change through intervention. g p y Conclusion Up to now, the introduction of the SwissDRG has no infl uence on patients’ discharge characteristics from a large ICU. Data assessment will continue and further data analysis has to be performed. Methods Adults (>17 years) admitted to the ICU, Princess Alexandra Hospital, Australia following injury were prospectively followed. Data were collected on demographics, pre-injury health, injury characteristics and acute care factors. Psychosocial measures (self- effi cacy (SE), illness perception (IP), post-traumatic stress disorder (PTSD) symptoms and psychological distress) and health status (SF-36) were collected via questionnaire 1, 6, 12, and 24 months post injury. Outcomes of interest were the Physical Function (PF) and Mental Health (MH) subscales of the SF-36. Regression models were used to estimate predictors of physical function and mental health over a 2-year period. Do ICU patients’ discharge characteristics change with Swiss Diagnosis Related Groups? Do ICU patients’ discharge characteristics change with Swiss Diagnosis Related Groups? Do ICU patients’ discharge characteristics change with Swiss Diagnosis Related Groups? G Rauchenstein-Ziltener, CA Rüst, P Fodor, S Blumenthal Tiemli City Hospital, Zurich, Switzerland Critical Care 2013, 17(Suppl 2):P483 (doi: 10.1186/cc12421) g G Rauchenstein-Ziltener, CA Rüst, P Fodor, S Blumenthal Tiemli City Hospital, Zurich, Switzerland Critical Care 2013, 17(Suppl 2):P483 (doi: 10.1186/cc12421) Results Preliminary results, based upon data from 175 included patients, reveal an ICU mortality of 12.6% and 30-day mortality of 22.9%. No patients were lost to follow-up. When the Physical Component of SF-36 (PCS) was used as an estimate of preadmission quality of life, the area under the curve for model B (AUC = 0.80) was comparable with model E (AUC = 0.81), and better than model A (AUC = 0.85). The General Health item of SF-36 (GH), used as an estimate of preadmission quality of life, gave an AUC = 0.76 (model D). All models were controlled for sex and age. Introduction Swiss Diagnosis Related Groups (SwissDRG) have been eff ective since 1 January 2012. The infl uence of this new system on patients’ discharge characteristics from a large ICU is not known. With the introduction of the DRG we expect patients to be discharged after a shorter length of stay on the ICU and with higher severity of illness. Methods The ICU of the City Hospital Triemli in Zurich has an inter- disciplinary organization with surgical and internal medical patients, with a maximum occupancy of 18 beds and a center function for the surrounding hospitals. In this ongoing prospective observational study, we collect and analyze the anonymized data of all patients discharged from our ICU prior to and after the introduction of the SwissDRG. The primary endpoint was the length of stay on the ICU in hours. The secondary endpoints were the severity of illness of the patients at the time of discharge, detected by the scoring system SAPS II as well as measured by the number of readmissions to the ICU. Initially all patients were analyzed and in a second step only patients within percentiles 6 to 94 were considered. We also analyzed the subgroups of patients referred internally, patients sent back to referring hospital and patients regionalized to a homebase hospital. The statistics have been done with SPSS and P <0.05 was considered signifi cant. P481i P481 Signifi cance of preadmission quality of life for mortality in the ICU: a prospective cohort study RB Bukan1, A Moeller1, M Henning1, K Mortensen2, T Klausen1, T Waldau1 1Herlev University Hospital, Herlev, Denmark; 2University of Copenhagen, Denmark Critical Care 2013, 17(Suppl 2):P481 (doi: 10.1186/cc12419) Signifi cance of preadmission quality of life for mortality in the ICU: a prospective cohort study p p y RB Bukan1, A Moeller1, M Henning1, K Mortensen2, T Klausen1, T Waldau1 1Herlev University Hospital, Herlev, Denmark; 2University of Copenhagen, Denmark 1. Levy MM, et al.: 2001 SCCM/ESICM/ACCP/ATS/SIS International Sepsis Defi nitions Conference. Intensive Care Med 2003, 29:530-538. Levy MM, et al.: 2001 SCCM/ESICM/ACCP/ATS/SIS International Sei 2. Vincent JL, et al.: The SOFA (Sepsis-Related Organ Failure Assessment) score to describe organ dysfunction/failure. On behalf of the Working Group on Critical Care 2013, 17(Suppl 2):P481 (doi: 10.1186/cc12419) S179 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Introduction Assessing whether a critically ill patient should be admitted to an ICU remains diffi cult and mortality amongst ICU patients is high. To render intensive care with no prospect of success is an immense emotional burden for both patient and relatives, and a great socioeconomic burden for society as well. Therefore, validated strategies that can help identify patients who will benefi t from intensive care are in demand. This study seeks to investigate whether preadmission quality of life can act as a predictor of mortality amongst patients admitted to the ICU. Predictors of PF included IP (β = –1.5, 95% CI = –3.1 to –1.1, P <0.01), SE (β = 1.8, 95% CI = 1.3 to 2.6, P <0.01), hospital length of stay (β = –1.7, 95% CI = –2.0 to –0.8, P <0.01), never having been married (β = 1.8, 95% CI = 0.3 to 5.5, P = 0.03), and having injury insurance (β = –2.7, 95% CI = –6.9 to –1.9, P <0.01). Predictors of MH included PTSD symptoms (β = –2.4, 95% CI –3.4 to –1.4, P <0.01), psychological distress (β = –6.9, 95% CI = –8.9 to –5.2, P <0.01), SE (β = 0.6, 95% CI = 0.2 to 1.1, P <0.01), and unemployment (β = –2.3, 95% CI = –5.0 to –0.2, P = 0.04). P481i Predictors of PF included IP (β = –1.5, 95% CI = –3.1 to –1.1, P <0.01), SE (β = 1.8, 95% CI = 1.3 to 2.6, P <0.01), hospital length of stay (β = –1.7, 95% CI = –2.0 to –0.8, P <0.01), never having been married (β = 1.8, 95% CI = 0.3 to 5.5, P = 0.03), and having injury insurance (β = –2.7, 95% CI = –6.9 to –1.9, P <0.01). Predictors of MH included PTSD symptoms (β = –2.4, 95% CI –3.4 to –1.4, P <0.01), psychological distress (β = –6.9, 95% CI = –8.9 to –5.2, P <0.01), SE (β = 0.6, 95% CI = 0.2 to 1.1, P <0.01), and unemployment (β = –2.3, 95% CI = –5.0 to –0.2, P = 0.04). p y β Conclusion Trauma ICU patients experience compromised physical function and mental health 24 months after injury. Psychological distress, self-effi cacy and illness perception infl uence outcomes and are potentially amenable to change in response to interventions initiated during hospital stay. p Methods All patients (>18 years) admitted to the ICU for more than 24 hours are included. In order to assess preadmission quality of life, the patient or close relatives complete the Short-Form 36 (SF-36) within 72 hours after ICU admission. Mortality is evaluated from ICU admission until 30 days hereafter. Logistic regression and receiver operating characteristic analyses are employed to assess predictive value for mortality using fi ve models: SF-36 Physical Component Summary (PCS) and APACHE II (model A), SF-36 PCS (model B), SF-36 General Health (GH) and APACHE II (model C), SF-36 GH (model D), and APACHE II (model E). Classifi cation tables are composed in order to assess sensitivity, specifi city, positive and negative predictive values and likelihood ratios. Do ICU patients’ discharge characteristics change with Swiss Diagnosis Related Groups? Conclusion Preliminary results indicate that the SF-36 GH and the SF-36 PCS are as good as APACHE II to predict mortality 30 days after ICU admission. However, in order to conclude whether preadmission quality of life can contribute to triage, by successfully identifying patients suitable for intensive care, fi nal analyses, due in 2013, are awaited. These results will clarify whether future randomized controlled trials, in which preadmission quality of life acts as a supplement to triage, are justifi able. P484 Do Diagnosis Related Groups change admission practice to a large Swiss ICU? K Homburg, CA Rüst, P Fodor, S Blumenthal Triemli City Hospital, Zurich, Switzerland Critical Care 2013, 17(Suppl 2):P484 (doi: 10.1186/cc12422) P484 Do Diagnosis Related Groups change admission practice to a large Swiss ICU? K Homburg, CA Rüst, P Fodor, S Blumenthal Triemli City Hospital, Zurich, Switzerland Critical Care 2013, 17(Suppl 2):P484 (doi: 10.1186/cc12422) Predicting physical function and mental health in trauma intensive care patients 2 years after hospitalisation A subject-specifi c intercept in a mixed model was used to account for repeated data from participants over time. P484 Do Diagnosis Related Groups change admission practice to a large Swiss ICU? Introduction The new Swiss fi nancing system by means of Diagnosis Related Groups (DRG), eff ective 1 January 2012, forms the basis of a performance-based system to reimburse general hospital services. There are only few data on the infl uence of DRG on ICU patients [1,2]. We expect that the introduction of the DRG in Switzerland will change the number of admissions from external hospitals to a large ICU with a centre function and will infl uence the severity of disease of the admitted patients. Introduction The new Swiss fi nancing system by means of Diagnosis Related Groups (DRG), eff ective 1 January 2012, forms the basis of a performance-based system to reimburse general hospital services. There are only few data on the infl uence of DRG on ICU patients [1,2]. We expect that the introduction of the DRG in Switzerland will change the number of admissions from external hospitals to a large ICU with a centre function and will infl uence the severity of disease of the admitted patients. p p p Results Participants (n = 123) were young (median 37, IQR 22 to 55 years), predominantly male (83%) and spent on average 3 days in the ICU and 3 weeks in hospital. Response rates were over 55% at each follow-up, with responders similar to nonresponders except for being generally older. PF and MH scores improved over time, although the averages remained below the Australian norms at 24 months. S180 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Methods The ICU of the Triemli City Hospital in Zurich has an inter- disciplinary organisation with surgical and internal medical patients, with a maximum occupancy of 18 beds and a centre function for the surrounding hospitals of the region. In this prospective ongoing observational study, we collect and analyse the anonymised data of all patients admitted to our ICU from an external hospital during 12 months prior to (1 January to 31 December 2011) and after (1 January to 31 December 2012) the introduction of the DRG in Switzerland. Exclusion criteria are admissions by the emergency department, self-assignments into the hospital and internal relocations. The primary endpoint is the number of admissions from an external hospital to our ICU. References References 1. McLaughlin AM, et al.: Intensive Care Med 2009, 35:2135-2140. 2. Rossi C, et al.: Intensive Care Med 2006, 32:545-552. References 1. McLaughlin AM, et al.: Intensive Care Med 2009, 35:2135-2140. 2. Rossi C, et al.: Intensive Care Med 2006, 32:545-552. P486 Variable cost of a Greek ICU E Tsigou1, D Karabatsou1, E Boutzouka1, V Psallida1, G Baltopoulos1, M Tsironi2 1A. Anargiroi Hospital, Athens, Greece; 2University of Peloponnisos, Sparti, Greece Critical Care 2013, 17(Suppl 2):P485 (doi: 10.1186/cc12423) Critical Care 2013, 17(Suppl 2):P485 (doi: 10.1186/cc12423) Introduction The ICU consumes almost 20% of total hospital resources, despite accounting for less than 10% of hospital beds. Medical cost can be divided into a fi xed part, consisting of personnel and accommodation expenses, and a variable part, determined by the needs of each patient (medications, consumables, examinations). In order to rationalize medical cost, estimation of expenses must be real and individualized. The aim of this study was to assess the variable cost of critically ill patients in a new, seven-bed, adult, general ICU, using bottom-up costing methodology. Conclusion The latest audit follows introduction of a referral system directly to the ICU consultant and may account for the reduction in numbers of referrals attended by junior doctors. ED/medicine persist as the main source of referral to the ICU. Discussion with the referring team consultant may reduce inappropriate referrals. ICU staffi ng should not be reduced. Methods All 138 patients who were treated during 2011, and stayed for at least 24 hours, were included in the study. Data were retrospectively collected from patient records and included demographics, cause of admission, APACHE II score at admission, length of stay (LOS) and outcome. Cost was recorded for every day and for every patient, based on a hospital-specifi c cost catalogue and on national agreements. Data are presented as mean ± SEM. Analysis of data was carried out using Graph Pad Prism 5.0., applying Student’s t test. References 1. Ahmad M, et al.: Chest 1988, 93:176-179. 2. Thomas F, et al.: Chest 1987, 91:418-423. 1. Ahmad M, et al.: Chest 1988, 93:176-179. Results There were 105 occasions when a junior doctor left the unit in 2011, totalling 118 hours and 38 minutes (5 days), a continuation of the upward trend from the previous 2 years. In 2012 this reduced to 47 occasions totalling 40 hours and 55 minutes. Most attended referrals were during the daytime, 54% in 2011 and 68% in 2012. The majority of referrals originated from the ED/medicine, combined numbers 58% in 2011 and 38% in 2012. In 2011 only 30% of ED referrals had been discussed with a consultant of the referring team and only 32% of medical referrals. Just two of the 14 inappropriate ED referrals were discussed with the referring consultant, and none of the six inappropriate medical referrals were discussed with a medical consultant. In 2012 just 33% of ED/medical referrals had prior discussion with a consultant from the referring team. At the point of leaving, the ICU was left without a doctor on 22 occasions in 2011 and six occasions in the 2012 audit. Most occasions occurred at either night shift/weekend (86% in 2011/100% in 2012). Referral resulted in a patient being admitted to the ICU on 20 occasions in 2011 and 12 in 2012. 2. Thomas F, et al.: Chest 1987, 91:418-423. Audit of the medical staffi ng of and referrals to a mixed adult ICU in a teaching hospital g p A Needham, T Lyndham, D O’Neil, J Greiff University Hospitals of Leicester, UK Critical Care 2013, 17(Suppl 2):P486 (doi: 10.1186/ Introduction Leicester Royal Infi rmary is a (funded) 15-bed ICU, annual admission 1,102 patients per year (ICNARC data 2011 to 2012). It houses a broad range of specialty, has over 1,000 beds and an emergency department (ED) responsible for 297 admissions over the 2011 to 2012 period. Unit guidelines state that a doctor should be immediately available to the unit at all times, ideally physically present on the unit. This audit looked in detail at each occasion a junior doctor left the unit, the timings, reason, and outcome of each episode. Results We present the preliminary data for 10 months (in each case January to October) before and after the introduction of the DRG. We observed an increase of 9.2% (391 vs. 427 patients) of admissions to our ICU after the introduction of the DRG. The severity of disease determined by the SAPS II score is unchanged (mean 26.7 vs. 26.0 points, P = 0.466). The severity of disease determined by the APACHE II score is signifi cantly lower (15.4 vs. 14 points, P = 0.017). We also noted that after the introduction of the DRG the patients were earlier transferred from an external hospital to our ICU (mean time until transfer 29.9 vs. 18.7 hours), but this value was not signifi cant (P = 0.55). Conclusion Up to now the introduction of the DRG in Switzerland has had a complex infl uence on the number and the kind of patients admitted from an external hospital to an ICU with centre function. Data assessment and analysis will continue. g p Methods Between 17 October 2011 and 14 November 2011, and between 2 July 2012 and 30 July 2012, junior doctors on the ICU completed a data-capture form after each occasion that they left the unit for a clinical need. Data collected were matched to the objectives. The 2011 audit results were communicated at the University Hospitals of Leicester Trust ICU Audit Meeting. A system was introduced so that daytime referrals were directed to the ICU consultant. The impact of this intervention was assessed by the 2012 re-audit. Do Diagnosis Related Groups change admission practice to a large Swiss ICU? Secondary endpoints are the severity of the disease of the admitted patients, detected by the scoring systems SAPS II and APACHE II as well as the length of stay in external hospitals before admission. The statistical analysis is descriptive. variable cost of intensive care treatment. Among drugs, antibiotics accounted for the largest part. A medical cause of admission was correlated with higher costs. variable cost of intensive care treatment. Among drugs, antibiotics accounted for the largest part. A medical cause of admission was correlated with higher costs. Impact of the time elapsed between ICU request and actual admission on mortality and length of stay Introduction The World Bank has warned that the rapid growth of the world’s urban population can only be accommodated safely if cities adequately develop key infrastructure, such as the provision of acute care resources. Yet, even basic descriptive information on urban acute care supply and demand is extremely limited. We therefore conducted a pilot assessment across seven diverse urban settings across the world. Methods We selected a convenience sample of seven large cities with varying geographical and socioeconomic characteristics: Boston, Paris, Bogota, Recife, Liaocheng, Chennai, and Kumasi. To estimate acute care supply, we developed an instrument to collect data on acute and critical care infrastructure. We collected data from municipal authorities and local research collaborators. We expressed the burden of acute disease as the number of deaths due to acute illnesses, estimated from the 2008 Global Burden of Disease Study. Results were expressed as acute care supply and acute deaths per 100,000 population and acute care supply per 100 acute deaths. Introduction The World Bank has warned that the rapid growth of the world’s urban population can only be accommodated safely if cities adequately develop key infrastructure, such as the provision of acute care resources. Yet, even basic descriptive information on urban acute care supply and demand is extremely limited. We therefore conducted a pilot assessment across seven diverse urban settings across the world. Methods We selected a convenience sample of seven large cities with varying geographical and socioeconomic characteristics: Boston, Paris, Bogota, Recife, Liaocheng, Chennai, and Kumasi. To estimate acute care supply, we developed an instrument to collect data on acute and critical care infrastructure. We collected data from municipal authorities and local research collaborators. We expressed the burden of acute disease as the number of deaths due to acute illnesses, estimated from the 2008 Global Burden of Disease Study. Results were expressed as acute care supply and acute deaths per 100,000 population and acute care supply per 100 acute deaths. Results In total, 413 patients were included, 300 of which belonged to the LWP group (65.4%). For the entire cohort, the mean APACHE II score was 19 ± 7, the mean age was 52 ± 22 years, and 211 patients were male (51.1%). The LWP group did not show diff erence in the APACHE II score (19 ± 7 vs. 18 ± 8, P = 0.13), but was older (55 ± 20 vs. Impact of the time elapsed between ICU request and actual admission on mortality and length of stay 49 ± 23, P = 0.01). LWP also had a higher incidence of primary bloodstream infection (23.8% vs. 10.4%, P = 0.01) and catheter-associated urinary tract infection (10.2% vs. 1.9%, P = 0.01). LWP patients had higher mortality (37.8% vs. 25.9%, P = 0.02) and longer ICU LOS (21 ± 47 vs. 14 ± 18 days, P = 0.01). Relative risk for death in the LWP was 1.74 (95% CI: 1.11 to 2.72). Conclusion Despite showing no signifi cant diff erences on APACHE II scores from the SWP group, patients from the LWP group presented greater incidence of primary bloodstream infection, catheter- associated urinary tract infection, higher mortality outcomes and longer ICU LOS. pp y p Results The supply of hospital beds varied from 72.4/100,000 population in Kumasi to 245.8/100,000 in Boston. ICU beds with capacity for invasive mechanical ventilation and intensive nursing services ranged from 0.4/100,000 in Kumasi to 19/100,000 population in Boston. The number of ambulances varied 70-fold between cities. The gap between cities widened when demand was estimated based on disease burden, with a 70-fold diff erence between cities in ICU beds/acute deaths. In general, most of the data were unavailable from municipal authorities. P490f P490 Diff erences in severity and resource utilization for medical and surgical ICU patients BH Nathanson1, WT McGee2, E Lederman2, TL Higgins2 1OptiStatim LLC, Longmeadow, MA, USA; 2Baystate Medical Center, Springfi eld, MA, USA Critical Care 2013, 17(Suppl 2):P490 (doi: 10.1186/cc12428) Impact of the time elapsed between ICU request and actual admission on mortality and length of stay GM Filho1, TA Silva1, AR Santana1, FB Soares1, LJ Almeida1, LG Godoy1, TA Rodrigues1, MO Maia2, JA Neto2, AP Amorim3, EB Moura2, FF Amorim1 1Escola Superior de Ciências da Saúde, Brasília, Brazil; 2Unidade de Terapia Intensiva Adulto do Hospital Santa Luzia, Brasilia, Brazil; 3Liga Academica de Medicina Intensiva do Distrito Federal (LIGAMI), Brasilia, Brazil Critical Care 2013, 17(Suppl 2):P487 (doi: 10.1186/cc12425) Results The age of participants (84 men and 54 women, 71 medical and 67 surgical) was 68.75 ± 1.18, APACHE II score was 18.64 ± 0.61, LOS was 18.46 ± 2.54, and mortality was 19%. The majority of the patients (61%) were mechanically ventilated. The total days of ICU stay were 2,548 and the total variable cost was €1,460,465 (€573.18 per patient and per day). Cost per patient was subdivided as follows: medication (including drugs, fl uids, blood products, nutrition): 56.49%, examinations (including laboratory, microbiological assays and diagnostic procedures): 22.23%, consumables: 21.26%. As for medication cost, the largest part was comprised of antibiotics (45.69%), followed by blood products (17.61%) and cardiovascular drugs (12.39%). Costs for medical patients were signifi cantly higher than those for surgical patients (P <0.0001). Conclusion The total average cost per patient and per day was found to be €573.18. Medication expenditure was responsible for the highest Introduction Measures to ensure an appropriate early treatment for critically ill patients result in signifi cant decreases in mortality [1,2]. This study aims to evaluate the impact of the time elapsed from request until admission to the ICU on mortality and ICU length of stay (LOS). Methods A retrospective cohort study performed on patients in the ICU of Hospital Regional de Samambaia over a period of 4 years, from January 2008 to December 2011. The patients were allocated into two groups: patients who waited longer than 6 hours, long waiting period S181 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 (LWP, n = 300); and patients whose waiting time was equal to or less than that period, short waiting period (SWP, n = 113). (LWP, n = 300); and patients whose waiting time was equal to or less than that period, short waiting period (SWP, n = 113). Diff erences in severity and resource utilization for medical and surgical ICU patients BH Nathanson1, WT McGee2, E Lederman2, TL Higgins2 1OptiStatim LLC, Longmeadow, MA, USA; 2Baystate Medical Center, Springfi eld, MA, USA Methods Several disasters are studied. The Fukushima Daiichi Nuclear plant explosion after the Higashinihon earthquake 2011 was medically reviewed based on the total 30-day stay on-site in addition to several days around the site. The Chernobyl incident 1986 was inspected 15 years after the incident. Other nuclear disasters are included. p gi Critical Care 2013, 17(Suppl 2):P490 (doi: 10.1186/cc12428) Introduction Medical and surgical patients use the ICU diff erently. Resources may not always be allocated by severity of illness, but by custom or habit, particularly if diff erent groups administer bed control and triage. Specialty-specifi c diff erences may exist even when a single team controls triage. Variability in resource utilization has important implications for cost-containment and triage. Results Many serious problems were revealed in the medical teams, which are as follows: inappropriate basic preparedness against large special disasters, including nuclear disaster; lack of appropriate education and training for medical teams against nuclear disaster – that is, most members of Japan DMAT or the disaster medical assistance team are still laypersons; incorrect standard/rules of Japan DMAT, which were excessively focused upon cure of the usual type of injury and planned short period or nearly 48 hours, which should be abandoned; and insuffi cient consideration to the weak/vulnerable people or CWAP, children, (pregnant) women, aged people, and the poor people/sicker patients. Many of them died because of an insuffi cient emergency transportation system from their contaminated houses or hospital. Methods Patients admitted to a single, closed medical/surgical ICU with full-time intensivists and unifi ed triage control in a large, university-affi liated hospital were evaluated during 2011 to 2012. Patients who died in the ICU were excluded. The day of discharge (D/C) and severity using APACHE IV and its related Acute Physiology Score Figure 1 (abstract P490). y Conclusion In order to cope with the special disasters, such as NBC or nuclear, biological and chemical disaster, it is insuffi cient to take makeshift measures or use cheap tricks. Working out the systematization of disaster medicine, based upon the academic viewpoints and philosophy/reliability, is essential to protect the people and the nation. g References 1. Amorim FF, et al.: J Clin Med Res 2012, 4:410-414. 1. Amorim FF, et al.: J Clin Med Res 2012, 4:410-414. 2. Rivers E, et al.: N Engl J Med 2001, 345:1368-1377. 1. Amorim FF, et al.: J Clin Med Res 2012, 4:410-414. 2. Rivers E, et al.: N Engl J Med 2001, 345:1368-1377. 2. Rivers E, et al.: N Engl J Med 2001, 345:1368-1377. Medical problems: review from the major nuclear incidents Medical problems: review from the major nuclear incidents Y Haraguchi1, Y Tomoyasu2, H Nishi3, M Hoshino4, M Sakai5, E Hoshino6 1Disaster Medicine Compendium Team, Japan, Tokyo, Japan; 2Shirahigebashi Hospital, Tokyo, Japan; 3Tokyo Disaster Medical Center, Tokyo, Japan; 4Shisei Hospital, Saitama, Japan; 5Tokyo Women’s Medical College, Tokyo, Japan; 6Chiba Nursing Association, Chiba, Japan Critical Care 2013, 17(Suppl 2):P488 (doi: 10.1186/cc12426) Medical problems: review from the major nuclear incidents Y Haraguchi1, Y Tomoyasu2, H Nishi3, M Hoshino4, M Sakai5, E Hoshino6 1Disaster Medicine Compendium Team, Japan, Tokyo, Japan; 2Shirahigebashi Hospital, Tokyo, Japan; 3Tokyo Disaster Medical Center, Tokyo, Japan; 4Shisei Hospital, Saitama, Japan; 5Tokyo Women’s Medical College, Tokyo, Japan; 6Chiba Nursing Association, Chiba, Japan Critical Care 2013, 17(Suppl 2):P488 (doi: 10.1186/cc12426) Conclusion The provision of acute care services, a key aspect of urban infrastructure, varied substantially across the seven diverse urban settings we studied. Furthermore, the local municipal authorities generally appeared to have little knowledge of their acute care infrastructure, with implications for future planning and development. Introduction Intensivists are expected to have many roles during and after a major disaster/catastrophe; that is, triage, intensive care, education for people, and so forth. The roles of intensivists against special disaster or nuclear disaster are studied based on actual experiences. P489 Variation in acute care burden and supply across diverse urban settings S Murthy1, S Austin2, H Wunsch3, NK Adhikari4, V Karir2, K Rowan5, ST Jacob6, J Salluh7, F Bozza8, B Du9, Y An10, B Lee2, F Wu2, C Oppong11, R Venkataraman12, V Velayutham13, D Angus2 1Hospital for Sick Children, Toronto, Canada; 2University of Pittsburgh, PA, USA; 3Columbia University, New York, NY, USA; 4University of Toronto, Canada; 5Intensive Care National Audit & Research Centre, London, UK; 6University of Washington, Seattle, WA, USA; 7D’OR Institute for Research and Education, Rio de Janeiro, Brazil; 8Oswaldo Cruz Foundation, Rio de Janeiro, Brazil; 9Peking Union Medical College Hospital, Beijing, China; 10Peking University People’s Hospital, Beijing, China; 11Komfo Anokye Teaching Hospital, Kumasi, Ghana; 12Apollo Hospital, Chennai, India; 13Stanley Medical College Hospital, Chennai, India Critical Care 2013, 17(Suppl 2):P489 (doi: 10.1186/cc12427) Figure 1 (abstract P490). S182 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Introduction The management of emergency medical admissions has been a subject of recent clinical incidents. There was a high percentage of patients that were referred to the ICU by staff in training, and 21% of referrals were made by junior doctors. Consultant physicians had no knowledge of the case in 57% of referrals. Introduction The management of emergency medical admissions has been a subject of recent clinical incidents. There was a high percentage of patients that were referred to the ICU by staff in training, and 21% of referrals were made by junior doctors. Consultant physicians had no knowledge of the case in 57% of referrals. (APS) component were calculated daily for the fi rst 7 days. Trend was assessed across days by Cuzick’s test. y y Results A total of 719 surgical and 925 medical patients met inclusion criteria. In total, 20.2% of surgical and 21.3% of medical patients had an ICU LOS <1; P = 0.58. Admission severity was correlated with length of stay, P = 0.014 for both medical and surgical patients. Medical patients are sicker on admission and D/C from the ICU than surgical patients (P <0.05) (Figure 1). g Methods A prospective study of 21 cases of referrals and admissions to the ICU was conducted at the Glasgow Victoria Infi rmary Hospital from 8 to 21 September 2012. P489 A questionnaire was produced relating to the referrals, admissions, seniority involvement, cause of referral, and time of patient review by the ICU consultant after ICU admission. They were distributed to specialist registrars and the ICU consultants. All data were electronically recorded into an Excel database. Questionnaires that were not completely fi lled were further investigated using patient clinical notes and contact with medical staff . Information that may identify a patient or clinician was removed from the questionnaire for confi dentiality purposes. g Conclusion ICU utilization diff ered by patient type even with unifi ed triage control within a single unit. Surgical patients were less severely ill on admission to and D/C from the ICU. A signifi cant percentage of medical and surgical patients are D/C within 1 day and may be more effi ciently served in a less resource-intensive environment. The reasons for the diff erences in ICU utilization for surgical versus medical patients require clarifi cation and may have implications for both resource utilization and cost. Results Twenty-one complete questionnaires were collected. Fifty- seven percent (12/21) of cases involved admission to the ICU. Nine percent of the cases involved contacting either a specialist registrar or ICU consultant intensivist for assistance in practical procedures. Of the patients admitted to the ICU, 33% (4/12) were from medical wards, 33% were admitted from A&E. Consultants were the most common professionals who referred patients to critical care (48%; 10/21). Fourteen percent of cases (3/21) involved the referral of patients into ICU by a junior doctor, but only one of the referrals was accepted by the ICU intensivist. Consultants referred or were aware of the referral in 71% (15/21) of cases. Of admissions, 58% (7/12) were accepted by the ICU consultant and the remaining by the specialist registrars. All accepted were acknowledged by the ICU consultant. After admission all of the patients were reviewed by the ICU consultant and the time of review after admission was on average 1 hour 23 minutes (25 minutes to 3 hours 45 minutes). Educational eff ectiveness of introductory training for the Rapid Response System in Japan p y p T Kodama1, M Nakagawa2, E Kawamoto3, S Fujiwara4, H Imai3, S Fujitani5, K Atagi6 y T Kodama1, M Nakagawa2, E Kawamoto3, S Fujiwara4, H Imai3, S Fujitani5, K Atagi6 g 1The University of Texas Southwestern Medical Center at Dallas, TX, USA; 2Social Insurance Kinan Hospital, Tanabe-City, Japan; 3Mie University Hospital, Tsu-City, Japan; 4NHO Ureshino Medical Center, Ureshino-City, Japan; 5Tokyobay UrayasuIchikawa Medical Center, Urayasu-City, Japan; 6Osaka City General Hospital, Osaka-City, Japan Critical Care 2013, 17(Suppl 2):P491 (doi: 10.1186/cc12429) 1The University of Texas Southwestern Medical Center at Dallas, TX, USA; 2Social Insurance Kinan Hospital, Tanabe-City, Japan; 3Mie University Hospital, Tsu-City, Japan; 4NHO Ureshino Medical Center, Ureshino-City, Japan; 5Tokyobay UrayasuIchikawa Medical Center, Urayasu-City, Japan; 6Osaka City General Hospital, Osaka-City, Japan Critical Care 2013, 17(Suppl 2):P491 (doi: 10.1186/cc12429) Introduction Interest in safety and clinical outcomes of inpatients has been growing in Japan, because the 100,000 Lives Campaign was introduced under the Japanese Patient Safety Act in 2008. In this act, an introduction of the Rapid Response System (RRS) was one of the mainstreams to inpatients’ care. However, many Japanese healthcare providers cannot understand how to achieve the introduction of the RRS, because there are few who have knowledge of the system. Therefore, we developed a new introductory training course for the RRS. The educational eff ectiveness was analyzed through the surveillance questionnaires after the course. Conclusion There is still an issue with junior doctors referring patients to the ICU without the acknowledgement of consultant physicians, resulting in unnecessary admissions and decreased time that ICU trainees spend in the ICU. There are more appropriate ICU admissions when there is involvement with seniority. Contact with ICU staff to perform practical procedures outside the ICU and not about admissions should be explored further. Methods The educational program includes a lecture series con cern- ing the outline and management methods, introduction of facilities that have already deployed, small group discussions, and teaching methods-of-training for the medical emergency team using a simulator. Evaluation was made in the fi ve-point scale by 82 participants (58 physicians, 16 nurses and eight other professions) throughout seven courses. The questionnaires are: A. understanding of RRS, B. knowledge acquisition about patient safety, C. expectation for decreasing the cardiopulmonary arrest by RRS, and D. expectation for decreasing the psychological burden by RRS. P491 P491 Educational eff ectiveness of introductory training for the Rapid Response System in Japan T Kodama1, M Nakagawa2, E Kawamoto3, S Fujiwara4, H Imai3, S Fujitani5, K Atagi6 1The University of Texas Southwestern Medical Center at Dallas, TX, USA; 2Social Insurance Kinan Hospital, Tanabe-City, Japan; 3Mie University Hospital, Tsu-City, Japan; 4NHO Ureshino Medical Center, Ureshino-City, Japan; 5Tokyobay UrayasuIchikawa Medical Center, Urayasu-City, Japan; 6Osaka City General Hospital, Osaka-City, Japan Critical Care 2013, 17(Suppl 2):P491 (doi: 10.1186/cc12429) Educational eff ectiveness of introductory training for the Rapid Response System in Japan Reference 1. Gillbe C, Gunning K: Standards for Consultant Staffi ng of Intensive Care Units. ICS & IBTICM Standards. Intensive Care Society; 2006. P493 Knowledge of confi dentiality, consent and information disclosure is not aff ected by position or experience in adult critical care M Lowings1, A Molokhia2, C Anderson3, S Cowman4, T Dixson5, K El-Boghdadly6, L Williams2 1St Thomas’ Hospital, London, UK; 2Lewisham University NHS Trust, London, UK; 3King’s College Hospital, London, UK; 4Great Ormond Street Hospital, London, UK; 5Queen Elizabeth Hospital, Woolwich, UK; 6Princess Royal University Hospital, Bromley, UK Critical Care 2013, 17(Suppl 2):P493 (doi: 10.1186/cc12431) y g y Results Seventy-three participants (89.0%) answered the question- naires. The numbers of participants who scored more than four points were as follows: A. was 71 (97.2%), B. was 70 (95.9%), C. was 64 (87.7%), and D. was 68 (93.2%), respectively. The majority of participants obtained the correct knowledge, and had a solid understanding for the RRS. It was evident that providing abundant material and didactic lectures traced from the introduction to management, and collecting and resolving the questions, promoted comprehension. However, there is a limitation of whether or not the participants introduce the RRS into their own institutions. It is essential to improve the course and continue to support the activities of the participants. Introduction Teaching of medical ethical issues including confi den- tia lity and consent have long been a small part of the medical curriculum. These issues are more complex in an ICU where patients may lack capacity. Documents such as Good Medical Practice 1995, Confi dentiality 2009 and the Mental Capacity Act 2005 give guidance to medical professionals in these matters in the UK. Conclusion Our training course may promote the introduction and dissemination of the RRS in Japan. Methods A questionnaire was distributed amongst staff in four ICUs in South London. Results were analysed according to level of experience and background (medical/nursing or allied health professional (AHP)). Results Of 225 questionnaires distributed, the response rate was 66% (31% doctors, 56% nurses and 13% AHP). Staff with either less than 1 year experience or greater than 10 years experience had the greatest exposure to the Mental Capacity Act and Data Protection Act, suggesting a gap in knowledge in staff with intermediate experience. Knowledge of the Caldicott principles were unaff ected by experience, with many experienced respondents having ‘No Idea’. The majority of respondents (unaff ected by experience) felt that References 1. Leape LL, et al.: JAMA 2005, 293:2384-2390. 2. Jones D, et al.: N Engl J Med 2011, 365:139-146. References 1. Schiza et al.: J Clin Sleep Med 2012, 8:21-26. 2. Kaw et al.: Br J Anaesth 2012, 109:897-906. 1. Schiza et al.: J Clin Sleep Med 2012, 8:21-26. Survey of alcohol-related admissions to critical care and the resource and fi nancial implications Survey of alcohol-related admissions to critical care and the resource and fi nancial implications M Slattery, P Temblett, M Heatley, N Singatullina, B Jones ABMU University Trust, Swansea, UK Critical Care 2013, 17(Suppl 2):P494 (doi: 10.1186/cc12432) Introduction Alcohol-related hospital and ICU admissions are known to have a huge impact on healthcare resources in the UK. Excessive use of alcohol is independently associated with sepsis, septic shock and hospital mortality among ICU patients. This study assesses the relationship between alcohol abuse and intensive care resource utilisation in a mixed medical, surgical and neurosurgical ICU. gi p p y Conclusion Undiagnosed SDB is highly prevalent among cardiac surgical patients. Clinical trajectories of individuals with severe SDB are described by a prolonged recovery of pulmonary function, delayed weaning and a pronounced infl ammatory response after surgery. Screening for SDB might identify patients that are susceptible for a complicated postoperative course. Methods A prospective survey of emergency alcohol-related admissions over a 1-year period was undertaken at a tertiary university adult general and neurosurgical ICU. All patients were screened for acute and chronic alcohol abuse on admission. Acute alcohol abuse was defi ned as being intoxicated with alcohol at the time of admission and chronic alcohol abuse was defi ned as chronic alcohol use exceeding recommended UK national guidelines on consumption. The amount of alcohol consumption was obtained, diagnosis on admission, ICU and hospital mortality, length of stay, and total cost were recorded. All patients were screened for alcohol-related comorbidities. Comparative retrospective data were obtained for the same time period for nonalcohol-related emergency ICU admissions. Data were analyzed using SPSS. P492 Standards of referrals and admissions of critically ill patients to the ICU A Ng Glasgow Victoria Infi rmary Hospital, Glasgow, UK Critical Care 2013, 17(Suppl 2):P492 (doi: 10.1186/cc12430) A Ng Glasgow Victoria Infi rmary Hospital, Glasgow, UK Critical Care 2013, 17(Suppl 2):P492 (doi: 10.1186/cc12430) S183 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 is signifi cantly less than previously reported. Our study reveals interspecialty variations in demographic data, APACHE II scores, mortality and cost of admission. Neurosurgical alcohol-related admissions bear higher mortality and result in greater resource utilisation relative to nonalcohol-related neurosurgical admissions. Alcohol continues to burden both our patients and critical care. when giving information to relatives face to face, relatives should be kept fully informed. When giving information over the telephone, most doctors felt the response should be tailored to the knowledge of the person being spoken to whilst nurses were split between tailoring the response, giving full information, setting up a password system and not giving any information at all. Most respondents felt date of birth and hospital number constituted ‘Patient Identifi able Information’. However, experienced staff did not appreciate the importance of unusual diagnosis and clinical photographs as also being able to identify patients. Similarly, the majority knew that the patient themselves identifi ed the ‘Next of Kin’ but 7% (unaff ected by experience) felt this was decided by the family and felt the family could decide on resuscitation status. When consent is required for an elective procedure in a patient who lacks capacity, doctors tended to have a better understanding of the need to delay the procedure where possible than nurses, the majority of which felt this could be decided by the next of kin or two consultant doctors. Most doctors felt that ‘Acting in the Patient’s Best Interests’ would mean doing what would give the patient the best outcome rather than doing what the patient would have wanted (unaff ected by experience). The majority of staff , on answering this questionnaire, felt that they lacked suffi cient knowledge on the subject and most felt annual reminders would be useful. P495 P495 95 Association of sleep disordered breathing with clinical trajectories in patients undergoing cardiac surgery J Roggenbach, B Tan, E Von der Leyen, A Weymann, M Karck, E Martin, S Hofer University of Heidelberg, Germany Critical Care 2013, 17(Suppl 2):P495 (doi: 10.1186/cc12433) Introduction The prevalence of sleep disordered breathing (SDB) is presumably high among individuals with cardiac diseases [1], nonetheless SDB remains predominantly undiagnosed. However, unrecognized SDB might have relevant impact on the postoperative course of patients undergoing cardiac surgery [2]. g g g y Methods Polygraphic recordings of 181 patients, without previous diagnosis of SDB, undergoing standard cardiac surgical procedures with extracorporeal circulation were obtained during a preoperative night. The apnea–hypopnea index (AHI – the number of apneas, hypopneas per hour recorded) was determined and compared with clinical characteristics and postoperative course. Conclusion The ICU is an environment where issues of consent, confi dentiality and disclosure of information occur daily. Staff feel they lack knowledge in these areas that is unaff ected by their experience. We need to ensure that all staff have the necessary knowledge to deal with these situations. p p Results The prevalence of SDB was considerably high among all examined patients. Median AHI was 20.8 (interquartile range, 10.6 to 36.4). Preoperative AHI was >30 in 32% of all examined individuals. During the fi rst three postoperative days, preoperative AHI >30 was associated with a prolonged weaning time, a reduced oxygenation index (arterial pO2/FiO2), an impaired kidney function, an augmented infl ammatory response and an overall increased length of stay in the ICU. The observed association of high preoperative AHI values with postoperative clinical characteristics remained statistically signifi cant throughout the fi rst three postoperative days. Can massage therapy help cardiac patients? A literature review plus a case study A Fenlon St Georges NHS Trust, London, UK Critical Care 2013, 17(Suppl 2):P496 (doi: 10.1186/cc12434) Introduction A literature review was performed to assess whether massage benefi ts patients postoperatively following coronary bypass grafts (CABG) and or valve replacement/repair. A case study on a patient who had suff ered a hypoxic brain post cardiac arrest was conducted. Methods A review on MEDLINE and Cochrane using search terms massage, cardiac and ICU identifi ed nine research papers on the benefi ts of massage postoperatively for the aforementioned patient group. Other papers were listed but unrelated to cardiac surgery. None of the nine papers identifi ed for this review were ICU specifi c in the title but the ICU was mentioned in the main text body. For the purpose of this review the selected papers are researching the eff ects of massage on physiological parameters, anxiety, pain, calm and perceived stress indicators in the CABG and/or valve repair/replacement. Out of these nine papers, one is British (2002). Five are American (2006 to 2012), two are Brazilian (2010) and one is an Indian paper (2010). All papers are randomised control trials (RCTs). Papers written prior to 1999 were excluded from this literature review. Results In total, 7.7% of patients were admitted with a history of acute/chronic alcohol excess. Sixty-seven per cent of alcohol- related admissions were due to acute alcohol excess. Neurosurgical patients admitted due to alcohol excess had higher ITU mortality than nonalcohol-related neurosurgical patients: 32.1% versus 14.39% (P = 0.02), respectively. Ninety-three per cent of alcohol-related neurosurgical admissions were caused by acute alcohol intoxication. The intensive care cost was signifi cantly higher for alcohol-related (£12,396 per patient) compared with nonalcohol-related neurosurgical admissions (£7,284 per patient). Of the medical patients admitted, 60% of these admissions were due to acute alcohol excess. The cost of intensive care treatment was lower for alcohol-related medical admissions. Conclusion This is one of the largest studies of alcohol-related admissions to critical care. Our survey confi rms that alcohol-related admissions to the ICU are commonplace; however, our frequency S184 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 educational program can reduce VAP incidence in the ICU [1]. In this research we aimed to observe the reduction of VAP incidence after the implementation of VAP care bundles to ICU medical personnel. Can massage therapy help cardiac patients? A literature review plus a case study A Fenlon Results Research from 1999 states there are methodological errors in prior papers and very few large-scale studies prior to this date, destabilising the validity and reliability of research from the papers written before 1999. The later research suggests that any change in the measured physiological parameters of blood pressure, heart rate and respiratory rate are insignifi cant (P >0.05). Pain, anxiety, rest and a calm score perform better with P >4.71 overall. Another American study showed that the length of stay is reduced if a patient receives healing touch postoperatively. However, in one RCT pain and anxiety increases and in some case SpO2 decreases. A case study was chosen by the author and the results support the benefi ts of massage with the cardiac patient group.i Methods Inclusion criteria: all adult surgical patients (>18 years old) who are on ventilatory support in the surgical ICU at Siriraj Hospital. There are two groups, divided into pre-educational group (group I) and post-educational group (group II) (n = 220/group). We also observed the adherence rate to VAP care bundles according to the educational program. The pretest and post-test to determine the effi cacy of the educational program were done. The VAP care bundles consisted of weaning according to weaning protocol, sedation vacation, head- of-bed elevation, measurement of cuff pressures four times/day, 2% chlorhexidine use for mouth care and emptying of ventilator circuit condensate. Conclusion There appears to be some benefi ts but larger-scale studies are required within this and other ICUs in Britain. The author will be inquiring at her workplace whether further studies can be performed and whether she can initiate the research. She will identify further case studies and trial massage on select patients. The author recommends training for nurses interested in massage therapy so a bigger caseload can be identifi ed. Results There were 45.38 and 25.25 episodes of VAP per 1,000 ventilator- days in group I and group II, respectively (P = 0.020). The incidence of VAP was 21.82% in group I and 9.09% in group II (P = 0.000). There was signifi cant reduction in the length of ventilatory support per person (group I = 2, group II = 1 (median), P = 0.013, 95% CI = 0.319 to 0.936) and mortality rate (group I = 15.5%, group II = 8.2%, P = 0.017). There was no signifi cant diff erence in LOI, LOH and ATB cost. References 1. Apisarnthanarak A, Pinitchai U, Thongphubeth K, Yeukyen C, Warren DK, Zack JE, et al.: Eff ectiveness of an educational program to reduce ventilator- associated pneumonia in a tertiary care center in Thailand: a 4-year study. Clin Infect Dis 2007, 45:704-711. Ischemic stroke in diff erent ICU type M Damasceno, F Rodriguez, R Turon Adherence rate to VAP care bundles Bundle Pre-education Post-education P value Wean 95.04 96.08 0.723 Sedate 92.26 95.28 0.467 HOB 50.10 70.36 0.017 Cuff pressure 87.29 80.78 0.278 M care 89.60 80.86 0.204 Drain 95.42 99.42 0.130 Results A total of 3,854 admissions were registered in the period in both ICUs, with 257 (6.7%) being by ischemic stroke – 49 in GICU and 208 in NICU. Mean age of patients: 73 years in GICU and 70.1 years in NICU. Admissions from emergency unit: 44 (89.8%) GICU, 181 (87%) NICU. Mean SAPS 3 score: 50.7 (29 to 71) GICU, 50.6 (29 to 98) NICU. Patients admitted with infection: 6 (12.2%) GICU, 10 (4.81%) NICU. Mean Charlson comorbidity index points: 1.61 (median 1.0) GICU, 1.1 (median 1.0) NICU. Mean length of ICU stay: 4.8 days (median 3) GICU, 4.3 days (median 3) NICU. Mean length of hospital stay: 12.2 days (median 8) GICU, 14.5 days (median 8) NICU. Predicted hospital mortality (mean ± SD): 20.07% ± 13.48 GICU, 20.4% ± 13.83 NICU. Hospital mortality rate: 12.5% GICU, 5.39% NICU. Observed-to-expected (O/E) mortality ratios: 0.62 GICU, 0.26 NICU. Conclusion The educational program and the implementation of VAP care bundles can reduce the incidence of VAP, length of ventilatory support and mortality rate in the ICU. Reference y Conclusion Despite the similar proportions numbers for patients in both ICUs, the mortality rate and the O/E mortality ratio for ischemic stroke were higher in patients of the GICU when compared with the NICU. Can massage therapy help cardiac patients? A literature review plus a case study A Fenlon The pretest scores were 15.53 and 17.53 on average from 40 medical personnel in group I and group II, respectively (P = 0.000). The head-of-bed elevation adherence rate was improved after the educational program (group I = 50.1%, group II = 70.36%, P = 0.017). But the adherence to other bundles was not improved. See Tables 1 and 2. Ischemic stroke in diff erent ICU type M Damasceno, F Rodriguez, R Turon Hospital de Clinicas de Niterói, Rio de Janeiro, Brazil Critical Care 2013, 17(Suppl 2):P497 (doi: 10.1186/cc12435) Table 1 (abstract P498). Impact of the educational program on outcomes Outcome Pre-education Post-education P value VAP 45.38 25.25 0.020 Ventday 2 1 0.013 LOI 3 3 0.054 LOH 17 17 0.960 ATB cost 2,842.50 2,098.00 0.994 % death 15.5 8.2 0.017 Table 2 (abstract P498). Adherence rate to VAP care bundles Bundle Pre-education Post-education P value Wean 95.04 96.08 0.723 Sedate 92.26 95.28 0.467 HOB 50.10 70.36 0.017 Cuff pressure 87.29 80.78 0.278 M care 89.60 80.86 0.204 Drain 95.42 99.42 0.130 Conclusion The educational program and the implementation of VAP care bundles can reduce the incidence of VAP, length of ventilatory support and mortality rate in the ICU. Reference 1. Apisarnthanarak A, Pinitchai U, Thongphubeth K, Yeukyen C, Warren DK, Zack JE, et al.: Eff ectiveness of an educational program to reduce ventilator- associated pneumonia in a tertiary care center in Thailand: a 4-year study. Clin Infect Dis 2007, 45:704-711. P499 Development of a severe infl uenza critical care curriculum and training materials for resource-limited settings JV Diaz1, P Lister2, JR Ortiz3, NK Adhikari4, N Adhikari5 1World Health Organization, San Francisco, CA, USA; 2Great Ormond Street Hospital, London, UK; 3University of Washington, Seattle, WA, USA; 4Sunnybrook Health Sciences Centre and University of Toronto, Canada; 5Health Sciences Centre and University of Toronto, Canada Critical Care 2013, 17(Suppl 2):P499 (doi: 10.1186/cc12437) Table 1 (abstract P498). Impact of the educational program on outcomes Introduction In our hospital there are a general intensive care unit (GICU) and a neurointensive care unit (NICU). Despite the preference for the NICU, in both there are admissions for ischemic stroke. There are diff erent staff for each ICU, with the same physician’s leadership. We have decided to evaluate the performance of both ICUs, analysing whether there are diff erences in results as some authors publish best results in specialized ICUs. Methods Using prospectively collected data, we undertook a retrospective evaluation of all patients admitted to the GICU and NICU of our hospital with the diagnosis of ischemic stroke, from December 2010 to November 2012. In both ICUs there are intensivists, but in the NICU the intensivists have special expertise in neuroscience. Data were collected from Epimedmonitor. Table 2 (abstract P498). P497 Ischemic stroke in diff erent ICU type M Damasceno, F Rodriguez, R Turon 1. Varelas PN, et al.: Neurocrit Care 2008, 9:293-299. 2. Berchad EM, et al.: Neurocrit Care 2008, 9:287-292. P499 Development of a severe infl uenza critical care curriculum and training materials for resource-limited settings j p CH Pisitsak, O Chaiwat Siriraj Hospital, Bangkok, Thailand JV Diaz1, P Lister2, JR Ortiz3, NK Adhikari4, N Adhikari5 1World Health Organization, San Francisco, CA, USA; 2Great Ormond Street Hospital, London, UK; 3University of Washington, Seattle, WA, USA; 4Sunnybrook Health Sciences Centre and University of Toronto, Canada; 5Health Sciences Centre and University of Toronto, Canada Critical Care 2013, 17(Suppl 2):P499 (doi: 10.1186/cc12437) Introduction VAP has continued to be a major cause of morbidity and mortality in critically ill patients in Thailand for decades. Previous research found that the implementation of VAP care bundles and the S185 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Introduction The 2009 H1N1 pandemic caused surges of severely ill patients with viral pneumonia requiring ventilation and particularly aff ected high-dependency medical services such as ICUs in settings that lack suffi cient personnel and resources to provide optimal care. Ministries of Health of several countries asked the World Health Organization (WHO) for clinical management guidance. Introduction The 2009 H1N1 pandemic caused surges of severely ill patients with viral pneumonia requiring ventilation and particularly aff ected high-dependency medical services such as ICUs in settings that lack suffi cient personnel and resources to provide optimal care. Ministries of Health of several countries asked the World Health Organization (WHO) for clinical management guidance. Figure 2 (abstract P500). Compliance with pre-ICU resuscitation bundle. g ( ) g g Methods We developed (2009 to 2011) and piloted (2011 to 2012) curriculum and training materials targeted at clinicians without formal critical care training who care for adult and paediatric patients with severe acute respiratory infections in ICUs in resource-limited settings. Results With contributions from 37 global experts, we developed a 3-day course including 14 learning units in early recognition, patho- physiology, oxygen therapy, infl uenza diagnostics, infection control, resuscitation of septic shock, antimicrobial therapy, monitoring, lung- protective ventilation for acute respiratory distress syndrome, sedation, weaning, preventive care, quality improvement, and ethics. Teaching techniques are appropriate for adult learners and include short slideshow-based lectures, interactive small-group role-play sessions and a toolkit with practical resources such as checklists. With funding from the WHO and local Ministries of Health, the training course was piloted among 67 clinician participants in the Caribbean and Indonesia (29 and 38, respectively) and taught by external critical care specialists. Participants’ daily evaluations rated all units as at least very good. P501 Safety climate perception for staff nurses in an ICU RL Rivera-Romero, M Torres-Campos, MJ Delgado-Amaya, E Curiel-Balsera, JG Quesada-García Hospital Regional Carlos Haya, Málaga, Spain Critical Care 2013, 17(Suppl 2):P501 (doi: 10.1186/cc12439) Introduction Following our study of severe sepsis care across three centres [1], we aimed to introduce a rapid feedback mechanism into our rolling audit programme. Whilst previous audits raised awareness of severe sepsis, only whole organisation performance was reported and no feedback was given to individual clinicians. It is recognised that such feedback loops can improve clinical practice [2]. Introduction When we talk about safety culture, we speak of being aware that things can go wrong. We must be able to recognize mistakes and learn from them, sharing that information fairly and impartially to try to prevent its recurrence. Organizations such as the Agency for Healthcare Research and Quality (AHRQ) have developed tools to help organizations measure their safety culture and there is little information about our country. Methods Patients admitted to critical care (58 beds, four units) with a primary admission diagnosis of infection were screened for severe sepsis. Pre-ICU care was then audited against the Surviving Sepsis Guidelines [3]. Time zero is defi ned as when criteria for severe sepsis were fi rst met. An individualised traffi c-light report was then generated and emailed to the patient’s consultant and other stakeholders involved in care (Figure 1). We aimed to report cases within 7 days of critical care admission. A cumulative report is generated monthly to track organisation-wide performance. Methods A descriptive survey study. We sent the Spanish version of the questionnaire on patient safety culture (AHRQ) to the nursing staff of a polyvalent ICU of 42 beds in a tertiary hospital. Results The questionnaire was sent to 179 nurses, receiving correctly answered 88 surveys (response rate of 49.16%). On a scale of 0 to 10, 6.97 points was obtained to estimate the safety climate for staff respondents. The item best scored was teamwork in the unit (65.9%). Detected as a fortress, ‘communication between nurses at shift changes’ (76.1% positive responses). The worst rating was obtained in the section on human resources, followed by management support in the fi eld of patient safety.f Figure 1 (abstract P500). Example report. Figure 1 (abstract P500). Example report. Conclusion The perception of safety culture in an ICU by nursing staff is far from optimal levels. Quality assurance in severe sepsis: an individualised audit/feedback system results in substantial improvements at a UK teaching hospital p M Simmonds, E Blyth, W Robson, M Chikhani Nottingham University Hospitals NHS Trust, Nottingham, UK Critical Care 2013, 17(Suppl 2):P500 (doi: 10.1186/cc12438) P499 Development of a severe infl uenza critical care curriculum and training materials for resource-limited settings Test scores to assess participant critical care knowledge improved signifi cantly from before the training to immediately after (Caribbean, 58 to 80%; Indonesia, 56 to 75%; P <0.001 for both). Results Since November 2011, 153 cases of severe sepsis have been audited and reported back to clinicians. Compliance with antibiotics in <1 hour has risen from 35 to 75% and compliance with the pre- ICU elements of the resuscitation bundle has risen from 20 to 70% (Figure 2). Feedback from clinicians has been encouraging as our reports highlight both positive and negative examples of practice. Conclusion Individualised feedback on sepsis care has led to substantial improvements in guideline compliance. This concept could be translated to other time-dependent patient pathways. References 1. Simmonds MJR, Chikhani M, Smith P, et al.: Multi-departmental system analysis is needed for evaluation of severe sepsis care: a multi-centre study [abstract]. Presented at the BMJ International Forum on Quality and Safety in Healthcare; 2011; Amsterdam. Conclusion It was feasible to develop and deliver an advanced critical care curriculum and related training materials in a short, interactive workshop for noncritical care trained clinicians in resource-limited settings. However, the small-scale programme was labour and time intensive. Widespread dissemination of these materials requires identifi cation of target countries, engagement of Ministries of Health, and secure funding. Longer-term evaluation is necessary to determine whether such programmes improve processes of care and clinical outcomes for critically ill patients. 2. Jamtvedt G, Young JM, Kristoff ersen DT, et al.: Audit and feedback: eff ects on professional practice and health care outcomes. Cochrane Database Syst Rev 2006, 2:CD000259. 3. Dellinger RP, Levy MM, Carlet JM, et al.: Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock: 2008. Crit Care Med 2008, 36:296-327. First-hour protocol clarifi es resource management and nursing staff education in the ICU First-hour protocol clarifi es resource management and nursing staff education in the ICU M Arbelius-Iltanen, R Siren, J Heinilä, P Korhonen, T Sutinen, V Jalkanen, T Ahonen, S Karlsson Tampere University Hospital, Tampere, Finland Critical Care 2013, 17(Suppl 2):P503 (doi: 10.1186/cc12441) Results A 10% increase in the proportion of ICU nurses with a bachelor’s degree or higher was associated with 2% lower odds of death while controlling for patient and hospital characteristics. Patients cared for in better work environments experienced 11% lower odds of risk- adjusted death than those cared for in poorer ICU work environments. Conclusion Patients cared for in hospitals with a greater proportion of bachelor’s prepared ICU nurses and in better ICU work environments experienced signifi cantly lower odds of death. As the demand for critical care services increases, attention to the education level of ICU nurses and ICU work environment may be warranted to optimize currently available resources and potentially yield better outcomes. M Arbelius-Iltanen, R Siren, J Heinilä, P Korhonen, T Sutinen, V Jalkanen, T Ahonen, S Karlsson Tampere University Hospital, Tampere, Finland Critical Care 2013, 17(Suppl 2):P503 (doi: 10.1186/cc12441) Introduction The fi rst-hour protocol determines the patient-specifi c resources for the start of an ICU stay [1]. Staff resources are decided through triage. Task charts direct the start of intensive care. Our primary goal is to improve patient care. g p p Methods A triage method (red, yellow, green) is used to manage ICU resources according to the severity of illness. For example, one doctor and one nurse would admit a stable (green) patient coming from the operating room for postoperative ICU care. A patient in septic shock with multiple organ disorder (red), on the other hand, would be admitted by a team of two doctors and three nurses. Each staff member has a task chart in a checking-list format. Also, an admission chart is used to improve data collection. The use of the protocol started as a pilot study in early 2012. Simulation education for staff members started in August 2012 and has included video recordings and debriefi ng of each simulated ICU admission. Primary goal-directed therapy goals have been mean arterial pressure (MAP >65 mmHg), SpO2 >94%, timing of the laboratory tests, start of antibiotics, and blood glucose level 6 to 8 mmol/l. Quality indicators have been followed from the data provided by The Finnish Intensive Care Consortium. P504l Methods In a survey, we evaluated the study course. Therefore, a questionnaire was distributed among all students including the topics course contents, learning materials, time management, supervision, and overall impression. The students were asked to score their agreement to the statements ‘content is well structured’, ‘content extent is appropriate’ and ‘content is relevant for medical purposes’ on a scale ranging from 1 (fully disagree) to 5 (fully agree). P502 Physico-technical medicine: teaching technical skills to the intensivist Introduction The requirements for the intensivist in handling medical technology are constantly growing. It appears necessary to acquire technological competences particularly within the fi elds of medical technology and physics. In the master’s degree program ‘MasterOnline Physico-Technical Medicine’, such technical authority is conveyed. To cope with the intensive vocational situation of the physician, this study course follows the blended-learning concept; that is, it is conceived as an online study course with small portions of intermittent presence phases. Within the fi rst year, technical basic skills such as ‘measurement technique’, ‘informatics’, and ‘advanced physics’ are covered. Subsequently, two of various advanced courses in diff erent fi elds of medical technology (‘technology in intensive care medicine’, ‘technology in surgery’, ‘technical cardiology’, ‘radiology’, and other) are selected. Conclusion The fi rst-hour protocol has helped us in resource management, start of the patients’ intensive care and education of nursing staff . Reference 1. Brown R, et al.: Aust Crit Care 2012, 25:178-187. 1. Brown R, et al.: Aust Crit Care 2012, 25:178-187. 1University of Pittsburgh, PA, USA; 2University of Pennsylvania, Philadelphia, PA, USA Critical Care 2013, 17(Suppl 2):P504 (doi: 10.1186/cc12442) Introduction Demand for critical care services is increasing yet a comprehensive understanding of how critical care nurses – the largest group of ICU direct care providers – impact outcomes remains unclear. The purpose of this study was to determine how critical care nurse education (hospital proportion of bachelor’s prepared ICU nurses) and ICU work environment infl uenced 30-day mortality of mechanically ventilated older adults. Results The students participated actively in this study course with highest motivation and large commitment. The students’ workload was in the targeted range of about 10 hours/week. Content structure was scored with 4.3 ± 0.1, content extent with 4.1 ± 0.2 and medical relevance with 4.3 ± 0.2. Conclusion The blended-learning concept fulfi lls the requirements for occupation-accompanying continued medical education, since it off ers the possibility to study self-employed accessing text documents, lecture recordings, and electronic lectures and to convert in concentrated presence phases this knowledge into practical exercises. Methods A multi-state cross-sectional nurse survey was linked to hospital administrative data and Medicare claims (2006 to 2008). The fi nal sample included 55,159 mechanically ventilated older adults in 303 hospitals. Logistic regression modeling was employed to jointly assess the relationship of critical care nurse education, work environment and staffi ng on 30-day mortality while adjusting for hospital and patient characteristics and accounting for clustering. First-hour protocol clarifi es resource management and nursing staff education in the ICU Questionnaires for the staff members have been used to evaluate opinions about the fi rst- hour protocol. P505 Infl uence of critical care nurse education and work environment on outcomes of mechanically ventilated older adults DM Kelly1, LH Aiken2 1University of Pittsburgh, PA, USA; 2University of Pennsylvania, Philadelphia, PA, USA P501 The team work dimension was identifi ed as the most valued by workers, with the transmission of information on shift changes the most valued item. Figure 1 (abstract P500). Example report. S186 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 P502 Physico-technical medicine: teaching technical skills to the intensivist J Seifried, S Richter, J Guttmann, S Schumann University Medical Center Freiburg, Germany Critical Care 2013, 17(Suppl 2):P502 (doi: 10.1186/cc12440) nurses considered that the protocol has no eff ect, and none thought it to be adverse for patient care. Corresponding numbers for our ICU doctors were 87% (benefi cial n = 13/15), 13% (no eff ect n = 2/15) and 0% (adverse). Furthermore, 82.5% (n = 66/80) of the nurses replied that education of new nurse staff members has improved because of the fi rst-hour protocol. A total of 17.5% (n = 14/80) thought there has been no eff ect, and none considered the protocol harmful for education. For ICU doctors the protocol did not bring either clear educational advantages or disadvantages. The variable life-adjusted display curves (The Finnish Intensive Care Consortium) have shown improvement in our patient care after the implementation of the fi rst-hour protocol. However, we cannot determine whether it is a signifi cant factor in our intensive care results.i Are general wards suffi ciently staff ed to care for level 1 patients? G Rajendran, C Tjen, S Hutchinson, S Fletcher Norfolk & Norwich University Hospital, Norwich, UK Critical Care 2013, 17(Suppl 2):P506 (doi: 10.1186/cc12444) y gi Results The survey was completed by 129 ICU workers. The global prevalence of ISQ in ICU was 67.4%. ISQ was observed in 45% of physicians and 82% of nurses and nurses assistant (P <0.001). Sleep medication was used by 13.3% of the ICU team. Univariate analysis showed that ISQ was signifi cantly associated with sex (73% vs. 43%, P = 0.03 in women and men, respectively), marital status (84% vs. 61%, P = 0.01 in single and couple workers, respectively), more than 60 hours working in the last week (76% vs. 61%, P = 0.07) and less than 6 sleeping hours (95% vs. 54%, P <0.0001). Multivariable analysis demonstrated that a sleep duration less than 6 hours was independently associated with ISQ (OR = 24.5; 95% CI = 5.2 to 115.8; P <0.0001). Furthermore, pathologic sleepiness was present in 59.3% of ICU workers. Sleepiness was independently associated with use of sleep medication (OR = 5.9; 95% CI = 1.2 to 28.5; P = 0.025). Introduction There are several defi nitions of level 1 (L1) care, all refer to a group at risk of clinical deterioration on the ward [1-3]. There is evidence that ward patients who become acutely unwell often receive suboptimal care [4]. A regional study commissioned by Norfolk, Suff olk & Cambridgeshire Critical Care Network (NSCCCN) found that a majority of ward patients may be of L1 dependency and death rates appear to be correlated with L1 status. We aim to examine the relationship between the ward distribution of illness acuity, staffi ng and patient outcome. fi Methods Data were collected as part of NSCCCN’s observational prevalence study in 2010. Ward surveys included acuity of illness, staffi ng levels and skill mix. Secondary data were obtained from the Patient Administration System. Emergency, oncology, paediatric and maternity units were excluded. Conclusion The prevalence of ISQ and sleepiness is very high among ICU workers. Those disturbances are independently associated with a sleep duration less than 6 hours, and sleep medication use, respectively. These results highlights that strategies to decrease ISQ and sleepiness in ICU clinicians are urgently needed to improve work performance, improve quality of care provided and prevent adverse events. Results Complete datasets were obtained from 1,402 patients in 22 wards in our university hospital over two seasons. This constitutes 98.3% of inpatients from those wards. P505 Activity and case-mix changes in a medical ICU after the geographical transfer of a third-level university hospital JC Cebrián, FM Monsalve, JB Bonastre Hospital Universitario y Politecnico La Fe, Valencia, Spain Critical Care 2013, 17(Suppl 2):P505 (doi: 10.1186/cc12443) Introduction Information about big hospital geographical transfer is scarce in the medical literature. On 20 February 2011 our hospital (in fact, a big university complex) was transferred from their previous location in the north-center of our city towards a new southern peripheral, geographical location. This transfer has been done without any changes in assisted population or nursing/medical staff . The only change was a slight increase in bed number (21 to 24). Our aim is to analyze changes in activity indexes (length of stay, occupancy rate, and so forth) and case mix (origin, previous quality of life and NYHA score, main diagnostic groups, severity scores, in-ICU and in-hospital mortality). Results According to the questionnaire replies, 80% (n = 64/80) of our nurses estimate that the fi rst-hour protocol has improved the starting process of our patients’ intensive care. Twenty percent (n = 16/80) of the S187 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Methods To compare our number of admissions, related activity and case-mix indicators 1 year before and after the geographical change was done. We analyzed our whole number of patients admitted to the ICU. We used the chi-square test for categorical variables and one-way analysis of variance for quantitative data. Minitab and Statbas statistical programs were used. We plotted activity data using the Barber– Johnson 1 diagram. [http://www.nice.org.uk/cg50] 3. AUKUH Acuity/Dependency Tool. Association of UK University Hospitals; 2007. [http://www.aukuh.org.uk/index.php/affi liate-groups/directors-of-nursing/ patient-care-portfolio] [http://www.nice.org.uk/cg50] 3. AUKUH Acuity/Dependency Tool. Association of UK University Hospitals; 2007. [http://www.aukuh.org.uk/index.php/affi liate-groups/directors-of-nursing/ patient-care-portfolio] 4. 4. An Acute Problem? NCEPOD; 2005. [http://www.ncepod.org.uk/2005report/index.html] Results A total of 2,774 cases (63% males; mean age 61 years) were admitted to our ICU during the period (1 year before and after the transfer). No diff erences between both groups were founded in demographic data, Knaus score and NYHA status. Regarding their origin, we found more patients admitted from other hospital centers (20 vs. 29%; P <0.001). APACHE II score increased from 17.24 to 19.08% (P <0.001) and a slight increase change in SAPS 3 score was also found (52.29 to 53.75; P <0.01). Work-related stress amongst doctors in intensive care, anaesthetics, accident and emergency and general medicine g y g Tuthill, MS Ahmed, G Mathew, AC Bolton, AA Molokhia JI Tuthill, MS Ahmed, G Mathew, AC Bolton, AA Molokhia University Hospital Lewisham, London, UK Introduction Work-related stress is a potential problem among doctors and is associated with anxiety, depression, reduced job satisfaction, days off work, errors and near misses [1]. To compare stress levels between diff erent groups of doctors and identify causes of stress, we conducted a survey at University Hospital Lewisham using the UK Health and Safety Executive’s Management Standards (HSEMS). HSEMS is a validated tool developed to identify work conditions that warrant interventions to reduce stress levels across organisations [2]. y y Conclusion Better outcome with improved SPr may be unsurprising, although if proven conclusively would signifi cantly inform workforce planning. Lack of correlation between staffi ng levels and occupancy or acuity is also interesting given that we know L1 criteria are associated with worse outcome. P505 Our in-ICU mortality remains lower (15.5 to 15.6%) whereas observed mortality decreased (22.37 to 19.88%; P <0.001). An increase in our neurologic patients has been the most consistent change regarding diagnostic groups. The activity indexes show a slightly decrease in occupancy rate (79.2 vs. 76.8). 1. Levels of Critical Care for Adult Patients. Intensive Care Society; 2002. [http://www.ics.ac.uk/professional/standards_safety_quality/standards_and_ guidelines/levels_of_critical_care_for_adult_patients] 2. Acutely Ill Patients in Hospital. NICE Guideline 50. NICE; 2007. P508 Work-related stress amongst doctors in intensive care, anaesthetics, accident and emergency and general medicine JI Tuthill, MS Ahmed, G Mathew, AC Bolton, AA Molokhia University Hospital Lewisham, London, UK Critical Care 2013, 17(Suppl 2):P508 (doi: 10.1186/cc12446) Work-related stress amongst doctors in intensive care, anaesthetics, accident and emergency and general medicine 1. Yates J: Hospital Beds: A Problem for Diagnostic and Management. Heinemann Medical Books; 1982. Are general wards suffi ciently staff ed to care for level 1 patients? G Rajendran, C Tjen, S Hutchinson, S Fletcher Norfolk & Norwich University Hospital, Norwich, UK Critical Care 2013, 17(Suppl 2):P506 (doi: 10.1186/cc12444) The mean ward occupancy rate was 94% (10th to 90th percentile: 85% to 100%). At least one L1 acuity criterion was scored by 898 (64%) patients, with 25% from geriatrics followed by orthopaedics (17%) and general surgery (10%). Each ward had an average of eight qualifi ed nursing staff (range: 4 to 12) equating to an average staff :patient ratio (SPr) of 0.253. There was no correlation between ward occupancy and nursing staff (Pearson correlation, corr: 0.55), nor between prevalence of L1 criteria and staffi ng (corr: 0.34). The admission rate to intensive care was noted to be higher if the patients were nursed in a ward with lower than average SPr compared with higher SPr (2.7% vs. 1.2%, P = 0.058 Fisher’s exact), but this was not statistically signifi cant. Senior nursing (Band 6) staff were part of the skill mix on only nine of 44 ward surveys. P508 Reference 1. Yates J: Hospital Beds: A Problem for Diagnostic and Management. Heinemann Medical Books; 1982. Methods A survey was conducted in six Uruguayan ICUs. The sleep quality was evaluated on the basis of the Pittsburgh score (PS), and the sleepiness was identifi ed by the Epworth scale. ISQ was defi ned as PS greater than 5 points and sleepiness by an Epworth scale higher than 6 points. ICU’s, patient’s, and clinician’s characteristics were assessed for their association with the prevalence of ISQ. All variables with P <0.2 in univariate analysis were included in a model of ordinal regression. P <0.05 was considered statistically signifi cant. P507 Sleep quality among ICU workers h 1 k h 1 h 2 Sleep quality among ICU workers G Burghi1, I Serkochian1, B Frache2, P Alzugaray2, K Goinheix1, M Rodriguez Verde3, H Bagnulo1, E Azoulay4 1Hospital Maciel, Montevideo, Uruguay; 2Sanatorio Americano, Montevideo, Uruguay; 3Hospital de Paysandú, Uruguay; 4Hôpital Saint Louis, Paris, France Critical Care 2013, 17(Suppl 2):P507 (doi: 10.1186/cc12445) Introduction Prolonged shifts, workload, stress, and diff erent con- fl icts are associated with burnout, loss of psychological wellbeing, and probably with an inadequate sleep quality (ISQ). This relevant disturbance leads to deterioration of the work performance, may impair quality of care provided to patients and increases the incidence of serious adverse events. The objective was to determine the prevalence of ISQ and sleepiness among Uruguayan ICU workers, and to evaluate risk factors associated with ISQ. Conclusion According to the previous data our ICU seems to perform better in the new location with a decrease in the standardized mortality rate. On the other hand, we are admitting more patients transferred from other hospitals. A better occupancy rate was found. f P506 A P506 Are general wards suffi ciently staff ed to care for level 1 patients? G Rajendran, C Tjen, S Hutchinson, S Fletcher Norfolk & Norwich University Hospital, Norwich, UK Critical Care 2013, 17(Suppl 2):P506 (doi: 10.1186/cc12444) Infl uence of adverse drug events on the mortality and the length of hospital stay in ICUs in Japan: the JADE Study Y Ohta1, M Sakuma1, D Bates2, T Morimoto1 1Kinki University, Osaka, Japan; 2Harvard University, Boston, MA, USA Critical Care 2013, 17(Suppl 2):P510 (doi: 10.1186/cc12448) og dad y , J o , O oc e 1Princess Royal University Hospital, London, UK; 2University Hospital Lewisham, London, UK Critical Care 2013, 17(Suppl 2):P509 (doi: 10.1186/cc12447) Critical Care 2013, 17(Suppl 2):P509 (doi: 10.1186/cc12447) Introduction South-east London (SEL) presents unique challenges to healthcare providers due to its diverse demographic. The high levels of poverty, immigration and psychiatric illness impact delivery of obstetric care. These were identifi ed as risk factors for poor outcome in the latest CMACE report [1]. The Intensive Care National Audit and Research Centre (ICNARC) produced data on obstetric critical care admissions in 2007 [2]. We reviewed the obstetric critical care admissions in three SEL hospitals and compared this with the national average determined in the ICNARC and CMACE data. Introduction Adverse drug events (ADEs) are associated with a substantial increase in morbidity and mortality in any setting. Because patients in ICUs were critically ill with complex diseases and varied organ dysfunction, the incidence of ADEs on such patients is much more crucial than the counterparts. We thus assessed the nature of ADEs and their infl uence in ICUs. l Methods We conducted a prospective cohort study at ICUs at three large tertiary-care hospitals in Japan. Trained research nurses reviewed all medical charts, incident reports and reconciliations from the pharmacy to identify suspected ADEs as well as the background of patients. ADEs are any injuries that result from the use of a drug. After suspected ADEs are collected by research nurses, physician reviewers independently evaluated them and classifi ed them as ADEs or rule violations. We used the validated methodology [1]. Methods All critical care admissions in three high-risk obstetric units in SEL (1 August 2009 to 31 July 2011) were screened for patients who were currently or recently pregnant. We compared local results with national data by ICNARC and CMACE. Figure 1 (abstract P509). Causes for critical care admission in SEL, CMACE and ICNARC. Results We included 459 patients with 3,231 patient-days. The median age was 70 years and the median length of stay was 3 days. In total, 70 patients (15%) had at least one ADE during their stay in the ICU. The median ICU stay in patients who had ADEs was 14 days while 2 days in patients who had no ADEs (P <0.0001). The median length of the ADE onset days since admission was 3 days. References Methods We conducted an anonymous survey of doctors working in anaesthetics, intensive care, general medicine and accident and emergency (A&E) departments over 6 weeks using the HSEMS S188 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 question naire. We also surveyed awareness of the Trust’s stress management services and whether staff had a designated supervisor or mentor. Results were analysed using the HSEMS Analysis Tool, which rates stressors with a score from 1 to 5 (5 represents the lowest amount of stress). We compared the Trust’s results against HSEMS national standards. Results There were 68 obstetric critical care admissions in the SEL hospitals within the audited time frame. The mean age was 30.05 in ICNARC data compared with 33.93 in SEL. Average APACHE II scores were lower in SEL compared with the ICNARC data, but length of stay was greater in SEL (2.72 days) compared with ICNARC (1.5 days). Haemorrhage was the most common reason for admission in SEL, whilst sepsis was the leading cause of death according to the latest CMACE report (Figure 1). Results Seventy-two doctors completed the survey. Lowest stress levels were found in doctors working in intensive care (n = 12, mean 3.63, SD 0.39). This was followed by medicine (n = 26, mean 3.55, SD 0.47), anaesthetics (n = 27, mean 3.40, SD 0.44), and A&E (n = 7, mean 3.11, SD 0.65), which had the highest stress levels. There was no signifi cant diff erence in stress levels between diff erent grades of doctors. When compared with HSEMS targets, staff relationships and peer support exceeded national standards. However, management of organisational change and demands at work need improvement. The majority of doctors (82%) had no idea what stress management services were provided by the Trust. Seventy-nine per cent of doctors had an allocated supervisor or mentor, 91% of those felt able to approach their supervisor. g Conclusion Data from national audits may guide protocol, but services must be tailored to local circumstances. SEL has unique population characteristics and obstetric critical care admissions diff er signifi cantly from national statistics; in particular, haemorrhage is over-represented in our region. Critical care services were generally required for a short period of time; during this period, routine postpartum care may be omitted as treatment priorities diff er. Morimoto T, et al.: Qual Saf Health Care 2004, 13:306-314. References 1. Kerr et al.: HSE management standards and stress-related work outcomes. Occup Med (Lond) 2009, 59:574-579. 1. Kerr et al.: HSE management standards and stress-related work outcomes. Occup Med (Lond) 2009, 59:574-579. 2. Health and Safety Executive Management Standards [http://www.hse.gov. uk/stress/standards/index.htm] 2. Health and Safety Executive Management Standards [http://www.hse.gov. uk/stress/standards/index.htm] References Dedicated critical care services on the labour ward may be a way to combine postnatal care with transient high-dependency requirements. This may enhance patient experience and prove cost-eff ective. References Conclusion These survey results provide reassurance that stress levels in intensive care compare well, despite critically unwell patients and higher mortality rates. We identifi ed areas that need improvement within the Trust and will present these results to all relevant departments. With the support of hospital management we will initiate HSEMS-validated measures to reduce stress. 1. Centre for Maternal and Child Enquiries: Saving mothers’ lives: reviewing maternal deaths to make motherhood safer: 2006–08. The Eighth Report on Confi dential Enquiries into Maternal Deaths in the United Kingdom. BJOG 2011, 118(Suppl 1):1-203. 2. Female Admissions (Aged 16–50 Years) to Adult, General Critical Care Units in England, Wales and Northern Ireland, Reported as ‘Currently Pregnant’ or ‘Recently Pregnant’ [http://www.oaa-anaes.ac.uk/assets/_ managed/editor/File/Reports/ICNARC_obs_report_Oct2009.pdf] References P510l Infl uence of adverse drug events on the mortality and the length of hospital stay in ICUs in Japan: the JADE Study Y Ohta1, M Sakuma1, D Bates2, T Morimoto1 1Kinki University, Osaka, Japan; 2Harvard University, Boston, MA, USA Critical Care 2013, 17(Suppl 2):P510 (doi: 10.1186/cc12448) P509 Audit of obstetric critical care admissions in a high-risk population K El-Boghdadly1, J Aron2, DN Onwochei1 1Princess Royal University Hospital, London, UK; 2University Hospital Lewisham, London, UK Critical Care 2013, 17(Suppl 2):P509 (doi: 10.1186/cc12447) P511 Attitude and knowledge of intensive care staff concerning donation in Hungary: it is the fi rst step to change g yi p A Smudla1, S Mihály2, J Fazakas1 A Smudla , S Mihály , J Fazakas 1Semmelweis University, Budapest, Hungary; 2Hungarian National Blood Transfusion Service, Budapest, Hungary Critical Care 2013, 17(Suppl 2):P511 (doi: 10.1186/cc12449) Conclusion Introduction of a SN:OD and a clinical pathway has led to the identifi cation of previously missed potential organ donors in the ED. Several patients have subsequently been admitted to critical care solely to facilitate organ donation. Introduction In Hungary, despite the high level of social support, the number of organ recoveries from deceased donors has not changed signifi cantly. The donation activity shows a positive relationship with the level of education of staff in ICUs as well as with their attitude towards transplantation. The aim of this cross-sectional study is to estimate the attitude and knowledge of intensive care specialists and nurses as regard donation and transplantation. Infl uence of adverse drug events on the mortality and the length of hospital stay in ICUs in Japan: the JADE Study Y Ohta1, M Sakuma1, D Bates2, T Morimoto1 1Kinki University, Osaka, Japan; 2Harvard University, Boston, MA, USA Critical Care 2013, 17(Suppl 2):P510 (doi: 10.1186/cc12448) Regarding the mortality, 73 patients (16%) were dead during their ICU stay: 12 deaths (17%) in patients who had ADEs and three of 12 deaths were caused by an ADE, and 61 deaths (16%) in counterparts (P = 0.8). There were no signifi cant diff erences of patients’ characteristics between patients with ADEs and without ADEs (Table 1). Conclusion ADEs were associated with longer stay and caused a part of death in ICU (4%) although they did not increase the mortality. Because the characteristics of patients were not associated with ADEs, early detection and intervention for ADEs could be important to improve the morbidity and reduce the death caused by ADEs in ICUs. Reference Figure 1 (abstract P509). Causes for critical care admission in SEL, CMACE and ICNARC. Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S189 Table 1 (abstract P510). Patient characteristics Patients Patients with without ADEs ADEs Characteristic (n = 70) (n = 389) P value Age ≥65 years, n (%) 49 (70) 227 (58) 0.08 Male, n (%) 40 (57) 250 (64) 0.2 Department Medicine 35 (50) 228 (59) Surgery 35 (50) 161 (41) 0.2 Unconsciousness, GCS ≤8 13 (19) 91 (23) 0.4 Heart failure, NYHA = 4 37 (53) 216 (56) 0.7 Respiratory failure 44 (62) 262 (67) 0.5 Kidney failure 6 (9) 58 (15) 0.2 Clinical laboratory measurements, median (25%, 75%) Blood urea nitrogen (mg/dl) 21 (15, 39) 19 (14, 31) 0.2 Creatinine (mg/dl) 0.8 (0.5, 1.5) 0.9 (0.7, 1.7) 0.8 Aspartate aminotransferase (IU/l) 27 (20, 47) 30 (22, 48) 0.5 Alanine transaminase (IU/l) 20 (13, 42) 20 (12, 33) 1.0 Total protein (g/dl) 6.7 (5.9, 7.4) 6.6 (5.9, 7.3) 0.8 Number of medications on admission ≥4, 38 (54) 223 (57) 0.6 n (%) Number of medications on admission, 5 (3, 7) 5 (4, 8) 0.7 median (25%, 75%) GCS, Glasgow Common Scale; NYHA, New York Heart Association. Table 1 (abstract P510). Patient characteristics knowledge regarding the law and ethics of donation (P <0.01), donor management (P <0.01), living and deceased donor transplantation (P <0.01) and joining Eurotransplant (P <0.01). Older professionals had more information about all fi elds (P <0.01). Nurses had less knowledge concerning donor management (P <0.01), law and ethics (P <0.01) and deceased donor transplantation (P <0.01) than doctors. y Reference 1. UK Organ Donation Taskforce: Organs for Transplants: A Report from the Organ Donation Taskforce. Department of Health; 2008. Specialist nurse for organ donation in an emergency department will increase organ donation Introduction In the UK, three people die each day awaiting trans plan- tation, due to the unavailability of donor organs. Traditionally, donor identifi cation has been restricted to the ICU. However, following the UK Organ Donation Taskforce report in 2008 [1], a number of emergency departments (EDs) have been working with specialist nurses for organ donation (SN:OD) to identify potential donors and approach their families for consent in the ED. We present our initial experience after the introduction of a SN:OD to an Irish teaching hospital’s ED. Methods We conducted a retrospective review of deaths in our ED during a 28-month period. For those who died in the ED, case notes were reviewed to identify those suitable for organ donation. Referral and donation rates were compared in two cohorts, pre and post introduction of a SN:OD. Fisher’s exact test was used to assess diff erences between groups. f Results Ninety-one deaths occurred in the study period. Following introduction of the SN:OD, referrals increased from zero to eight. Of the eight referred, three received consent and were transferred to the ICU, two of whom became successful donors. The number of missed potential donors fell from six to one (P = 0.009). Infl uence of adverse drug events on the mortality and the length of hospital stay in ICUs in Japan: the JADE Study Y Ohta1, M Sakuma1, D Bates2, T Morimoto1 1Kinki University, Osaka, Japan; 2Harvard University, Boston, MA, USA Critical Care 2013, 17(Suppl 2):P510 (doi: 10.1186/cc12448) knowledge regarding the law and ethics of donation (P <0.01), donor management (P <0.01), living and deceased donor transplantation (P <0.01) and joining Eurotransplant (P <0.01). Older professionals had more information about all fi elds (P <0.01). Nurses had less knowledge concerning donor management (P <0.01), law and ethics (P <0.01) and deceased donor transplantation (P <0.01) than doctors. Conclusion Education about organ donation needs to be part of specialist training of intensive care staff , and refresher courses every fi fth year as well. The course should include knowledge regarding brain death, donor management and communication with family. This is the fi rst step to improve the number of transplantations. Reference Sepsis in HIV patients admitted to the ICU d l 1 l 1 l Sepsis in HIV patients admitted to the ICU P Vidal-Cortés1, P Lameiro-Flores1, M Mourelo-Fariña2, A Aller-Fernández2, R Gómez-López1, P Fernández-Ugidos1, M Alves-Pérez1, E Rodríguez-García1 1CHU Ourense, Spain; 2CHU A Coruña, Spain Critical Care 2013, 17(Suppl 2):P513 (doi: 10.1186/cc12451) Sepsis in HIV patients admitted to the ICU P Vidal-Cortés1, P Lameiro-Flores1, M Mourelo-Fariña2, A Aller-Fernández2, R Gómez-López1, P Fernández-Ugidos1, M Alves-Pérez1, E Rodríguez-García1 1CHU Ourense, Spain; 2CHU A Coruña, Spain Critical Care 2013, 17(Suppl 2):P513 (doi: 10.1186/cc12451) nurses as regard donation and transplantation. Methods The self-completed questionnaire that consisted of 20 items was completed at the Congress of Hungarian Society of Anesthesiology and Intensive Therapy in 2011. Besides the epidemiological data, the intensive care specialists (n = 179) and nurses (n = 103) were asked about donation activity, participation in an organ donation course, self- reported knowledge of joining Eurotransplant, donor management, legislation, and transplantation. The data were analyzed by SPSS 17.0. Results A total of 53.6% of physicians and 16.7% of nurses attended an earlier organ donation course (P <0.01). The average age of those who participated in training was signifi cantly higher among doctors (P <0.01). Fifty-nine percent of doctors and 65.1% of nurses did not even want to participate in such training. Donation activity was higher among staff who joined training (P <0.01). Independently from accepting the presumed consent legislation (91.1%), 66% of physicians agreed with the hospital practice that requests the adult donor’s relatives to consent to organ recovery. This standpoint did not depend on donation activity, participation in an organ donation course, opinion about legislation and the nature of staff . A total 95.4% of participants consented to their organ retrieval after death. The staff who participated in an organ donation course had more g Methods The self-completed questionnaire that consisted of 20 items was completed at the Congress of Hungarian Society of Anesthesiology and Intensive Therapy in 2011. Besides the epidemiological data, the intensive care specialists (n = 179) and nurses (n = 103) were asked about donation activity, participation in an organ donation course, self- reported knowledge of joining Eurotransplant, donor management, legislation, and transplantation. The data were analyzed by SPSS 17.0. Introduction Our objective was to analyze septic HIV patients admitted to intensive care. Introduction Our objective was to analyze septic HIV patients admitted to intensive care. Table 2 (abstract P515). Unit and hospital mortality Methods A retrospective study analyzed children with hematological disorders admitted to our ICU between April 2005 and June 2012. All of the included children required emergency admission and invasive mechanical ventilation. Those who did not need intubation, or required intubation only for therapeutic intervention and died within 24 hours of ICU admission were excluded. The survival group was defi ned as patients who were discharged from the ICU, and the nonsurvival group was defi ned as those who died in the ICU or within 7 days after discharge from the ICU. The PELOD score and PIM-II were applied as morbidity scoring systems Patients ICU mortality, Hospital mortality, (n) n (%) n (%) Haematology 163 52 (31.9%) 81 (49.7%) Controls 237 73 (30.8%) 99 (41.8%) P value 0.827 0.126 Conclusion Unit mortality of critically ill patients with HM was similar to those without HM. Hospital mortality in patients with HM was higher than those without HM, although not statistically signifi cant. Severity of illness at presentation to critical care is the main determinant of outcome in patients with HM. Results Twenty-seven patients, including 18 males and nine females, with a median age of 6.1 years (range, 0.2 to 16.6 years) were analyzed. Sixteen patients had leukemia, fi ve had hemophagocytic syndrome, six had solid tumors. The average predicted mortality rate was 31.3% in PIM-II. The survival group included 15 patients (56%) and the nonsurvival group included 12 patients (44%). When the survival group was compared with the nonsurvival group, there were no signifi cant diff erences in the systolic blood pressure (101.3 ± 13.9 mmHg vs. 92.8 ± 25.4 mmHg; P = 0.15), the proportion of patients requiring continuous renal replacement therapy (33.3% vs. 50.0%; P = 0.30), and PELOD score (15.5 ± 10.4 vs. 21.8 ± 15.4; P = 0.22). In the nonsurvival group, the PIM-II was higher than that in the survival group (27.9 ± 10.4 vs. 35.7 ± 9.0; P = 0.06); the PaO2/FiO2 (272.5 ± 136.7 vs. 153.3 ± 123.3; P = 0.03) and oxygenation index (6.7 ± 8.1 vs. 14.1 ± 9.5; P = 0.04) were signifi cantly worse in the nonsurvival group than in the survival group. Conclusion The data show that respiratory failure is more strongly associated with mortality than other organ failures in pediatric hematology patients requiring intensive care. Sepsis in HIV patients admitted to the ICU d l 1 l 1 l Mean viral load: 4.57 ± 3.25 log (vs. 2.257 ± 1.96, P = 0.001). Of sepsis patients, 62.1% were on their CD4 nadir (vs. 34.5% in nonseptic patients, P = 0.009). Mean albumin levels were 2.3 ± 0.53 g/ dl (vs. 2.92 ± 0.94, P <0.001). APACHE II at admission was 21.98 ± 7.97 (vs. 18.15 ± 8.47, P = 0.046). At admission, 52.8% were on severe sepsis and 44.3% on septic shock. The lung was the most frequent source of infection (65.6%) followed by CNS (16.4%), UTI (4.9%) and IE (4.9%). The most common pathogen isolated on these patients was S. pneumoniae (28.8%), followed by P. jirovecii (13.6%), toxoplasma (8.5%), E. coli (5.1%) and H. infl uenzae (5.1%). In total, 62.9% needed vasopressors (vs. 28.6% in non-infected patients, P = 0.001), 79% mechanical ventilation (vs. 42.85%, P <0.001) and 19.4% renal replacement (9.5% in no septic patients, P = 0.173). Mean ICU and hospital LOS was 10.43 ± 10.52 and 34.76 ± 29.64 days in septic patients versus 6.04 ± 8.45 (P = 0.026) and 20.54 ± 27.93 days (P = 0.016). ICU mortality: 33.9% (19% in nonseptic patients, P = 0.098), hospital mortality: 41.9% (vs. 23.8%, P = 0.057). Conclusion Sepsis is a common reason for admission to the ICU in HIV patients and is accompanied by high mortality. Pneumonia is the most frequent source of infection. Septic patients are less frequently under HAART and have a worse inmune status (lower CD4 count and higher viral load). Despite a higher APACHE II, and a higher need for hemodynamic and respiratory support, there is no statistically signifi cant diff erence in ICU and hospital mortality between septic and nonseptic patients. (2.4% in non-infected patients, P = 0.223) and 40.3% were under HAART (64.3% in patients admitted without infection, P = 0.016). Mean CD4 count at admission: 219.62 ± 353.93 cells/mm3 (vs. 370.22 ± 362.56, P = 0.048). Mean viral load: 4.57 ± 3.25 log (vs. 2.257 ± 1.96, P = 0.001). Of sepsis patients, 62.1% were on their CD4 nadir (vs. 34.5% in nonseptic patients, P = 0.009). Mean albumin levels were 2.3 ± 0.53 g/ dl (vs. 2.92 ± 0.94, P <0.001). APACHE II at admission was 21.98 ± 7.97 (vs. 18.15 ± 8.47, P = 0.046). At admission, 52.8% were on severe sepsis and 44.3% on septic shock. Table 2 (abstract P515). Unit and hospital mortality These results also suggest that mechanical ventilation intervention in patients with respiratory failure must occur earlier to improve the outcomes for these patients. Sepsis in HIV patients admitted to the ICU d l 1 l 1 l The lung was the most frequent source of infection (65.6%) followed by CNS (16.4%), UTI (4.9%) and IE (4.9%). The most common pathogen isolated on these patients was S. pneumoniae (28.8%), followed by P. jirovecii (13.6%), toxoplasma (8.5%), E. coli (5.1%) and H. infl uenzae (5.1%). In total, 62.9% needed vasopressors (vs. 28.6% in non-infected patients, P = 0.001), 79% mechanical ventilation (vs. 42.85%, P <0.001) and 19.4% renal replacement (9.5% in no septic patients, P = 0.173). Mean ICU and hospital LOS was 10.43 ± 10.52 and 34.76 ± 29.64 days in septic patients versus 6.04 ± 8.45 (P = 0.026) and 20.54 ± 27.93 days (P = 0.016). ICU mortality: 33.9% (19% in nonseptic patients, P = 0.098), hospital mortality: 41.9% (vs. 23.8%, P = 0.057). References 1. Hampshire PA, et al.: Crit Care 2009, 13:R137. 2 M i PB t l C it C M d 2002 30 2260 2 References References References 1. Tamburro RF, et al.: Pediatr Crit Care Med 2008, 9:270-277. 2. Chima RS, et al.: Pediatr Crit Care Med 2012, 13:e336-e342. P515 Survival of critically ill patients with haematological malignancies compared with patients without haematological malignancy R Pugh1, P Hampshire2, P Hajimichael3 1Glan Clwyd Hospital, Rhyl, UK; 2Royal Liverpool University Hospital, Liverpool, UK; 3Christie Hospital, Manchester, UK Critical Care 2013, 17(Suppl 2):P515 (doi: 10.1186/cc12453) P515 Survival of critically ill patients with haematological malignancies compared with patients without haematological malignancy R Pugh1, P Hampshire2, P Hajimichael3 1Glan Clwyd Hospital, Rhyl, UK; 2Royal Liverpool University Hospital, Liverpool, UK; 3Christie Hospital, Manchester, UK Critical Care 2013, 17(Suppl 2):P515 (doi: 10.1186/cc12453) Introduction Critically ill patients with haematological malignancies (HM) have high hospital mortality [1]. Severity of illness scores may underestimate mortality in such patients [2]. Introduction Critically ill patients with haematological malignancies (HM) have high hospital mortality [1]. Severity of illness scores may underestimate mortality in such patients [2]. Methods Data collection was conducted at three hospitals from 2008 to 2011. Patients with any active HM condition were matched with two control patients at two hospitals and with one control at Christie Hospital. Control patients had the same APACHE II (within 2 points) and admission diagnosis, but no HM. Readmissions and planned surgical cases were excluded. p p y Conclusion Sepsis is a common reason for admission to the ICU in HIV patients and is accompanied by high mortality. Pneumonia is the most frequent source of infection. Septic patients are less frequently under HAART and have a worse inmune status (lower CD4 count and higher viral load). Despite a higher APACHE II, and a higher need for hemodynamic and respiratory support, there is no statistically signifi cant diff erence in ICU and hospital mortality between septic and nonseptic patients. Results A total of 163 patients with HM were compared with 237 control patients. Seventy-four admissions with HM were identifi ed at two hospitals, and each was matched with two control patients. Eighty- nine admissions with HM from Christie Hospital were identifi ed. These were matched with 89 controls. Patients with HM spent signifi cantly longer in hospital before ICU admission (Table 1). Unit and hospital mortality rates were not statistically diff erent between patients with HM and without HM (Table 2). Mortality related to respiratory failure among pediatric hematology patients requiring intensive care H Okuno, K Atagi, H Shimaoka Osaka City General Hospital, Osaka, Japan Critical Care 2013, 17(Suppl 2):P514 (doi: 10.1186/cc12452) Table 1 (abstract P515). Patient characteristics Haematology Controls P value Age, mean (SD) 56.5 (15.1) 57.1 (15.7) 0.717 APACHE II, mean (SD) 22.7 (6.1) 23.1 (6.0) 0.484 ICU LOS (median) 3.2 (1.8 to 6.2) 3.1 (1.3 to 8.4) 0.948 LOS prior (median) 6.5 (2 to 17) 2.0 (0 to 7) <0.0001 bl ( b P ) i d h i l li Table 1 (abstract P515). Patient characteristics Table 1 (abstract P515). Patient characteristics H Okuno, K Atagi, H Shimaoka Introduction Although recent reports show an improvement in out- comes for pediatric hematology patients requiring intensive care [1,2], respiratory failure remains one of the major risks of pediatric mortality. This study was conducted to assess our hypothesis that mortality associated with respiratory failure is higher than that for other organ failures in pediatric hematology patients admitted to our ICU. Table 2 (abstract P515). Unit and hospital mortality Sepsis in HIV patients admitted to the ICU d l 1 l 1 l Methods A retrospective study of HIV patients admitted to our ICU between January 2005 and December 2009. We identify patients admitted to the ICU with sepsis and analyze demographic factors, etiology, organ failure and outcome, and we compare immune status, frequency of organ failure and outcome between these patients and those admitted for other reasons. We use Student’s t test to compare quantitative variables, and the chi-square test for qualitative data. Methods A retrospective study of HIV patients admitted to our ICU between January 2005 and December 2009. We identify patients admitted to the ICU with sepsis and analyze demographic factors, etiology, organ failure and outcome, and we compare immune status, frequency of organ failure and outcome between these patients and those admitted for other reasons. We use Student’s t test to compare quantitative variables, and the chi-square test for qualitative data. Results A total of 104 HIV patients were admitted to our ICU during the study period, 62 with sepsis (71% men, mean age: 40.59 ± 8.12). Of sepsis patients, 56.5% were admitted from the ER and 38.7% from a medical ward; 66.1% had history of intravenous drugs use, other comorbidities: COPD (9.7%), cirrhosis (8.1%), solid or hematologic malignancy (12.9%); 8.1% patients were unaware of their condition Results A total of 53.6% of physicians and 16.7% of nurses attended an earlier organ donation course (P <0.01). The average age of those who participated in training was signifi cantly higher among doctors (P <0.01). Fifty-nine percent of doctors and 65.1% of nurses did not even want to participate in such training. Donation activity was higher among staff who joined training (P <0.01). Independently from accepting the presumed consent legislation (91.1%), 66% of physicians agreed with the hospital practice that requests the adult donor’s relatives to consent to organ recovery. This standpoint did not depend on donation activity, participation in an organ donation course, opinion about legislation and the nature of staff . A total 95.4% of participants consented to their organ retrieval after death. The staff who participated in an organ donation course had more S190 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 (2.4% in non-infected patients, P = 0.223) and 40.3% were under HAART (64.3% in patients admitted without infection, P = 0.016). Mean CD4 count at admission: 219.62 ± 353.93 cells/mm3 (vs. 370.22 ± 362.56, P = 0.048). Automated, electronic monitoring for early detection of sepsis S Rauch1, M Fischer1, C Martin2, A Sablotzki3 Critical Care 2013, 17(Suppl 2):P517 (doi: 10.1186/cc12455) Introduction In the year 2009, the organizational structure of the ICU in the Kanazawa University Hospital changed from an open to a semi- closed format ICU. The objective of this study was to evaluate the eff ect of this organizational change on outcome in high-risk surgical patients. Methods The medical records of all consecutive high-risk surgical patients admitted to the ICU from 2006 to 2009 (open format, n = 1,598) and from 2009 to 2012 (semi-closed format, n = 1,521) were reviewed. Parameters studied were mortality and ICU length of stay. Introduction Early detection of sepsis is important for a suffi cient treatment to reduce mortality. We hypothesized that using modifi ed systemic infl ammatory response syndrome criteria over 1 hour using an electronic software program facilitates the clinical diagnosis of sepsis. Methods After IRB approval and informed consent we enrolled in this prospective, observational, single-center study 1,119 consecutive patients (age 68.6 ± 16.4, female/male 476/649) admitted over a 6-month period to a surgical ICU. A total 149 of them met modifi ed systemic infl ammatory response criteria. Patients were monitored by an electronic software program using live data from the laboratory and bedside monitors to detect modifi ed systemic infl ammatory response syndrome criteria persisting over 1 hour. The physicians were blinded to the software program alerts that notifi ed in real time when modifi ed systemic infl ammatory response syndrome criteria were detected and persisted over 1 hour, but did not provide treatment recommendations. Results There was a total of 149 modifi ed systemic infl ammatory response syndrome criteria alerts. Seventy-four were confi rmed as true sepsis cases by physicians. The overall incidence of sepsis was 7%. Patients were categorized into length of stay <24 hours, 24 to 96 hours and >96 hours. The overall sensitivity of our system for detecting sepsis was 68% and the specifi city was 91%. The positive predictive value is 34% and the negative predictive value is 98%. y g y Results Mortality of ICU patients was 9.9% in the open format group and 6.6% in the closed format group (P <0.05). The average length of hospital stay was 4.9 days in the open format group and 4.8 days in the closed format group. Outcome of critically ill patients with haematological malignancy admitted to the ICU as an emergency T Lofaro1, S Easdale2, S Rowe2, M Ostermann2, R Carr2 1Queen Elisabeth Hospital, Woolwich, UK; 2Guys’ and St Thomas’ Hospital, London, UK Introduction ICU admission policies regarding patients with haemato- logical malignancy still vary despite data showing an acceptable prognosis. Our aim was to review ICU and 6-month outcomes in this S191 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 group when requiring emergency admission to the ICU in a tertiary cancer centre. Figure 1 (abstract P518). Starch administration between November 2008 and November 2012. Figure 1 (abstract P518). Starch administration between November 2008 and November 2012. Methods A retrospective review of medical notes between 2004 and 2012. Results A total of 249 patients were admitted, of whom 54 had more than one admission. There were 310 episodes in total. Leukaemia n = 85; lymphoma n = 90; myeloma n = 36. We compared the characteristics of those who survived ICU admission with those who failed to survive to discharge from ICU. The two populations were similar (age 51 vs. 57; males 59% vs. 57%). Those who survived had a lower APACHE II score on admission (19 vs. 23; P <0.001), lower mean organ failure scores (1 vs. 2; P <0.05), lower requirements of inotropes (26% vs. 50%; P = 0.001), ventilation (31% vs. 64%; P = 0.001) and fi ltration (11% vs. 26%; P = 0.004). There was no diff erence in the prevalence of sepsis at the time of admission (64% vs. 70%). Both groups included patients with prior bone marrow transplant (38% vs. 40%). Of note, ICU and 6-month survival were 27% and 50%, respectively. These values are lower than those reported in the literature to date. Figure 1 (abstract P518). Starch administration between November 2008 and November 2012. Methods Our e-prescribing software enables users to prescribe intravenous fl uids from a series of menus. One of these is a template that has several fl uids available to use as a bolus when instructed by a clinician. We removed starch as an option from the template in April 2009. Starch could still be prescribed elsewhere on the prescribing system. Data on the use of starch from November 2008 to November 2012 were analysed as the mean volume of starch infused per patient per month. The mean of each set of parameters was then compared using a Student’s t test. Outcome of critically ill patients with haematological malignancy admitted to the ICU as an emergency Conclusion ICU and 6-month mortalities were 27% and 50%, respectively. Patients with haematological malignancy stand to benefi t from intensive care, and should be off ered admission based on clinical need. References 1. Cuthberson et al.: The outcome of haematological malignancy in Scottish intensive care units. J Intensive Care Soc 2008, 9:135-140. 2. Evison JM, et al.: Intensive care unit admission in patients with haematological disease: incidence, outcome and prognostic factors. Swiss Med Wkly 2001, 131:681-688. g Results The mean volume of starch per patient administered before and after electronic prescription options were altered was 480 ml and 21 ml, respectively (P = 0.004). See Figure 1. y 3. Beed et al.: Intensive care management of patients with haematological malignancy. Conti Edu An Crit Care Pain 2010, 10:167-171. 4. Kleber et al.: Comorbidity as a prognostic variable in multiple myeloma: comparative evaluation of common comorbidity scores and use of a novel MM–comorbidity score. Blood Cancer J 2011, 1:e35. Conclusion Despite clinicians intending to reduce the use of starch it was still regularly administered on our ICU. The removal of a default prescribing option dramatically reduced the volume of starch used whilst not restricting the ability to make a conscious choice to prescribe it. Adjusting default options has potential to infl uence clinical decisions and ensure more reliable, evidence-based care. Reference 5. McGrath S, et al.: ICU and 6-month outcome of oncology patients in the intensive care unit. QJM 2010, 103:397-403. P517f 1. Schultz MJ, et al.: J Crit Care 2005, 20:199-204. Eff ect of organizational structure of the ICU on the prognosis: open format versus semi-closed format T Taniguchi, M Okajima Kanazawa University Hospital, Kanazawa, Japan Critical Care 2013, 17(Suppl 2):P517 (doi: 10.1186/cc12455) T Taniguchi, M Okajima P518 Use of defaults on an electronic prescribing tool infl uences the type of fl uid received by patients L Herbert, C Bordeaux, M Thomas, L Burrows, J Bewley, T Gould Bristol Royal Infi rmary, Bristol, UK Critical Care 2013, 17(Suppl 2):P518 (doi: 10.1186/cc12456) Use of defaults on an electronic prescribing tool infl uences the type of fl uid received by patients Automated, electronic monitoring for early detection of sepsis S Rauch1, M Fischer1, C Martin2, A Sablotzki3 Conclusion Our results suggest that a semi-closed format is a more favorable setting than an open format to improve mortality in the ICU and to warrant safe outcome in this patient group. P519 Kanazawa University Hospital, Kanazawa, Japan Automated, electronic monitoring for early detection of sepsis S Rauch1, M Fischer1, C Martin2, A Sablotzki3 1Alb Fils Kliniken, Göppingen, Germany; 2Fa. Löser, Leipzig, Germany; 3Klinikum Sankt Georg, Leipzig, Germany Critical Care 2013, 17(Suppl 2):P519 (doi: 10.1186/cc12457) Use of defaults on an electronic prescribing tool infl uences the type of fl uid received by patients l y p L Herbert, C Bordeaux, M Thomas, L Burrows, J Bewley, T Gould Bristol Royal Infi rmary, Bristol, UK Critical Care 2013, 17(Suppl 2):P518 (doi: 10.1186/cc12456) l y p L Herbert, C Bordeaux, M Thomas, L Burrows, J Bewley, T Gould Bristol Royal Infi rmary, Bristol, UK yi y Critical Care 2013, 17(Suppl 2):P518 (doi: 10.1186/cc12456) Introduction Many evidence-based interventions are not delivered to patients [1]. This may not be due to a clinician’s intentional decisions. The aim of this project was to compare the use of starch before and after removing it as an option from an e-prescribing template. Conclusion Real-time alerts using an automated, electronic monitoring of modifi ed systemic infl ammatory response syndrome criteria facili- tate the clinical diagnosis of sepsis. S192 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 P520 score correlations between three raters were excellent (r = 0.80 to 0.94). Internal consistency in 98 videotaped patient presentations was acceptable (α = 0.78). Application to 22 IDRs led by 14 diff erent intensivists in three ICUs in two hospitals demonstrated that indicators could be unambiguously rated. The staff and management of all three ICUs that were rated had considered their IDRs to be adequately performed, and they were surprised by these study results. score correlations between three raters were excellent (r = 0.80 to 0.94). Internal consistency in 98 videotaped patient presentations was acceptable (α = 0.78). Application to 22 IDRs led by 14 diff erent intensivists in three ICUs in two hospitals demonstrated that indicators could be unambiguously rated. The staff and management of all three ICUs that were rated had considered their IDRs to be adequately performed, and they were surprised by these study results. Conclusion This study showed that the quality of IDRs can be reliably assessed for patient plan of care and process. The IDR-Assessment Scale had satisfactory inter-rater reliability, excellent overall item score correlations, and acceptable internal consistency. Our instrument may provide feedback for ICU professionals and managers to develop adjustments in quality of care. Testing the IDR-Assessment Scale in other ICUs may be required to establish general applicability. Reference P520 Admission to the ICU overnight: is it really a bad thing? Leadership training and quality improvement of interdisciplinary rounds in the ICU EC Ten Have, JE Tulleken Universitair Medical Center Groningen, the Netherlands Critical Care 2013, 17(Suppl 2):P522 (doi: 10.1186/cc12460) Introduction The development of patient-centered care by inter- disciplinary teams in the ICU has focused attention on leadership behavior. The purpose of this intervention study was to measure the eff ect of leadership training on the quality of performed inter- disciplinary rounds (IDRs) in the ICU. Methods In this nonrandomized intervention study, participants included nine intensive care medicine fellow trainees (intervention group) and 10 experienced intensivists (control group). Participants in the intervention and control groups previously were untrained in leading IDRs in the ICUs. After each participant led an IDR that was videotaped, the fellow trainees participated in a 1-day leadership training, which was consistent with principles of adult learning and behavioral modeling. After training, each fellow trainee led another IDR that was videotaped. Quality of the performed IDRs was measured by review of videotapes of the 19 IDRs lead by 19 intensivists, including 198 patient discussions subdivided into four ICUs, and assessment with the IDR-Assessment Scale. Conclusion There is no signifi cant diff erence between the mortality of patients admitted overnight and patients admitted during the day to our unit. The hospital length of stay is increased in patients who are admitted overnight to intensive care; however, ICU length of stay is not aff ected. Adjustment for other confounders such as current bed occupancy and staffi ng ratios during the entire patient stay may help to understand the diff erences seen in the hospital length of stay. Reference 1. Kuijsten HA, et al.: Intensive Care Med 2010, 36:1765-1771. f References References y Methods A literature search was performed to identify criteria for instruments about assessing team processes in the ICU. Then, 10 videotaped patient presentations led by diff erent intensivists were analyzed by Delphi rounds. Appropriate and inappropriate behaviors were highlighted. The IDR-Assessment Scale was developed and statistically tested. The inter-rater reliability was evaluated by rating nine randomly selected videotaped patient presentations by three raters. Finally, the scale was applied to 98 videotaped patient presentations during 22 IDRs in three ICUs for adults in two hospitals in Groningen. 1. Ten Have ECM, et al.: Assessing the quality of interdisciplinary rounds in the intensive care unit. J Crit Care 2013, in press. 1. Ten Have ECM, et al.: Assessing the quality of interdisciplinary rounds in the intensive care unit. J Crit Care 2013, in press. 2. Miller A, et al.: Uni- and interdisciplinary eff ects on round and handover content in intensive care units. Hum Factors 2009, 51:339-353. 3. Pronovost PJ, et al.: Perspective: physician leadership in quality. Acad Med 2009, 84:1651-1656. Reference 1. Reader TW, et al.: Developing a team performance framework for the intensive care unit. Crit Care Med 2009, 37:1787-1793. y g y Results Crude ICU and hospital mortality rates in patients admitted during the day and overnight were examined. There was no signifi cant diff erence in unit mortality (day 22.3% vs. night 22.7%, OR = 1.02, 95% CI = 0.91 to 1.16, P = 0.718) or hospital mortality (day 30.7% vs. night 29.1%, OR = 0.93, 95% CI = 0.83 to 1.04, P = 0.203). The mean unit length of stay showed no diff erence in patients admitted during daytime compared with those admitted overnight (4.27 days vs. 4.09 days, P = 0.162). The mean hospital length of stay was decreased in patients admitted during daytime compared with patients admitted overnight (19.3 days vs. 21.7 days, P = 0.004). The average age of patients was less in those admitted out of hours (night 56.5 years vs. day 59.2 years, P = <.0001). There was no signifi cant diff erence in APACHE II scores of patients between the groups (day 19 vs. night 19, P = 0.580).if Assessing the quality of interdisciplinary rounds EC Ten Have, JE Tulleken Universitair Medical Center Groningen, the Netherlands Critical Care 2013, 17(Suppl 2):P521 (doi: 10.1186/cc12459) Results Comparison of the intervention versus control groups shows that the intervention group has more yes scores on the IDR-Assessment Scale than the control group. This diff erence was signifi cant in 12 of the, in total, 19 quality indicators. g , Critical Care 2013, 17(Suppl 2):P521 (doi: 10.1186/cc12459) Conclusion Quality of leadership will be reliably trained and measured in the context of IDRs in ICUs. Training in a simulation environment, with real-life IDR scenarios including confl icting situations, and workplace- based feedback in the preparation and feedback phases, appears to be eff ective to train leadership behaviour. Introduction Interdisciplinary rounds (IDRs) in the ICU are increasingly recommended to support quality improvement and to reduce confl icts, but uncertainty exists about assessing the quality of IDRs. We developed, tested, and applied a scoring instrument to assess the quality of IDRs in ICUs. Use of defaults on an electronic prescribing tool infl uences the type of fl uid received by patients M Adams, P Dean, K MacDowall, P Stenhouse, A Mackay Victoria Infi rmary, Glasgow, UK Critical Care 2013, 17(Suppl 2):P520 (doi: 10.1186/cc12458) Introduction Admission to hospital overnight has been shown to increase mortality and decrease hospital length of stay [1]. The objective of this study was to determine whether this relationship is valid in patients admitted to our ICU, and whether length of stay was aff ected. y y y Conclusion This study showed that the quality of IDRs can be reliably assessed for patient plan of care and process. The IDR-Assessment Scale had satisfactory inter-rater reliability, excellent overall item score correlations, and acceptable internal consistency. Our instrument may provide feedback for ICU professionals and managers to develop adjustments in quality of care. Testing the IDR-Assessment Scale in other ICUs may be required to establish general applicability. Reference f Methods A retrospective data collection identifi ed 5,827 patients admitted to a fi ve-bed ICU from April 1994 to November 2012. Data regarding patient age, sex, APACHE II score and ICU admission date and time were collected along with the length of stay in the unit and hospital. Defi nitions of day and night were set to local ICU standards of 9:00 am to 8:00 pm. Patients were then separated into two groups and analysed using Analyse-It software for Excel. Let’s go round again! Quality improvement through intentional roundingi g Results The IDR-Assessment Scale had 19 quality indicators, subdivided into two domains: Patient Plan of Care, and Process. The domain Patient Plan of Care refl ects the technical performance from the initial identifi cation of a goal to the evaluative phase. The domain Process refl ects the team processes that are important to ensure that the appropriate plan of care is agreed, understood, and executed as planned by all care providers. Indicators were essential or supportive. The inter-rater reliability of nine videotaped patient presentations among three raters was satisfactory (κ = 0.85). The overall item P Doyle, F Cox, R Tollyfi eld, A Serajii P Doyle, F Cox, R Tollyfi eld, A Serajii Harefi eld Hospital, Harefi eld, UK ii Critical Care 2013, 17(Suppl 2):P523 (doi: 10.1186/cc12461) Introduction Harefi eld Hospital is a 150-bed cardiothoracic tertiary referral centre with transplantation, artifi cial heart, ECMO and primary angioplasty services. Our 35-bed critical care department consists of 18 intensive therapy unit, seven recovery and 10 high-dependency S193 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 (572.08 ± 214.68 vs. 168.6 ± 97.27 seconds, P <0.001) but are also associated with higher distractions particularly during evening shifts. Conclusion A higher percentage of nurses received handover training; nursing handovers are longer and more inclusive of other components of patient management; perceived importance of components of handover varies among healthcare professionals; distractions are common during handovers and associated with longer duration, by nurses and in the evening shifts; and higher confi dence level in patient’s management following the handover is associated with better satisfaction. (572.08 ± 214.68 vs. 168.6 ± 97.27 seconds, P <0.001) but are also associated with higher distractions particularly during evening shifts. beds. Intentional rounds or proactive patient rounds were recognised by the Royal College of Physicians and the Royal College of Nursing [1] as structured, evidence-based processes for nurses to carry out regular checks with individual patients at set intervals. The senior nursing team decided to adapt this initiative to the intensive care setting in order to address clinical challenges and provide guidance for shift leaders to focus on key elements of care. Methods Our intentional rounds, performed once per shift (twice daily), include two components. First, pressure area care – this component involves the shift leader checking whether key elements of pressure sore prevention have been performed. P524 Prospective observational study of handover in a medical ICU A Mukhopadhyay1, B Leong1, A Lua2, R Aroos2, J Wong2, N Koh2, N Goh2, K See1, J Phua1, Y Kowitlawakul3 1National University Health System, Singapore; 2National University of Singapore, Singapore; 3Alice Lee School of Nursing, National University of Singapore, Singapore Critical Care 2013, 17(Suppl 2):P524 (doi: 10.1186/cc12462) Results Over a 6-month period, 131 teleconsultations (114 patients) were done. Mean age was 50.1 years, 57.1% was male and mean APACHE II score was 24.3. A total of 64.8% originated from the ICU and 35.2% from the ED. Main consultation diagnoses were sepsis (31.3%); stroke (29.8%); survival from cardiac arrest (6.1%); trauma (6.1%); and acute myocardial infarction (5.3%). TM improved diagnosis in 14.5% and infl uenced the clinical management in 85.5% of the consultations. Invasive procedures were indicated in 61.1%. Life-saving procedures were TM related in seven patients (6.1%): stroke thrombolysis (n = 6) and limb amputation (n = 1). Seven patients (6.1%) were transferred and submitted to surgical procedures (heart surgery (n = 2), neurosurgery (n = 4) and liver transplantation (n = 1)). The majority of the patients remained at HMMD and were discharged. g p g p Critical Care 2013, 17(Suppl 2):P524 (doi: 10.1186/cc12462) Introduction Handovers are often associated with poor communi ca- tion. ICU patients with multiple complex problems are ideal to study naturally occurring handovers. However, few studies have been conducted in the ICU. Methods We conducted questionnaires of physicians and nurses involved and observed handovers in real time of medical ICU patients over 1 month. Results We interviewed 580 of 672 physicians and nurses involved (86.3%) and observed 90 real-time handovers (45 patients, 26.8%) of 168 patients. Mean duration of handover was 391.3 (± 263.6) seconds, 78.5% were face to face and 1.26 (± 1.75) distractions per handover were noted, person-to-person calling being the commonest mode of distraction (46.7%). Nurses received training during induction in signifi cantly higher numbers, covered allied specialties more and reviewed the patients early (all P <0.05). Perception of the relative importance of diff erent components of the handover varied signifi cantly between donors, recipients, physicians and nurses. P525 Impact of knowledge transfer through the implementation of a telemedicine program in a community hospital in Brazil CA Abreu Filho1, M Steinman1, A Andrade2, R Cal1, N Akamine1, J Teixeira2, E Silva1, A Kanamura1, M Cenderoglo1, C Lottenberg1 1Hospital Israelita Albert Einstein, São Paulo, Brazil; 2Hospital Municipal Dr. Moysés Deutsch, São Paulo, Brazil Critical Care 2013, 17(Suppl 2):P525 (doi: 10.1186/cc12463) Results The incidence of pressure ulcers in the 4 months since the initiative began has averaged 2.25 per month compared with 7.8 per month prior to commencement of intentional rounding. Added to the rounding tool at the end of September 2012, RRT rates in the preceding 4 months averaged 31.5 ml/kg/hour over 24 hours, an 11.9% reduction from the previous average of 35.75 ml/kg/hour. If the pattern of RRT was to continue, this could equate to a cost saving of UK£40,000 per annum. 1Hospital Israelita Albert Einstein, São Paulo, Brazil; 2Hospital Municipal Dr. Moysés Deutsch, São Paulo, Brazil C l C (S l ) P (d / ) Introduction Emergency survival rates vary signifi cantly according to the quality of care, which depends on human and technological resources. Emergency and critical care medicine physicians must make fast decisions; the presence of experienced consultants improves survival. In developing countries, there is a shortage of skilled doctors. The aim is to describe the fi rst Brazilian initiative of real- time teleconferencing telemedicine (TM) providing 24/7 emergency department (ED) and ICU coverage. Introduction Emergency survival rates vary signifi cantly according to the quality of care, which depends on human and technological resources. Emergency and critical care medicine physicians must make fast decisions; the presence of experienced consultants improves survival. In developing countries, there is a shortage of skilled doctors. The aim is to describe the fi rst Brazilian initiative of real- time teleconferencing telemedicine (TM) providing 24/7 emergency department (ED) and ICU coverage. Conclusion The use of a modifi ed targeted intentional rounding tool by the nursing shift leader can help ensure that best practice guidelines are adhered to. This strategy can improve patient outcomes and provide potentially signifi cant fi scal benefi ts. f 1. RCP, RCN: Ward Rounds in Medicine. Principles for Best Practice. London: Royal College of Physicians, Royal College of Nursing; 2012. 1. RCP, RCN: Ward Rounds in Medicine. Principles for Best Practice. London: Royal College of Physicians, Royal College of Nursing; 2012. References 1. Ye K, et al.: Handover in the emergency department: defi ciencies and adverse eff ects. Emerg Med Australas 2007, 19:433-441 1. Ye K, et al.: Handover in the emergency department: defi ciencies and adverse eff ects. Emerg Med Australas 2007, 19:433-441 2. Apker J, et al.: Communicating in the ‘gray zone’: perceptions about emergency physician hospitalist handoff s and patient safety. Acad Emerg Med 2007, 14:884-894. Let’s go round again! Quality improvement through intentional roundingi These include completion of the Waterlow risk assessment tool [2], noting the frequency of repositioning, use of lateral positioning and pressure-relieving pads. Second, renal replacement therapy rates – this element was identifi ed as an area for focus after we established that our haemofi ltration fl uid use per hour of therapy was twice that of a near identical clinical setting. This pattern continued even after adopting similar therapy guidelines. The shift leader was guided to check whether therapy rates had been adjusted in line with latest biochemical results. P525 g Methods Since May 2012 a TM program has been implemented at two hospitals in São Paulo, Brazil – Hospital Municipal Dr. Moysés Deutsch (HMMD), a public, secondary hospital, and Hospital Israelita Albert Einstein (HIAE), a tertiary private philanthropic entity – due to a partnership with the Brazilian Health Ministry. TM Central Command was located at HIAE with Endpoint 97 MXP Cisco® Solution and a mobile Intern MXP ISDN/IP Cisco® for the remote hospital (HMMD) via dedicated GB/sec connection. Imaging examinations were evaluated using PACS technology. Every recruited patient was assessed by the Central Command through TM with an experienced consultant. 2. Waterlow J: The importance of accurate risk assessment and appropriate intervention in tissue viability. Br J Nurs 2009, 18:1090. 2. Waterlow J: The importance of accurate risk assessment and appropriate intervention in tissue viability. Br J Nurs 2009, 18:1090. P524 Both physicians and nurses seldom (39.7%) reviewed the available electronic past medical records of the patient before handover, which in addition to training in handover and overall confi dence level in the management following handover are signifi cantly associated with better satisfaction in univariate analysis; only the confi dence level in patient management remained signifi cant after multivariate analysis. However, agreement between donor and recipient on overall satisfaction was poor (P >0.05). Nursing handovers were signifi cantly longer than physicians’ Conclusion A TM program is feasible to be implemented in a community hospital. The major benefi t is expertise medical transfer from the tertiary hospital to the community setting, improving diagnosis and management of critical care patients, and avoiding routine transfer to a major urban center. P526 P526 Utilization of telemedicine to improve burn care in a developing country J Knittel, G Fuzaylov Massachusetts General Hospital, Boston, MA, USA Critical Care 2013, 17(Suppl 2):P526 (doi: 10.1186/cc12464) Alternative to improve palliative care for all patients and families in critical care units: development and preliminary evaluation following MRC guidance of the King’s Psychosocial, Assessment and Care toolf Results Since 2011 we have provided consultation on 14 patients in Lviv, Ukraine, ranging in age from 15 months to 63 years. Each patient had an average of six consultations. We present two of these cases as examples of the capabilities of our telemedicine program. The fi rst case involved a 15-month-old female with 40% TBSA from scald injury, where telemedicine was instrumental in the primary assessment as well as to arrange a direct assessment from a nearby burn surgeon. The second case resulted from a house fi re with multiple casualties, where physicians in Boston were able to utilize telemedicine to guide the initial resuscitation and airway management of three critically burned children, as well as to arrange for transport of one of the victims, an 11-year-old male with 87% TBSA, from Ukraine to the USA for acute management. Multiple diffi culties were overcome in implementing the system between the two countries including: time zone diff erences, language barrier, and diff erent approaches to patient care. I Higginson, C Rumble, J Koff man, P Hopkins, S Heenen, W Prentice, R Burman, S Leonard, O Dampier, J Noble, M Morgan, C Shipman King’s Health Partners AHSC, London, UK Critical Care 2013, 17(Suppl 2):P528 (doi: 10.1186/cc12466) Introduction More than one in fi ve people admitted to an ICU will die there. Research has highlighted concerns about support for patients and families and decision-making in this context [1,2]. Here, we describe the development and evaluation of a tool to improve palliative care in a 32-bed general ICU in a central London teaching hospital. Methods Medical Research Council guidance for complex interventions Phase 0 to I comprised literature review, theoretical modelling, obser- vation and qualitative interviews and focus groups with staff and families exploring concerns and views of interventions identifi ed in the literature review. Phase II comprised intervention development, implementation and evaluation of tool feasibility and eff ects using staff survey, observation, audit of records and relative survey.f f Conclusion We have established a telemedicine program linking physicians in Boston, MA, USA with City Hospital #8 in Lviv, Ukraine to improve care in pediatric and adult burn patients. Our program has provided consultation on 14 patients since 2011, and it highlights the capabilities of telemedicine for acute consultation as well as triage and transport of critically ill patients to tertiary-care facilities. Diff erent aspects of therapy limitation: a comparative study of the nurse’s view É O Szűcs1, G Élő1, L Szabó1, C Varga2, J Gál1, L Zubek1 1Semmelweis University, Budapest, Hungary; 2University of Kaposvár, Hungary Critical Care 2013, 17(Suppl 2):P527 (doi: 10.1186/cc12465) O Szűcs1, G Élő1, L Szabó1, C Varga2, J Gál1, L Zubek1 1Semmelweis University, Budapest, Hungary; 2University of Kaposvár, Hungary Critical Care 2013, 17(Suppl 2):P527 (doi: 10.1186/cc12465) Introduction During the last few years the frequency of end-of- life decisions (EOLD) signifi cantly increased in ICUs. The method of nurse involvement in making EOLD is diff erent worldwide [1,2]. The purpose of this study was to analyze opinions of nurses about therapy restriction. We have examined with a multicenter study the opinions of the medical stuff about end-of-life care in Hungarian ICUs. Methods We performed a questionnaire evaluation among physicians and nurses of ICUs about infl uencing factors of therapy restriction, the method of the decision-making process, and the frequency of diff erent EOLD. The questionnaire, containing 21 questions, was delivered electronically to Hungarian ICUs, and then we analyzed the responses anonymously. The retrieved 302 answers (191 physicians, 102 nurses) were analysed using a nonparametric Student’s test. Conclusion K-PACE is a feasible tool to improve the palliative care of patients and their families in the ICU. Further refi nement is needed and planned, with consideration of roll-out into the wider medical centre. References 1. Azoulay E, et al.: Half the families of ICU patients experience inadequate communication with physicians. Crit Care Med 2000, 28:3044-3049 1. Azoulay E, et al.: Half the families of ICU patients experience inadequate 1. Azoulay E, et al.: Half the families of ICU patients e communication with physicians. Crit Care Med 2000, 28:3044-3049.l Results A total 71% of the nurse responders work in university clinics, 2% in regional centrum, 24% in municipal hospital, 3% in other ICUs. The nurses found both human (2.72/5 vs. 1.98/5) and material (2.81/5 vs. 2.12/5) resources more restrictive factors during patient admission than physicians (P = 0.025, P = 0.0024). Nurses working in municipal hospital were more strongly infl uenced by lack of material and human resources (3.34/5, 3.3/5) than nurses working in university clinics (2.2/5, 2.43/5), P = 0.01, P = 0.025. Younger nurses (working between 6 and 10 years) were more interested in the patient’s or surrogate’s wishes than older nurses (working more than 10 years). P528 P528 Alternative to improve palliative care for all patients and families in critical care units: development and preliminary evaluation following MRC guidance of the King’s Psychosocial, Assessment and Care tool I Higginson, C Rumble, J Koff man, P Hopkins, S Heenen, W Prentice, R Burman, S Leonard, O Dampier, J Noble, M Morgan, C Shipman King’s Health Partners AHSC, London, UK Critical Care 2013, 17(Suppl 2):P528 (doi: 10.1186/cc12466) country y J Knittel, G Fuzaylov Massachusetts General Hospital, Boston, MA, USA Critical Care 2013, 17(Suppl 2):P526 (doi: 10.1186/cc12464) Introduction Our objective is to present our experience from Shriner’s Hospital and Massachusetts General Hospital in Boston, MA, USA in S194 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 physicians slightly involved them in the end-of-life decision-making process (2.1/5 vs. 2.4/5 P = 0.0001). using telemedicine to provide acute burn and critical care consultation on pediatric and adult burn patients in Lviv, Ukraine, as well as in triage and transport of critically ill patients from Lviv to a tertiary-care facility in the USA for further management. using telemedicine to provide acute burn and critical care consultation on pediatric and adult burn patients in Lviv, Ukraine, as well as in triage and transport of critically ill patients from Lviv to a tertiary-care facility in the USA for further management. Conclusion We found that the workplace, level of medical attendance, godliness, work experience, and position in medical staff strongly infl uenced making EOLD. While limitation of the therapy should be team work, nurses felt their opinions were hardly taken into consideration, although nurses seemed to be more realistic in the decision-making process. Methods Using a new telemedicine learning center established at City Hospital #8 in Lviv, Ukraine, consultations regarding acutely injured burn victims occurred between physicians in Ukraine and physicians at Shriners Hospital and Massachusetts General Hospital in Boston. After the initial presentation, each patient was reviewed on a daily basis by physicians in Boston. Skype, an Internet-based communication tool, was used in communication with the Burn Center in Lviv. Radiographic images were scanned and digitalized using an electronic scanner, and JPEG image compression was used to facilitate the transmission of radiographic images and patient charts. Informed consent and HIPPA guidelines were followed in transmitting any patient-related information. Diff erent aspects of therapy limitation: a comparative study of the nurse’s view É Religion did not infl uence patient admission and forego therapy; however, religious nurses compared with atheists and nonpracticing believers preferred to prolong therapy against the patient’s will (P = 0.04). Nurses felt that communication with physicians. Crit Care Med l 2. Asch DA, et al.: Confl icts between physicians’ practices and patients’ wishes. Am J Respir Crit Care Med 1995, 151:288-292. P527f P527 Diff erent aspects of therapy limitation: a comparative study of the nurse’s view O Szűcs1, G Élő1, L Szabó1, C Varga2, J Gál1, L Zubek1 1Semmelweis University, Budapest, Hungary; 2University of Kaposvár, Hungary Critical Care 2013, 17(Suppl 2):P527 (doi: 10.1186/cc12465) Diff erent aspects of therapy limitation: a comparative study of the nurse’s view É References References . Prendergast et al.: A national survey of end-of-life care for critical 1. Prendergast et al.: A national survey of end-of-life care for critic 1. Prendergast et al.: A national survey of end-of-life care for critically ill patients. Am J Respir Crit Care Med 1998, 158:1165-1167. 2. Banbenishty et al.: Nurse involvement in end-of-life decision making: the ETHICUS Study. Intensive Care Med 2006, 32:129-132. 2. Banbenishty et al.: Nurse involvement in end-of-life decision making: the ETHICUS Study. Intensive Care Med 2006, 32:129-132. Alternative to improve palliative care for all patients and families in critical care units: development and preliminary evaluation following MRC guidance of the King’s Psychosocial, Assessment and Care toolf y y Results Phase I: 47 staff and 24 family members were interviewed. The short time between decisions for treatment withdrawal and death, plus concerns for support management, communication and decision-making, highlighted a need to ensure excellent psychosocial assessment for all. Phase II: as part of integrated care guidelines, we developed the King’s Psychosocial Assessment and Care tool (K-PACE). K-PACE is used for all patients entering the ICU, completed within 24 hours of admission. It contains psychosocial assessment of the family and patient needs, and identifi es key individuals for contact. Educational training was supported by K-PACE and was implemented in two waves. Post-implementation survey of 95 ICU staff found that most (80%) were aware of K-PACE. Eighty-two per cent of nurses but only 17% of doctors had completed the tool. In total, 158/213 (74%) family members responded to the survey (additionally three patients responded). There were high levels of satisfaction for symptom control and psychosocial care but concerns continued regarding explanation of treatment and care. P529 d End-of life thoughts in the ICU: results of a survey A Yaguchi, M Namiki, N Saito, R Nagai, M Takeda, T Harada, R Moroi Tokyo Women’s Medical University, Tokyo, Japan Critical Care 2013, 17(Suppl 2):P529 (doi: 10.1186/cc12467) End-of life thoughts in the ICU: results of a survey A Yaguchi, M Namiki, N Saito, R Nagai, M Takeda, T Harada, R Moroi Tokyo Women’s Medical University, Tokyo, Japan Critical Care 2013, 17(Suppl 2):P529 (doi: 10.1186/cc12467) y y y p Critical Care 2013, 17(Suppl 2):P529 (doi: 10.1186/cc12467) Introduction The decision of terminal care in the ICU is a very tough issue because the law, ethics, traditions and futility should S195 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 that withholding and withdrawing of treatment were ethically equal. A DNR would be used most often. Withdrawal of inotropes or antibiotics would be used more often than withdrawal of mechanical ventilation or extubation. Termination of artifi cial hydration would be rarely used in practice. be concerned involving the family’s will. Especially, stopping or withdrawing therapy is a quite diffi cult operation in Japan because of legal issues. Our hypothesis is that some diff erence exists in thoughts between physicians and nurses for terminal patients in the ICU. The aim of this study is to know their real thoughts. y g Methods A questionnaire survey was performed on physicians and nurses in our medico-surgical ICU. The questionnaire consists of 11 questions with fi ve optional answers related to the thoughts of participants about treatment of hopeless or brain death patients. Concretely, the questions were; whether to withhold therapy or not, whether to accept to withdraw therapy or not and with family’s will, whether to accept to immediately stop therapy and with family’s will, whether to positively or not donate organs from a brain death patient, necessity of ICU care for brain death patients, and feeling guilty and stress for stopping or withdrawing therapy. The optional answer has fi ve gradations from ‘Yes’ to ‘No’ for all questions. The participants were asked to answer the questionnaire by expressing themselves without regarding legal issues or the consensus. It was guaranteed to be anonymous for them in the data analysis. The answers were compared between physicians and nurses. The Mann–Whitney U test was used for statistical analysis. P <0.05 was considered statistically signifi cant. P529 d P531 Attitudes of intensivists in the UK to withdrawal of futile therapy M Mariyaselvam, M Irvine, J Carter, M Blunt, P Young Queen Elizabeth Hospital, Kings Lynn, UK Critical Care 2013, 17(Suppl 2):P531 (doi: 10.1186/cc12469) Introduction We aimed to determine the current practice and attitudes of consultants in intensive care medicine when withdrawing futile life-sustaining therapy. Published guidelines suggest variation in withdrawal of futile life-sustaining therapy and are therefore not prescriptive [1]. Although there is an awareness of diff ering practices, the extent of these variations is not established. Methods We surveyed a convenience sample of delegates at the Intensive Care Society (UK) State of the Art Meeting (2012) on attitudes and practice regarding withdrawal practice. Anonymised data were collected using surveymonkey.com. i Results There were in total 52 participants (response rate 98.1%) with 20 physicians and 32 nurses. Withdrawing therapy was signifi cantly accepted in nurses than in physicians (83% vs. 55%, P = 0.039), when the family well understood. Withholding therapy should not be operated for brain death patients for physicians (65%), while it seemed a diffi cult judgement for nurses (23%, P = 0.021). ICU care for brain death patients is less necessary for physicians than nurses (80% vs. 53%, P = 0.016). There were no signifi cant diff erences in other questions between physician and nurses such as feeling guilty or stress for stopping or withdrawing therapy.f Results Of 457 consultant attendees from the UK, 149 completed the survey (33%). For 58% of consultants there was no formal institutional protocol for withdrawal of futile therapy. When deciding to withdraw therapy, 57% of consultants routinely seek and document a second opinion. Regarding donation after cardiac death (DCD), 93% of consultants were happy to delay withdrawal to facilitate successful donation, 85% have already done so in their practice and 14% routinely withdraw therapy in theatres rather than on the ICU. Even if it would impact on the care of other patients, 48% would delay withdrawal of therapy to facilitate DCD. For patients accepted for DCD, 36% think that some intensivists withdraw more aggressively (in essence, hasten death) in the hope of improving the likelihood of a successful organ donation and 29% have felt pressurised to withdraw therapy more quickly than their usual practice. Furthermore, 45% experienced pressure to refer a patient for DCD when it they felt it was not appropriate. End-of-life decisions in Slovenian ICUs: a cross-sectional survey S Grosek1, M Orazem2, M Kanic2, G Vidmar3, U Groselj1 Conclusion This survey confi rms variation in the practice and attitudes to withdrawal of futile therapy amongst UK consultant intensivists. Formal protocols were frequently unavailable to guide withdrawal and second opinions were often not sought. Nearly one-half of the intensivists delay withdrawal to facilitate donation, even if this may impact on the care of other patients. Many intensivists have felt pressure to refer for donation when they feel this is inappropriate and there is a perception that some intensivists may withdraw care more aggressively in those who are accepted for DCD to improve the likelihood of a successful donation. This survey may help inform debate in this ethically challenging area. Critical Care 2013, 17(Suppl 2):P530 (doi: 10.1186/cc12468) Introduction The purpose of our study was to assess the attitudes of Slovenian intensivists towards end-of-life (EOL) decision-making and to analyze the decision-making process in their clinical practice. Methods A cross-sectional survey among Slovenian intensivists and intensive care medicine residents from 35 diff erent ICUs was performed using a questionnaire containing 43 questions about views on EOL decision-making. Fisher’s exact test and the Fisher–Freeman–Halton test were applied to cross-tabulated data; signifi cance level was set at P ≤0.001 due to the large number of tested hypotheses. Reference Reference g yp Results The response rate was 72.1% (267 questionnaires were returned out of 370 distributed), which represented roughly the same percentage of all Slovenian intensivists. Termination of futile treatment was assessed as ethically acceptable (P <0.001). The statement that there is no ethical distinction between withholding and withdrawing of treatment could not be confi rmed (the answers ‘there is a diff erence’ and ‘undecided’ were less frequent, but not statistically signifi cant; P = 0.216). A do-not-resuscitate order (DNR) was used more often than other withholding treatment limitations (P <0.001). A DNR was used most frequently in internal medicine ICUs (P <0.001; compared with paediatric and surgical ICUs). Withdrawal of inotropes or antibiotics was used more often than withdrawal of mechanical ventilation or extubation (66.7% vs. 12.0%; P <0.001). Withdrawal of mechanical ventilation or extubation was more often used in the paediatric ICUs (21.7%) as compared with the internal medicine ICUs (19.6%) and the surgical ICUs (3%) (P <0.001). Over two-thirds (70.6%) of intensivists were against termination of hydration, which would be more often used in the internal medicine ICUs (P <0.001). Thirty-one percent of intensivists used written DNR orders. 1. Organ Donation after Circulatory Death 1. Organ Donation after Circulatory Death [http://www.ics.ac.uk/professional/standards_and_guidelines/dcd] 1. Organ Donation after Circulatory Death [http://www.ics.ac.uk/professional/standards_and_guidelines/dcd] P529 d Conclusion Some of end-of-life thoughts in the ICU showed diff erences between physicians and nurses. P530 P530 End-of-life decisions in Slovenian ICUs: a cross-sectional survey S Grosek1, M Orazem2, M Kanic2, G Vidmar3, U Groselj1 1University Medical Centre Ljubljana, Slovenia; 2 University of Ljubljana, Slovenia; 3University Rehabilitation Institute, Republic of Slovenia, Ljubljana, Slovenia Critical Care 2013, 17(Suppl 2):P530 (doi: 10.1186/cc12468) Feasibility of neuromuscular electrical stimulation in critically ill patients p J Segers, G Hermans, F Bruyninckx, G Meyfroidt, D Langer, R Gosselink UZ/KU Leuven, Belgium Critical Care 2013, 17(Suppl 2):P535 (doi: 10.1186/cc12473) J Segers, G Hermans, F Bruyninckx, G Meyfroidt, D Langer, R Gosselink UZ/KU Leuven, Belgium Critical Care 2013 17(Suppl 2):P535 (doi: 10 1186/cc12473) Results Seven terminally ill patients wishing to go home for dying were transferred home. Diagnoses varied: end-stage lung disease, cancer, surgical complications. Ages ranged from 68 to 84 years. All patients survived transport home, and time at home varied from a few hours to 4 days. Later contacts with patients’ families indicated that both patient and family were grateful, and that they did not experience the patient having pain or dyspnea at home. Introduction Survivors of critical illness often have a prolonged stay on the ICU. These patients may suff er from ICU-acquired weakness. It has been shown that reduction in muscle mass and muscle strength occurs early after admission to the ICU. However, in the very early stage on the ICU, patients are often sedated and not able to participate in any active mobilizations. Therefore the use of neuromuscular electrical stimulation (NMES) is becoming a treatment of interest in the ICU. The aim was to study the feasibility and safety of NMES in a surgical and medical ICU of a large, tertiary referral university hospital. Conclusion Sending critically ill patients home to die is not common. Anyhow, our experiences doing that are only positive. Terminally ill patients, awake to make a decision of their own, and in a condition making it possible, should have the choice to go home to die, with our help in logistics, planning and transportation. Methods Fifty patients with an expected prolonged stay on the ICU of 5 more days (judged on day 3) with no trauma or neurological disease were included. They then received daily a NMES session (DUO 500; Gymna, Belgium) for 25 minutes on the quadriceps bilaterally during their entire stay on the ICU. The main outcome was the ability to produce a contraction of the quadriceps through NMES. The muscle contraction was quantifi ed on a 5-point scale: 1 (no contraction palpable and visible) up to 5 (contraction very well palpable and visible). Patients were classifi ed as responders when an adequate muscle bulk was obtained in ≥75% of the sessions. Getting critical care patients home for end-of-life care LS Nielsen Kolding Sygehus, Kolding, Denmark Critical Care 2013, 17(Suppl 2):P533 (doi: 10.1186/cc12471) Introduction Despite our eff orts in making patients healthy and going home, critical illness has a mortality, in Danish ICUs, of 10 to 12%. Approximately 90% of deaths in ICUs happen after life-sustaining therapy has been withheld or withdrawn. Although trying to provide patients and family with what we suppose is a good death, most patients would prefer dying at home, and sometimes patients and family ask for the possibility of doing that. The last 2 years we transferred seven patients from our unit to end-of-life care in their own home. Conclusion The 8-week exercise intervention resulted in statistically signifi cant improvements in fi tness at 9 weeks while focus group participants highlighted the positive eff ects of the exercise intervention leading to enhanced energy levels, motivation and achievement. Psychological benefi ts of the exercise programme are apparent from the focus group, emphasising the important link between physical and mental health. Methods After making the decision of withholding or withdrawing intensive care therapy, the care of the patient changes from an active, medical, technological treatment to relief and care. In that period we determine whether the patient’s condition is stable enough to go home. We try to fi nd out if it is a wish for the patient to go home, and if the family has resources to take care of the patient at home. If that is the case, we start planning care at home, arrange for transportation, and contact the primary care physician and nurse. Due to the patient’s condition on the day of transferring, we plan following the patient by either a nurse or a doctor. P535 Parent’s perception on end-of-life care in Brazil P Lago, J Piva, G Halal, M Halal Hospital de Clinicas de Porto Alegre, Brazil Critical Care 2013, 17(Suppl 2):P532 (doi: 10.1186/cc12470) Parent’s perception on end-of-life care in Brazil P Lago, J Piva, G Halal, M Halal Hospital de Clinicas de Porto Alegre, Brazil Critical Care 2013, 17(Suppl 2):P532 (doi: 10.1186/cc12470) Introduction The aim of the study is evaluate the perceptions of parents of children who died in two Brazilian pediatric ICUs. Introduction The aim of the study is evaluate the perceptions of parents of children who died in two Brazilian pediatric ICUs. Methods An exploratory-descriptive study with a qualitative approach in the PICU of Hospital São Lucas and Hospital de Clinicas de Porto Alegre involving 15 parents of children who died. Data collection was performed through three steps: (a) the researchers contacted the parents through a telephone call to invite them to attend the hospitals; (b) at the hospital, the doctors who assisted the children clarifi ed doubts about the therapy off ered; and (c) an interview was carried out by two researchers not involved in the care. Data analysis was performed using the technique of thematic content analysis. Conclusion Termination of futile treatment was found to be ethically acceptable for Slovenian intensivists, although they were not convinced S196 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 arm of the study, we examined the patient experience after critical illness, their views of the exercise programme and the eff ects on their HRQoL. Results and conclusion The research shows that the diffi culty of com- muni cation is a factor that impacts negatively on the grieving process. Moreover, it stresses the importance for parents to rediscuss the moment of their child’s death with health professionals. Methods A randomised controlled trial was undertaken in adult survivors of ICU admission. They were allocated to receive an 8-week in-hospital supervised aerobic programme consisting of two cycle ergometry and one unsupervised session per week (exercise group) or no exercise (control group). Primary outcomes were the anaerobic threshold (in ml O2/kg mass/minute), physical function and mental health scores (SF-36 questionnaire), measured at weeks 9 and 26. Participants were then allocated to focus groups where the interpretation of experiences was compared with outcomes from the PIX study. 1. Meyer E, Ritholz M, Burns J, Truog R: Improving the quality of end-of-life care in the pediatric intensive care unit: parents priorities and recommendations. Pediatrics 2006, 3:469-457. 2. Meyer E: On speaking less and listening more during end-of-life conferences. Feasibility of neuromuscular electrical stimulation in critically ill patients The potential factors associated with the feasibility were: gender, age, body mass index (BMI), diagnosis of sepsis, Barthel index prior to admission to the hospital, APACHE II score, Glasgow Coma Scale (GCS), fi ve questions for adequacy, stimulus intensity and leg edema. A multiple regression analysis was performed to identify the factors determining whether or not a contraction could be expected in a patient. Safety of NMES was assessed through heart rate, blood pressure, oxygen saturation and respiratory rate. 1. Lusardi P, et al.: The going home initiative: getting critical care patients home with hospice. Crit Care Nurse 2011, 31:46-57. P534 Project PIX (Post Intensive care eXercise): impact on physical fi tness and focus group analysis of quality of life following exercise rehabilitation Parent’s perception on end-of-life care in Brazil P Lago, J Piva, G Halal, M Halal Hospital de Clinicas de Porto Alegre, Brazil Critical Care 2013, 17(Suppl 2):P532 (doi: 10.1186/cc12470) Crit Care 2004, 32:1609-1611. 3. Piva, JP, Lago P, Othero J, Garcia PC: Evaluating end of life in ten Brazilian pediatric and adults intensive care units. J Med Ethics 2010, 36:344-348. 3. Piva, JP, Lago P, Othero J, Garcia PC: Evaluating end of life in ten Brazilian pediatric and adults intensive care units. J Med Ethics 2010, 36:344-348. 4. Meert, K et al.: Parents’ perspectives on physician-parent communication near the time of a child’s death in the pediatric intensive care unit. Pediatr Crit Care 2008, 9:2-7. 4. Meert, K et al.: Parents’ perspectives on physician-parent communication near the time of a child’s death in the pediatric intensive care unit. Pediatr Crit Care 2008, 9:2-7. y Results Fifty-nine patients were recruited to the study. The anaerobic threshold increased at week 9 in the exercise group by a clinically and statistically signifi cant amount of 2 ml O2/kg mass/minute (90% CI, 1 to 3 ml/kg/minute). There was further improvement in fi tness levels in both groups by week 26 (although no signifi cant diff erence between groups). No signifi cant diff erence in HRQOL measures between groups was demonstrated; however, the exercise group did show an improvement in their mental health scores. The focus groups centred on feelings of isolation, abandonment, vulnerability, dependency and reduced physical activity post hospital discharge. Many reported a lack of social inclusion as they did not have the energy or confi dence to venture outside. However, those in the exercise group felt that the rehabilitation programme was motivating, built up confi dence, improved fi tness, helped social interaction and gave them a sense of achievement. P533 G i Getting critical care patients home for end-of-life care LS Nielsen Kolding Sygehus, Kolding, Denmark Critical Care 2013, 17(Suppl 2):P533 (doi: 10.1186/cc12471) Implementation of an ICU follow-up clinic: outcomes and patient satisfaction after 1 year Implementation of an ICU follow-up clinic: outcomes and patient satisfaction after 1 year Implementation of an ICU follow-up clinic: outcomes and patient satisfaction after 1 year y Results Sixty-two patients matched inclusion criteria and were contacted. Thirty-three patients (90.9% males; age 38.7 ± 14.3 years; mean SAPS II 33.75 ± 11.79) were interviewed. Mean ICU stay was 25.7 ± 15.3 days. Fifteen patients (45.5%) had undergone temporary tracheostomy. Twenty-six patients were employed at the moment of trauma: of these, 11 (42.3%) stated to be jobless and one was pensioned at follow-up. In the whole cohort, the unemployment rate increased from 12.1 to 45.6%. Mean PCS was 43.59 ± 6.43, mean MCS was 41.04 ± 7.69. At follow-up, the 14 employed patients had a lower MCS versus the 19 jobless patients (37.94 ± 7.14 vs. 43.33 ± 7.44; P = 0.04), while no diff erences concerning PCS were found. The three patients that were living on their own when the trauma occurred showed, at follow-up, a lower mean MCS score towards the rest of the cohort (32.2 ± 0.47 vs. 41.93 ± 7.51; P <0.001). Introduction The aim was to analyse the outcomes and patient satisfaction of a recently implemented ICU follow-up clinic. These clinics are National Institute for Clinical Excellence recommended [1]. Introduction The aim was to analyse the outcomes and patient satisfaction of a recently implemented ICU follow-up clinic. These clinics are National Institute for Clinical Excellence recommended [1]. Methods A retrospective analysis of prospective collected data from January to December 2012. The clinic is run monthly by an ICU consultant and a critical care outreach sister. Criteria to be invited to the clinic are mechanical ventilation ≥3 days. Patients fi lled an anonymous satisfaction survey after the clinic. Methods A retrospective analysis of prospective collected data from January to December 2012. The clinic is run monthly by an ICU consultant and a critical care outreach sister. Criteria to be invited to the clinic are mechanical ventilation ≥3 days. Patients fi lled an anonymous satisfaction survey after the clinic. y Results Our attendance rate is 50% (26 patients), which is similar to other series reported in the literature. Those patients who attended the clinic required a longer length of mechanical ventilation (5.3 days vs. 7.1) and a longer length of stay in the ICU (7.6 vs. 13) and in hospital (14 vs. 28). Follow-up and counseling service in trauma patients: needs and goals – a preliminary study DL Grieco, M Biancone, SL Cutuli, G Pintaudi, MT Santantonio, ES Tanzarella, F Toni, AG De Belvis, MG Bocci, C Sandroni, M Antonelli Catholic University School of Medicine, Rome, Italy Critical Care 2013, 17(Suppl 2):P536 (doi: 10.1186/cc12474) g p y y DL Grieco, M Biancone, SL Cutuli, G Pintaudi, MT Santantonio, ES Tanzarella, F Toni, AG De Belvis, MG Bocci, C Sandroni, M Antonelli Catholic University School of Medicine, Rome, Italy Critical Care 2013, 17(Suppl 2):P536 (doi: 10.1186/cc12474) Conclusion This survey demonstrates that the provision of ICU clinics in the UK is not well established, with only 48% currently providing a regular service. Currently 67% of clinics are not funded and further resources should be employed so this service becomes an integral part of the ICU pathway. Despite recent evidence demonstrating that diaries reduce new-onset PTSD, only 44% of ICUs in our study provide this service. Ninety-one per cent of ICUs do not provide family support groups; similarly, it appears that fi nancial constraints are the limiting factor. Introduction Trauma is the most common cause of morbidity in young people. It has a high social impact both because of the high cost of the acute treatments and because of the physical and psychological consequences that it may cause. A prospective, observational, single- center study on quality of life 12 to 24 months after trauma was carried out. The aim of the study is to evaluate life quality after trauma and to identify the most important needs of the patients, in order to improve the level of care after an ICU stay and to implement a faster and more eff ective reintegration into the active and productive society. References 1. DH and Modernisation Agency: The National Outreach Report. London: NHS Modernisation Agency; 2003. 2. Jones C, et al.: Crit Care Med 2010, 14:168. 3. Orcutt TA:. Nurs Crit Care 2010, 5:33-37. 1. DH and Modernisation Agency: The National Outreach Report. London: NHS Modernisation Agency; 2003. f Methods All patients admitted to the 18-bed ICU of our university hospital between 5 May 2010 and 28 February 2011 because of a trauma who stayed in the ICU for at least 48 hours were eligible for the study. Patients were contacted and invited to attend a multidisciplinary interview. Data concerning family, working and economic status, main disabilities and medical needs were collected. The SF12 questionnaire was administered to defi ne physical component summary (PCS) and mental component summary (MCS) scores. SPSS 20.0 was used to perform statistical analysis. Project PIX (Post Intensive care eXercise): impact on physical fi tness and focus group analysis of quality of life following exercise rehabilitation Critical Care 2013, 17(Suppl 2):P534 (doi: 10.1186/cc12472) Introduction This study assessed the eff ect of a hospital-based aerobic exercise programme on physical fi tness and health-related quality of life (HRQoL) for survivors of ICU admission. Including the qualitative Results In 48% of the patients we were able to achieve adequate muscle contractions in more than 75% of the sessions. GCS (P = 0.047), edema S197 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 (P = 0.001) and sepsis (P = 0.010) were signifi cantly diff erent between responders and nonresponders. Responders had a lower mean GCS (7 ± 3 vs. 9 ± 3), lower amount of edema and were less likely to have had sepsis. In a multiple regression analysis, sepsis, edema, BMI and age explained 51% of the variance. As for safety, none of the parameters changed signifi cantly. incidence of new-onset PTSD [2]. Family support groups may also play a pivotal role [3]. We performed a national survey via the Intensive Care Society (ICS) to determine the provision of follow-up clinics, patient diary services and family support groups. Methods An online structured questionnaire was sent via the ICS to all ICU linkmen at 124 hospitals in the UK. Responses were received from 77 ICUs, a 62% response rate. Conclusion In patients with a better neurological condition, sepsis and/ or leg edema it was more diffi cult to obtain an adequate quadriceps contraction with NMES. NMES is safe to apply on the ICU. Results Out of 77 ICUs, 37 (48%) run a follow-up clinic. The majority of clinics (51%) only invite patients who have been admitted for more than 3 to 4 days. Only 10 clinics (30%) receive funding from the ICU budget or PCT, with the majority (67%) receiving no funding at all. Only 44% (34) of ICUs use patient diaries, mostly as a nonfunded service (68%). Additionally, 91% of ICUs do not run family support groups; from the minority that do, these are mostly held quarterly and are largely not funded (55%). Implementation of an ICU follow-up clinic: outcomes and patient satisfaction after 1 year We identifi ed a wide range of physical and nonphysical morbidities on these patients (Figure 1). We referred them to the appropriate specialities. Patients were very satisfi ed with this new service (Figure 2). Conclusion Trauma is associated with an increase of the unemployment rate in young people. Patients that are employed at follow-up seemed to have a worse mental health status towards the unemployed ones. Conversely, the employed patients did not show any trend to a lower physical health status. Thus a counseling service that provides psychological care would be useful to implement more eff ective work reintegration for trauma patients. Figure 1 (abstract P538). Physical and nonphysical morbidity. Follow-up and counseling service in trauma patients: needs and goals – a preliminary study Follow-up and counseling service in trauma patients: needs and goals – a preliminary study DL Grieco, M Biancone, SL Cutuli, G Pintaudi, MT Santantonio, ES Tanzarella, F Toni, AG De Belvis, MG Bocci, C Sandroni, M Antonelli Catholic University School of Medicine, Rome, Italy Critical Care 2013, 17(Suppl 2):P536 (doi: 10.1186/cc12474) Introduction Optimal patient evaluations of ICU rehabilitation therapy remain unclear. Introduction Optimal patient evaluations of ICU rehabilitation therapy remain unclear. Methods One hundred ICU patients with acute respiratory failure were randomized to receive early rehabilitation (ER) or usual-care (UC). Cohort 1 (n = 50) received ER as one physical therapy (PT) session/day versus UC; Cohort 2 (n = 50) received ER as 2 PT/day with the second session resistance training, versus UC. UC was without ER. Blood was drawn for cytokines through day 7. Cohort 2 underwent strength and physical functional assessments using the Short Physical Performance Battery (SPPB), a valid and reliable measure of physical function consisting of walking speed, balance, and repeated chair stands. It is a well-studied composite measure in older persons, but has not been used in ICU survivors. Small changes of 0.5 to 0.6 points in the SPPB have been shown to be clinically meaningful. Methods This study is a prospective randomised controlled trial with a repeated-measures crossover design. Eligible participants, requiring mechanical ventilation for 4 or more days, completed two exercise activities routinely used in early critical care rehabilitation, a passive chair transfer (PCT) and active sitting on the edge of the bed (SOEOB). The oxygen consumption and cardiovascular parameters were measured to quantify and compare the exercise response between the two activities. Results Data are presented as the median (interquartile range). Data for fi ve patients have been collected, aged 68 years (23), with an ITU stay of y g Results Baseline parameters were similar between groups. Median days from enrollment to fi rst PT were 4 (IQR 1 to 7.25). Deaths occurred in eight UC subjects and four in ER (P = 0.22). For both arms, ventilator days, ICU days and hospital days were not statistically diff erent. ER had ventilator-free days of 22, 95% CI = 19.9 to 24.6, where UC had 22.3 days, 19.9 to 24.6, P = 0.99. ICU-free days for ER was 21, 95% CI = 19.1 to 23.6, and that for UC was 21.0, 18.8 to 23.2, P = 0.84. Similarly, hospital days for ER was 16.7, 95% CI = 11.8 to 21.4, and for UC was 18.2, 13.8 to 22.7, P = 0.45. TNF, IL-6 and IL-8 through day 7 were not diff erent between groups. Despite similar baseline acuity and infl ammatory profi les, Cohort 2 ER group strength scores were numerically but not statistically higher. P537 Post-traumatic stress disorder – are we doing all we can? A UK national-based survey X Watson Ashford and St Peter’s Hospital, Surrey, UK Critical Care 2013, 17(Suppl 2):P537 (doi: 10.1186/cc12475) Introduction Post-traumatic stress disorder (PTSD) is a well-recognised complication in patients discharged from the ICU. ICU clinics have been recommended to treat physical and psychosocial problems post discharge, with guidelines issued by the Department of Health UK [1]. Recent evidence has advocated the use of patient diaries to reduce the Figure 1 (abstract P538). Physical and nonphysical morbidity. Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S198 Critical Care 2013, Volume 17 Suppl 2 http://ccforum com/supplements/17/S2 pp http://ccforum.com/supplements/17/S2 Figure 2 (abstract P538). Patient satisfaction survey. Figure 2 (abstract P538). Patient satisfaction survey. Conclusion Our follow-up clinic has enabled us to identify a wide range of complications related to ICU admission and coordinate their future management. This clinic improved patient satisfaction. Reference 15 days (10.5) and duration of mechanical ventilation 8 days (12), at the point of intervention. Exercise response results are reported (Table 1). Conclusion Intensive care patients with prolonged mechanical ventilation demonstrate a higher rate of oxygen consumption when actively sitting on the edge of the bed, compared with a passive chair transfer. This may have important consequences for early mobilisation of the critically ill. 1. Rehabilitation after Critical Illness [www.nice.org.uk/CG83] References References 1. Unroe et al.: Ann Intern Med 2010, 153:167-175. 2. Thomas: Phys Ther Rev 2011, 16:46-57. Acute exercise response in the critically ill N Collings1, R Young2 g g 1University Hospital Southampton, UK; 2Sheffi eld Hallam University, Sheffi eld, UK Randomized, controlled pilot study of early rehabilitation strategies in acute respiratory failure Critical Care 2013, 17(Suppl 2):P539 (doi: 10.1186/cc12477) Introduction This study aims to quantify the acute exercise response to early passive and active activities in order to inform exercise prescription when designing rehabilitation programmes for the critically ill. Critical care survival is often associated with a poor functional outcome [1], with recent investigations presenting the case for early rehabilitation in order to optimise functional recovery [2]. There, remains, however, a scarcity of research investigating the immediate response to exercise and subsequent exercise prescription, in the acute phase following critical illness. p y D Files, P Morris, S Shrestha, S Dhar, M Young, J Hauser, E Chmelo, C Thompson, L Dixon, K Murphy, B Nicklas, M Berry Wake Forest University School of Medicine, Winston Salem, NC, USA Critical Care 2013, 17(Suppl 2):P540 (doi: 10.1186/cc12478) D Files, P Morris, S Shrestha, S Dhar, M Young, J Hauser, E Chmelo, C Thompson, L Dixon, K Murphy, B Nicklas, M Berry Wake Forest University School of Medicine, Winston Salem, NC, USA Critical Care 2013, 17(Suppl 2):P540 (doi: 10.1186/cc12478) Introduction Optimal patient evaluations of ICU rehabilitation therapy remain unclear. Grip strength, as a percentage of predicted for ER was 66 versus Table 1 (abstract P539) Parameter PCT (n = 5) SOEOB (n = 5) Oxygen consumption (ml/minute) 270 (46.5) 334 (121) Carbon dioxide production (ml/minute) 142 (45.5) 185 (121) Heart rate (bpm) 92 (24) 77.5 (29) Mean arterial pressure (mmHg) 100.3 (20) 97.3 (13) Minute ventilation (l/minute) 11.0 (3.5) 12.1 (3.4) S199 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 39 for UC, P = 0.06. Dynamometry for ER was 211 versus 181 lbs for UC, P = 0.124. Although the diff erence in SPPB values for ER versus UC (5 vs. 3, P = 0.172) was not statistically diff erent, it was greater than the minimal clinically signifi cant diff erence. There were no diff erences in adverse events. or alleviate this stress [4]. To action this it is important to identify the main stressors from the patient’s perspective. A systematic review was performed to provide a list of what patients consider stressors in intensive care. These were then ranked in order to provide an identifi cation tool that can be used to shape appropriate care. Conclusion In this pilot study, early ICU rehabilitation was safe, and was associated with numerically although not statistically shorter hospital stay, greater strength and improved functional scores. Particularly, the SPPB demonstrated discriminatory ability in groups of ICU survivors with low physical function. Future early ICU rehabilitation studies should consider ICU survivor assessments using the SPPB due to its ease, reproducibility and discriminatory ability following ICU and hospital discharge. i Methods A systematic review was performed using MEDLINE, CINAHL, Psych INFO and Academic Search Complete. Grey literature was included and searches were not restricted to type of intensive care or country. Criteria were used to fi lter those articles that identifi ed the patients’ views of their stressor, not the patient experience. Eligible articles were critiqued using the Critical Appraisal Skills Programme for qualitative studies [5] and brought together using a narrative synthesis. All of the reviewed studies used a questionnaire as a means to identify what elements on the intensive care patients found stressful. A list of the top-10 stressors could then be expressed for each study and compared. From this information, a set of guidelines for best practice were devised. y References y p References 1. Baker C, et al.: Physical rehabilitation following critical illness. J Intensive Care Soc 2008, 9:166-169. 2. Jones C, et al.: Long term outcome from critical illness. Anaesth Intensive Care Med 2006, 7:155-156. Results In six hospitals the CQI ‘R-ICU’ is sent to relatives after receiving informed consent (n = 441), 55.1% of the respondents are the patient’s partner. Respondents seem to be most satisfi ed with the presence of a professional at fi rst entrance to the ICU. The highest need for improvement scores relate to information about meals, parking and other disciplines (for example, social worker, spiritual worker or psychologist). Factor analysis shows that quality of care is determined by four clusters of items: Support, Communication, General Information and Organisation. The reliability of the CQI ‘R-ICU’ is suffi ciently high, only Communication and Support are signifi cant predictors of total quality judgement of relatives (adj. R2 = 0.74). In addition, there is a signifi cant diff erence in mean total quality judgement between the six hospitals as well as between the four wards within Erasmus MC. None 3. CG83 Critical Illness Rehabilitation: Guideline 2009 [www.nice.org.uk/CG83] P543 Quality of care in the ICU from the perspective of relatives M Van Mol1, E Bakker2, A Rensen3, I Menheere3, L Verharen3 1Erasmus MC, Rotterdam, the Netherlands; 2OU, Heerlen, the Netherlands; 3HAN, Nijmegen, the Netherlands Critical Care 2013, 17(Suppl 2):P543 (doi: 10.1186/cc12481) P543 Quality of care in the ICU from the perspective of relatives M Van Mol1, E Bakker2, A Rensen3, I Menheere3, L Verharen3 1Erasmus MC, Rotterdam, the Netherlands; 2OU, Heerlen, the Netherlands; 3HAN, Nijmegen, the Netherlands Critical Care 2013, 17(Suppl 2):P543 (doi: 10.1186/cc12481) Introduction This study describes the development and validation of the Consumer Quality Index Relatives in ICUs (CQI ‘R-ICU’), which aims to measure the satisfaction of relatives and to identify aspect of care that need improvement in the ICU in a reliable and valid way. According to the quality standards of the Dutch Society of Intensive Care, every ICU needs to record the satisfaction of relatives [1]. At this moment there is insuffi cient insight into the quality of care off ered to relatives on the ICU because an evidence-based Dutch measurement instrument is missing. Results At baseline measurements, there were no statistical diff erences in age, gender, length of stays or APACHE II scores between the two groups. Results indicate that the exercise group (n = 10) had signifi cantly greater improvements in cardiopulmonary fi tness (P <0.001) and balance (P <0.05) compared with the control group (n = 10). Greater improvements were also evident in anxiety, depression and grip strength in the exercise group, although not statistically signifi cant. i Conclusion This pilot study highlights that a 6-week supervised exercise programme can signifi cantly improve cardiopulmonary fi tness and balance in post-ICU patients. Further recruitment to the study and 6-month/1-year follow-up is needed. Methods The CQI ‘R-ICU’ has been developed based on a scientifi c and standardised method [2]. A mixed design method is used, consisting of qualitative and quantitative survey studies. Factor analyses are carried out to determine the underlying structure of the newly developed questionnaire. Multiple regression analysis is used to explore the relationship between demographic variables and the perceived quality of care. P541 P541 Early results of a 6-week exercise programme in post-ICU patients C Battle1, K James1, P Temblett1, H Hutchings2 1Morriston Hospital, ABMU Health Board, Swansea, UK; 2Swansea University, Swansea, UK Critical Care 2013, 17(Suppl 2):P541 (doi: 10.1186/cc12479) Early results of a 6-week exercise programme in post-ICU patients C Battle1, K James1, P Temblett1, H Hutchings2 1Morriston Hospital, ABMU Health Board, Swansea, UK; 2Swansea University, Swansea, UK Critical Care 2013, 17(Suppl 2):P541 (doi: 10.1186/cc12479) Results A total 1,424 articles were systematically assessed for suitability and applicability. Of these, 14 articles remained eligible for review. The stressors were ranked by their frequency in the individual studies’ top- 10 lists. In rank order: 1, Tubes. 2, Pain. 3, Sleep Diffi culties. 4, Thirst. 5, Lack of Patient Understanding. Overall, stressors were found to be similar throughout all of the studies.i Early results of a 6-week exercise programme in post-ICU patients C Battle1, K James1, P Temblett1, H Hutchings2 g 1Morriston Hospital, ABMU Health Board, Swansea, UK; 2Swansea University, Swansea, UK 1Morriston Hospital, ABMU Health Board, Swansea, UK; 2Swansea University, Swansea, UK Introduction The aim of this study was to investigate the eff ect of a 6-week exercise programme on outcomes in post-ICU patients. With improvements in intensive care medicine, increasing numbers of patients are surviving catastrophic illness [1]. Severe weakness is common in patients with prolonged critical illness and results in considerable morbidity, mortality and healthcare costs [2]. The NICE83 guidelines Rehabilitation in Critical Care recommend follow-up for post-ICU patients and that further research is needed in this fi eld [3]. Conclusion The review identifi ed a list of the most pertinent common stressors. Awareness of these and ranking in priority may enable plans of care to be instigated to eff ectively alleviate patient stress. References References 1. Jones C, et al.: Clin Intensive Care 1998, 9:199-205. 2. Scragg P, et al.: Anaesthesia 2001, 56:9-14. 3. Perrins J, et al.: Intensive Crit Care Nurs 1998, 14:108-116. 4. Ballard KS: Issues Ment Health Nurs 1981, 3:89-108. 5. Public Health Resource Unit: Critical Appraisal Skills Programme. 10 Questions to Help You Make Sense of Qualitative Research. [http://www.medev.ac.uk/ static/uploads/workshop_resources/166/166_Qualitative_Appraisal_Tool.pdf] References 1. Jones C, et al.: Clin Intensive Care 1998, 9:199-205. 2. Scragg P, et al.: Anaesthesia 2001, 56:9-14. 3. Perrins J, et al.: Intensive Crit Care Nurs 1998, 14:108-116. 4. Ballard KS: Issues Ment Health Nurs 1981, 3:89-108. 5. Public Health Resource Unit: Critical Appraisal Skills Programme. 10 Questions to Help You Make Sense of Qualitative Research. [http://www.medev.ac.uk/ static/uploads/workshop_resources/166/166_Qualitative_Appraisal_Tool.pdf] 1. Jones C, et al.: Clin Intensive Care 1998, 9:199-205. i Methods Patients who have been discharged home from hospital following an ICU stay of 48 hours or more were recruited to the study. Patients were only excluded if they were not considered safe for exercise. Baseline measurements were completed prior to stratifi ed (age, gender, APACHE II score) random allocation to either the exercise or control group. Outcome measures included cardiopulmonary fi tness (6-minute walk test), balance (Berg Balance Scale), grip strength (JAMAR grip dynamometer) and hospital anxiety and depression (HAD score). The exercise group completed a 6-week supervised exercise programme, twice a week for up to 1 hour. In the seventh week, all patients repeated the baseline measurements. An unpaired Student’s t test was used to compare any diff erences between the control and exercise groups.f Introduction Optimal patient evaluations of ICU rehabilitation therapy remain unclear. Acknowledgement Supported by NIH grants 1R011186-01 and P30 AG21332. Family satisfaction in the ICU: enhancing patient experience E Kursumovic, J Bilinska, A Molokhia University Hospital Lewisham, London, UK Critical Care 2013, 17(Suppl 2):P544 (doi: 10.1186/cc12482) p p J Othero, AB Cavalcanti, JC Mouro, K Normilio-da-Silva, R Pusch, F Moreira, AA Kodama, O Berwanger Hospital do Coração – HCor, São Paulo, Brazil Critical Care 2013, 17(Suppl 2):P545 (doi: 10.1186/cc12483) Introduction Following recommendations made by the Scottish Intensive Care Society [1], we have introduced an annual Family Satisfaction Survey in the intensive care unit (FS-ICU), in anticipation that this will become an integral part of how we measure, improve and strive to advance patient care in the future. We aim to recognise factors impacting patient care and highlight areas for improvement. Improving understanding of patients’ needs is currently a UK national goal for CQUIN 2011/2012. An estimated £6.9 billion per year can be saved by reaching these goals [2] and a positive patient experience results in improved long-term outcomes and shorter hospital stays [2]. Methods We performed a study to assess Family Satisfaction over a 10- week period in a 16-bed critical care unit (CCU). A modifi ed version of an FS-ICU published in US and Canadian studies [3] was distributed to up to two family members per patient. Five-point Likert scale responses were linearly transformed to give percentage scores. Higher values represented a greater degree of satisfaction. Introduction We are conducting a cluster randomized trial with two parallel arms to evaluate strategies to improve family satisfaction with the care that themselves and their critically ill relatives receive in the ICUs of nonacademic Brazilian public hospitals. Here we report the results of the baseline phase of this trial. Introduction We are conducting a cluster randomized trial with two parallel arms to evaluate strategies to improve family satisfaction with the care that themselves and their critically ill relatives receive in the ICUs of nonacademic Brazilian public hospitals. Here we report the results of the baseline phase of this trial. Methods In this baseline phase, we interviewed the family member most closely involved with the care of critically ill patients who stayed in the ICU for at least 72 hours. We applied a form with 24 questions divided into four domains: overall ICU experience, communication, decision-making, and questions related to end-of-life care for patients who died in the ICU. Each question scored from 0 (very poor) to 100 (excellent). The form was adapted from the Family Satisfaction with Care in the ICU (FS-ICU 24). Recognition of the primary stressors aff ecting intensive care patients: a systematic review p y S Birch, S Elliot S Birch, S Elliot Leeds Teaching Hospitals, Leeds University Teaching Hospitals Trust, Leeds, UK Critical Care 2013, 17(Suppl 2):P542 (doi: 10.1186/cc12480) , Leeds Teaching Hospitals, Leeds University Teaching Hospitals Trust, Leeds, UK Critical Care 2013, 17(Suppl 2):P542 (doi: 10.1186/cc12480) Introduction Intensive care patients suff er psychological and physiological distress that may have debilitating and long-lasting eff ects [1-3]. Healthcare professionals are in a position to help avoid S200 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 References 3. Wall et al.: Refi nement, scoring, and validation of the Family Satisfaction in the Intensive Care Unit (FS-ICU) survey. Crit Care Med 2007, 35:271-279. 1. Vos et al.: Quality measurement at intensive care units: which indicators should we use? J Crit Care 2007, 22:267-274. 2. Sixma et al.: Handboek CQI Ontwikkeling: richtlijnen en voorschriften voor de ontwikkeling van een CQI meetinstrument. Utrecht: Nivel; 2008. 2. Sixma et al.: Handboek CQI Ontwikkeling: richtlijnen en voorschriften voor de ontwikkeling van een CQI meetinstrument. Utrecht: Nivel; 2008. References of the demographic variables such as sex, age, education, race and length of stay had an eff ect on perceived quality of care. 1. Quality Indicators for Critical Care in Scotland [http://www.sicsag.scot.nhs. uk/SICSQIG-report-2012-120209.pdf] 1. Quality Indicators for Critical Care in Scotland [http://www.sicsag.scot.nhs. uk/SICSQIG-report-2012-120209.pdf] 1. Quality Indicators for Critical Care in Scotland [http://www.sicsag.scot.nhs. uk/SICSQIG-report-2012-120209.pdf] f Conclusion The CQI ‘R-ICU’ turned out to be a valid, reliable, sensitive and feasible instrument. Large-scale implementation is recommended. Acknowledgements Thanks to researchers of Ziekenhuis Gelderse Vallei, Kennemer Gasthuis, Catharinaziekenhuis, Ziekenhuis Rivieren- land and Scheperziekenhuis. References 2. Using the Commissioning for Quality and Innovation (CQUIN) Payment Framework – Guidance on National Goals for 2011/12 [http://www.dh.gov. uk/prod_consum_dh/groups/dh_digitalassets/@dh/@en/@ps/documents/ digitalasset/dh_123009.pdf] 2. Using the Commissioning for Quality and Innovation (CQUIN) Payment Framework – Guidance on National Goals for 2011/12 [http://www.dh.gov. uk/prod_consum_dh/groups/dh_digitalassets/@dh/@en/@ps/documents/ digitalasset/dh_123009.pdf]i Family satisfaction with patient care in ICUs of nonacademic Brazilian public hospitals Family satisfaction with patient care in ICUs of nonacademic Brazilian public hospitals J Othero, AB Cavalcanti, JC Mouro, K Normilio-da-Silva, R Pusch, F Moreira, AA Kodama, O Berwanger Hospital do Coração – HCor, São Paulo, Brazil Critical Care 2013, 17(Suppl 2):P545 (doi: 10.1186/cc12483) Family satisfaction in the ICU: enhancing patient experience E Kursumovic, J Bilinska, A Molokhia University Hospital Lewisham, London, UK Critical Care 2013, 17(Suppl 2):P544 (doi: 10.1186/cc12482) As many questions assessed the quality of intensivist care or communication, the interview was applied by a psychologist or a nurse. y g Results Families of 564 patients were interviewed. A total 45/564 (8.3%) died in the ICU. Most respondents were satisfi ed with overall ICU experience (mean ± SD score 85.5 ± 11.9). However, family satisfaction with communication (67.8 ± 18.0) and decision-making (69.5 ± 21.1) resulted in somewhat lower scores. Most families of patients who died in the ICU (38/45 (82.6%)) considered that their relative’s life was neither extended nor shortened unnecessarily. Also, most of the families believed that their relative did not suff er or suff ered little in the ICU (37/46 (80.4%)) and felt supported by the healthcare team (40/46 (87.0%)).i Results We received and analysed 32 completed surveys. Seventy- eight per cent of relatives reported that the treatment of the patient was excellent, including symptom control such as pain, breathlessness and agitation (72%, 69%, 100%, respectively). Ninety-one per cent felt the care and frequency of communication provided by nurses was excellent or very good, compared with 81% by doctors. Only 9% of family members were off ered spiritual support during their CCU experience. Forty-seven per cent felt they would have liked more involvement in the decision-making process. Conclusion Most families were satisfi ed with the care themselves and their critically ill relatives received in the ICU. Also, most relatives of patients who died in the ICU felt that end-of-life care was adequate. Although we believe there is much room for improvement in communication, decision-making and support critically ill patients and their families, as their baseline satisfaction with patient care is quite high, it may be hard to demonstrate substantial improvement after interventions. Conclusion The vast majority of relatives rated their overall experience on CCU as excellent or very good. The study highlighted two main areas for improvement: provision of spiritual support as well as family and patient involvement in the decision-making process. We will arrange multi-disciplinary teaching sessions focusing on the positive impact, and therefore importance of patient satisfaction. A poster providing information about available spiritual support will be displayed around our CCU. Further studies are required to evaluate these measures.
https://openalex.org/W4366687130	https://www.researchsquare.com/article/rs-2489878/latest.pdf	English	null	The first chick brain with non-invasively embedded beads: a foundation for the automation of brain research	Bulletin of the National Research Centre/Bulletin of the National Research Center	2,023	cc-by	5,707	The first chick brain with non-invasively embedded beads: a foundation for the automation of brain research Akari Yoshimura Tohoku Medical and Pharmaceutical University Masayuki Seki (  seki@tohoku-mpu.ac.jp ) Tohoku Medical and Pharmaceutical University Akari Yoshimura Tohoku Medical and Pharmaceutical University Masayuki Seki (  seki@tohoku-mpu.ac.jp ) Tohoku Medical and Pharmaceutical University Research Article License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Page 1/16 Page 1/16 Conclusion With this technique, beads are embedded inside the brain without skull incision, promising a novel non- invasive method that overcomes the drawbacks associated with traditional invasive brain manipulation. B k d With this technique, beads are embedded inside the brain without skull incision, promising a novel non- invasive method that overcomes the drawbacks associated with traditional invasive brain manipulation. Results Fluorescent micro-beads that mimic probes were inoculated into the future brain area of chick embryos, and 20% of the manipulated embryos hatched, with 71% of the hatched chicks containing multiple beads in their brains. Background The automation of biotechnology, such as next-generation DNA sequencing, revolutionarily provides massive amounts of data and integrates various research fields. By contrast, many non-automated brain research fields are not interconnected with one other. In this study, we developed a basis for the automation of brain research. Two major technical barriers for the automation of brain research in vertebrates are the necessity for skull incision and a precise inoculation system for probes, devices, and electrodes in defined brain locations. Methods The former barrier in the background was overcome by inoculating probes into the future brain area of chick embryos before skull formation. Preparation of fluorescent beads Fluorescent Nile Red Particles (size: 5.0–7.9 mm; FP-6056-2) were purchased from Spherotech. Bead suspensions (100 mL) were mixed with 1 mL of sterilized phosphate-buffered saline (PBS). The mixture was centrifuged, and the supernatant was discarded. Then a needle was soaked in the remaining sterilized dense bead suspension for bead attachment (Fig. 2B(a)). Background In order to facilitate studying vertebrate behavior, we will propose here the concept on automation of brain research. Sanger and Mullis manually operated DNA sequencing and PCR, respectively (Supplementary Fig.S1(a)). Automatic DNA sequencing technology combining their principals has been developed (Supplementary Fig.S1 (b)), leading to the completion of various genome projects on model organisms. Furthermore, next- generation sequencing based on non-Sanger methods has generated massive DNA sequences of many organisms and individuals (Supplementary Fig.S1 (c)), revolutionarily expanding and integrating numerous biological research fields. Page 2/16 Page 2/16 By contrast, an integrated approach to brain research is lacking because a standardized automated system has not been established yet (Fig. 1). With the aim to automate brain research, a robot was developed that could implant 3,072 electrodes, mimicking a MEA (multielectrode array), into the rat brain (Fig. 1(a)). In total, 96 threads (each containing 32 electrodes) are implanted, with a rate of six threads per minute [1]. As such, it takes 16 min to complete this robotic surgery. Instead of this invasive robotic approach, we here propose a non-invasive automated system using vertebrate embryos. This approach is based on the inoculation of probes into the future brain area of developing chick embryos before skull formation. After the manipulated chicks hatch, the probes in their brains can communicate with helmet transmitters sending and receiving near-infrared (NIR) light from outside the skull (Fig. 2A(a)), similar to the existing near-infrared spectroscopy employed in humans (Fig. 1(b)). Numerous NIR-driven probes for brain research have already been developed, including probes that emit blue light to stimulate optogenetically manipulated neurons [2], sense catecholamine [3], record neuronal activity [4], stimulate neurons [5], suppress neurons [6], and deliver drugs as well as release a variety of macromolecules [7]. However, currently, all these probes must be invasively and manually inoculated into the brain. The development of NIR-driven brain micro-probes could be accelerated if hundreds of probes could be automatically, accurately, reproducibly, and non-invasively embedded into the brain. Here, we inoculated fluorescent micro-beads that mimic brain micro-probes into the head regions of chick embryos (Fig. 2B), thus obtaining the first animals with non-invasively inoculated multiple beads in their brains at birth (Fig. 3). The application of our successful technique in integrating brain research and applying to study animal behavior, is presented (Fig. 4, 5) Developmental stages of chick embryos The future brain area of chick embryos is visible at developmental stage 10, and the hard skull forms by day 13 [8]. Since the formation of the vascular network between the embryo and yolk after stage 18 hinders inoculation experiments, beads must be inoculated before stage 18 (see stages 13 and 18 in Fig. 3A(a) and (b), respectively). Since no commercially available brain probes, devices, or electrodes that are suitable for inoculating chick embryos exist, we inoculated 0.4–50 mm sized red or green fluorescent beads, instead of probes. Transparent treatment of the whole brain The hatched chicks were euthanatized by exposure to isoflurane, the occipital region of the skull was incised, and the whole brain was removed and placed into a 12-well plate (Greiner Bio-One). The brain was soaked in 4% paraformaldehyde in PBS (Wako) at 4 ℃ overnight. The fixed brain was washed three times with PBS and soaked in transparent reagent (CUBIC-L; Tokyo Kasei), according to the manufacturer’s protocol. CUBIC-L was changed twice within 48 h and then changed every 48 h for 6 days. Afterwards, the brain was washed three times with PBS and then soaked in 50% CUBIC-R (Tokyo Kasei; the original solution was diluted with distilled water) at room temperature overnight. Finally, the brain was soaked in 100% CUBIC-R for 24 h. The transparent whole brain was examined under a fluorescent stereomicroscope (Leica MZ10 F). Inoculation of fluorescent beads into chick embryos Fertilized chicken (Gallus gallus) eggs were purchased from the Yamagishi poultry farm (Gunma, Japan). The fertilized eggs were wiped once with 70% ethanol and incubated at 37.6°C (humidity 65 ± 5%) in an incubator (PH-3; Showa Furanki) until reaching the developmental stages 11, 13, or 18 [8]. The age of embryo in each stage as follows; stage11 (40–45 hour), stage 13 (48–52 h), and stage 18 (65–69 h). During incubation, fertilized eggs were manually rotated twice per day. After reaching the appropriate Page 3/16 Page 3/16 developmental stages, a window (diameter, 5 mm) of eggshell was cracked open. After removing 0.7 mL of egg white, the head area of the chick embryo was stabbed with a needle (Reactsystem, 35G) coated with fluorescent beads. After bead implantation, the window of eggshell was sealed with adhesive tape (Nichiban No. 405-1P). The manipulated embryos were incubated (at 37.6°C and 65 ± 5% humidity) in a fully automatic egg incubator (Maxi II EX; Brinsea Products Inc), which automatically and intermittently rotates manipulated eggs, until the 16th day. Finally, manipulated eggs were transferred to the PH-03 incubator (at 37.6°C and 65 ± 5% humidity) and incubated without egg rotation, from the 17th day to their hatching. developmental stages, a window (diameter, 5 mm) of eggshell was c of egg white, the head area of the chick embryo was stabbed with a with fluorescent beads. After bead implantation, the window of eggs (Nichiban No. 405-1P). The manipulated embryos were incubated (a fully automatic egg incubator (Maxi II EX; Brinsea Products Inc), whi rotates manipulated eggs, until the 16th day. Finally, manipulated eg incubator (at 37.6°C and 65 ± 5% humidity) and incubated without eg hatching. Chicks From Embryos Manipulated At Stage 18 Successfully Hatched The tip of the 35G needle (outer diameter, 0.23 mm) coated with 5.0–7.9 mm sized fluorescent beads was stabbed into the future brain area of stage-18 embryos without using a stereomicroscope. Since the head regions of stage-18 embryos were large enough to be recognized by the naked eye (Fig. 3A(b)), injecting black ink for visualization was unnecessary. After inoculation (Fig. 3B(a)), embryos were incubated until they hatched, and 26.4% of embryos hatched (Fig. 3C, Exp. 1). A photograph of one of the hatched chicks is shown (Fig. 3B(b)). Two additional independent experiments using stage-18 embryos yielded hatch rates of 10.0% and 20.8% (Fig. 3C, Exp. 2 and 3). A total of 38 hatched chicks out of 190 manipulated embryos were obtained (“YES” in Figs. 2B(b) and 3C). The average hatch rate of the manipulated embryos was thus 20.0%. The Chicks Had Beads Inside Their Brains At Birth To examine the presence of inoculated beads in the brains of hatched chicks (Fig. 2B(c)), all 38 hatched chicks were euthanatized, and their brains were removed. Each whole brain was fixed with paraformaldehyde (Fig. 3D(a)) and then soaked in transparent reagent (Fig. 3D(b)). To detect the fluorescent beads, the transparent brains were observed under a fluorescent stereomicroscope (Fig. 3E(a)). We detected multiple beads in a small, restricted region of the brain (Fig. 3E(b)(c)). Among the 38 hatched manipulated chicks, 27 chicks had fluorescent beads in their brains (Fig. 3F, “YES” in Fig. 2B(c)). The average rate of bead detection was 71.1%. Of note, multiple beads were always detected (Fig. 3E(b)(c)), reflecting the unlikelihood of picking up only one bead with the needle. In other words, dozens of beads mimicking micro-probes can be simultaneously embedded into chick embryos. Similar results were obtained with 0.4–0.6 mm sized fluorescent beads (Supplementary Fig.S2). Inoculation Of Chick Embryos At Stages 11 And 13 Is Fatal Chick embryos at stages 10–11 might be appropriate for the inoculation of beads because the brain primordium was shown to be swapped between males and females at stage 10 [9]. We initially used embryos at stages 11 and 13 for bead implantation. Since the head regions of embryos at stages 11 and 13 were still small (Fig. 3A(a)), they had to be visualized by a sub-blastodermal injection of black ink [9]. Under a stereomicroscope, the head regions of the embryos were stabbed with 33G needles (outer diameter, 0.25 mm) coated with 50 mm fluorescent beads (Fig. 2B(a)). The manipulated embryos were then incubated; however, they all died before hatching (“NO” in Fig. 2B(b)). Page 4/16 Page 4/16 Page 4/16 Stabbing the needle (outer diameter, 0.25 mm) into the head region (width, 1 mm; Fig. 3A(a)) of embryos at stages 11 and 13 was speculated to lethally damage the embryos. Furthermore, the 50 mm fluorescent beads were probably too large and thus unsuitable for implantation at embryonic stages 11 and 13. Although the exact causes of death were uncertain, we simultaneously changed the following three experimental conditions: embryonic stage, needle size, and fluorescent bead size. Discussion Since first manual experiments on DNA sequencing and PCR were reported, industries developed automatic-machines based on original principal (Supplementary Fig.S1). Thus, we will mainly discuss four perspectives; (1) How to develop machine based on the results of Fig. 3 (Fig. 4), (2) What sort of inoculatable devices to be developed (Fig. 2), (3) Application of automatic-brain research to a variety of vertebrates (Fig. 5), (4) Facilitation of study on animal behavior via automatic-brain research. Perspectives, (1)(2)(3) will be developed by industries, just like automatic-DNA sequencing. Achievement Page 5/16 Page 5/16 of (1)(2)(3) will lead us to legendary item, “King Solomon’s Ring” [10], which enables us to communicate with a variety of vertebrates. Thus, individual researchers could study animal behavior using such symbolic Ring in future. First, we propose how to automatically inoculate probes into chick brains. Although we succeeded in non- invasively generating animals whose brains contain multiple beads at birth, accurately embedding the beads in specific regions of the brain was not achieved. To overcome this problem, it is essential to develop automatic robotic inoculation systems (Fig. 2A(b)). The following four different industrial systems (Fig. 4(a–d)) exist: (a) the traditional pharmaceutical vaccine production line (which automatically sterilizes fertilized chick eggs and bores a hole in the eggshell), an automatic precise probe inoculation system combining (b) an integrated-circuit electronic production line and (c) an automatic staging and recording stereomicroscope system (utilized in various biological and medical sciences to record digital information of the x_y_z position of the object), and (d) poultry farming for the massive production of chicks. Combining these industries will enable a mass production line of chicks whose brains contain hundreds of beads (probes) (Fig. 2A(b), Fig. 4). Moreover, (e) computed tomography scanning of chick brains could individually, precisely, and digitally record the x_y_z position of each embedded bead in the brain. Finally, (f) artificial intelligence-mediated deep learning of the inoculation position in the embryo (c, g) and the real position of the beads embedded in the brain (e) guarantees an optimized program that inoculates probes into pre-defined brain regions. Since existing surgical robots have names such as “da Vinci”, the entire system of this putative automatic robot (Fig. 4) could be called “Spemann”, referring to the developmental biologist who manipulated vertebrate embryos and found “the organizer” [11]. Discussion 4(h)) will synergistically accelerate and integrate a variety of top-down and bottom-up brain research (Fig. 2A). Once such positive feedback begins (Fig. 4(h)), big data and the industrialization of neuroscience [13] will be accelerated under Moore’s law. Thus, the experimental results in Fig. 3 will be a key foundation for the transition from the brain research in Fig. 1 to that in Fig. 2A, via both the development of “Spemann” (Fig. 4) and new functional probes (Fig. 2A and Fig. 4(h)). If so, we predict that the “emperor” of neuroscience research [14] will wear a “new wardrobe” (i.e., the chick as a model [15]) in the near future. Importantly, such a putative “Spemann” system (Fig. 4) could reproducibly and simultaneously embed multiple probes in the brain, enabling the reproduction of any experiment, anytime and anywhere. Furthermore, all digital data could be opened, shared, analyzed, and utilized by everyone, similar to the free accessibility of existing DNA sequence data banks (Supplementary Fig.S1). In a similar manner to the recent establishment of automatic DNA sequencing (Supplementary Fig.S1(b)(c)), positive feedback between continuous technological improvements that accurately inoculate brain probes and the development of various functional probes (Fig. 4(h)) will synergistically accelerate and integrate a variety of top-down and bottom-up brain research (Fig. 2A). Once such positive feedback begins (Fig. 4(h)), big data and the industrialization of neuroscience [13] will be accelerated under Moore’s law. Thus, the experimental results in Fig. 3 will be a key foundation for the transition from the brain research in Fig. 1 to that in Fig. 2A, via both the development of “Spemann” (Fig. 4) and new functional probes (Fig. 2A and Fig. 4(h)). If so, we predict that the “emperor” of neuroscience research [14] will wear a “new wardrobe” (i.e., the chick as a model [15]) in the near future. Third, we propose application of the technology on chicks toward studies on a variety of vertebrate behavior. This method of producing chicks with multiple probes embedded in the brain in a non-invasive manner could be applied to any amniote. In chick embryos, stage 18 (Figs. 3A(b) and 5(a)) is similar to the pharyngula stage (Fig. 5(b)), sketched by Haeckel nearly 150 years ago [16]. The pharyngula embryonic stage of reptiles and mammals is remarkably similar to the embryo in Fig. 5(b), noted by Haeckel [16]. Thus, the proposed automatic “Spemann” system (Fig. Discussion 4) could be applied to any chick-type eggs derived from birds and reptiles. Thus, the behaviors and brain activities of many animals, such as penguins, hummingbirds, chameleons, and turtles (Fig. 5(c)–(f)), could be analyzed (Fig. 2A), tremendously facilitating comparative brain research. A robot embedding multiple electrodes in the rat brain has been developed (Fig. 1(a)) [1]. Thus, a robot that could operate small mammals (e.g., mice and rats) and embed probes into the head region of embryos in utero could be developed (Fig. 5(g)) using the “Spemann” method, yielding mice or rats with probes inside their brains at birth (Fig. 5(h)). Third, we propose application of the technology on chicks toward studies on a variety of vertebrate behavior. This method of producing chicks with multiple probes embedded in the brain in a non-invasive manner could be applied to any amniote. In chick embryos, stage 18 (Figs. 3A(b) and 5(a)) is similar to the pharyngula stage (Fig. 5(b)), sketched by Haeckel nearly 150 years ago [16]. The pharyngula embryonic stage of reptiles and mammals is remarkably similar to the embryo in Fig. 5(b), noted by Haeckel [16]. Thus, the proposed automatic “Spemann” system (Fig. 4) could be applied to any chick-type eggs derived from birds and reptiles. Thus, the behaviors and brain activities of many animals, such as penguins, hummingbirds, chameleons, and turtles (Fig. 5(c)–(f)), could be analyzed (Fig. 2A), tremendously facilitating comparative brain research. A robot embedding multiple electrodes in the rat brain has been developed (Fig. 1(a)) [1]. Thus, a robot that could operate small mammals (e.g., mice and rats) and embed probes into the head region of embryos in utero could be developed (Fig. 5(g)) using the “Spemann” method, yielding mice or rats with probes inside their brains at birth (Fig. 5(h)). Discussion “Spemann” could meet the academic and industrial demand for brain probes, devices, and electrodes that are controlled via NIR from outside the skull (Fig. 2A(a) and Fig. 4(h)). Second, we propose candidate probes and integrating whole brain research. Some existing methods in brain research (Fig. 1) can be replaced with the technology of non-invasively embedding brain probes that are individually driven by NIR (Fig. 2A(0)). Such technologies (Fig. 2A(1)–(13)) include probes that (1) destroy the surrounding neurons in a temporally and spatially specific manner, like surgical resection; (2) sense oxygen or glucose, like fMRI or PET; (3) sense a variety of molecules including neurotransmitters, (4) simultaneously or individually stimulate or suppress neuronal activities, like MEA; (5) simultaneously or individually record neuronal activities, like MEA; and (6) deliver drugs in a temporally and spatially specific manner, instead of drug injection. Furthermore, (7) massive digital signals of a particular animal that are interconnected with machines, robots, and other animals via the internet according to the brain- net concept could be employed. Upon such detailed in vivo top-down probe analyses (1–7), (8) biopsies of the corresponding probes can enable a variety of biochemical analyses, and (9) the neuronal network as a connectome [12] can be visualized by neurons that take up retrograde-type dyes released from the probes. Furthermore, after euthanizing animals, (10–11) neurons and brain sections can be analyzed in vitro by stimulating, suppressing, and recording their activity via the probe; (12) and the signal transduction of neurons and brain sections can be analyzed in vitro around the probe. These bottom-up Page 6/16 Page 6/16 analyses (8–12) could be functionally and mutually linked to top-down analyses (1–7), integrating all brain research. Finally, when using transgenic animals or recombinant virus-infected animals for optogenetics, (13) blue light can be supplied from the NIR-driven probe [2]. Importantly, such a putative “Spemann” system (Fig. 4) could reproducibly and simultaneously embed multiple probes in the brain, enabling the reproduction of any experiment, anytime and anywhere. Furthermore, all digital data could be opened, shared, analyzed, and utilized by everyone, similar to the free accessibility of existing DNA sequence data banks (Supplementary Fig.S1). In a similar manner to the recent establishment of automatic DNA sequencing (Supplementary Fig.S1(b)(c)), positive feedback between continuous technological improvements that accurately inoculate brain probes and the development of various functional probes (Fig. Consent for publication Not applicable. Author’s contributions MS designed the study. AY conducted the experiments and the data analysis. AY and MS wrote the manuscript. All authors reviewed the manuscript. Availability of data and materials Since all data we have are shown in main and supplementary materials, there is no data deposition along with the manuscript. The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. Funding Not applicable. Ethics approval and consent to participate All experiments were carried out according to the ARRIVE guidelines and with the institutional ethical approval. Briefly, the experimental procedures described in “Methods” were approved by the institutional animal care and use committee of Tohoku Medical and Pharmaceutical University based on the National Institutes of Health guide for the care and use of laboratory animals. Conclusion With the technique developed here (Fig. 3), beads are embedded inside the brain without skull incision, promising a novel non-invasive method that overcomes the drawbacks associated with traditional invasive brain manipulation. Such novel animals with beads in their brains at birth will open completely new avenues toward the automation of brain research (Figs. 2, 4, 5), leading to enormous applications on studying a variety of animal behaviors (Fig. 5). Beside brain researches, the automation of developmental biology could also be achieved with the “Spemann” method (details in Supplementary Fig.S3 and its legend). Thus, our proposal in this study could accelerate all brain researches, studies of a variety of vertebrate behavior, and studies of developmental biology in many amniotes. Page 7/16 Page 7/16 Competing interests The authors declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. References 1. Musk E. (2019) An integrated brain-machine interface platform with thousands of channels. J Med Internet Res. 2019; doi: 10.2196/16194. 1. Musk E. (2019) An integrated brain-machine interface platform with thousands of channels. J Med Internet Res. 2019; doi: 10.2196/16194. 1. Musk E. (2019) An integrated brain-machine interface platform with thousands of channels. J Med Internet Res. 2019; doi: 10.2196/16194. 2. Chen S, Weitemier AZ, Zeng X, He L, Wang X, Tao Y, Huang AJY, et al. Near-infrared deep brain stimulation via upconversion nanoparticle-mediated optogenetics. Science. 2018;359:679–84. 2. Chen S, Weitemier AZ, Zeng X, He L, Wang X, Tao Y, Huang AJY, et al. Near-infrared deep brain stimulation via upconversion nanoparticle-mediated optogenetics. Science. 2018;359:679–84. 3. Beyene AG, Delevich K, Bonis-O’Donnell JTD, O’Donnell B, Piekarski DJ, Lin WC, et al. Imaging striatal dopamine release using a nongenetically encoded near infrared fluorescent catecholamine nanosensor. Sci Adv. 2019. 10.1126/sciadv.aay3108. 3. Beyene AG, Delevich K, Bonis-O’Donnell JTD, O’Donnell B, Piekarski DJ, Lin WC, et al. Imaging striatal dopamine release using a nongenetically encoded near infrared fluorescent catecholamine nanosensor. Sci Adv. 2019. 10.1126/sciadv.aay3108. 4. Liu J, Zhang R, Shang C, Zhang Y, Feng Y, Pan L, et al. Near-infrared voltage nanosensors enable rea time imaging of neuronal activities in mice and zebrafish. J Am Chem Soc. 2020;142:758–67. 4. Liu J, Zhang R, Shang C, Zhang Y, Feng Y, Pan L, et al. Near-infrared voltage nanosensors enable real- time imaging of neuronal activities in mice and zebrafish. J Am Chem Soc. 2020;142:758–67. 5. Ma Y, Bao J, Zhang Y, Li Z, Zhou X, Wan C, et al. Mammalian near-infrared image vision through injectable and self-powered retinal nanoantennae. Cell. 2019;177:243–55. 5. Ma Y, Bao J, Zhang Y, Li Z, Zhou X, Wan C, et al. Mammalian near-infrared image vision through injectable and self-powered retinal nanoantennae. Cell. 2019;177:243–55. 6. Lee JW, Jung H, Cho HH, Lee JH, Nam Y. Gold nanostar-mediated neural activity control using plasmonic photothermal effects. Biomaterials. 2018;153:59–69. 7. Li W, Luo R, Lin X, Jadhav AD, Zhang Z, Yan L, et al. Remote modulation of neural activities via near- infrared triggered release of biomolecules. Biomaterials. 2015;65:76–85. 8. Bellairs R, Osmond M. The atlas of chick development. 2nd ed. London: , Osmond M. The atlas of chick development. 2nd ed. London: Academic pr 9. Maekawa F, Sakurai M, Yamashita Y, Tanaka K, Haraguchi S, Yamamoto K, et al. Acknowledgements Since the practical and theoretical research was based on numerous studies done by past and present undergraduate students, we thank the following students: Yunome M, Chiba H, Kimura Y, Furuichi M, Yamazaki Y, Ono A, Sasaki S, Kuroda T, Yaegashi W, Ohtomo Y, Torigoe M, Furuta E, Yashima A, Hironobu M, Itoh N, Yoshida S, Sato K. We thank Drs. Nakamura M, Tamura K, Homma K, Hamazaki H for various advice on chick embryo manipulation. We thank Drs. Ando R, Enomoto T for enabling us to perform this study. We thank Drs. Ish-Horowicz D, Henrique D, Lewis J, Stern C, Hatada Y for inspiring the authors to analyze brain function by using developing chick embryos. We thank our colleagues, Drs. Ambo A, Nakabayashi Y for continuously encouraging this study. We thank Ms. Seki M for illustrating most of the figures. Since this study was financially supported for many years by the Tohoku Medical and Page 8/16 Page 8/16 Pharmaceutical University, whose founding sprit is “We will open the gate of truth”, we thank Dr. Takayanagi M, chairman of the University. Pharmaceutical University, whose founding sprit is “We will open the gate of truth”, we thank Dr. Takayanagi M, chairman of the University. References A genetically female brain is required for a regular reproduction cycle in chicken brain chimeras. Nat Commun. 2013. 10.1038/ncomms2372. 10. Lorenz KZ. King Solomon’s Ring. Translated by Wilson MK. London: Met 11. Spemann H, Mangold H. Induction of embryonic primordia by implantation of organizers from a different species. 1923. Int J Dev Biol. 2001;45:13–38. 12. Winnubst J, Bas E, Ferreira TA, Wu Z, Economo MN, Edson P, et al. Reconstruction of 1,000 projection neurons reveals new cell types and organization of long-range connectivity in the mouse brain. Cell. 2019;179:268–81. 13. Frégnac Y. Big data and the industrialization of neuroscience: A safe roadmap for understanding the brain? Science. 2017;358:470–77. 14. Yartsev MM. The emperor's new wardrobe: Rebalancing diversity of animal models in neuroscience research. Science. 2017;358:466–69. 15. Rogers LJ. The development of brain and behavior in the chicken. UK: CAB international, Wallingford;; 1992. 15. Rogers LJ. The development of brain and behavior in the chicken. UK: CAB international, Wallingford;; 1992. 16. Richardson MK. Heterochrony and the phylotypic period. Dev Biol. 1995;172:412–21. 16. Richardson MK. Heterochrony and the phylotypic period. Dev Biol. 1995;172:412–21. Page 9/16 Page 9/16 17. Rothschild RM. Neuroengineering tools/applications for bidirectional interfaces, brain-computer interfaces, and neuroprosthetic implants – a review of recent progress. Front Neuroeng. 2010. 10.3389/freng.2010.00112. 18. Miranda PC. Physics of effects of transcranial brain stimulation. Handb Clin Neurol. 2013;116:353– 66. 19. Lebedev MA, Carmena JM, O'Doherty JE, Zacksenhouse M, Henriquez CS, Pricipe JC, et al. Cortical ensemble adaptation to represent velocity of an artificial actuator controlled by a brain-machine interface. J Neurosci. 2005;25:4681–93. 19. Lebedev MA, Carmena JM, O'Doherty JE, Zacksenhouse M, Henriquez CS, Pricipe JC, et al. Cortical ensemble adaptation to represent velocity of an artificial actuator controlled by a brain-machine interface. J Neurosci. 2005;25:4681–93. 20. Simeral JD, Hosman T, Saab J, Flesher SN, Vilela M, Franco B, et al. Home use of a percutaneous wireless intracortical brain-computer interface by individuals with tetraplegia. IEEE Trans Biomed Eng. 2021;68:2313–25. 20. Simeral JD, Hosman T, Saab J, Flesher SN, Vilela M, Franco B, et al. Home use of a percutaneous wireless intracortical brain-computer interface by individuals with tetraplegia. IEEE Trans Biomed Eng. 2021;68:2313–25. Figures Page 10/16 Figure 1 Figure 1 Page 10/16 The key concepts of our research. (A) Our vision for the automation of brain research. (A) Our vision for the automation of brain research. An illustration of a chick, whose brain contains probes, wearing a blue helmet representing a near-infrared (NIR) transmitter (0), as in Fig. 1(b). (a) NIR light should reach the deep brain of chicks [2]. (b) If hundreds of probes, devices, and electrodes are embedded in a defined area of vertebrate brain, the probes could individually communicate with the outside of the skull via NIR (0). Some existing brain research in Fig. 1 can be replaced with the NIR-driven probes (1)-(13) mentioned in the text. (B) The aim of this study. Lack of automation in brain research. The various methods in brain research described below are not automatized or technically interconnected with each other. Two approaches exist in vivo: non-invasive and invasive approaches. Non-invasive technologies, such as positron emission tomography (PET), computed tomography (CT), functional magnetic resonance imaging (fMRI), magnetoencephalography (MEG), near-infrared spectroscopy (NIRS), and electroencephalography (EEG), measure brain activities via different principals [17], whereas transcranial magnetic stimulation (TMS), transcranial direct current stimulation (tDCS), and transcranial alternating current stimulation (tACS), broadly stimulate the brain [18]. These non-invasive technologies are not interconnected and cannot evaluate or stimulate individual neuronal activity. Genetically manipulated animals, including knockout, knockdown, or overexpressed mice, and pharmacological approaches are also categorized as in vivo non-invasive brain research. Conversely, electrocorticography (ECoG), an in vivo invasive technology, simultaneously stimulates and records broad areas of the brain cortex [17]. Other in vivo invasive approaches, such as microelectrode arrays (MEA), electrical cortical stimulation (ECS), and deep brain stimulation (DBS), enable the direct stimulation and recording of neuronal activities [17]. Optogenetic manipulation is categorized as in vivo invasive technology that directly stimulates or suppresses neuronal activities, because blue light, which is necessary for optogenetics, must be supplied under the skull incision [2]. Of note, digital data obtained by ECoG or MEA can be interconnected with data from other animals and robots via the Internet [19]. Furthermore, the brown wireless device (BWD), a wireless broadband intracortical brain-computer interface (iBCI), non- invasively records and decodes broadband field potentials and spiking activity from individuals with tetraplegia [20]. Various in vitro brain research methods exist, including constructing the connectome [12], stimulating, suppressing, and detecting the activities of neurons and brain sections via electrodes or pharmacological methods, dissecting signal transduction inside neurons and brain sections, and various biochemical analyses (of DNA, RNA, proteins, and metabolites, including neurotransmitters). (a) 3,072 electrodes mimicking MEA are robotically implanted into the rat brain [1]. (b) NIRS is utilized for functional brain imaging. Near-infrared (NIR) penetrates into human brain tissues through the skull [17]. Page 11/16 Figure 2 The key concepts of our research. (A) Our vision for the automation of brain research Figure 2 The key concepts of our research. (B) The aim of this study. (a) Fluorescent beads mimicking micro-probes were inoculated into the future brain region of chick embryos. (b) After incubating the manipulated eggs, we examined whether they would hatch. (c) Upon Page 12/16 hatching, the whole brain was removed from the skull. The brains were treated with transparent reagent and examined for fluorescent beads. Figure 3 Manual implantation of fluorescent beads into the head area of chick embryos. (A) Stage-13 and -18 embryos. Chick embryos at stages 13 (a) and 18 (b), taken from the textbook [8]. (B) A photograph of one of the first hatched chicks after inoculation. (a) After implanting the micro-beads into the embryos, the eggshells were sealed with tape. Then the manipulated embryos were incubated until hatching. (b) The first animal to have micro-beads in its brain at birth. (a) After implanting the micro-beads into the embryos, the eggshells were sealed with tape. Then the manipulated embryos were incubated until hatching. (b) The first animal to have micro-beads in its brain at birth. Page 13/16 (C) The hatch rate of the inoculation experiments. Page 13/16 (C) The hatch rate of the inoculation experiments. Page 13/16 Results of three independent experiments using stage-18 embryos. Results of three independent experiments using stage-18 embryos. (D) Transparent treatment of the whole brain. (D) Transparent treatment of the whole brain. Photographs of (a) the whole brain fixed with 4% paraformaldehyde and (b) the transparent whole brain. (E) Multiple beads were detected in the brains. Photographs of (a) the whole brain fixed with 4% paraformaldehyde and (b) the transparent whole brain. (E) Multiple beads were detected in the brains. Fluorescent beads in the transparent brain tissue were photographed; (a) a large part of the whole brain (bright field), (b) the restricted region (open rectangle in (a)) (relief contrast), and (c) the same region as in (b) (fluorescence). Fluorescent beads in the transparent brain tissue were photographed; (a) a large part of the whole brain (bright field), (b) the restricted region (open rectangle in (a)) (relief contrast), and (c) the same region as in (b) (fluorescence). (F) The number of brains with beads among the hatched chicks. (F) The number of brains with beads among the hatched chicks. In three independent experiments (Fig. 3C), the numbers of manipulated brains with fluorescent beads were counted. Figure 4 Page 14/16 Page 14/16 The necessity to develop the robot “Spemann” Four existing industrial technologies (a–d; described in the text) can be combined to create a robot that automatically inoculates individually controllable probes into the head region of developing chick embryos, resulting in a mass production of chicks with non-invasively embedded probes. The position of inoculated probes in an embryo (c, g) and in the brain of a hatched chick (e) can be precisely recorded. Both positions can be determined by deep learning artificial intelligence (AI) (f), enabling the mass production and reproducibility of implanted probes in defined regions of chick brains. The system described above will be called “Spemann”. (h) The development of “Spemann” could trigger the explosive development of various near-infrared (NIR)-driven probes. Application to amniotic embryos. (a) Stage-18 chick embryos (opposite side view of Fig. 3A(b)) utilized for bead inoculation in this study. (b) Sk t h f H k l’ h l t hi k b [16] Ill t ti f ( ) i (d) (a) Stage-18 chick embryos (opposite side view of Fig. 3A(b)) utilized for bead inoculation in this study. (b) Sketches of Haeckel’s pharyngula-stage chick embryo [16]. Illustrations of (c) a penguin, (d) (a) Stage-18 chick embryos (opposite side view of Fig. 3A(b)) utilized for b (b) Sketches of Haeckel’s pharyngula-stage chick embryo [16]. Illustrations of (c) a penguin, (d) b) Sketches of Haeckel’s pharyngula-stage chick embryo [16]. Illustrations of (b) Sketches of Haeckel’s pharyngula-stage chick embryo [16]. Illustrations of (c) a penguin, (d) Page 15/16 hummingbird, (e) chameleon, and (f) turtle. (g) The abdomen of small mammals (e.g., mice and rats) can be robotically opened, and then probes can be inoculated into the head region of embryos in utero by “Spemann”. (h) An illustration of a mouse whose brain has multiple probes. All animals (c–f, h) are wearing a blue helmet representing a NIR transmitter, as in Fig. 1(b) and Fig. 2A(a). hummingbird, (e) chameleon, and (f) turtle. (g) The abdomen of small mammals (e.g., mice and rats) can be robotically opened, and then probes can be inoculated into the head region of embryos in utero by “Spemann”. (h) An illustration of a mouse whose brain has multiple probes. All animals (c–f, h) are wearing a blue helmet representing a NIR transmitter, as in Fig. 1(b) and Fig. 2A(a). SupplementarymaterialsFig.S1S3.pdf Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Page 16/16
https://openalex.org/W3010275413	https://www.nature.com/articles/s41598-020-60760-6.pdf	English	null	Optimization of concurrent production of xylanolytic and pectinolytic enzymes by Bacillus safensis M35 and Bacillus altitudinis J208 using agro-industrial biomass through Response Surface Methodology	Scientific reports	2,020	cc-by	9,698	Methodology Vihang S. Thite 1,2, Anuradha S. Nerurkar 1 & Nandita N. Baxi 1 Application of crude xylanolytic and pectinolytic enzymes in diverse industrial processes make these enzymes commercially valuable and demand their production process to be cost-effective. Out of four different agrowaste biomass, wheat bran (WB) and citrus peel (CP), when amended as fermentation substrates, respectively induced the highest xylanolytic enzymes and pectinolytic enzymes from both, B. safensis M35 and B. altitudinis J208. Further, the simultaneous amendment of WB and CP yielded concurrent production of these cellulase free xylanolytic and pectinolytic enzymes. Hence, the quadratic model was developed using the Central Composite Design of Response Surface Method (CCD-RSM). The model gave the concentration values for WB and CP substrates to be amended in one single production medium for obtaining two optimized predicted response values of xylanase activity and pectinase activity units, which were further practically validated for the xylanase and pectinase production responses from the optimized production medium (OPM). These practically obtained response values from OPM were found to be in accordance with a range of 95% predicted intervals (PI) values. These observations verified the validity of the predicted quadratic model from RSM and suggested that both xylanase and pectinase enzymes can be induced concurrently from both of the bacterial strains. Xylanases and pectinases are the commercially important industrial groups of enzymes, members of which exhibit diverse xylanolytic and pectinolytic enzymatic activities. These groups of enzymes harbor a huge com- mercial potential as their biotechnological applications span broad spectra in diverse industries such as biofuels, pulp-paper, food, animal feed, textile, fiber, etc. Out of these, biofuel industries demand these xylanolytic and pectinolytic enzymes play their accessory role to the core cellulase enzymes for improving plant biomass sac- charification. Whereas, animal feed industries require combination of cellulase, xylanase and or pectinase for improving the nutrition quality of grain and feed. On the other hand, pulp and paper as well as textile industries need the cellulase free xylanase and pectinase enzymes for their respective applications, i.e., to prepare hemicel- lulose free cellulose papers, to remove pectin (rhamnogalacturonan) coating from cotton and denim fibers, Food and beverage industries also need xylanase and pectinase enzymes to clarify pectin polymeric fraction from fruit juices and to improve the tea flavor1,2. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Methodology These applications append the worth to the organisms which can produce the xylanase and pectinase enzymes and the cellulase free nature of such enzymes is an add-on benefit to this. 1Department of Microbiology and Biotechnology Centre, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara, Gujarat, 390002, India. 2Department of Biological Sciences and Biotechnology, Institute of Advanced Research, Koba institutional area, Gandhinagar, Gujarat, 382426, India. email: thitevihang87@yahoo.com Scientific Reports \| (2020) 10:3824 \| https://doi.org/10.1038/s41598-020-60760-6 www.nature.com/scientificreports/ Three Bacillus strains M35, R31, and J208 have been isolated from camel, bull and buffalo dung samples and have been identified as B. safensis M35, B. altitudinis R31 and B. altitudinis J208 during our earlier work3. These strains have been shown to produce an endo-xylanase, and three types of endo-pectinases i.e., polygalacturo- nate hydrolase (PGase), pectate lyase (PL) and pectin lyase (PL). Studies on the physico-chemical properties of these enzymes during our earlier work revealed diverse characteristics along with their cellulase free feature imparting industrial importance to these enzymes3,4. The accessory potential of these cellulase free xylanase and pectinase enzymes to the commercial cellulase has been proven through the enzyme cocktail mediated sacchar- ification of raw and pretreated agrowaste biomass5,6. As many industrial applications require both xylanase and pectinase enzymes at the same time, it would be economical and convenient to obtain both the enzymes from a single isolate in the single optimized production medium (OPM). The use of pure polysaccharide substrates as enzyme inducers in such OMP requires a preliminary purification step for these polysaccharide substrates. The current methods employed for obtaining pure xylan and pectin substrates consume time, resources and capi- tal investments. Moreover, such purified substrates can induce only one enzyme at a time. On the other hand, agrowaste biomass, which is cheap and easily available in bulk as well as contains cellulose, hemicellulose (xylan) and rhamnogalacturonan (pectin) as its cell wall constituents, can induce more than one enzyme at a time and can become a good alternative in the production media leading to cost-effective process. In literature also, such crude agrowaste material has been used for the cost-effective production of individual enzymes. Methodology Therefore, the replacement of these pure polysaccharide substrates in the conventional medium with their inexpensive counter- parts would make the enzyme production process cost-effective7.h p y p pf There are several reports available on optimization of media components as well as physical parameters of growth conditions for the production of either xylanase or pectinase enzymes individually. Increased production of xylanase has been optimized from B. pumilus 3GAH and B. mojavensis UEB-FK in two different studies8,9. Optimized pectinase production from B. licheniformis has been reported from orange peel10. The reports on the optimized production of both, xylanase and pectinase enzymes, together in one single medium are very scanty for some of the Bacillus species in the literature. B. pumilus AJK was reported for concurrent production of cellulase free xylanase and pectinase enzymes on fermentation medium containing 2% of each, wheat bran and mosambi peel11,12. While B. firmus SDB9 was reported for concomitant production of xylanase, pectinase and cellulase enzymes from submerged fermentation of pectin salt medium13. The literature lacks the mention of B. safensis as well as B. altitudinis regarding the optimization of inducer substrate concentrations for concurrent production of cellulase free xylanolytic and pectinolytic enzymes. y y p y y Hence, the work was taken up to screen diverse crude industrial agrowaste biomass substrates for their abilities to induce xylanolytic and pectinolytic enzymes simultaneously from two different Bacillus isolates, i.e., B. safensis M35 and B. altitudinis R31, which is a novelty of the studies and not reported so far. For this purpose, diverse crude agrowaste biomass were used as submerged fermentation substrates and for the first time, screening and evaluation of their xylanase and/or pectinase inducing potential was based on the growth kinetic as well as enzyme production kinetic parameters for both the isolates in the present studies. The concentration of selected inducer substrate in the production medium was further optimized through central composite design (CCD) of response surface methodology (RSM) in design expert software for concurrent production of cellulase free xylanase-pectinase enzymes from M35 and J208 strains and theoretically predicted values were practically validated. www.nature.com/scientificreports/ enzymes were further referred to as M35 xylanase and J208 xylanase as well as M35 pectinase and J208 pectinase throughout the studies. enzymes were further referred to as M35 xylanase and J208 xylanase as well as M35 pectinase and J208 pectinase throughout the studies. Growth measurement. Growth in terms of OD600 nm was estimated in aliquots withdrawn from the flasks in a 24 well microtiter plate on Tecan Infinite M200 Pro using i-Control software based on normalization with uninoc- ulated media control. Protein estimation. The amount of secretory protein in CFS was estimated using Bradford method14. Samples were diluted properly using DW to 1.0 ml volume. 1.0 ml of Bradford reagent was added and the reaction mixture was incubated in dark for 10 min and followed by measurement of A595 nm. Using bovine serum albumin (BSA) as a standard the amount of protein equivalent to BSA was calculated. Xylanase (xylan hydrolase) activity. Fifty microliters of appropriately diluted crude CFS from the individual isolate was added to 250 µl of 50 mM Tris-Cl pH 8.0 buffered 0.5%w/v xylan and incubated in water-bath at 40 °C for 10 min. The reaction was terminated by the addition of 300 µl dinitro salicylic acid (DNS) reagent followed by boiling in water-bath for 10 min. Once the system was cooled down to room temperature, DW was added to make volume up to 1.5 ml and A540 nm was measured. One unit of xylanase activity was defined as the amount of xylanase required to release an end product equivalent to one µmol of D-xylose in reaction mixture per unit time in optimum incubation conditions3,15. Pectinase (pectin lyase) activity. Fifty microliters of appropriately diluted crude CFS from the individual isolate was added to 250 µl of 50 mM Tris-Cl pH 8.0 buffered 0.5%w/v pectin and incubated in water-bath at 50 °C for 30 min. The reaction was terminated by adding 500 µl 1 N NaOH followed by incubation at 76 °C for 10 min. To this 600 µl of 1 N HCl followed by 500 µl of 0.04 M 2-thiobarbituric acid were added and incubated at 76 °C for 10 min. Developed pink coloration was estimated as A550 nm. One unit of pectin lyase activity was defined as the amount of enzyme required to increase the A550 nm of the reaction mixture by 0.01 value per unit time in optimum incubation conditions4,16. Kinetic characteristics for enzyme production. Material and Methods Crude and pure polysaccharide substrates and other chemicals. Citrus peels (CP) were purchased from the local market, freeze-dried, and finely ground in a grinder. Wheat bran (WB), cotton seed cake (CSC) and rice bran (RB) were purchased from local industries (near Vadodara, Gujarat, India) and stored in an airtight container at room temperature. Molasses (M) was collected from the local market and stored at −20 °C to avoid microbial growth. These five agro-industrial residues were used as a source of crude complex polysaccharide substrates and explored for enzyme production. Whereas, Birchwood xylan (Xn, Sigma-Aldrich, Missouri, USA) and Citrus pectin (Pn, HiMedia, Mumbai, India) were used as commercially extracted pure complex polysaccha- ride substrates for enzyme production. Henceforth, these agro-industrial residues and commercially extracted polysaccharides will be referred to as crude and pure substrates respectively. Glucose, a pure carbon source, was used as control substrate for growth and enzyme production. All other chemicals used for media preparations and enzyme quantification assays were of analytical grade and purchased from HiMedia (Mumbai, India) or Sigma- Aldrich (Missouri, USA). Bacterial strains and media used for enzyme production. Bacillus strains B. safensis M35 (MCC3305) and B. altitudinis J208 (MCC3307) were studied for concurrent production of xylanase and pectinase enzymes. Pure cultures were maintained by inoculation and incubation on nutrient agar (NA) plates at 37 °C for 24 h fol- lowed by storage at 4–6 °C. In 250 ml Erlenmeyer flask, 100 ml of basal production medium BHM-YEP compris- ing (g/1000 ml) Bushnell Haas Medium (BHM) 3.27, Yeast extract (YE) 0.25 and Peptone (P) 0.75 was prepared, amended with pure or crude polysaccharide substrates (as separately mentioned in upcoming sections for indi- vidual experiments) and sterilized by autoclaving at 10 lbs for 20 min. This sterilized medium was inoculated with freshly grown 0.2 OD600 nm set culture in nutrient broth (NB) and incubated at 37 °C at 160 rpm till up to 96 h and studied for the following parameters. Parameters analyzed. After the samples were collected, part of it was used for microbial growth measure- ment. The remaining sample was centrifuged at 10,000 rpm for 10 min to obtain the cell-free supernatant (CFS) and subjected to quantify secretory protein as well as xylanase and pectinase activities through colorimetric esti- mations on Tecan Infinite M200 Pro Nanoquant multimode reader. The obtained crude xylanase and pectinase Scientific Reports \| (2020) 10:3824 \| https://doi.org/10.1038/s41598-020-60760-6 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Kinetic characteristics for the enzyme production process were estimated in terms of the volumetric rate of enzyme production as well as the specific rate of enzyme produc- tion (or economic coefficient). These parameters were compared to find the best substrate for efficient enzyme production17,18. • Volumetric rate of the enzyme production process (Qp): It is defined as total units of enzyme produced in one hour when the organism was grown in 1 L medium (IUlit−1h−1).ifii • Volumetric rate of the enzyme production process (Qp): It is defined as total units of enzyme produced in one hour when the organism was grown in 1 L medium (IUlit−1h−1).ifii g g Specific rate of the enzyme production or Economic coefficient (Yp/s): It is defined as total units of enzyme produced per gram of introduced substrate (IU/g−1). Effect of polymeric substrates on growth and enzyme production. Zero point five percent (v/v) inoculum of freshly grown 0.2 OD600 set culture was transferred to 250 ml Erlenmeyer flask containing100 ml sterilized BHM-YEP medium amended individually with 0.5% (v/v or w/v) of pure or crude fermentation sub- strates i.e., citrus peel (CP), cotton seed cake (CSC), molasses (M), pectin (Pn), rice bran (RB), wheat bran (WB) and xylan (Xn). Glucose (G) was used as control. This inoculated medium was incubated at 37 °C for 72 h at 160 rpm. Samples were collected after every 12 h and quantified for microbial growth, secretory protein, xylanase, and pectinase activities. Parameters of enzyme production kinetics on these substrates were evaluated. Screening for enzyme induction. To discriminate between the enzyme-inducing and growth-enhancing nature of various complex substrates, a comparison for normalized growth ratio as well as normalized activity ratio for xylanase and pectinase enzymes were carried out for each isolate. Normalization was done using the values of growth, xylanase and pectinase activities obtained on individual pure or crude complex fermentation substrate (CP, CSC, M, Pn, RB, WB, and Xn) to the simple fermentation substrate Glucose (G) as mentioned below. Results and Discussionf Effect of polymeric substrates on growth and enzyme production. Growth of B. safensis M35 and B. altitudinis J208 in terms of OD600 nm is depicted in Fig. 1a,b respectively. In comparison with basal BHM-YEP medium alone or basal BHM-YEP amended with glucose (G), supplementation of each polysaccharide substrate to basal BHM-YEP individually resulted in increased growth for both the isolates. Among all supplemented substrates the least growth was observed on glucose (G) supplementation throughout the incubation period. The growth further increased in this order, citrus peel (CP), rice bran (RB), cotton seed cake (CSC), molasses (M), wheat bran (WB), pectin (Pn) and resulted in highest on supplementation of xylan (Xn). Only CSC supplemen- tation was earmarked for the late stationary phase at ~36 h with extended lag and log phase. y p g g p Figure 1c,d represent xylanase activity units and Fig. 1e,f represent pectinase activity units exhibited by Bacillus strains M35 and J208 respectively on BHM-YEP medium with and without carbohydrate supplemen- tations. Commercial beechwood xylan (Xn) and crude substrate WB, when individually amended in media, resulted in 6–8 units and 3.5–5 units of xylanase activities respectively within 12 h of incubation. Pectinase activ- ities were detected within 12 h of incubation from WB and CP supplemented media, and later, after 48 h of incu- bation, commercial citrus pectin (Pn) supplemented media exhibited the highest pectinase activities in the range of 220–280 units. Supplementation of CSC and RB resulted in moderate activities of xylanase after 36 h and pecti- nase after 24 h of incubation. In literature also Streptomyces sp. QG-11-3 has been reported to produce xylanase and pectinase enzymes after supplementing Horikoshi medium supplemented with diverse crude agrowaste i.e., wheat bran, rice bran, cottonseed cake20.h Thus, it can be stated that all these crude and commercial polysaccharide substrates supported the luxuri- ant growth of bacterial isolates during the submerged fermentation process, but only some of them could sup- port the xylanase and/or pectinase production. Molasses (M) and glucose (G) yielded negligible xylanase and pectinase enzymes. WB, CSC, and RB could concurrently produce both the xylanases and pectinases. In litera- ture, several spp. of bacteria and fungi such as Aureobasidium pullulans Y-2311-1, Bacillus sp. DT7, Aspergillus niger LB-02-SF, Streptomyces sp. P12–137, have been used for the separate production of xylanase and pectinase enzymes from such crude polysaccharide substrates21–24. www.nature.com/scientificreports/ Xylanase activity ratio Xylanase units obtained on polysaccharide Xylanase units obtained on glucose (1) = = Pectinase activity ratio Pectinase units obtained on polysaccharide Pectinase units obtained on glucose (2) = Growth OD ratio Growth OD obtained on substrate Growth OD obtained on glucose ( ) ( ) ( ) (3) 600 600 600 Xylanase activity ratio Xylanase units obtained on polysaccharide Xylanase units obtained on glucose (1) = Xylanase activity ratio Xylanase units obtained on polysaccharide Xylanase units obtained on glucose (1) = = Pectinase activity ratio Pectinase units obtained on polysaccharide Pectinase units obtained on glucose (2) = Growth OD ratio Growth OD obtained on substrate Growth OD obtained on glucose ( ) ( ) ( ) (3) 600 600 600 (1) (2) (3) Cumulative effect of inducer substrates on enzyme production. To study the cumulative effect of selected inducer substrates on B. Safensis M35 and B. altitudinis J208 for concurrent production of xylanases and pectinases, 0.5%w/v of each, CP, and WB were amended simultaneously in BHM-YEP media and all the param- eters were estimated as mentioned in Section 2.3. Scientific Reports \| (2020) 10:3824 \| https://doi.org/10.1038/s41598-020-60760-6 Scientific Reports \| (2020) 10:3824 \| https://doi.org/10.1038/s41598-020-60760-6 www.nature.com/scientificreports/ Optimization of inducer substrate concentration on enzyme production. In order to determine the optimum concentration of inducer substrate(s) in the production medium for concurrent production of xylanolytic and pectinolytic enzymes from Bacillus isolates, the relationship between the experimental independ- ent variable factors i.e., concentration of inducer substrates WB (F1) and CP (F2) to obtain the maximum response of xylanase (R1) and pectinase (R2) from each of M35 and J208 was analyzed through central composite design (CCD) using response surface methodology (RSM) which follows the second-order polynomial equation and allows to develop up to a cubic model19. In the present study, Design Expert Software (Version 7.1.6, State-Ease, Minneapolis, USA) was used to design and analyze the experiments containing a set of 11 runs. BHM-YEP medium (100 ml) was prepared, supplemented with different WB and CP concentrations (F1 and F2 as designed by software) in 250 ml Erlenmeyer flask and autoclaved. After transferring 0.5% v/v inoculum from freshly grown 0.2 OD600 set culture, media was incubated at 37 °C up to 72 h at 160 rpm. Samples were collected and subjected to estimations of xylanase and pectinase production responses as mentioned earlier in this section. www.nature.com/scientificreports/ Validation of optimized substrate concentrations for concurrent production of xylanase-pectinase enzymes. One hundred milliliters of optimized production medium (OPM) was prepared using basal pro- duction medium BHM-YEP amended with optimized concentrations of WB and CP in 250 ml Erlenmeyer flask and autoclaved. After transfer of 0.5% (v/v) inoculum from freshly grown 0.2 OD600 set culture, media was incu- bated at 37 °C up to 72 h at 160 rpm. After every 12 h, samples were collected and subjected to estimations as mentioned in section 2.3. Data analysis. All quantitative estimation experiments were performed individually in triplicates (n = 3) and result data along with error values are represented as Mean ± Standard Error of Mean (SEM) for each experiment either in GraphPad Prism 6.0 or Origin 2017 software. Statistical analysis was carried out using the Two-way ANOVA method in GraphPad Prism 6.0. Design-Expert Software version 7.0 was used to perform response surface studies using Central Composite Design for enzyme inducer optimization and to produce the contour graphs. Results and Discussionf Thus, it was necessary to screen these substrates for efficient concurrent induction of xylanase and pectinase enzymes. Screening for better enzyme inducer substrate(s). During the balanced growth, an increase in the biomass is accompanied by a comparable increase of all other measurable properties of the population, e.g., protein, RNA, DNA, and intracellular water, etc. During the balanced growth, components such as DNA, RNA, and proteins, which are being synthesized by the cells, increase at the same rate as cell number. Based on this phe- nomenon, it becomes easy to measure any of these components and determine the growth rate, either of which is dependent on the initial population used for inoculation25,26. Thus, in the normal condition of balanced growth, a cell produces an enzyme at a constant rate which is directly proportional to the cell number and growth rate. But the incorporation of the inducer compounds in the media results in the induced production of the enzymes by each of the bacteria. Hence, the amount of the enzyme produced per bacterium rises and subsequently levels off a value determined by the growth rate27. y g As the inoculum was constant for each culture, the normalized ratio of microbial growth, xylanase, and pecti- nase activities on complex substrate v/s simple substrate were compared to screen a better enzyme inducer sub- strate(s). Figure 2a,b represent such comparison for B. safensis M35 and B. altitudinis J208 respectively. Despite Scientific Reports \| (2020) 10:3824 \| https://doi.org/10.1038/s41598-020-60760-6 www.nature.com/scientificreports/ i Figure 1. Growth of Bacillus strains and production of xylanolytic and pectinolytic enzymes on BHM-YEP medium with and without supplementation of individual substrates. Growth of (a) B. safensis M35 and (b) B. altitudinis J208; Xylanase activity from CFS of (c) B. safensis M35 and (d) B. altitudinis J208; Pectinase activity from CFS of (e) B. safensis M35 and (f) B. altitudinis J208 - observed up to 72 h on BHM-YEP media supplemented individually with citrus peel (CP), cotton seed cake (CSC), glucose (G), molasses (M), pectin (Pn), rice bran (RB), wheat bran (WB) and xylan (Xn). Values plotted are Mean ± Standard Error of Mean (SEM) for n = 3. Figure 1 Growth of Bacillus strains and production of xylanolytic and pectinolytic enzymes on BHM YEP medium Figure 1. Growth of Bacillus strains and production of xylanolytic and pectinolytic enzymes on BHM-YEP medium with and without supplementation of individual substrates. Growth of (a) B. Results and Discussionf safensis M35 and (b) B. altitudinis J208; Xylanase activity from CFS of (c) B. safensis M35 and (d) B. altitudinis J208; Pectinase activity from CFS of (e) B. safensis M35 and (f) B. altitudinis J208 - observed up to 72 h on BHM-YEP media supplemented individually with citrus peel (CP), cotton seed cake (CSC), glucose (G), molasses (M), pectin (Pn), rice bran (RB), wheat bran (WB) and xylan (Xn). Values plotted are Mean ± Standard Error of Mean (SEM) for n = 3. higher growth ratio on Xn and Pn supplemented media, significantly higher xylanase and pectinase activity ratios (p < 0.0001) from the respective media suggested that both of these commercial polysaccharide substrates, i.e. Xn and Pn, are good growth enhancers as well as xylanase and pectinase inducers respectively. CP exhibited significant difference for pectinase ratio making it a good pectinase inducer while WB exhibited a significant higher growth ratio on Xn and Pn supplemented media, significantly higher xylanase and pectinase activity ratios (p < 0.0001) from the respective media suggested that both of these commercial polysaccharide substrates, i.e. Xn and Pn, are good growth enhancers as well as xylanase and pectinase inducers respectively. CP exhibited significant difference for pectinase ratio making it a good pectinase inducer while WB exhibited a significant Scientific Reports \| (2020) 10:3824 \| https://doi.org/10.1038/s41598-020-60760-6 Scientific Reports \| (2020) 10:3824 \| https://doi.org/10.1038/s41598-020-60760-6 www.nature.com/scientificreports/ p p Figure 2. Screening of substrates for induction of xylanases and pectinases individually. Comparison of normalized growth ratio with normalized activity ratio of xylanase (blue column) as well as pectinase (dotted column) for (a) B. safensis M35 and (b) B. altitudinis J208; Values plotted are Mean ± Standard Error of Mean (SEM) for n = 3; Asterisk marks depicts significant difference between xylanase (blue coloured ) as well as pectinase (black coloured ) activity ratio from growth ratio (* = p < 0.05, = p < 0.01, * = p < 0.001 and **** = p < 0.0001). Figure 2. Screening of substrates for induction of xylanases and pectinases individually. Comparison of normalized growth ratio with normalized activity ratio of xylanase (blue column) as well as pectinase (dotted column) for (a) B. safensis M35 and (b) B. Results and Discussionf Values presented are Mean ± (Standard Error of Mean) for n = 3. Table 2. Volumetric rate and Specific rate of enzyme production on WB, CP and their combination. Values presented are Mean ± (Standard Error of Mean) for n = 3. Standard Order Run Order F1:WB (g/100 ml) F2:CP (g/100 ml) B. safensis M35 B. altitudinis J208 R1:X R2:P R1:X R2:P 1 6 0.482 0.482 5.43 138.47 4.97 328.09 2 8 2.568 0.482 8.37 158.06 14.90 407.44 3 11 0.482 2.568 0.64 0.00 0.53 0.00 4 5 2.568 2.568 0.90 43.57 0.90 94.89 5 9 0.050 1.525 0.86 0.00 0.76 3.98 6 4 3.000 1.525 4.77 44.07 6.51 129.06 7 7 1.525 0.050 8.46 188.78 14.53 309.19 8 2 1.525 3.000 0.72 11.94 0.58 19.35 9 10 1.525 1.525 14.43 372.47 8.92 648.66 10 1 1.525 1.525 13.96 356.58 9.30 671.51 11 3 1.525 1.525 14.51 361.04 9.68 751.02 Table 3. CCD-RSM design to study the effects of WB and CP concentrations for concurrent production of xylanase and pectinase activities. F1:WB Factor 1- Wheat Bran; F2:CP = Factor 2 - Citrus Peel; R1:X = Response 1- Xylanase activity units; R2:P = Response 2 - Pectinase activity units. Table 3. CCD-RSM design to study the effects of WB and CP concentrations for concurrent production of xylanase and pectinase activities. F1:WB Factor 1- Wheat Bran; F2:CP = Factor 2 - Citrus Peel; R1:X = Response 1- Xylanase activity units; R2:P = Response 2 - Pectinase activity units. Screening of effective concentration range of WB and CP for enzyme production. In a separate experiment, a broad concentration range (0.5–10.0% w/v) of WB and CP was individually supplemented to basal production medium and the effective range of concentration for WB and CP was figured out to optimize the production of xylanase as well as pectinase enzymes. A bell shape pattern was clearly visible for xylanase as well as pectinase production as an effect of different concentrations of WB (Supplementary Fig. 1a,b respectively). After reaching a maximum value at 2% WB concentration, both the activities started decreasing and this decrease was significant over 3% concentration of WB making it a limiting concentration for the concurrent production of enzymes. Similarly, a bell-shaped pattern was clearly visible for xylanase and pectinase production as an effect of different concentrations of CP (Supplementary Fig. 1c,d respectively). Results and Discussionf Scientific Reports \| (2020) 10:3824 \| https://doi.org/10.1038/s41598-020-60760-6 www.nature.com/scientificreports/ BHM-YEP media Volumetric rates (IUL−1h−1) Specific rates (IU/g substrate) For xylanase production For pectinase production For xylanase production For pectinase production B. safensis M35 B. altitudinis J208 B. safensis M35 B. altitudinis J208 B. safensis M35 B. altitudinis J208 B. safensis M35 B. altitudinis J208 Supplemented with CP 4.7 ± 0.9 6.9 ± 0.8 22718.0 ± 2376.3 62182.8 ± 1726.5 68.0 ± 13.4 99.2 ± 11.7 327139.7± 34219.0 895432.2 ± 24861.0 CSC 18.3 ± 3.8 21.5 ± 0.8 3256.8 ± 888.4 8195.1 ± 393.7 263.5 ± 54.9 309.3 ± 11.5 46897.8 ± 12792.3 118008.9 ± 5669.6 Pn 3.5 ± 0.2 6.4 ± 0.2 56437.0 ± 1955.6 71321.3 ± 782.0 50.8 ± 2.9 91.8 ± 2.9 812693.3 ± 28160.1 1027026.7 ± 11260.6 RB 18.1 ± 1.7 19.1 ± 1.1 2948.8 ± 269.3 4117.3 ± 712.8 260.5 ± 23.8 275.4 ± 16.3 42462.3 ± 3878.1 98488.9 ± 10264.6 WB 48.3 ± 0.9 60.1 ± 0.9 5091.1 ± 693.3 11343.8 ± 391.3 695.8 ± 13.5 865.0 ± 12.4 73312.3 ± 9984.1 183351.1 ± 5634.3 Xn 83.4 ± 0.6 65.3 ± 0.9 0.0 ± 0.0 0.0 ± 0.0 1201.0 ± 8.6 940.8 ± 13.0 0.0 ± 0.0 0.0 ± 0.0 Table 1. Volumetric rate and Specific rate of enzyme production on different inducer substrates. Values presented are Mean ± SEM (Standard Error of Mean) for n = 3. BHM-YEP medium Volumetric rates (IUL−1h−1) Specific rates (IU/g substrate) For xylanase production For pectinase production For xylanase production For pectinase production B. safensis M35 B. altitudinis J208 B. safensis M35 B. altitudinis J208 B. safensis M35 B. altitudinis J208 B. safensis M35 B. altitudinis J208 Supplemented with 0.5% WB 91.0 ± 4.6 103.3 ± 2.0 48100.4 ± 1931.2 79601.0 ± 3856.4 436.9 ± 22.0 496.1 ± 9.8 230881.7 ± 9269.9 382084.6 ± 18510.9 0.5%WB + 0.5% CP 113.2 ± 4.2 116.1 ± 2.3 109877.8 ± 5520.9 198742.6 ± 14288.5 543.5 ± 20.2 557.5 ± 11.1 527413.3 ± 26500.3 953964.4 ± 68584.9 0.5% CP 6.6 ± 0.3 16.2 ± 1.5 63029.6 ± 3013.7 146573.9 ± 829.9 31.5 ± 1.3 77.7 ± 7.3 302542.2 ± 14466.0 703554.7 ± 3983.5 Table 2. Volumetric rate and Specific rate of enzyme production on WB, CP and their combination. Values presented are Mean ± (Standard Error of Mean) for n = 3. Results and Discussionf BHM-YEP media Volumetric rates (IUL−1h−1) Specific rates (IU/g substrate) For xylanase production For pectinase production For xylanase production For pectinase production B. safensis M35 B. altitudinis J208 B. safensis M35 B. altitudinis J208 B. safensis M35 B. altitudinis J208 B. safensis M35 B. altitudinis J208 Supplemented with CP 4.7 ± 0.9 6.9 ± 0.8 22718.0 ± 2376.3 62182.8 ± 1726.5 68.0 ± 13.4 99.2 ± 11.7 327139.7± 34219.0 895432.2 ± 24861.0 CSC 18.3 ± 3.8 21.5 ± 0.8 3256.8 ± 888.4 8195.1 ± 393.7 263.5 ± 54.9 309.3 ± 11.5 46897.8 ± 12792.3 118008.9 ± 5669.6 Pn 3.5 ± 0.2 6.4 ± 0.2 56437.0 ± 1955.6 71321.3 ± 782.0 50.8 ± 2.9 91.8 ± 2.9 812693.3 ± 28160.1 1027026.7 ± 11260.6 RB 18.1 ± 1.7 19.1 ± 1.1 2948.8 ± 269.3 4117.3 ± 712.8 260.5 ± 23.8 275.4 ± 16.3 42462.3 ± 3878.1 98488.9 ± 10264.6 WB 48.3 ± 0.9 60.1 ± 0.9 5091.1 ± 693.3 11343.8 ± 391.3 695.8 ± 13.5 865.0 ± 12.4 73312.3 ± 9984.1 183351.1 ± 5634.3 Xn 83.4 ± 0.6 65.3 ± 0.9 0.0 ± 0.0 0.0 ± 0.0 1201.0 ± 8.6 940.8 ± 13.0 0.0 ± 0.0 0.0 ± 0.0 Table 1. Volumetric rate and Specific rate of enzyme production on different inducer substrates. Values presented are Mean ± SEM (Standard Error of Mean) for n = 3. Table 1. Volumetric rate and Specific rate of enzyme production on different inducer substrates. Values presented are Mean ± SEM (Standard Error of Mean) for n = 3. BHM-YEP medium Volumetric rates (IUL−1h−1) Specific rates (IU/g substrate) For xylanase production For pectinase production For xylanase production For pectinase production B. safensis M35 B. altitudinis J208 B. safensis M35 B. altitudinis J208 B. safensis M35 B. altitudinis J208 B. safensis M35 B. altitudinis J208 Supplemented with 0.5% WB 91.0 ± 4.6 103.3 ± 2.0 48100.4 ± 1931.2 79601.0 ± 3856.4 436.9 ± 22.0 496.1 ± 9.8 230881.7 ± 9269.9 382084.6 ± 18510.9 0.5%WB + 0.5% CP 113.2 ± 4.2 116.1 ± 2.3 109877.8 ± 5520.9 198742.6 ± 14288.5 543.5 ± 20.2 557.5 ± 11.1 527413.3 ± 26500.3 953964.4 ± 68584.9 0.5% CP 6.6 ± 0.3 16.2 ± 1.5 63029.6 ± 3013.7 146573.9 ± 829.9 31.5 ± 1.3 77.7 ± 7.3 302542.2 ± 14466.0 703554.7 ± 3983.5 Table 2. Volumetric rate and Specific rate of enzyme production on WB, CP and their combination. Results and Discussionf altitudinis J208; Values plotted are Mean ± Standard Error of Mean (SEM) for n = 3; Asterisk marks depicts significant difference between xylanase (blue coloured ) as well as pectinase (black coloured ) activity ratio from growth ratio (* = p < 0.05, = p < 0.01, * = p < 0.001 and **** = p < 0.0001). difference for both xylanase and pectinase suggesting it as a good inducer for both the enzymes. CSC and RB exhibited pectinase and xylanase ratios significantly higher than growth ratios suggesting that these substrates served as moderate to good xylano-pectinolytic enzyme inducers. In literature, B. subtilis and B. megaterium have been reported to exhibit xylanase production from the molasses amended media28. In present studies, molasses served as growth substate as it enhanced the growth of strains B. safensis M35 and B. altitudinis J208, but it could not induce the xylanase and pectinase production from the isolates. Enzyme production kinetics on individual plant polysaccharide substrates. Comparison of vol- umetric and specific rate of xylanase and pectinase production for diverse media supplementation to select the better inducer substrate(s) out of the four crude substrates (CP, CSC, RB, and WB) along with two commercial substrates (Pn and Xn) for concurrent production of xylanase and pectinase by Bacillus strains M35 and J208 has been shown in Table 1. The comparison suggested that commercially purified xylan and pectin are the best inducers for xylanase and pectinase respectively. Crude WB and CP induced the highest xylanase and pecti- nase enzymes respectively among all crude substrates. Moreover, WB also induced pectinase higher than RB and CSC. The richness of pectin in composition makes CP the best pectinase inducer. Whereas, the richness of arabinoxylan in the composition attributes in making WB as the best inducer substrate for xylanase production. Moreover, the presence of glucan-galactan also contributes to pectinase induction by WB which was higher than RB and CSC29–32. Although CSC and RB supported production of xylanolytic and pectinolytic enzymes, their production rates were comparatively lower than WB and CP amongst crude polysaccharides. Moreover, the cost for commercial preparation of xylan and pectin becomes a major limiting factor and hence, CP and WB were the two crude polysaccharide substrates selected for further optimization studies for concurrent production of xylano-pectinolytic enzymes. Results and Discussionf Unlike WB, different effects of CP were earmarked on the induction of xylanase and pectinase. Xylanase was produced at all CP concentrations (maxi- mum activity at 5%) but not pectinase. Lower concentration (up to <2%) induced pectinase activity (maximum at 1%) but above 2% pectinase production ceased making it a limiting factor for the concurrent production of enzymes. These results pointed towards the use of narrow range (0.05–3.0%) of both substrates during media optimization for obtaining concurrent induction of xylanase and pectinase enzymes. Scientific Reports \| (2020) 10:3824 \| https://doi.org/10.1038/s41598-020-60760-6 www.nature.com/scientificreports/ Figure 3. Contour plots, Desirability plots and Overlay pots for response surface p plots exhibiting effects of WB and CP on xylanase production responses from (a) B altitudinis J208; pectinase production responses from (c) B. safensis M35 and (d) B. represented in each box beside the contour line represent the respective enzyme act for n = 3; (e–h) Desirability and Overlay plots for optimized responses of xylanase X and Y-axis represents concentration (in % w/v) of WB and CP respectively; For d safensis M35 and (f) B. altitudinis J208, value in each box beside contour line repres obtained through numerical optimization; For overlay plots of (g) B. safensis M35 a value in large box represents the predicted maximum responses at mentioned varia th h hi l th d Y ll l d i i l l t ( h) i di t t Figure 3. Contour plots, Desirability plots and Overlay pots for response surface prediction. (a–d) Contour plots exhibiting effects of WB and CP on xylanase production responses from (a) B. safensis M35 and (b) B. altitudinis J208; pectinase production responses from (c) B. safensis M35 and (d) B. altitudinis J208. The values represented in each box beside the contour line represent the respective enzyme activity units and are Mean for n = 3; (e–h) Desirability and Overlay plots for optimized responses of xylanase and pectinase production. X and Y-axis represents concentration (in % w/v) of WB and CP respectively; For desirability plots of (e) B. safensis M35 and (f) B. altitudinis J208, value in each box beside contour line represents the desirability value obtained through numerical optimization; For overlay plots of (g) B. safensis M35 and (h) B. Results and Discussionf This simultaneous sup- plementation significantly increased xylanase and pectinase enzyme production when compared with individu- ally amended WB and CP substrates (p < 0.0001). Moreover, increased volumetric and specific rates of enzyme production in case of simultaneous supplementation are earmarked than individual supplementation of WB and CP from the data in Table 2. In literature, B. pumilus AJK has been reported for the production of xylanase and pectinase after growing on 2% WB and 2% Mosambi Peels11. Enhancement of pectinase activities by Aspergillus niger LB-02-SF has been reported after the supplementation of wheat bran to the orange residues containing media23. Statistical optimization of CP and WB concentration using CCD-RMS. Effect of the experimental variables (i.e., different concentrations of both inducer substrates WB and CP) and their interactions on the responses (i.e., induction of xylanase and pectinase enzymes) was calculated by the second-order polynomial equation for response surface method (RSM) using CCD. The optimum concentration for two inducers i.e., WB and CP each was determined in basal production medium for concurrent production of xylanase and pectinase from B. safensis M35, B. altitudinis J208. Table 3 represents the CCD of 11 randomized runs with corresponding xylanase and pectinase production responses by the two isolates. As can be seen from the table, standard run orders 9 to 11 (center point type) with 1.525% WB, 1.525% CP combination, exhibited a maximum 14.51 units of xylanase, and 372.47 units of pectinase activities for B. safensis M35. The highest values of xylanase activities for B. altitudinis J208 were observed was 14.90 units from 2.568% WB and 0.482% CP concentrations in std order 2 (corner point type). Whereas, the highest values of pectinase activities for B. altitudinis J208 were found to be 751.02 units from 1.535% WB and 1.525% CP concentrations in std order 9–11 (center point type). Model analysis. During the analysis of response data, the suitable model was selected through a fit summary report using design expert software (DX7). For xylanase and pectinase production response from each of the two isolates, Fit summary plots, ANOVA and model diagnostic plots analyses suggested that the quadratic model fits with each of these four responses (Supplementary Data). For all cases, the quadratic model was significantly above the 99.96% confidence level with p-value < 0.0004. Similarly, the Lack of Fit F-value for the quadratic model was >0.11 suggesting that the Lack of Fit was not significant. Results and Discussionf altitudinis J208, value in large box represents the predicted maximum responses at mentioned variable concentrations obtained through graphical method; Yellow-coloured region in overlay plots (g,h) indicates the range of predicted highest response. Figure 3. Contour plots, Desirability plots and Overlay pots for response surface prediction. (a–d) Contour plots exhibiting effects of WB and CP on xylanase production responses from (a) B. safensis M35 and (b) B. altitudinis J208; pectinase production responses from (c) B. safensis M35 and (d) B. altitudinis J208. The values represented in each box beside the contour line represent the respective enzyme activity units and are Mean for n = 3; (e–h) Desirability and Overlay plots for optimized responses of xylanase and pectinase production. X and Y-axis represents concentration (in % w/v) of WB and CP respectively; For desirability plots of (e) B. safensis M35 and (f) B. altitudinis J208, value in each box beside contour line represents the desirability value obtained through numerical optimization; For overlay plots of (g) B. safensis M35 and (h) B. altitudinis J208, value in large box represents the predicted maximum responses at mentioned variable concentrations obtained through graphical method; Yellow-coloured region in overlay plots (g,h) indicates the range of predicted highest response. Scientific Reports \| (2020) 10:3824 \| https://doi.org/10.1038/s41598-020-60760-6 www.nature.com/scientificreports/ Media composition and incubation conditions Organisms B. safensis M35 B. altitudinis J208 Media components (g/100 ml) BHM 0.327 0.327 Yeast Extract 0.025 0.025 Peptone 0.075 0.075 Inducer substrate (g/100 ml) WB 1.57 1.90 CP 1.26 0.62 Physical parameters for incubation Initial pH Default (5.8) Default (6.2) Temperature (°C) 37 37 Agitation rate (rpm) 160 160 Inoculum (%v/v) 0.5 0.5 Response 1 Xylanase 95% PI Low 13.38 12.49 Prediction 14.77 13.94 95% PI High 16.16 15.39 Observed 15.01 14.27 Response 2 Pectinase 95% PI Low 324.44 376.16 Prediction 371.54 601.01 95% PI High 418.64 823.84 Observed 411.58 728.74 Table 4. Validation of predicted points from the selected quadratic model. Observed response values presented as Mean, for n = 3. Table 4. Validation of predicted points from the selected quadratic model. Observed response values presented as Mean, for n = 3. Effect of simultaneous supplementation of WB and CP on concurrent xylanase and pectinase production. During the simultaneous supplementation of WB and CP in BHM-YEP, their cumulative effects on concurrent production of xylanase and pectinase are shown in Supplementary Fig. 2. Scientific Reports \| (2020) 10:3824 \| https://doi.org/10.1038/s41598-020-60760-6 Results and Discussionf Further, for the quadratic model, values of correlation coefficient R2 as well as adjusted R2 values were above 0.92 and predicted R2 values were above 0.77 for all four responses. The thumb rule that all R2 should be >0.7 and the difference between the predicted R2 and adjusted R2 should not be >0.2 was followed here indicating a good correlation between the predicted and experimental R2. Predicted Residual Error Sum of Squares (PRESS) is a measure of how a model fits into each design point. PRESS values for the chosen model were observed to be less when compared for other models under consideration in case of all four-response data as per the rules. Adequate precision is a measure of a signal to noise ratio and its value above 4 indicates a preference for the model. Further, ANOVA analysis for selected factors and their interactions in a quadratic model of each response were carried out and they were found to be highly significant with confidence interval >99.93 indicating the significance of the model terms. Further detailed explanations on the above-mentioned terms for each of the four responses have been provided in separate analy- sis data in Supplementary Data. Based on this information, the effects of inducer substrates and their interactions on responses of xylanase and pectinase enzyme production were analyzed further. 9 Scientific Reports \| (2020) 10:3824 \| https://doi.org/10.1038/s41598-020-60760-6 www.nature.com/scientificreports/ Figure 4. Validation of predicted concurrent enzyme production responses by selected Bacillus strains and their growth on optimized production medium. Observation of (a) Growth (b) Xylanase activity and (c) Pectinase activity. From B. safensis M35 (dotted line), B. altitudinis J208 (intact line); (d) Comparison of normalized growth ratio (red line) with normalized xylanase activity ratio (blue column) as well as normalized pectinase activity ratio (dotted column) for optimized production media (OPM). Values plotted are Mean ± Standard Error of Mean (SEM) for n = 3. Figure 4. Validation of predicted concurrent enzyme production responses by selected Bacillus strains and their growth on optimized production medium. Observation of (a) Growth (b) Xylanase activity and (c) Pectinase activity. From B. safensis M35 (dotted line), B. altitudinis J208 (intact line); (d) Comparison of normalized growth ratio (red line) with normalized xylanase activity ratio (blue column) as well as normalized pectinase activity ratio (dotted column) for optimized production media (OPM). Values plotted are Mean ± Standard Error of Mean (SEM) for n = 3. Results and Discussionf Response surface plot for xylanase and pectinase responses. Contour plots of xylanase and pectinase production responses on WB and CP variables from two Bacillus strains M35 and J208 are presented in Fig. 3(a–d) respec- tively. The highest xylanase and pectinase response values were traced through axis adjustments for each contour plot. It was observed that moderate concentration of WB (~1.6 g) and CP (~1.2 g) yielded the highest xylanase response from B. safensis M35 (Fig. 3a), whereas high WB (~2.3 g) and low CP (~0.5 g) concentrations yielded the highest xylanase responses from both B. altitudinis J208 (Fig. 3b). But, the moderate concentration of WB (~1.5–1.6 g) and CP (~1.2–1.3 g) yielded the highest pectinase response from both isolates (Fig. 3c,d). Thus, the combination of polysaccharides containing raw agrowaste biomass such as CP and WB in combinations can induce the production of xylano-pectinolytic enzymes in one single setup which can relieve the production cost. Further, to achieve the production of both in an optimum amount, the point prediction analysis was performed. Desirability plots and point prediction. Initially numerical as well as graphical optimization for xylanase and pectinase production responses were carried out in Design-Expert software through desirability plots and based on this further point prediction was performed. Numerical optimization was performed with the same range of WB and CP concentrations which was used in CCD experiments for maximized production response of xyla- nase and pectinase. This analysis generated desirability plots for combined production of xylanase and pectinase where desirability values approaching 1.0 were most preferable for concurrent xylanase and pectinase produc- tion (Fig. 3e,f). Analysis through axis adjustments of each contour plots suggested desirability values 0.99 and 0.98 for M35 and J208 respectively. The corresponding concentrations of WB and CP variables for X and Y axis values at the highest desirability value, on the combination, can induce the maximum xylanase and pectinase Scientific Reports \| (2020) 10:3824 \| https://doi.org/10.1038/s41598-020-60760-6 10 www.nature.com/scientificreports/ production response. The region of highest desirability values was observed in the range of variables which produced 90–100% of maximum response observed during CCD studies. And hence, 90–100% of maximum response obtained was considered as the margin of xylanase and pectinase production response for graphical optimization of media and the overlay plots for both responses were generated. As shown in Fig. 3g,h, the limits of both responses were marked as contours in graphs. Results and Discussionf Both xylanase and pectinase production responses for each isolate were overlapped. The overlapped graphs of both responses for individual isolates are shown in Fig. 3g,h and the yellow-colored region from the overlapped graphs corroborated with the region of highest prediction. From this analysis concentration values of WB and CP were predicted and validated further. Validation of predicted points. The predicted values of F1 and F2 for production media to be validated along with the predicted responses R1 and R2 are listed in Table 4 along with other incubation parameters. Figure 4 represents the growth as well as xylanase and pectinase activities obtained from optimized production media (OPM) along with their normalized ratios calculated as mentioned in Materials and Methods section A compar- ison of OD600 and growth ratios of OPM (Fig. 4d) with those on WB and CP containing media (Fig. 2) revealed that the OPM enhanced. the growth of bacterial isolates several folds than individual WB or CP containing medium. Further comparison of xylanase and pectinase ratios with growth ratios confirmed that though the OPM acted as a growth medium, the xylanase ratio was 3 to 4 times more than the growth ratio and the pectinase ratio was 25–30 times more than the growth ratio. The observed experimental values for xylanase and pectinase production responses during validation studies were 15.10 U (M35-xylanase), 14.27 U (J208-xylanase), 411.58 U (M35-pectinase) and 728.74 U (J208-pectinase). As shown in Table 4, all these four responses were found to be in the range of predicted interval (at 95% PI) as given by the software. These results verified the validity of the model and the existence of the optimal points indicating the adequacy of the RSM data. Co-production of xylanase (4.31 U/mL) and alkaline protease (3.66 U/mL) by Bacillus spp have been optimized under submerged fermentation from WB33. Received: 7 November 2019; Accepted: 17 February 2020; Published: xx xx xxxx Received: 7 November 2019; Accepted: 17 February 2020; Published: xx xx xxxx References 1. Kumar, R. & Wyman, C. E. Effect of xylanase supplementation of cellulase on digestion of corn stover solids prepared by leading pretreatment technologies. Bioresour. Technol. 100, 4203–4213 (2009). 1. Kumar, R. & Wyman, C. E. Effect of xylanase supplementation of cellulase on digestion of corn stover solids prepared by leading pretreatment technologies. Bioresour. Technol. 100, 4203–4213 (2009). p g 2. Rebello, S. et al. Recent advancements in the production and application of microbial pectinases: an overview. Rev. Environ. Sci. Biotechnol. 16, 381–394 (2017).h p g 2. Rebello, S. et al. Recent advancements in the production and application of microbial pectinases: an overview. Rev. Environ. Sci Biotechnol. 16, 381–394 (2017).h 3. Thite, V. S. & Nerurkar, A. S. Xylanases of Bacillus spp. isolated from ruminant dung as potential accessory enzymes for agro-waste saccharification. Lett. Appl. Microbiol. 60, 456–466 (2015).h i pp ( ) 4. Thite, V. S. & Nerurkar, A. S. Physicochemical characterization of pectinase activity from Bacillus spp. and their accessory role in synergism with crude xylanase and commercial cellulase in enzyme cocktail mediated saccharification of agrowaste biomass. J. Appl. Microbiol. 124, 1147–1163 (2018).h i pp 4. Thite, V. S. & Nerurkar, A. S. Physicochemical characterization of pectinase activity from Bacillus spp. and their accessory role in synergism with crude xylanase and commercial cellulase in enzyme cocktail mediated saccharification of agrowaste biomass. J. Appl. Microbiol. 124, 1147–1163 (2018).h , ( ) 5. Thite, V. S. & Nerurkar, A. S. Crude Xylanases and Pectinases from Bacillus spp. Along with Commercial Cellulase Formulate an Efficient Tailor-Made Cocktail for Sugarcane Bagasse Saccharification. BioEnergy Res. https://doi.org/10.1007/s12155-019-10050-5 (2019). ( ) 6. Thite, V. S. & Nerurkar, A. S. Valorization of sugarcane bagasse by chemical pretreatment and enzyme mediated deconstruction. Sci Rep. 9, (2019). p ( ) 7. Gupta, A. & Verma, J. P. Sustainable bio-ethanol production from agro-residues: A review. Renew. Sustain. Energy Rev. 41, 550–567 (2015). ( ) 8. Kallel, F. et al. Statistical optimization of low-cost production of an acidic xylanase by Bacillus mojavensis UEB-FK: Its potential applications. Biocatal. Agric. Biotechnol. 5, 1–10 (2016). pp g ( ) 9. Kaur, A., Singh, A., Patra, A. K. & Mahajan, R. Cost-effective scouring of flax fibers using cellulase-free xylano-pectinolytic synergism from a bacterial isolate. J. Clean. Prod. 131, 107–111 (2016). y g ( ) 0. Bibi, N., Ali, S. & Tabassum, R. Scientific Reports \| (2020) 10:3824 \| https://doi.org/10.1038/s41598-020-60760-6 Conclusion Among a variety of agro-industrial waste biomass, screened for their potential to induce these xylano-pectinolytic enzymes, CP induced pectinase and WB induced xylanase as well as pectinase. Therefore, the study demonstrates a novel approach where different concentrations of WB and CP were optimized in one single media based on a quadratic model developed through CCD-RSM for concurrent production of cellulase free xylanase and pecti- nase enzymes from strains B. safensis M35 and B. altitudinis J208 individually. In the case of, B. safensis M35, the productivity of the OPM (containing 1.57% WB and 1.26% CP) was found to be 15.10 U for xylanase and 411.58 U for pectinase enzymes. Whereas, in the case of B. altitudinis J208, the productivity of the OPM (containing 1.9% WB and 0.62% CP) was 14.27 U of xylanase and 728.74 U of pectinase enzymes. The present studies can fur- ther be extended to the optimization of other physicochemical parameters like inoculum size, C and N source, pH of media, incubation conditions like temperature, aeration speed (rpm), and incubation time and can be scaled up at bioreactor level to validate the commercial practicability of the process. Ethical statement. This article does not contain any studies with human participants or animals performed by any of the authors. Received: 7 November 2019; Accepted: 17 February 2020; Published: xx xx xxxx References Fixed-bed pyrolysis of cottonseed cake: Product yields and compositions. Energy Sources 19, 905–915 (1997). 31. 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Acknowledgements g VST acknowledges the University Grants Commission (UGC, India) for providing Research Fellowship in Science for Meritorious Students under the Basic Scientific Research Scheme (UGC-BSR-RFSMS, Grant No. F.4- 1/2006 (BSR)/7-128/2007(BSR)) towards the support of the laboratory work. g VST acknowledges the University Grants Commission (UGC, India) for providing Research Fellowship in Science for Meritorious Students under the Basic Scientific Research Scheme (UGC-BSR-RFSMS, Grant No. F.4- 1/2006 (BSR)/7-128/2007(BSR)) towards the support of the laboratory work. References h h d l d d h f h bl l d 20. Beg, Q. K., Bhushan, B., Kapoor, M. & Hoondal, G. S. Production and characterization Streptomyces sp. QG-11-3. J. Ind. Microbiol. Biotechnol. 24, 396–402 (2000). 20. Beg, Q. K., Bhushan, B., Kapoor, M. & Hoondal, G. S. Production and characterization of thermostable xylanase and pectinase Streptomyces sp. QG-11-3. J. Ind. Microbiol. Biotechnol. 24, 396–402 (2000). 21. 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Cellulase production from Aspergillus niger MS82: effect of temperature and pH N. Biotechnol. 25, (2009). 8. 2 Kinetics of microbial growth and product formation. in Progress in Industrial Microbiology (ed. Sikyta, B.) 31, 33–61 (Science Direct, 1995). 19. Montgomery, D. C. Design and analysis of experiments. (Wiley, 2009). 19. Montgomery, D. C. Design and analysis of experiments. (Wiley, 2009). Additional information Supplementary information is available for this paper at https://doi.org/10.1038/s41598-020-60760-6. Supplementary information is available for this paper at https://doi.org/10.1038/s41598-020-60760-6. Correspondence and requests for materials should be addressed to V.S.T. Correspondence and requests for materials should be addressed to V.S.T. Reprints and permissions information is available at www.nature.com/reprints. Reprints and permissions information is available at www.nature.com/reprints. 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https://openalex.org/W4387601146	https://www.jclinmedcasereports.com/articles/OJCMCR-2034.pdf	English	null	Large right heart thrombus caused by nuss bar displacement	Open journal of clinical & medical case reports	2,023	cc-by	2,412	Corresponding Author: Han-Yu Lin Department of Anesthesiology, Taipei Tzu Chi Hospital, 289, Jianguo Rd., Sindian District, New Taipei City 23142, Taiwan. Tel: +886-2-66289779x2639; Email: bbkeric@gmail.com Large right heart thrombus caused by nuss ba Jian-You Huang; Chun-Yu Chang; Han-Yu Lin Abstract We reported a case of a 43-year-old woman with a funnel chest who presented with paroxysmal atrial fibrillation resulting from bar flipping after the Nuss procedure. During the Nuss bar removal surgery, a large floating right heart thrombus was detected on the intraoperative transesophageal echocardiography. Surgical thrombectomy was performed by the cardiac surgeon. During the operation, we found that her right heart was compressed by the dislocated bar, creating an adhesion band resulting in local constriction of the right cardiac chambers. Arrhythmia combined with local constriction and structural anomaly of the right heart could cause blood stasis and turbulent blood flow. The disturbed blood flow with low wall shear stress may modify endocardial endothelium structure and function of the right heart. According to Virchow’s venous thromboembolism triad, these changes are likely to be the mechanism underlying large intracardiac thrombosis. Thus, the surgical team must make every effort to prevent bar dislocation. It is also crucial for clinicians to familiar with echocardiographic skills for intracardiac thrombus identification. Although intracardiac thrombosis is a very rare complication after Nuss procedure, early diagnosis and aggressive management are of great importance because of its high mortality rate. Open Journal of Clinical & Medical Case Reports Open Journal of Clinical & Medical Case Reports Volume 9 (2023) Issue 15 Case Presentation A 43-year-old woman presented with occasional chest discomfort since adolescence. She came to the outpatient clinic for medical consultation, where pectus excavatum was diagnosed (Haller index of 7.5 and sternovertebral distance of 6.2 cm), and the Nuss procedure was arranged by a thoracic surgeon. She had no known past medical history, and the cardiac workup did not reveal dysrhythmias or any structural abnormalities, including intracardiac thrombi. During the operation, two stainless steel bars were inserted and fixed with wire sutures, leaving the lower stabilizer (Figure 1A). Her postoperative clinical course was uneventful, so she was discharged on the eighth day after the surgery. However, intermittent palpitations and chest discomfort occurred approximately one month after discharge. She was hospitalized again, and pertinent examinations revealed that she had paroxysmal atrial fibrillation. A cardiologist was consulted, and antiarrhythmic and anticoagulant medications were administered. After her symptoms subsided, she was discharged and followed up regularly in the outpatient clinic. Two months later, her palpitations did not improve, and she also experienced chest discomfort, back pain, dyspnea, and even symptoms of heart failure. Under the suspicion of pectus bar displacement, she was hospitalized, and lower Nuss bar flipping was confirmed (Figure 1B). The patient was then referred for metal bar removal surgery. The operation was performed under general anesthesia, and hemorrhage from the surgical wound was noticed after removing the bars and wires. Because cardiac perforation was highly suspected, TEE examination was ordered. It revealed an intact cardiac surface, but a floating Right Atrial (RA) thrombus extending to the Right Ventricle (RV) and Right Ventricular Outflow Tract (RVOT) with moderate Tricuspid Regurgitation (TR) was incidentally found (Figures 2 and 3). Anticoagulant therapy with low-molecular-weight heparin was given postoperatively in the Surgical Intensive Care Unit (SICU). Coagulation workup did not reveal a hypercoagulable state. Computed tomography angiography did not find pulmonary embolism, and deep venous thrombosis was also excluded by compression ultrasound examination on deep veins of bilateral lower extremities. Because RHT remained a potential risk for serious thromboembolic events and needed to be addressed accordingly, further surgical management with open thrombectomy was scheduled three days after the last surgery. During the operation, a severe band-like adhesion around the pericardial space was noticed, which resulted in a local constriction of the right heart [3]. Cardiotomy was performed following the initiation of cardiopulmonary bypass. Vol 9: Issue 15: 2034 geal Echocardiography (TEE) in a patient undergoing bar removal surgery after a previous Nuss procedure. Vol 9: Issue 15: 2034 geal Echocardiography (TEE) in a patient undergoing bar removal surgery after a previous Nuss procedure. Introduction The Nuss procedure is a minimally invasive surgery for the correction of pectus excavatum. This procedure remodels the chest wall by employing one or more curved metal bars behind the sternum with out resection of the costal cartilage [1]. Complications related to the Nuss procedure are not uncommon. Cardiac perforation is the most serious complication, and there are other complications, including major vascular trauma, pneumothorax, bar displacement, infection, pericardial effusion, and pleural effusion [2]. We describe a rare case of a Right Heart Thrombus (RHT) incidentally found on intraoperative Transesopha Lin HY Vol 9: Issue 15: 2034 geal Echocardiography (TEE) in a patient undergoing bar removal surgery after a previous Nuss procedure. Case Presentation Vol 9: Issue 15: 2034 Case Presentation On inspection, there was a large RA thrombus extending to the Tricuspid valve (TV), RV and RVOT, which was organized and easily fragmented and had broad-based attachments to the RA and RV free walls. After thrombectomy, TV perforation and RA defects were found, and TV replacement and bovine pericardial patch reconstruction were performed. Afterward, modified reconstruction of pectus excavatum, i.e., combined Ravitch and Nuss procedures, was performed by the thoracic surgeon (Figure 1C). The patient was then transferred to the SICU after the surgery. Her postoperative course was uneventful, and she was discharged one week after the last surgery. Page 2 Vol 9: Issue 15: 2034 Figure 1: Lateral chest radiographs. (A) Two 13-inch stainless steel bars were fixed with wire sutures and left lower stabili zer on the first postoperative day. (B) Obvious upward flipping of the lower metal bar one month after the Nuss procedure. (C) Image after surgical thrombectomy, combined Ravitch and Nuss procedure with one supporting bar for pectus excavatum reconstruction, and tricuspid valve replacement. Figure 1: Lateral chest radiographs. (A) Two 13-inch stainless steel bars were fixed with wire sutures and left lower stabili zer on the first postoperative day. (B) Obvious upward flipping of the lower metal bar one month after the Nuss procedure. (C) Image after surgical thrombectomy, combined Ravitch and Nuss procedure with one supporting bar for pectus excavatum reconstruction, and tricuspid valve replacement. reconstruction, and tricuspid valve replacement. Figure 2: Transesophageal echocardiography image with a floating thrombus in the right atrium crossing into the right ven tricle. Midesophageal RV inflow-outflow view at 58° showing the right ventricular portion of the clot of at least 2.30 × 1.95 cm. AV: Aortic Valve; RV: Right Ventricle; Th: thrombus; TR: Tricuspid Regurgitation; TV: Tricuspid Valve. Figure 3: Midesophageal modified bicaval view of transesophageal echocardiography image at 120° with color flow Doppler showing the right atrial portion of the clot and moderate tricuspid regurgitation. LA: Left Atrium; RA: Right Atrium; RV: Right Ventricle; Th: Thrombus; TR: Tricuspid Regurgitation; TV: Tricuspid Valve. Figure 2: Transesophageal echocardiography image with a floating thrombus in the right atrium crossing into the right ven tricle. Midesophageal RV inflow-outflow view at 58° showing the right ventricular portion of the clot of at least 2.30 × 1.95 cm. AV: Aortic Valve; RV: Right Ventricle; Th: thrombus; TR: Tricuspid Regurgitation; TV: Tricuspid Valve. Discussion Intracardiac thrombi may generate de novo in any cardiac chambers (type B) or form elsewhere in the body and be identified in the heart while in transit (type A) [4]. However, regardless of the etiology, the development of venous thrombosis mostly demonstrates components of Virchow’s triad: stasis of blood, endothelial injury, and a hypercoagulable state [5]. In our case, paroxysmal AF developed after the first Nuss procedure, which predisposes patients to atrial blood stasis and may lead to a subsequent procoagu lant status. In addition, a band-like adhesion around the right pericardial space due to malposition of the metal bar could further bring about local constriction of the heart chamber and structural anomaly. This phenomenon may cause turbulent blood flow in the right heart with low wall shear forces, and it may mo dify the structure and function of the endocardial endothelium of the right heart [6]. These pathophysiolo gical mechanisms potentially facilitate the development of large intracardiac thrombosis. To our knowledge, this is the first case of thrombogenic cardiac abnormalities caused by bar displacement, eventually giving rise to a floating RHT formation. Thus, the surgical team must make every effort to reduce the occurrence of bar displacement, including the use of bar stabilizers, suturing, or wiring the pectus bar and stabilizers, as well as adding additional novel fixation devices. Moreover, because the mortality rate in patients with RHT is approximately 28%, early diagnosis and aggressive treatment are of great importance [7]. In this case, we use cardiovascular ultrasound to diagnose the RHT and to guide the surgical plan intraoperatively. It is crucial for clinicians to familiar with echocardiographic skills for intracardiac thrombus identification and measurement. Case Presentation Figure 2: Transesophageal echocardiography image with a floating thrombus in the right atrium crossing into the right ven tricle. Midesophageal RV inflow-outflow view at 58° showing the right ventricular portion of the clot of at least 2.30 × 1.95 cm. AV: Aortic Valve; RV: Right Ventricle; Th: thrombus; TR: Tricuspid Regurgitation; TV: Tricuspid Valve Figure 2: Transesophageal echocardiography image with a floating thrombus in the right atrium crossing into the right ven tricle. Midesophageal RV inflow-outflow view at 58° showing the right ventricular portion of the clot of at least 2.30 × 1.95 cm. tic Valve; RV: Right Ventricle; Th: thrombus; TR: Tricuspid Regurgitation; TV: Tricuspid Valve. AV: Aortic Valve; RV: Right Ventricle; Th: thrombus; TR: Tricuspid Regurgitation; TV: T Figure 3: Midesophageal modified bicaval view of transesophageal echocardiography image at 120° with color flow Doppler showing the right atrial portion of the clot and moderate tricuspid regurgitation. LA: Left Atrium; RA: Right Atrium; RV: Right Ventricle; Th: Thrombus; TR: Tricuspid Regurgitation; TV: Tricuspid Valve. Figure 3: Midesophageal modified bicaval view of transesophageal echocardiography image at 120° with color flow Doppler showing the right atrial portion of the clot and moderate tricuspid regurgitation. LA: Left Atrium; RA: Right Atrium; RV: Right Ventricle; Th: Thrombus; TR: Tricuspid Regurgitation; TV: Tricuspid Valve. Page 3 Page 3 Vol 9: Issue 15: 2034 Declarations Acknowledgments: We offer special thanks to Dr. Shih-Ching Wang and Yung-Chu Teng for their excellent assistance in writing and revising the draft of this manuscript. We are also express our gratitude to Prof. Yeung-Leung Cheng and Dr. Chan-Yang Hsu, who performed the surgery and cared for the patients in this study. Author Contributions: All authors reviewed and approved the final manuscript. Informed Consent: Written informed consent was obtained from the patient for the publication of this report. Informed Consent: Written informed consent was obtained from the patient for the publication of this report. Ethics approval statement: Ethical approval was waived by the Institutional Review Board of Taipei Tzu- Chi Hospital in view of the observational nature of the study. Ethics approval statement: Ethical approval was waived by the Institutional Review Board of Taipei Tzu- Chi Hospital in view of the observational nature of the study. Declaration of conflicting interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Declaration of conflicting interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Funding: The author(s) received no financial support for the research, authorship, and/or publication of this article. Funding: The author(s) received no financial support for the research, authorship, and/or publication of this article. Page 4 Vol 9: Issue 15: 2034 References References 1. Nuss D, Kelly RE, Croitoru DP, Katz ME. A 10-year review of a minimally invasive technique for the correction of pectus excava tum. J Pediatr Surg. 1998; 33: 545-552. 1. Nuss D, Kelly RE, Croitoru DP, Katz ME. A 10-year review of a minimally invasive technique for the correction of pectus excava tum. J Pediatr Surg. 1998; 33: 545-552. 2. Nuss D, Croitoru DP, Kelly RE, Goretsky MJ, Nuss KJ, Gustin TS. Review and discussion of the complications of minimally inva sive pectus excavatum repair. Eur J Pediatr Surg. 2002; 12: 230-234. 2. Nuss D, Croitoru DP, Kelly RE, Goretsky MJ, Nuss KJ, Gustin TS. Review and discussion of the complications of minimally inva sive pectus excavatum repair. Eur J Pediatr Surg. 2002; 12: 230-234. 3. Hsu CY, Cheng YL, Wang SC, Shen TC. Massive right heart thrombus after the Nuss procedure: A case report. JTCVS Tech. 2022; 12: 69-71. 3. Hsu CY, Cheng YL, Wang SC, Shen TC. Massive right heart thrombus after the Nuss procedure: A case report. JTCVS Tech. 2022; 12: 69-71. 4. The European Working Group on Echocardiography, Kronik G. The European Cooperative Study on the clinical significance of right heart thrombi. Eur Heart J. 1989; 10: 1046-1059. 5. Egolum UO, Stover DG, Anthony R, Wasserman AM, Lenihan D, et al. Intracardiac thrombus: diagnosis, complications and man agement. Am J Med Sci. 2013; 345: 391-395. 6. Chiu JJ, Chien S. Effects of disturbed flow on vascular endothelium: pathophysiological basis and clinical perspectives. Physiol Rev. 2011; 91: 327-387. 7. Otoupalova E, Dalal B, Renard B. Right heart thrombus in transit: a series of two cases. Crit Ultrasound J. 2017; 9: 14. Manuscript Information: Received: March 31, 2023; Accepted: May 05, 2023; Published: May 08, 2023 Authors Information: Jian-You Huang1; Chun-Yu Chang1; Han-Yu Lin1,2* Authors Information: Jian-You Huang1; Chun-Yu Chang1; Han-Yu Lin1,2* 1Department of Anesthesiology, Taipei Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, New Taipei City, Taiwan. 2School of Medicine, Tzu Chi University, Hualien, Taiwan. 1Department of Anesthesiology, Taipei Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, New Taipei City, Taiwan. 2School of Medicine, Tzu Chi University, Hualien, Taiwan. Citation: Huang JY, Chang CY, Lin HY. Large right heart thrombus caused by nuss bar displacement. Open J Clin Med Case Rep. 2023; 2034. Citation: Huang JY, Chang CY, Lin HY. Large right heart thrombus caused by nuss bar displacement. Open J Clin Med Case Rep. References 2023; 2034. Copy right statement: Content published in the journal follows Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0). © Lin HY (2023) Copy right statement: Content published in the journal follows Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0). © Lin HY (2023) About the Journal: Open Journal of Clinical and Medical Case Reports is an international, open access, peer reviewed Journal focusing exclusively on case reports covering all areas of clinical & medical sciences. Visit the journal website at www.jclinmedcasereports.com For reprints and other information, contact info@jclinmedcasereports.com Page 5 Page 5
https://openalex.org/W3033191966	https://europepmc.org/articles/pmc7271449?pdf=render	English	null	In vitro effects of febrifugine on Schistosoma mansoni adult worms	Tropical medicine and health	2,020	cc-by	6,535	© The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Mitsui et al. Tropical Medicine and Health (2020) 48:42 https://doi.org/10.1186/s41182-020-00230-x Mitsui et al. Tropical Medicine and Health (2020) 48:42 https://doi.org/10.1186/s41182-020-00230-x Mitsui et al. Tropical Medicine and Health https://doi.org/10.1186/s41182-020-00230-x (2020) 48:42 In vitro effects of febrifugine on Schistosoma mansoni adult worms Yoshinori Mitsui1* , Mitsumasa Miura2 and Kentaro Kato2 Abstract Background: Reports on the antischistosomal effect of several antimalarial drugs such as artesunate, mefloquine, and amodiaquine suggest that febrifugine, which exerts an antimalarial effect, can also be expected to possess antischistosomal potential. The present study investigates the antischistosomal effects of febrifugine. Methods: In experiment 1, Schistosoma mansoni adult worm pairs were incubated in a medium alone as a control or supplemented with febrifugine at 0.05, 0.1, 0.2, and 0.5 μg/ml for 14 days. The morphology of the worms and the egg production of the female worms were observed simultaneously. In experiment 2, the incubation was conducted as in experiment 1, except that the febrifugine concentrations were reduced to 0.005, 0.01, and 0.02 μg/ ml. In addition, S. mansoni adult worms were incubated with either 0.5 μg/ml febrifugine or none as a control for 5 days and stained with neutral red dye. Results: Febrifugine significantly reduced the survival of S. mansoni male and female worms at concentrations of 0.02–0.5 μg/ml following incubation for 14 days and remarkably inhibited the daily egg output of the female worms. The non-treated male and female worms remained morphologically normal within the period of 14 days, whereas male and female worms treated with febrifugine at different concentrations gradually twisted and subsequently died. In addition, S. mansoni adult worms were incubated with either 0.5 μg/ml febrifugine or none as a control for 5 days and stained with neutral red dye. Non-treated male worms were morphologically normal and stained dark red with neutral red, while febrifugine-treated male worms appeared similar to those in the control group and were stained at a slightly lower level of dark red than the non-treated male worms. Non-treated female worms were morphologically normal, and their intestinal tract and vitellaria were stained deep red and dark red, respectively. In contrast, febrifugine-treated female worms were morphologically damaged, and their intestinal tract and vitellaria remained mostly unstained and stained dark red, respectively. Conclusion: Febrifugine exerts potent antischistosomal effects and can be expected to contribute to the development of a novel antischistosomal drug. Keywords: Febrifugine, Dichroa febrifuga, Antischistosomal drug, Schistosoma mansoni, Antimalarial drug for schistosomiasis. Chemotherapy with PZQ is the mainstay of strategies to control schistosomiasis [2], but the extensive reliance on PZQ raises concerns about the emergence of drug-resistant parasites [3, 4]. Therefore, the development of an antischistosomal drug in addition to or as an alternative to PZQ is urgently needed. Background S hi i i Schistosomiasis persists as a serious parasitic disease in 78 countries in both tropical and subtropical areas around the world [1]. Some 61 million people were treated with praziquantel (PZQ) by 2014, but an esti- mated 260 million people continue to require treatment Artemisinin derivatives such as artemether and artesu- nate (ART) are well-known antimalarial drugs that exert an antischistosomal effect [5, 6]. Other antimalarial drugs in current use, such as mefloquine (MQ), * Correspondence: ymitsui@nagasaki-u.ac.jp 1Department of Parasitology, Institute of Tropical Medicine, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan Full list of author information is available at the end of the article * Correspondence: ymitsui@nagasaki-u.ac.jp 1Department of Parasitology, Institute of Tropical Medicine, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan Full list of author information is available at the end of the article Parasite strain A Puerto Rican strain of S. mansoni (NIH-Sm-PR-1) was routinely maintained by passage through 6-week-old fe- male ICR mice (Japan SLC, Inc. Hamamatsu, Japan) and Biomphalaria glabrata snails (Newton’s NIH Puerto Rican/Brazilian M-line) in the Laboratory Animal Centre. At 8 weeks after infection with 200 S. mansoni cercariae, adult worms were collected by the perfusion technique as previously described by Smithers and Terry [21] and washed twice with NCTC 135 medium. Mitsui et al. Tropical Medicine and Health (2020) 48:42 Page 2 of 7 added to medium NCTC 135 (Sigma-Aldrich Co.) con- taining a 0.5% solution of antibiotics (penicillin 5000 units and streptomycin 5000 mg/l; Gibco, Langley, Okla- homa, USA) at concentrations of 0.005–0.5 μg/ml. A NR dye was purchased from Nacalai Tesque, Inc. (Kyoto, Japan) and dissolved in DW at a concentration of 0.1% as a stock solution (pH 3.1). amodiaquine (AQ), and primaquine (PQ), also display antischistosomal effects [7, 8], further suggesting that compounds possessing antimalarial activity may display antischistosomal potential. p Febrifugine (Feb) was isolated from the roots and leaves of Dichroa febrifuga Lour [9, 10], a plant used for centuries in China to treat malarial fevers [11]. Subse- quently, Feb was also isolated from the leaves of certain Hydrangea species [12]. Henderson et al. (1949) demon- strated the antimalarial effect of Feb against Plasmodium lophurae infection in ducks, P. relictum in canaries, and P. cynomolgi in rhesus monkeys in experimental studies [13]. Furthermore, Hewitt et al. (1952) reported that Feb was approximately 100 times more effective than quin- ine (QN) in counteracting P. lophurae infection in ducks [14]. Feb was on the verge of serving as a potent new antimalarial drug, but its severe side effects precluded its use as a clinical antimalarial drug for half a century [13, 15]. In recent years, a derivative of Feb called halofugi- none has attracted keen attention because of its wide range of beneficial biological activities, encompassing malaria and cancer as well as fibrosis-related and auto- immune diseases [16]. At the same time, Feb has come to light as a leading compound for the development of new antimalarial drugs [17, 18]. Feb is a quinazolinone alkaloid with a basic chemical structure different from that of the artemisinin derivatives and quinoline alka- loids observed in current antimalarial drugs such as QN, chloroquine, PQ, AQ, and MQ [19, 20]. Thus, Feb is a promising candidate for the development of a novel anti- malarial drug, and, in view of the mechanism of other antimalarial drugs, it may also exert antischistosomal ef- fects. To our knowledge, however, the effect of Feb on schistosomes has not been investigated to date. The as- sessment of the antischistosomal effects of Feb, there- fore, constitutes a new approach to the development of a novel antischistosomal drug. Incubation of Schistosoma mansoni adult worm pairs with febrifugine The incubation was conducted as previously described by Mitsui et al. (2009) [6], except for differences in the test drug, the drug concentration, and replacement of media over a period of 14 days. Before exposure to Feb, each pair of S. mansoni adult worms was preincubated for 1 day with 0.5 ml of NCTC 135 medium in a single well of a 24-well multi-well plate (Sumitomo Bakelite Co. Ltd, Osaka, Japan) in a 5% CO2 incubator at 37 °C. After preincubation, the adult worms were used for sub- sequent experiments. In experiment 1, 30 male/female S. mansoni adult worm pairs were randomly assigned to five groups of six pairs each. Then, each worm pair was transferred into a well containing 0.5 ml of NCTC 135 medium supple- mented with 0 as control, 0.05, 0.1, 0.2, or 0.5 μg/ml Feb. The plates were continuously incubated in a 5% CO2 incubator at 37 °C. The medium alone or medium supplemented with the drug was changed every 2 to 3 days for 14 days. The number of eggs produced daily was counted, and the morphological appearance of the worms was observed visually under a Nikon SMZ 800 stereoscopic microscope (Nikon Corporation, Tokyo, Japan). Assessment of the “dead or alive” status of adult worms was determined by the movement of the worm upon stimulation with a needle. Worms were classified as “dead” when they failed to respond to the needle stimulation. Images of worms treated with the drug were obtained using a digital Senamal C-mount camera (Microscope Network, Saitama, Japan) mounted on the stereoscopic microscope. In the present groundbreaking study, we evaluated the in vitro antischistosomal effects of Feb according to the modified method of Mitsui et al. (2009) [6]. The effects of the drug were examined with regard to (1) the egg production of female Schistosoma mansoni adult worms, (2) the survival of adult worms, (3) the gross morpho- logical alterations of the adult worms caused by the drug, and (4) the antischistosomal effect of Feb on S. mansoni adult worms assessed using neutral red (NR) non-fluorescent dye. Statistical analysis Data were analyzed using Epi-Info software (Centers for Disease Control and Prevention, Atlanta, GA, USA). The daily egg output per female adult worm was expressed as the arithmetic mean (± standard error). The Kruskal- Wallis or Mann-Whitney U test was used to compare daily egg outputs between the control and Feb-treated groups. The median survival time of S. mansoni adult worms was calculated. Comparison of worm survival time between the control and Feb-treated groups and between male and female worms in the Feb-treated groups was performed using the log-rank test. Materials and methods Chemicals and media Then, the coverslip was sealed w commercial fingernail polish. The images of the w were captured using an Olympus BX43 micro (Olympus Corporation, Tokyo, Japan) equipped w Table 1 Inhibitory effect of febrifugine on the daily egg o Febrifugine concentration (μg/ml) Mean daily egg output (± standard error) Day 0 Day 1 Day 2 Experiment 1 0 (control) 30.0 ± 9.9 115.3 ± 16.3 111.2 ± 1 0.05 20.2 ± 5.8 102.2 ± 13.8 81.0 ± 19 0.1 19.7 ± 9.2 101.3 ± 16.1 74.5 ± 7.8 0.2 24.7 ± 10.5 51.5 ± 17.5 68.8 ± 29 0.5 18.3 ± 6.0 85.3 ± 20.3 50.7 ± 15 Experiment 2 0 (control) 26.5 ± 12.6 94.2 ± 18.8 101.2 ± 2 0.005 20.3 ± 6.3 92.0 ± 18.3 69.0 ± 15 0.01 25.3 ± 10.1 86.5 ± 5.9 78.8 ± 15 0.02 16.8 ± 7.2 100.2 ± 28.5 89.5 ± 13 Mean daily egg output during the one-day preincubation (day 0) amon and 2 (Kruskal-Wallis test, P = 0.88 and 0.92 for all comparisons, respect aP < 0.05 bP < 0.01, compared with the corresponding control group according to P < 0.05 bP < 0.01, compared with the corresponding control group according to a Mann-Whitney U test Staining of febrifugine-treated Schistosoma mansoni adult worms with neutral red The antischistosomal effect of Feb on S. mansoni adult worms was assessed with NR dye according to a modified method previously described by Mitsui and Kato (2018) [22]. The male and female adult worms were randomly al- located into two groups of three worms each: control and 0.5 μg/ml Feb groups. Each group was incubated for 1 day with 2 ml of NCTC 135 medium alone in a 35 mm plastic dish (Sumitomo Bakelite Co. Ltd) in a 5% CO2 incubator at 37 °C. Subsequently, three male and female worms from each group were transferred to a new plastic dish contain- ing 2 ml of the medium alone as a control or supple- mented with 0.5 μg/ml Feb. The dishes were continuously incubated in a 5% CO2 incubator at 37 °C for 5 days with one change of the medium. Then, 40 μl of the stock NR solution was added to each dish (final concentration: 0.002%) and incubated in a 5% CO2 incubator at 37 °C for 1 ho. Then, the living male and female worms were indi- vidually placed in 20 μl of NETC 135 medium mounted on a regular microscope slide (76 × 26 × 0.9–1.2 mm; Matsunami Glass Industries, Ltd, Osaka, Japan) and sand- wiched with a coverslip (18 × 18 mm; Matsunami Glass Industries, Ltd). Then, the coverslip was sealed with a commercial fingernail polish. The images of the worms were captured using an Olympus BX43 microscope (Olympus Corporation, Tokyo, Japan) equipped with a an daily egg output during the one-day preincubation (day 0) among the control and febrifugine-treated groups was not significantly 2 (Kruskal-Wallis test, P = 0.88 and 0.92 for all comparisons, respectively) < 0 05 P < 0.05 bP < 0.01, compared with the corresponding control group according to a Mann-Whitney U test Materials and methods Chemicals and media Feb dihydrochloride was purchased from Sigma-Aldrich Co. (St. Louis, Missouri, USA) and diluted in deionized water (DW) to a concentration of 10 mg/ml as a free- base stock solution. This stock Feb solution was then The procedure in experiment 2 was identical to that of experiment 1, except for the concentration of Feb. That is, 24 adult worm pairs were randomly assigned to four Page 3 of 7 Mitsui et al. Tropical Medicine and Health (2020) 48:42 Mitsui et al. Tropical Medicine and Health digital camera (Sony Alpha NEX-5, Sony Corporation, Tokyo, Japan). digital camera (Sony Alpha NEX-5, Sony Corporation, Tokyo, Japan). digital camera (Sony Alpha NEX-5, Sony Corporation, Tokyo, Japan). groups of six worm pairs each, and each pair was con- tinuously incubated in the medium supplemented with 0 as control, 0.005, 0.01, or 0.02 μg/ml Feb for 14 days with replacement of the medium every 2 to 3 days. tinuously incubated in the medium supplemented w as control, 0.005, 0.01, or 0.02 μg/ml Feb for 14 with replacement of the medium every 2 to 3 days. Staining of febrifugine-treated Schistosoma mansoni worms with neutral red The antischistosomal effect of Feb on S. mansoni worms was assessed with NR dye according to a mo method previously described by Mitsui and Kato ( [22]. The male and female adult worms were random located into two groups of three worms each: contro 0.5 μg/ml Feb groups. Each group was incubated for with 2 ml of NCTC 135 medium alone in a 35 mm p dish (Sumitomo Bakelite Co. Ltd) in a 5% CO2 incu at 37 °C. Subsequently, three male and female worms each group were transferred to a new plastic dish con ing 2 ml of the medium alone as a control or su mented with 0.5 μg/ml Feb. The dishes were continu incubated in a 5% CO2 incubator at 37 °C for 5 days one change of the medium. Then, 40 μl of the stoc solution was added to each dish (final concentr 0.002%) and incubated in a 5% CO2 incubator at 37 ° 1 ho. Then, the living male and female worms were vidually placed in 20 μl of NETC 135 medium mou on a regular microscope slide (76 × 26 × 0.9–1.2 Matsunami Glass Industries, Ltd, Osaka, Japan) and wiched with a coverslip (18 × 18 mm; Matsunami Industries, Ltd). Gross morphological alterations of Schistosoma mansoni adult worms caused by febrifugine g In experiment 2, the mean daily egg output during the 1-day preincubation period was not significantly differ- ent among the control and 0.005, 0.01, and 0.02 μg/ml Feb-treated groups (P = 0.85 for all comparisons). Dur- ing the first 3 days of incubation, the mean daily egg output in the Feb-treated groups did not differ signifi- cantly from that in the control group, but on the fourth day of incubation, the mean daily egg output in the 0.02 μg/ml Feb-treated group was significantly lower than that in the control group (P < 0.05). On the other hand, during the incubation period of 7 days, the mean daily egg output in the 0.005 and 0.01 μg/ml Feb-treated groups was not significantly lower than that in the con- trol group. The morphological appearance of S. mansoni adult worm pairs untreated or treated with Feb was observed daily under a dissecting microscope. When paired adult worms were consecutively incubated in medium alone (control group) in experiment 1, all the male and female adult worms were still in pairs within three days (Fig. 1a). The black content in the body of male worms mostly disappeared within 3 days (Fig. 1a) and the black content in the body of separated female worms also faded over a period of 5 days (Fig. 1b). In the subsequent incubation, very little further morphological alteration was observed in either male or female worms (Fig. 1c, d). When paired adult worms were treated with 0.5 μg/ ml Feb for 3 days, no remarkable morphological alter- ation was observed in the body of the male worms or the separated female worms (Fig. 1e). However, on day 5 after exposure to Feb, although the male worms ap- peared similar to those in the control (Fig. 1f), the fe- male worms showed a visible twisting in the anterior part of the body (Fig. 1f). After subsequent incubation, all the male worms showed obvious twisting (Fig. 1g, h), and all the female worms were further twisted (Fig. 1g, h). Similar morphological alterations were observed in male and female worms treated with Feb at the other concentrations of 0.02, 0.05, 0.1, and 0.2 μg/ml during the incubation periods of 10–14, 6–13, 6–11, and 5–11 days, respectively (data not shown). Effect of febrifugine on the egg production of Schistosoma mansoni adult worm pairs Furthermore, on the fourth day of in- cubation, the mean daily egg output in the 0.05, 0.1, and Table 1 Inhibitory effect of febrifugine on the daily egg output of six paired Schistosoma mansoni adult worms within 7 days Febrifugine concentration (μg/ml) Mean daily egg output (± standard error) Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Experiment 1 0 (control) 30.0 ± 9.9 115.3 ± 16.3 111.2 ± 18.2 52.8 ± 14.3 47.2 ± 17.6 36.5 ± 12.9 7.3 ± 4.2 5.7 ± 3.3 0.05 20.2 ± 5.8 102.2 ± 13.8 81.0 ± 19.8 43.3 ± 3.8 4.8 ± 3.1a 0.3 ± 0.3b 0 0.1 19.7 ± 9.2 101.3 ± 16.1 74.5 ± 7.8 35.8 ± 3.0 1.2 ± 0.3b 0 0.2 24.7 ± 10.5 51.5 ± 17.5 68.8 ± 29.6 18.0 ± 9.1 0.8 ± 0.5b 0.2 ± 0.2b 0 0.5 18.3 ± 6.0 85.3 ± 20.3 50.7 ± 15.5 4.5 ± 2.5b 1.2 ± 0.8b 0 Experiment 2 0 (control) 26.5 ± 12.6 94.2 ± 18.8 101.2 ± 21.6 98.3 ± 18.9 57.5 ± 14.8 22.0 ± 6.6 8.7 ± 2.4 4.8 ± 1.9 0.005 20.3 ± 6.3 92.0 ± 18.3 69.0 ± 15.1 88.7 ± 29.0 66.2 ± 26.1 13.2 ± 6.1 6.7 ± 2.6 1.7 ± 0.8 0.01 25.3 ± 10.1 86.5 ± 5.9 78.8 ± 15.8 80.0 ± 15.4 48.0 ± 21.3 6.8 ± 5.7 1.7 ± 1.5 0.7 ± 0.4 0.02 16.8 ± 7.2 100.2 ± 28.5 89.5 ± 13.9 84.7 ± 29.8 12.0 ± 5.4a 1.7 ± 0.9a 0 Mean daily egg output during the one-day preincubation (day 0) among the control and febrifugine-treated groups was not significantly different in experiment 1 Mitsui et al. Tropical Medicine and Health (2020) 48:42 Mitsui et al. Tropical Medicine and Health Page 4 of 7 0.2 μg/ml Feb-treated groups was significantly lower than that in the control group (P < 0.05, P < 0.01, and P < 0.01), and on the fifth day, the mean daily egg output in the Feb-treated groups was zero or almost zero. difference was observed in the survival time between male and female worms in the 0.1 and 0.2 μg/ml Feb- treated groups. Effect of febrifugine on the survival time of Schistosoma mansoni adult worm pairs The survival time of S. mansoni adult worm pairs incu- bated with 0 (control), 0.05, 0.1, 0.2, and 0.5 μg/ml Feb in experiment 1 and with 0 (control), 0.005, 0.01, and 0.02 μg/ml Feb in experiment 2 is shown in Table 2. None of the worms died in the control groups during the 14-day incubation period in either experiment 1 or 2. On the other hand, although none of the worms died in the 0.005 and 0.01 μg/ml Feb-treated groups, all the male and female worms in the 0.02–0.5 μg/ml Feb- treated groups in experiments 1 and 2 died within the 14-day incubation period. Moreover, a significant Table 2 Effect of febrifugine on the survival time of six paired Schistosoma mansoni adult worms within 14 days Concentration of febrifugine (μg/ml) Median survival days (range) P valueb Male Female Experiment 1 0 (control) ND ND 0.05 12.0 (12–13)a 12.0 (11–12)a 0.18 0.1 11.0 (11)a 9.0 (9–11)a 0.00 0.2 10.5 (9–11)a 8.5 (8–9)a 0.00 0.5 8.0 (7–8)a 7.0 (7–8)a 0.09 Experiment 2 0 (control) ND ND – 0.005 ND ND – 0.01 ND ND – 0.02 13.0 (13–14)a 13.5 (13–14)a 0.57 A log-rank test was used for the statistical analysis ND death was not observed in any of the worms over the incubation period of 14 days aP value < 0.01, compared with the corresponding control group bCompared between male and female Table 2 Effect of febrifugine on the survival time of six paired Schistosoma mansoni adult worms within 14 days Table 2 Effect of febrifugine on the survival time of six paired Schistosoma mansoni adult worms within 14 days Effect of febrifugine on the egg production of Schistosoma mansoni adult worm pairs The effect of Feb on the daily egg output of female adult worms is shown in Table 1. In experiment 1, the mean daily egg output during the 1-day preincubation period was not significantly different among the control and 0.05, 0.1, 0.2, and 0.5 μg/ml Feb-treated groups (P = 0.88 for all comparisons). During the first 2 days of incuba- tion, the mean daily egg output in the 0.05–0.5 μg/ml Feb-treated groups was not significantly lower than that in the control group, but on the third day of incubation, the mean daily egg output in the 0.5 μg/ml Feb-treated group was significantly lower than that in the control group (P < 0.01). Staining of febrifugine-treated Schistosoma mansoni adult worms with neutral red dye y Within 5 days after male and female S. mansoni adult worms were treated with none as control or 0.5 μg/ml Feb, the worms were stained with NR. Images of the staining of non- and Feb-treated S. mansoni adult worms are presented in Fig. 2. When non-treated male and fe- male worms were stained with NR, the whole body of the males appeared dark red in color and the intestinal tract was stained deep red (Fig. 2a). With regard to the body of female worms, the status of NR staining was as follows: anterior intestinal tract, deep red; vitellaria, dark red; middle and posterior intestinal tract, deep red (Fig. 2b). On the other hand, the Feb-treated male worms were stained at a slightly lower shade of dark red than the non-treated male worms, and the accumulation of NR in the intestinal tract was less than that of the non- treated male worms (Fig. 2c). Furthermore, the staining status of Feb-treated female worms was as follows: an- terior intestinal tract, slightly stained deep red; vitellaria, Mitsui et al. Tropical Medicine and Health (2020) 48:42 Page 5 of 7 (2020) 48:42 Fig. 1 Effect of febrifugine on the gross morphological alteration of Schistosoma mansoni adult worms. Paired S. mansoni adult worms were continuously incubated in media alone for 3 (a), 5 (b), 7 (c), and 8 (d) days (control group), while paired adult worms were incubated with 0.5 μg/ml febrifugine for 3 (e), 5 (f), 7 (g), and 8 (h) days. Arrows indicate the localization of black contents. The symbols ♂and ♀indicate male and female worms, respectively. ant. anterior part of body, pos. posterior part of body. Scale bar = 1 mm Fig. 1 Effect of febrifugine on the gross morphological alteration of Schistosoma mansoni adult worms. Paired S. mansoni adult worms were continuously incubated in media alone for 3 (a), 5 (b), 7 (c), and 8 (d) days (control group), while paired adult worms were incubated with 0.5 μg/ml febrifugine for 3 (e), 5 (f), 7 (g), and 8 (h) days. Arrows indicate the localization of black contents. The symbols ♂and ♀indicate male and female worms, respectively. ant. anterior part of body, pos. posterior part of body. Scale bar = 1 mm dark red; middle and posterior intestinal tract, mostly unstained (Fig. 2d). achieve a clear understanding of the antischistosomal ef- fect of Feb. When pairs of S. Staining of febrifugine-treated Schistosoma mansoni adult worms with neutral red dye mansoni adult worms were treated with Feb at concentrations of 0.02–0.5 μg/ml, the daily egg output was significantly lower than that of the corre- sponding control groups after 3 or 4 days of incubation (Table 1), while the daily egg output of the 0.01 μg/ml Feb group was similar to that of the control group. Thus, the minimum concentration of Feb that effectively Discussion Several antimalarial drugs have been reported to exhibit an antischistosomal effect [6–8]. It follows that Feb, which exerts an antimalarial effect, may also possess antischistosomal potential, but the possibility has never been investigated. The present study was conducted to Fig. 2 Neutral red staining of Schistosoma mansoni adult worms incubated with febrifugine. Male and female S. mansoni adult worms were incubated with none (male: a, female: b) or 0.5 μg/ml febrifugine (male: c, female: d) for 5 days and stained with neutral red. it intestinal tract, vt vitellaria, ant. anterior, mid. middle, pos. posterior. Scale bar = 1 mm Fig. 2 Neutral red staining of Schistosoma mansoni adult worms incubated with febrifugine. Male and female S. mansoni adult worms were incubated with none (male: a, female: b) or 0.5 μg/ml febrifugine (male: c, female: d) for 5 days and stained with neutral red. it intestinal tract, vt vitellaria, ant. anterior, mid. middle, pos. posterior. Scale bar = 1 mm Mitsui et al. Tropical Medicine and Health (2020) 48:42 Page 6 of 7 Mitsui et al. Tropical Medicine and Health (2020) 48:42 Page 6 of 7 Mitsui et al. Tropical Medicine and Health worms suggests that Feb inflicts severe damage on fe- male worms, especially the intestinal tract. inhibits the daily egg output of adult worm pairs is pre- sumed to be 0.02 μg/ml. Furthermore, Feb significantly reduced the survival time of all adult worms at concen- trations of 0.02–0.5 μg/ml during the 14-day incubation period (Table 2), while no death of worms was observed in the 0.01 μg/ml Feb group, further suggesting that the minimum concentration of Feb effective in inhibiting the survival of adult worm pairs is also 0.02 μg/ml. In addition, the effect of Feb on the daily egg output and survival time of adult worm pairs was dependent on the concentration and duration of exposure to the drug. The significant difference in survival time between male and female worms treated with Feb at concentrations of 0.1 or 0.2 μg/ml (Table 2) suggested that female worms are more susceptible to Feb than male worms. However, since the survival time of female worms was similar to that of male worms at the lower concentration of 0.02 or 0.05 μg/ml, no definite conclusion could be drawn with regard to the difference between female and male worms in susceptibility to Feb. Discussion The antimalarial effect of halofuginone and other Feb derivatives is due to the inhibition of prolyl-tRNA syn- thetase (PRS) in the malaria parasite [18, 23]. Moreover, Jain et al. (2017) reported that synthetic derivatives of Feb selectively and potentially inhibit the PRS of not only malaria but also other protozoan parasites such as Plasmodium, Toxoplasma, Cryptosporidium, and Eimeria [24]. Hence, the antischistosomal effect of Feb may be attributable to a mode of action similar to that observed in the protozoan parasites. However, no inves- tigation related to the antischistosomal effect of Feb has been conducted to date, and the question remains as to whether other Feb derivatives exert an antischistosomal activity like that shown in the present study. Clearly, fur- ther studies are needed to elucidate the mode of action of Feb against S. mansoni adult worms. g The present study evaluated the antischistosomal ef- fect of Feb on egg production of S. mansoni female worms and the survivability of male and female worms and their morphological changes. We showed that the minimal concentration of Feb exhibiting anti- schistosomal activity is 0.02 μg/ml. However, since the present study focused broadly on the effect on S. mansoni worms as a way to evaluate the antischisto- somal activity of Feb, the cytotoxicity thereof remained to be examined. On the other hand, Jiang et al. (2005) and Kikuchi et al. (2014) evaluated the cytotoxicity of Feb against mammalian neuronal cells (NG108), macrophages (J774), and mouse L929 cells, and showed that the half-maximal inhibitory concen- trations were 0.064, 0.081, and 0.169 μg/ml, respect- ively [17, 25]. Thus, the therapeutic range of concentrations at which Feb is effective for treating schistosomiasis with acceptable side effects is pre- sumed to be narrow. However, despite severe side effects [13, 15], the antimalarial potency of Feb has encouraged medicinal chemists to develop new Feb analogues with potent antimalarial activity but negli- gible toxicity to humans [17, 19]. These analogues may also be expected to exert an antischistosomal effect and to play a role in the treatment of schistosomiasis. The non-treated male and female adult worms of S. mansoni remained morphologically normal within the period of 14 days, while the male and female worms treated with Feb at different concentrations gradually twisted and subsequently died (Fig. 1). The onset of morphological alteration in the Feb-treated worms was apparently dependent on the concentration and duration of exposure to the drug. Discussion Furthermore, the morphological alteration caused by Feb obviously occurred earlier in fe- male than in male worms (Fig. 1f). Subsequently, we applied NR as a non-fluorescent dye to better assess the antischistosomal effect of Feb on S. mansoni adult worms. A cationic non- fluorescent dye, NR is absorbed by living cells but not by dead or damaged cells. When S. mansoni adult worms were incubated with either 0.5 μg/ml Feb or none as a control for 5 days and stained with NR, the non-treated male worms were morphologically normal and stained dark red with NR (Fig. 2a), while the Feb-treated male worms appeared similar to those in the control group and were stained at a slightly lower level of dark red than the non-treated male worms (Fig. 2c), suggesting only slight damage caused by Feb. In contrast, the staining status of the 0.5 μg/ ml Feb-treated female worms differed remarkably from that of the non-treated female worms (Fig. 2b, d). The non-treated female worms were morphologic- ally normal, and their intestinal tract and vitellaria were stained deep red and dark red, respectively. On the other hand, the Feb-treated female worms were morphologically damaged, and their intestinal tract and vitellaria remained mostly unstained and stained dark red, respectively. The marked difference in stain- ing status between the non- and Feb-treated female Availability of data and materials Not applicable. 18. Jain V, Yogavel M, Oshima Y, et al. Structure of prolyl-tRNA synthetase- halofuginone complex provides basis for development of drugs against malaria and toxoplasmosis. Structure. 2015;23(5):819–29. Funding Thi 16. Pines M, Spector I. Halofuginone—the multifaceted molecule. Molecules. 2015;20(1):573-594. doi:https://doi.org/10.3390/molecules20010573. This research received no specific grant from any funding agency in either commercial or not-for-profit sectors. The present study was conducted on the basis of current budget allocations from Nagasaki University. 17. Jiang S, Zeng Q, Gettayacamin M, et al. Antimalarial activities and therapeutic properties of febrifugine analogs. Antimicrob Agents Chemother. 2005;49(3):1169–76. Abbreviations 6. Mitsui Y, Miura M, Aoki Y. In vitro effects of artesunate on the survival of worm pairs and egg production of Schistosoma mansoni. J Helminthol. 2009;83(1):7–11. ant.: Anterior or anterior part of body; AQ: Amodiaquine; ART: Artesunate; DW: Deionized water; Feb: Febrifugine; it: Intestinal tract; mid.: Middle; ant.: Anterior or anterior part of body; AQ: Amodiaquine; ART: Artesunate; DW: Deionized water; Feb: Febrifugine; it: Intestinal tract; mid.: Middle; MQ: Mefloquine; NR: Neutral red; pos.: Posterior or posterior part of body; PQ: Primaquine; PRS: Prolyl-tRNA synthetase; PZQ: Praziquantel; QN: Quinine; vt: Vitellaria; WHO: World Health Organization 7. Keiser J, Chollet J, Xiao SH, Mei JY, et al. Mefloquine—an aminoalcohol with promising antischistosomal properties in mice. PLoS Negl Trop Dis. 2009; 3(1):e350. MQ: Mefloquine; NR: Neutral red; pos.: Posterior or posterior part of body; PQ: Primaquine; PRS: Prolyl-tRNA synthetase; PZQ: Praziquantel; QN: Quinine; vt: Vitellaria; WHO: World Health Organization 8. Mitsui Y, Aoki Y. In vitro effect of current antimalarial drugs on the survival of paired Schistosoma mansoni adult worms and their egg production. Trop Med Health. 2010;38(2):69–73. Consent for publication Not applicable. Not applicable. Acknowledgements We would like to thank Prof. Yoshito Fujii, Department of Clinical Laboratory Sciences, Sanyo Women’s College and Prof. Satoshi Kaneko, Department of Eco-epidemiology, Institute of Tropical Medicine, Nagasaki University for en- couragement and support. We also thank Prof. Brian Burke-Gaffney, Depart- ment of Applied Information Technology, Nagasaki Institute of Applied Science, for helpful advice in the revision of the manuscript. We thank the In- stitute of Tropical Medicine, Nagasaki University for support in the payment of the processing charge of this manuscript. 9. Koepfli JB, Mead JF, Brockman JA Jr. An alkaloid with high antimalarial activity from Dichroa febrifuga. J Am Chem Soc. 1947;69(7):1837. 10. Koepfli JB, Mead JF, Brockman JA. Alkaloids of Dichroa febrifuga. I. Isolation and degradative studies. J Am Chem Soc. 1949;71(3):1048–54. 11. Jang CS, Fu FY, Huang KC, et al. Pharmacology of ch'ang Shan (Dichroa febrifuga) a Chinese antimalarial herb. Nature. 1948;161(4089):400–1. 11. Jang CS, Fu FY, Huang KC, et al. Pharmacology of ch ang Shan (Dichroa febrifuga) a Chinese antimalarial herb. Nature. 1948;161(4089):400–1. 12. Ablondi F, Gordon S, Morton J II, et al. An antimalarial alkaloid from hydrangea. II. Isolation. J Org Chem. 1952;17(1):14–8. g 12. Ablondi F, Gordon S, Morton J II, et al. An antimalarial alkaloid from hydrangea. II. Isolation. J Org Chem. 1952;17(1):14–8. 13. Henderson FG, Rose CL, Harris PN, et al. Gamma-Dichroine, the antimalarial alkaloid of ch'ang Shan. J Pharmacol Exp Ther. 1949;95(2):191–200. Conclusion The present study demonstrated that Feb significantly reduced the survival time of paired S. mansoni male and female adult worms at concentrations of 0.02–0.5 μg/ml during in vitro cultivation for 14 days and inhibited the egg production by female worms. The antischistosomal effect of Feb is a topic of keen interest and promises to contribute to the development of a novel antischistoso- mal drug. Page 7 of 7 Page 7 of 7 Mitsui et al. Tropical Medicine and Health (2020) 48:42 Mitsui et al. Tropical Medicine and Health (2020) 48:42 Mitsui et al. Tropical Medicine and Health (2020) 48:42 Authors’ contributions Mitsui designed this study, performed experiments, analyzed data, and prepared the first draft of the manuscript. Miura and Kato participated in the experiments and manuscript writing. All authors participated in data interpretation and preparation of the manuscript, and all authors read and approved the final manuscript. 14. Hewitt RI, Wallace WS, Gill ER, et al. An antimalarial alkaloid from hydrangea XIII. The effects of various synthetic quinazolones against plasmodium lophurae in ducks. Am J Trop Med Hyg. 1952;1(5):768–72. 15. Coatney GR, Cooper WC, Culwell WB, et al. Studies in human malaria. XXV. Trial of febrifugine, an alkaloid obtained from Dichroa febrifuga Lour against the Chesson strain of plasmodium vivax. J Natl Malar Soc. 1950;9(2):183–6. Competing interests 23. Jain V, Kikuchi H, Oshima Y, et al. Structural and functional analysis of the anti-malarial drug target prolyl-tRNA synthetase. J Struct Funct Genom. 2014;15(4):181–90. https://doi.org/10.1007/s10969-014-9186-x. 23. Jain V, Kikuchi H, Oshima Y, et al. Structural and functional analysis of the anti-malarial drug target prolyl-tRNA synthetase. J Struct Funct Genom. 2014;15(4):181–90. https://doi.org/10.1007/s10969-014-9186-x. The authors declare that they have no competing interests. Author details 1 24. Jain V, Yogavel M, Kikuchi H, et al. Targeting prolyl-tRNA synthetase to accelerate drug discovery against malaria, leishmaniasis, toxoplasmosis, cryptosporidiosis, and coccidiosis. Structure. 2017;25(10):1495–1505.e6. https://doi.org/10.1016/j.str.2017.07.015. 1Department of Parasitology, Institute of Tropical Medicine, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan. 2Department of Eco-epidemiology, Institute of Tropical Medicine, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan. 25. Kikuchi H, Horoiwa S, Kasahara R, Hariguchi N, Matsumoto M, Oshima Y. Synthesis of febrifugine derivatives and development of an effective and safe tetrahydroquinazoline-type antimalarial. Eur J Med Chem. 2014;76:10–9 https://doi.org/10.1016/j.ejmech.2014.01.036. 25. Kikuchi H, Horoiwa S, Kasahara R, Hariguchi N, Matsumoto M, Oshima Y. Synthesis of febrifugine derivatives and development of an effective and safe tetrahydroquinazoline-type antimalarial. Eur J Med Chem. 2014;76:10–9. https://doi.org/10.1016/j.ejmech.2014.01.036. Received: 23 April 2020 Accepted: 25 May 2020 Ethics approval and consent to participate 19. Kikuchi H, Tasaka H, Hirai S, et al. Potent antimalarial febrifugine analogues against the plasmodium malaria parasite. J Med Chem. 2002;45(12):2563–70. The authors assert that all procedures contributing to this work comply with the ethical standards of relevant national and institutional procedures for the care and use of laboratory animals. The present investigation was approved by the Ethics Review Committee for Animal Experimentation of Nagasaki University School of Medicine, and all animal experiments were performed according to the guidelines for animal experimentation in the Laboratory Animal Center for Biomedical Research at Nagasaki University. 20. Oliveira AB, Dolabela MF, Braga FC, et al. Plant-derived antimalarial agents: new leads and efficient phythomedicines. Part I. alkaloids. An Acad Bras Cienc. 2009;81(1):715–40. https://doi.org/10.1590/s0001-37652009000400011. 21. Smithers SR, Terry RJ. The infection of laboratory hosts with cercariae of Schistosoma mansoni and the recovery of the adult worms. Parasitology. 1965;55(4):695–700. 22. Mitsui Y, Kato K. Application of non-fluorescent dyes to assess the antischistosomal effect of antimalarial drugs on Schistosoma mansoni adult worms. Jpn J Infect Dis. 2018;71(5):382–7. https://doi.org/10.7883/yoken.JJID. 2018.034. 22. Mitsui Y, Kato K. Application of non-fluorescent dyes to assess the antischistosomal effect of antimalarial drugs on Schistosoma mansoni adult worms. Jpn J Infect Dis. 2018;71(5):382–7. https://doi.org/10.7883/yoken.JJID. 2018.034. Publisher’s Note 1. World Health Organization (WHO). Schistosomiasis: number of people treated worldwide in 2014. Wkly Epidemiol Rec. 2016;91(5):53–60 Available: http://www.who.int/wer/2016/wer9105/2en. 1. World Health Organization (WHO). Schistosomiasis: number of people treated worldwide in 2014. Wkly Epidemiol Rec. 2016;91(5):53–60 Available: http://www.who.int/wer/2016/wer9105/2en. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 2. World Health Organization (WHO). Helminth control in school-age children: a guide for managers of control programmes. 2nd ed. Geneva: WHO; 2011. Available: https://www.who.int/neglected_diseases/resources/9789241548267/en/. 3. Doenhoff MJ, Kusel JR, Coles GC, et al. Resistance of Schistosoma mansoni to praziquantel: is there a problem? Trans R Soc Trop Med Hyg. 2002;96(5): 465–9. 3. Doenhoff MJ, Kusel JR, Coles GC, et al. Resistance of Schistosoma mansoni to praziquantel: is there a problem? Trans R Soc Trop Med Hyg. 2002;96(5): 465–9. 4. Wang W, Wang L, Liang YS. Susceptibility or resistance of praziquantel in human schistosomiasis: a review. Parasitol Res. 2012;111(5):1871–7. 5. Lescano SZ, Chieffi PP, Canhassi RR, et al. Antischistosomal activity of artemether in experimental schistosomiasis mansoni. Rev Saude Publica. 2004;38(1):71–5.
https://openalex.org/W4225394263	https://kclpure.kcl.ac.uk/ws/files/180291144/Comparative_Genetic_Analysis_SOOMRO_Publishedonline4August2022_GOLD_VoR_CC_BY.pdf	English	null	Comparative Genetic Analysis of Psoriatic Arthritis and Psoriasis for the Discovery of Genetic Risk Factors and Risk Prediction Modeling	Arthritis & rheumatology	2,022	cc-by	9,176	Citation for published version (APA): the BADBIR Study Group, & the BSTOP study group (2022). Comparative Genetic Analysis of Psoriatic Arthritis and Psoriasis for the Discovery of Genetic Risk Factors and Risk Prediction Modeling. Arthritis and Rheumatology, 74(9), 1535-1543. https://doi.org/10.1002/art.42154 Citation for published version (APA): the BADBIR Study Group, & the BSTOP study group (2022). Comparative Genetic Analysis of Psoriatic Arthritis and Psoriasis for the Discovery of Genetic Risk Factors and Risk Prediction Modeling. Arthritis and Rheumatology, 74(9), 1535-1543. https://doi.org/10.1002/art.42154 Citing this paper Pl h C t g t s pape Please note that where the full-text provided on King's Research Portal is the Author Accepted Manuscript or Post-Print version this may differ from the final Published version. If citing, it is advised that you check and use the publisher's definitive version for pagination, volume/issue, and date of publication details. And where the final published version is provided on the Research Portal, if citing you are again advised to check the publisher's website for any subsequent corrections. Citation for published version (APA): the BADBIR Study Group, & the BSTOP study group (2022). Comparative Genetic Analysis of Psoriatic Arthritis and Psoriasis for the Discovery of Genetic Risk Factors and Risk Prediction Modeling. Arthritis and Rheumatology, 74(9), 1535-1543. https://doi.org/10.1002/art.42154 1Mehreen Soomro MSc, Michael Stadler, MSc, Farideh Jalali-najafabadi, PhD: Centre for Genetics and Genomics Versus Arthritis, Centre for Musculo- skeletal Research, Manchester Academic Health Science Centre, The University of Manchester, Manchester, UK; 2Nick Dand, PhD, Michael A. Simpson, PhD: King’s College London, London, UK; 3James Bluett, PhD, MBBS, Pauline Ho, Supported by Versus Arthritis (grants 21173, 21754, and 21755), the NIHR Cambridge Biomedical Research Centre (grant BRC-1215-20014), and Cam- bridge Arthritis Research Endeavour (CARE). Mr. Stadler’s work was sup- ported by an MRC Doctoral Training Partnership (DTP) Studentship (grant MR/N013751/1). Dr. Jalali-najafabadi’s work was supported by a Medical Research Council (MRC)/University of Manchester Skills Development Fellow- ship (grant MR/R016615). Dr. Marzo-Ortega’s work was supported by the Leeds NIHR Biomedical Research Centre (LBRC). Dr. Warren’s work was sup- ported by the Manchester NIHR Biomedical Research Centre. Dr. Barton’s work was supported by the NIHR. 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Download date: 24. Oct. 2024 PhD, MBBCh, John Bowes, PhD, Anne Barton, PhD, FRCP: Centre for Genetics and Genomics Versus Arthritis, Centre for Musculoskeletal Research, Manches- ter Academic Health Science Center, The University of Manchester, NIHR Man- chester Musculoskeletal Biomedical Research Unit, Manchester University NHS Foundation Trust, Manchester, UK; 4Deepak Jadon, PhD, MRCP, MBBCh: Uni- versity of Cambridge, Cambridge, UK; 5Michael Duckworth, BSc, Jonathan N. Barker, MD, FRCP, FRCPath: St John’s Institute of Dermatology, King’s College London, London, UK; 6Helena Marzo-Ortega, PhD, MRCP, Philip S. Helliwell, PhD, FRCP: NIHR Leeds Biomedical Research Centre, Leeds Teaching Hospitals Trust, Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, Leeds, UK; 7Anthony W. Ryan, PhD: Trinity Translational Medicine Institute, Trinity College Dublin and Genuity Science, Dublin, Ireland; 8David Kane, PhD, FRCPI: Tallaght University Hospital and Trinity College Dublin, Dub- lin, Ireland; 9Eleanor Korendowych, PhD, BMBCh, MRCP: Royal National Hospi- tal for Rheumatic Diseases and University of Bath, Bath, UK; 10Jonathan PhD, MBBCh, John Bowes, PhD, Anne Barton, PhD, FRCP: Centre for Genetics and Genomics Versus Arthritis, Centre for Musculoskeletal Research, Manches- ter Academic Health Science Center, The University of Manchester, NIHR Man- chester Musculoskeletal Biomedical Research Unit, Manchester University NHS Foundation Trust, Manchester, UK; 4Deepak Jadon, PhD, MRCP, MBBCh: Uni- versity of Cambridge, Cambridge, UK; 5Michael Duckworth, BSc, Jonathan N. Barker, MD, FRCP, FRCPath: St John’s Institute of Dermatology, King’s College London, London, UK; 6Helena Marzo-Ortega, PhD, MRCP, Philip S. Helliwell, PhD, FRCP: NIHR Leeds Biomedical Research Centre, Leeds Teaching Hospitals Trust, Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, Leeds, UK; 7Anthony W. Ryan, PhD: Trinity Translational Medicine Institute, Trinity College Dublin and Genuity Science, Dublin, Ireland; 8David Kane, PhD, FRCPI: Tallaght University Hospital and Trinity College Dublin, Dub- lin, Ireland; 9Eleanor Korendowych, PhD, BMBCh, MRCP: Royal National Hospi- tal for Rheumatic Diseases and University of Bath, Bath, UK; 10Jonathan PATIENTS AND METHODS PsA genome-wide association study (GWAS) cohort. A total of 4,072 patients with PsA were recruited from rheumatol- ogy centers in the UK, Ireland, and Switzerland, from the pro- spective Swiss Clinical Quality Management (SCQM) registry, and from Australia. Patients recruited in Manchester were diag- nosed by a rheumatologist based on the presence of both psori- asis and inﬂammatory peripheral arthritis, regardless of rheumatoid factor status. While the majority of patients satisﬁed the CASPAR (Classiﬁcation of Psoriatic Arthritis) classiﬁcation system (14), some were recruited prior to the introduction of this classiﬁcation system. All patients provided written informed con- sent (UK PsA National Repository Multicentre Research Ethics Committee reference no. 99/8/84). Samples from the Axial Dis- ease in Psoriatic Arthritis Study (ADIPSA) cohort were collected with ethics approval from the French Regional Ethics Committee (reference no. 12/SW/0110). The Leeds cohort comprises adult patients with a clinical diagnosis of PsA fulﬁlling the CASPAR clas- siﬁcation criteria who were recruited as part of an in-house bio- bank study investigating SNPs of immune response genes in patients with psoriasis, patients with PsA, and patients with anky- losing spondylitis and their relationship to disease susceptibility, articular and extraarticular manifestations, and response to treat- ment (Research Ethics Committee reference no. 04/Q1205/65, IRAS project no. 232680). All patients provided written informed consent. PsA is a typical complex disease in which susceptibility is inﬂuenced by a combination of environmental, lifestyle, and genetic risk factors. Previous family pedigree studies have esti- mated that the heritability of PsA far exceeds that of psoriasis alone, providing evidence of an increased genetic component which, once identiﬁed, could help to differentiate those patients at high risk of developing PsA by inclusion of genetic risk factors in clinical prediction models (4–6). However, the results of these family studies have been challenged by data from large-scale case–control studies analyzing variations in single-nucleotide polymorphisms (SNPs), in which only limited differences in herita- bility estimates have been demonstrated between patients with PsA and patients with psoriasis (7). Several studies have identiﬁed genetic risk factors that are speciﬁc to PsA, including amino acids within HLA–B and variants at the IL23R gene, and the current aim is to translate these genetic discoveries into improved clinical out- comes (8–12). A recent study demonstrated high performance in accurately distinguishing PsA from cutaneous-only psoriasis (PsC) using prediction models based on genetic risk factors. Packham, BM, DM, FRCP: Haywood Hospital and Midlands Partnership NHS Foundation Trust, Stoke on Trent, UK, and University of Nottingham, Notting- ham, UK; 11Ross McManus, PhD, FTCD: Trinity Translational Medicine Institute, Trinity College Dublin, Dublin, Ireland; 12Cem Gabay, MD: Geneva University Hospitals, University of Geneva, Geneva, Switzerland; 13Céline Lamacchia, PhD, Michael J. Nissen, MBBS, FRACP: Geneva University Hospitals, Geneva, Switzerland; 14Matthew A. Brown, MBBS, FRACP: King’s College London and Genomics England, London, UK; 15Suzanne M. M. Verstappen, PhD: NIHR Manchester Musculoskeletal Biomedical Research Unit, Man- chester University NHS Foundation Trust, Manchester Academic Health Science Centre, Centre for Epidemiology Versus Arthritis, Centre for Mus- culoskeletal Research, University of Manchester, Manchester, UK; 16Tjeerd Van Staa, PhD, MD: Health e-Research Centre, Health Data Research UK North, University of Manchester, Manchester, UK; 17Catherine H. Smith, MD, FRCP: St John’s Institute of Dermatology, Guy’s and St Thomas’ NHS Foundation Trust and King’s College London, London, UK; 18Oliver FitzGer- ald, MBBCh, FRCPI: Conway Institute of Biomolecular and Biomedical Comparative Genetic Analysis of Psoriatic Arthritis and Psoriasis for the Discovery of Genetic Risk Factors and Risk Prediction Modeling Mehreen Soomro,1 Michael Stadler,1 Nick Dand,2 James Bluett,3 Deepak Jadon,4 Farideh Jalali-najafabadi,1 Michael Duckworth,5 Pauline Ho,3 Helena Marzo-Ortega,6 Philip S. Helliwell,6 Anthony W. Ryan,7 David Kane,8 Eleanor Korendowych,9 Michael A. Simpson,2 Jonathan Packham,10 Ross McManus,11 Cem Gabay,12 Céline Lamacchia,13 Michael J. Nissen,13 Matthew A. Brown,14 Suzanne M. M. Verstappen,15 Tjeerd Van Staa,16 Jonathan N. Barker,5 Catherine H. Smith,17 the BADBIR Study Group, the BSTOP Study Group, Oliver FitzGerald,18 Neil McHugh,19 Richard B. Warren,20 John Bowes,3 and Anne Barton3 Objectives. Psoriatic arthritis (PsA) has a strong genetic component, and the identiﬁcation of genetic risk factors could help identify the ~30% of psoriasis patients at high risk of developing PsA. Our objectives were to identify genetic risk factors and pathways that differentiate PsA from cutaneous-only psoriasis (PsC) and to evaluate the performance of PsA risk prediction models. Methods. Genome-wide meta-analyses were conducted separately for 5,065 patients with PsA and 21,286 healthy controls and separately for 4,340 patients with PsA and 6,431 patients with PsC. The heritability of PsA was calculated as a single-nucleotide polymorphism (SNP)–based heritability estimate (h2 SNP) and biologic path- ways that differentiate PsA from PsC were identiﬁed using Priority Index software. The generalizability of previ- ously published PsA risk prediction pipelines was explored, and a risk prediction model was developed with external validation. Results. We identiﬁed a novel genome-wide signiﬁcant susceptibility locus for the development of PsA on chromo- some 22q11 (rs5754467; P = 1.61 × 10−9), and key pathways that differentiate PsA from PsC, including NF-κB signal- ing (adjusted P = 1.4 × 10−45) and Wnt signaling (adjusted P = 9.5 × 10−58). The heritability of PsA in this cohort was found to be moderate (h2 SNP = 0.63), which was similar to the heritability of PsC (h2 SNP = 0.61). We observed modest performance of published classiﬁcation pipelines (maximum area under the curve 0.61), with similar performance of a risk model derived using the current data. Conclusion. Key biologic pathways associated with the development of PsA were identiﬁed, but the investigation of risk classiﬁcation revealed modest utility in the available data sets, possibly because many of the PsC patients included in the present study were receiving treatments that are also effective in PsA. Future predictive models of PsA should be tested in PsC patients recruited from primary care. 1535 SOOMRO ET AL 1536 INTRODUCTION patients, and to explore differences in the genetic architecture of the 2 traits that could explain the progression to PsA. These data can be used to further our understanding of key genes and biologic pathways important in psoriatic disease using state-of-the-art bio- informatics tools and could be further used to explore the utility of genetic risk prediction models for classifying PsA. Psoriatic arthritis (PsA) is a chronic inﬂammatory condition characterized by the presence of peripheral arthritis, dactylitis, enthesitis, and axial spondyloarthritis (1). PsA affects between 14% and 30% of patients with psoriasis, leading to signiﬁcant dis- ability and a reduced quality of life (1–3). The ability to identify patients with psoriasis who are at a high risk of developing PsA is an important goal for clinical research, as this would allow early intervention to reduce the impact of PsA and ultimately lead to preventative treatments. Submitted for publication August 11, 2021; accepted in revised form April 28, 2022. Research, University College Dublin, Dublin, Ireland; 19Neil McHugh, MD: Royal National Hospital for Rheumatic Diseases, University of Bath, Bath, UK; 20Richard B. Warren, PhD, MBChB: Dermatology Centre, Salford Royal NHS Foundation Trust, Manchester NIHR Biomedical Research Centre, University of Manchester, Manchester, UK. Ms. Soomro and Mr. Stadler contributed equally to this work. Drs. Bowes and Barton jointly supervised this work. Author disclosures are available at https://onlinelibrary.wiley.com/action/ downloadSupplement?doi=10.1002%2Fart.42154&ﬁle=art42154-sup-0001- Disclosureform.pdf. Address correspondence to John Bowes, PhD, Centre for Genetics and Genomics Versus Arthritis, Division of Musculoskeletal and Dermatological Sciences, School of Biological Sciences, Faculty of Biology, Medicine and Health, The University of Manchester, AV Hill Building, Oxford Road, Man- chester, UK. Email: j.bowes@manchester.ac.uk. Submitted for publication August 11, 2021; accepted in revised form April 28, 2022. Address correspondence to John Bowes, PhD, Centre for Genetics and Genomics Versus Arthritis, Division of Musculoskeletal and Dermatological Sciences, School of Biological Sciences, Faculty of Biology, Medicine and Health, The University of Manchester, AV Hill Building, Oxford Road, Man- chester, UK. Email: j.bowes@manchester.ac.uk. Research, University College Dublin, Dublin, Ireland; 19Neil McHugh, MD: Royal National Hospital for Rheumatic Diseases, University of Bath, Bath, UK; 20Richard B. Warren, PhD, MBChB: Dermatology Centre, Salford Royal NHS Foundation Trust, Manchester NIHR Biomedical Research Centre, University of Manchester, Manchester, UK. Ms. Soomro and Mr. Stadler contributed equally to this work. Drs. Bowes and Barton jointly supervised this work. Author disclosures are available at https://onlinelibrary.wiley.com/action/ downloadSupplement?doi=10.1002%2Fart.42154&ﬁle=art42154-sup-0001- Disclosureform.pdf. PATIENTS AND METHODS Patients with psoriasis requiring systemic therapy who also consented to enrol- ment in the British Association of Dermatologists Biologics Inter- ventions Registry (a UK pharmacovigilance registry) were recruited to BSTOP from over 60 secondary and tertiary care out- patient dermatology departments throughout the UK including centers in London, Manchester, Nottingham, and Liverpool. All patients provided written informed consent (BSTOP Ethics refer- ence no. 11/H0802/7). Classiﬁcation of PsC in the BSTOP cohort is based on information collected at multiple follow-up consultan- cies (one every 6 months in the ﬁrst 3 years and then once annu- ally) during which a research nurse or clinician actively investigated the patient’s medical records for the presence of a PsA diagnosis made by a rheumatologist. On average, patients in this cohort had a psoriasis disease duration of 27 years without a recorded PsA diagnosis (see Supplementary Figure 1A, avail- able at http://onlinelibrary.wiley.com/doi/10.1002/art.42154) and had been participants in the British Association of Dermatologists Biologic and Immunomodulators Register (BADBIR) study for ~7 years (Supplementary Figure 1B) with an average of 8 follow- up consultancies. Imputation. Imputation was performed for the combined data set of PsA, PsC, and control samples described above. Prior to imputation, SNPs with ambiguous alleles (C/G and A/T) were excluded, and remaining SNPs were aligned to the Haplotype Reference Consortium (HRC) panel (version 1.1) using the HRC imputation preparation tool (https://www.well.ox.ac.uk/ ~wrayner/tools/). Imputation was performed using the Michigan Imputation server in which phasing was performed with Shapeit2 and imputation was performed with the HRC panel. Following imputation, SNPs were excluded based on a minor allele fre- quency (MAF) of <0.01 and imputation accuracy of r2 <0.5. UK Biobank. We accessed imputed genotype data from the UK Biobank (application number 799) for self-reported out- comes in 731 PsA patients and 3,197 psoriasis patients (16). Control population data were obtained using random sampling from the remaining cohort at a ratio of 4 controls to 1 patient to minimize inﬂation of test statistics due to case–control imbalance. All participants were selected from the subset of White patients of British ancestry. In addition, we created a data set based on Inter- national Statistical Classiﬁcation of Diseases and Related Health Problems, Tenth Revision (ICD-10) codes L40 and L405, which yielded a cohort of 795 psoriasis patients and 435 PsA patients. PsA Immunochip data set. PATIENTS AND METHODS Although this study demonstrated validity by internal cross- validation methods, assessment of these models for generaliz- ability in external data sets is still warranted (13). To help further our understanding of the genetic basis for PsA, we have constructed a large integrated genetic data set of PsA patients, PsC patients, and population controls imputed to the lat- est population reference panels. We supplemented this data set by performing a meta-analysis using UK Biobank data, allowing us to contrast PsA patients with population controls and PsC A total of 283 patients with PsA were recruited from St. Vincent’s University Hospital observational PsA cohort. All patients met the CASPAR criteria. The study protocol received approval from the local ethics committee of St. Vincent’s COMPARATIVE GENETIC ANALYSIS OF PsA AND PSORIASIS 1537 genotyping array. This was performed in accordance with the manufacturer’s instructions where genotype calling was per- formed using the GenCall algorithm in the GenomeStudio Data Analysis software platform (Genotyping Module version 1.8.4). Psoriasis samples were genotyped using the Illumina HumanOmniExpressExome-8v1-2_A array performed at King’s College London with quality control as previously described (15). The 3 data sets (PsA, PsC, and controls) were combined with the intersection of SNPs being retained; hereafter, this is referred as the PsA-BSTOP GWAS data set. Further details are provided in the Supplementary Materials and Methods and Supplementary Figure 2 (available at http://onlinelibrary.wiley.com/doi/10.1002/ art.42154). University Hospital. Written informed consent was obtained from all patients. In addition, a total of 272 patients with PsA were recruited from the prospective SCQM registry in which diagnosis was based on the CASPAR criteria. The study protocol received approval from the local ethics committee of the University Hospital of Geneva (protocol no. 10-089) and the SCQM Biobank Scien- tiﬁc Advisory Board and followed the Guidelines for Good Clinical Practice. Written informed consent was obtained from all patients. A summary of available clinical phenotype data for this cohort is given in Supplementary Table 1 (available on the Arthri- tis & Rheumatology website at http://onlinelibrary.wiley.com/doi/ 10.1002/art.42154). Psoriasis GWAS cohort. We had access to data from 2,086 psoriasis patient samples obtained through the Biomarkers of Systemic Treatment Outcomes in Psoriasis study (BSTOP) described previously (9). Analysis of patients was restricted to those with no previous diagnosis of PsA, and we refer to this sam- ple group as having cutaneous-only psoriasis (PsC). PATIENTS AND METHODS Genotype data were available for 1,962 PsA patients and 8,923 controls (controls were recruited from the 1958 Birth Cohort and the National Blood Ser- vice) (17). Sample overlap with the GWAS and UK Biobank data sets was determined using identity by descent analysis (Kinship- based Inference for GWAS software) and duplicate samples were excluded from the Immunochip data set, leaving a total of 725 PsA patients and 8,897 controls. Control population GWAS cohort. As controls, geno- type data were available for 9,965 general population subjects from the UK Household Longitudinal Study (https://www. understandingsociety.ac.uk/), accessed through the European Genotype-phenome Archive. Samples were genotyped at the Wellcome Trust Sanger Institute using the Illumina Inﬁnium Cor- eExome genotyping array. The quality control procedures applied to genotyping of control samples were consistent with those described below for patient samples. Association testing and meta-analysis. Case–control association analyses were performed with the SNPTEST software package (version 2.5.2) using their scoring method to account for imputation uncertainty. Meta-analyses were conducted using an inverse variance meta-analysis assuming ﬁxed effects with version Genotyping and statistical quality control. PsA sam- ples were genotyped using the Illumina Inﬁnium CoreExome 1538 SOOMRO ET AL and the Supplementary Materials and Methods for further details, available at http://onlinelibrary.wiley.com/doi/10.1002/art. 42154). 2.2.2 of the software package Genome-Wide Association Meta- Analysis (GWAMA) (18). Lambda genomic control (λgc) inﬂation fac- tor, corrected for sample size (λgc1000), was calculated to test for inﬂation of test statistics attributable to population stratiﬁcation, and potential inﬂation of test statistics from other sources. An over- view of these analyses is available in Supplementary Figure 3 and further details are provided in the Supplementary Materials and Methods (association testing and meta-analysis) (available at http://onlinelibrary.wiley.com/doi/10.1002/art.42154). Model development and validation. We developed a PsA prediction model using a set of 4,729,872 SNPs with a mini- mum imputation accuracy score of ≥0.9 and call rate of ≥0.99 in both the PsA-BSTOP GWAS and the UK Biobank GWAS ICD-10 data sets in which the PsA-BSTOP data set was used as the training data set and the UK Biobank ICD-10 data set was used for external validation (see Supplementary Figure 4B, available at http://onlinelibrary.wiley.com/doi/10.1002/art.42154). We utilized a lasso-penalized linear regression model using all post–quality control imputed SNPs where the penalty (L1) was tuned with 10 repetitions of 10-fold cross-validation implemented in the SparSNP software package (20). PATIENTS AND METHODS The best model was selected based on the maximal area under the curve (AUC) and classiﬁca- tion and calibration were evaluated in the validation data set. Heritability estimates. Heritability of PsA and PsC was estimated in the PsA-BSTOP GWAS data set using genome-wide complex trait analysis (GCTA software). SNPs were stratiﬁed into quartiles based on levels of linkage disequilibrium, and then fur- ther stratiﬁed into bins according to MAF values (see Supplemen- tary Materials and Methods). Calculations were performed with no prevalence speciﬁed and with a speciﬁed disease prevalence of 1% for comparison with previously reported estimates (7). Both calculations were repeated with SNPs excluded from the major histocompatibility complex (MHC). Data availability. Summary statistics of the GWAS ana- lyzed in the current study are available through the National Human Genome Research Institute-European Bioinformatics Institute GWAS Catalog at https://www.ebi.ac.uk/gwas/ downloads/summary-statistics. Control population data were obtained from the UK Household Longitudinal Study. Information on how to access the data can be found on the Understanding Society website at https://www.understandingsociety.ac.uk/. Gene and pathway prioritization. We prioritized key genes and pathways for psoriatic disease using the priority index (Pi) pipeline (19). Genes were prioritized based on the following criteria: 1) proximity of SNPs to genes and localization to their topologically associated domain (cell line GM12878); 2) physical interaction determined by chromatin conformation (monocytes, macrophages [M0, M1, M2], neutrophils, CD4 T cells [naive and total], CD8 T cells [naive and total], or B cells [naive and total]); 3) correlation with gene expression (monocytes [unstimulated, lipo- polysaccharide (LPS)–stimulated for 2 hours and 24 hours, interferon-γ (IFNγ)–stimulated for 24 hours], B cells, peripheral whole blood, CD4 T cells, CD8 T cells, neutrophils, or natural killer cells). Further scoring was based on gene ontologies for immune function, immune phenotype, and rare genetic immune diseases according to the OMIM. Enrichment in pathways was based on Reactome pathways. RESULTS Heritability estimates. We calculated the SNP-based heritability (h2 SNP) of PsA in the PsA-BSTOP GWAS data set of 3,609 patients and 9,192 controls. The estimated heritability of PsA in the full data set was h2 SNP = 0.63 (SD 0.04), while in anal- yses using non-MHC SNPs, the estimated heritability was h2 SNP = 0.61 (SD 0.04). In analyses in which the disease prevalence was speciﬁed to be 1% (in comparison to previous prevalence estimates [7]), the estimated heritability of PsA was h2 SNP = 0.43 (SD 0.03), while the heritability of PsA in analyses using non-MHC SNPs was h2 SNP = 0.41 (SD 0.03). Reproducing existing pipelines. A recent publication reported the performance of an analysis pipeline based on multi- ple machine learning approaches for the classiﬁcation of PsA in patients with psoriasis, referred to hereafter as the Michigan clas- siﬁcation pipeline (13). Based on the author recommendations for reproducing this pipeline, we trained 2 of the reported best per- forming machine learning algorithms (random forest and condi- tional inference forest) in the PsA-BSTOP GWAS data set to capture the cohort-speciﬁc parameters using the reported model parameters and the sets of genetics features (see Supplementary Table 2, available at http://onlinelibrary.wiley.com/doi/10.1002/ art.42154). The models were internally validated using k-fold cross-validation and were trained using the Machine Learning in R (MLR) package (see Supplementary Figure 4A for an overview The heritability of PsC in a population of 2,085 patients and 9,192 controls was estimated to be h2 SNP = 0.61 (SD 0.05), while in analyses using non-MHC SNPs, the estimated heritability of PsC was h2 SNP = 0.59 (SD 0.05). With a disease prevalence of 1%, the estimated heritability of PsC was found to be h2 SNP = 0.56 (SD 0.04), while the heritability of PsC in analyses using non-MHC SNPs was h2 SNP = 0.54 (SD 0.05). Association testing and meta-analysis. We performed a meta-analysis of GWAS summary statistics from a total of 5,065 PsA patients and 21,286 controls for a maximum of COMPARATIVE GENETIC ANALYSIS OF PsA AND PSORIASIS 1539 Table 1. Non-MHC loci with genome-wide signiﬁcance in the development of PsA identiﬁed through a meta-analysis of GWAS summary statis- tics from PsA patients and controls* Table 1. RESULTS Non-MHC loci with genome-wide signiﬁcance in the development of PsA identiﬁed through a meta-analysis of GWAS summary statis- tics from PsA patients and controls* SNP Chromosome Base position Notable genes Risk/non-risk allele RAF P OR (95% CI) P by Cochran’s Q test I2 rs33980500 6 111913262 TRAF3IP2 T/C 0.07 1.14 × 10−36 1.66 (1.54–1.8) 0.48 0 rs62377586 5 158766022 IL12B G/A 0.67 8.17 × 10−35 1.36 (1.3–1.43) 0.52 0 rs2111485 2 163110536 IFIH1 G/A 0.61 1.24 × 10−20 1.25 (1.19–1.31) 0.80 0 rs12044149 1 67600686 IL23R T/G 0.26 3.84 × 10−20 1.27 (1.2–1.33) 0.27 0.23 rs76956521 5 150464641 TNIP1 C/A 0.05 2.65 × 10−16 1.49 (1.36–1.64) 0.82 0 rs848 5 131996500 IL13 C/A 0.82 9.49 × 10−16 1.28 (1.21–1.36) 0.65 0 rs34536443 19 10463118 TYK2 G/C 0.95 1.16 × 10−14 1.71 (1.49–1.96) 0.70 0 rs17622208 5 131717050 SLC22A5 A/G 0.48 5.73 × 10−14 1.19 (1.14–1.24) 0.12 0.53 rs2020854 12 56743367 STAT2 T/C 0.93 1.26 × 10−13 1.43 (1.3–1.57) 0.01 0.78 rs3794767 17 26124605 NOS2 C/T 0.64 4.73 × 10−13 1.19 (1.14–1.25) 0.83 0 rs13203885 6 111995127 FYN C/T 0.12 1.55 × 10−11 1.26 (1.18–1.35) 0.74 0 rs1395621 1 25270572 RUNX3 C/T 0.48 6.48 × 10−11 1.17 (1.12–1.23) 0.65 0 rs5754467† 22 21985094 CCDC116 G/A 0.19 1.61 × 10−9 1.19 (1.13–1.27) 0.85 0 rs610604 6 138199417 TNFAIP3 G/T 0.32 7.76 × 10−9 1.15 (1.1–1.21) 0.14 0.50 * Inconsistency metrics (I2) describing the percentage of variation across studies due to heterogeneity were assessed for signiﬁcance by Cochran’s Q heterogeneity test. The threshold for genome-wide signiﬁcance was P = 5 × 10−8. MHC = major histocompatibility complex; GWAS = genome-wide association study; SNP = single-nucleotide polymorphism; RAF = risk allele frequency; OR = odds ratio; 95% CI = 95% con- ﬁdence interval. † Novel locus not previously identiﬁed as signiﬁcant in the development of psoriatic arthritis (PsA). nome-wide signiﬁcance in the development of PsA identiﬁed through a meta-analysis of GWAS summary statis- ls* * Inconsistency metrics (I2) describing the percentage of variation across studies due to heterogeneity were assessed for signiﬁcance by Cochran’s Q heterogeneity test. The threshold for genome-wide signiﬁcance was P = 5 × 10−8. MHC = major histocompatibility complex; GWAS = genome-wide association study; SNP = single-nucleotide polymorphism; RAF = risk allele frequency; OR = odds ratio; 95% CI = 95% con- ﬁdence interval. † Novel locus not previously identiﬁed as signiﬁcant in the development of psoriatic arthritis (PsA). RESULTS 22q11 in close proximity to the gene UBE2L3. We also found that the 2 previously reported PsA-speciﬁc susceptibility loci PTPN22 (rs2476601; P = 6.03 × 10−7) and chr5q31 (rs715285; P = 2.86 × 10−11) had genome-wide signiﬁcance for the develop- ment of PsA. 8,558,403 SNPs using data from the PsA-BSTOP, the UK Biobank, and the PsA Immunochip data sets (see Supplementary Figure 3A, available at http://onlinelibrary.wiley.com/doi/10.1002/ art.42154). The genomic control inﬂation factor λgc (λgc1000) for the PsA-BSTOP GWAS data set was estimated to be 1.1 (1.01), indi- cating minimal residual population stratiﬁcation based on inﬂation of test statistics. We identiﬁed 16 non-MHC loci with genome- wide signiﬁcance for the development of PsA (P = 5 × 10−8), 15 of which have previously been reported as signiﬁcant in PsA and one which is novel (Table 1 and Figure 1). This novel genome-wide association represents an association with the intergenic SNP rs5754467 (P = 1.61 × 10−9) on chromosome Next, we performed a meta-analysis of summary statistics for the comparison of PsA to PsC (PsA-BSTOP and UK Biobank data) to identify PsA-speciﬁc susceptibility loci using a population consisting of 4,340 PsA patients and 6,431 PsC patients (see Supplementary Figure 3B, available at http://onlinelibrary.wiley. com/doi/10.1002/art.42154). We identiﬁed signiﬁcant genome- wide association in 2 loci previously reported to be associated Figure 1. Manhattan plots showing the P values of genome-wide signiﬁcance from the meta-analysis of summary statistics obtained from psoriatic arthritis (PsA) patients compared to population controls (top), and PsA patients compared to cutaneous-only psoriasis (PsC) patients (bottom). The genome-wide signiﬁcance threshold was set at P = 5 × 10−8 and is indicated by the dashed lines. Each dot represents a single- nucleotide polymorphism (SNP). Red dots indicate the most signiﬁcant SNPs in both data sets. Figure 1. Manhattan plots showing the P values of genome-wide signiﬁcance from the meta-analysis of summary statistics obtained from psoriatic arthritis (PsA) patients compared to population controls (top), and PsA patients compared to cutaneous-only psoriasis (PsC) patients (bottom). The genome-wide signiﬁcance threshold was set at P = 5 × 10−8 and is indicated by the dashed lines. Each dot represents a single- nucleotide polymorphism (SNP). Red dots indicate the most signiﬁcant SNPs in both data sets. 1540 SOOMRO ET AL 1540 Table 2. RESULTS The random forest model was also found to be poorly calibrated, with a calibration-in-the-large (CITL) score of 0.27 (95% CI –0.13, 0.69) in internal validation and a noticeably worse CITL score of 1.2 (95% CI 0.92–1.51) in external validation. data set (PsA-BSTOP) and the validation data set (UK Biobank ICD-10), with C statistics of <0.6 by external validation (Figure 2). Each model was characterized by high sensitivity but low speciﬁc- ity, indicating a high rate of false positives (see Supplementary Table 7, available at http://onlinelibrary.wiley.com/doi/10.1002/ art.42154). In addition, calibration and clinical utility were found to be poor for both the random forest model and the conditional inference forest model (see Supplementary Figures 5 and 6). The best performing model based on accuracy of discrimination in the validation data set was the random forest model, where the C statistic was found to be 0.61 (95% conﬁdence interval [95% CI] 0.56–0.76) in internal validation, but which dropped consider- ably to 0.57 (95% CI 0.54–0.61) in external validation. The overall performance of the random forest model as measured by the Brier score was similar for internal and external validation, with Brier scores of 0.22 and 0.30, respectively, suggesting poor agreement in both data sets. The random forest model was also found to be poorly calibrated, with a calibration-in-the-large (CITL) score of 0.27 (95% CI –0.13, 0.69) in internal validation and a noticeably worse CITL score of 1.2 (95% CI 0.92–1.51) in external validation. with the development of PsA, namely the MHC region (rs1050414; P = 8.49 × 10−59) and the IL23R gene (rs72676069; P = 9.94 × 10−9). No other regions reached genome-wide signiﬁcance. However, 4 loci demonstrated evi- dence of signiﬁcant association in both data sets, with an overall P value in the meta-analysis of P < 5 × 10−6 (Table 2), giving us conﬁdence in the existence of additional PsA-speciﬁc loci. Gene and pathway prioritization. We utilized the recently described Pi bioinformatics pipeline to identify key genes and pathways in the development of PsA (19). Using summary statistics from the meta-analyses described above for PsA patients versus controls, we found that the most highly ranked gene with regard to PsA susceptibility based on the Pi was ICAM1, which has a role in epithelial cell adhesion (see Supple- mentary Table 3, available at http://onlinelibrary.wiley.com/doi/ 10.1002/art.42154). In addition, several genes involved in IFN regulation were highly ranked (IRF1, IRF5 and IRF7). RESULTS Loci showing the most signiﬁcant association with PsA or PsC from the PsA-STOP, UK Biobank, and meta-analysis data sets* SNPs rs17194140 rs11665266 rs76800961 rs306281 Chromosome 3 18 14 7 Base position 2198673 10441470 85656555 154785362 Notable genes CNTN4 None None PAXIP1 Risk/non‐risk allele T/C A/G A/C G/A P for association, PsA‐BSTOP data set 2.75 × 10–5 0.00304 3.33 × 10–5 1.81 × 10–4 P for association, UK Biobank data set 2.62 × 10–3 6.35 × 10–5 2.30 × 10–2 6.92 × 10–3 P for association, meta‐analysis data set 2.51 × 10–7 1.96 × 10–6 2.61 × 10–6 3.97 × 10–6 OR (95% CI) 1.2 (1.12–1.29) 1.34 (1.19–1.51) 1.39 (1.21–1.59) 1.17 (1.09–1.24) P by Cochran’s Q test 0.97 0.15 0.60 0.95 I2 0.00 0.53 0.00 0.00 * The overall P value for the meta-analysis was P = 5 × 10−6. Inconsistency metrics (I2) describing the percentage of variation across studies due to heterogeneity were assessed for signiﬁcance by Cochran’s Q heterogeneity test. See Table 1 for deﬁnitions. Table 2. Loci showing the most signiﬁcant association with PsA or PsC from the PsA-STOP, UK Biobank, an * The overall P value for the meta-analysis was P = 5 × 10−6. Inconsistency metrics (I2) describing the percentage of variation across studies due to heterogeneity were assessed for signiﬁcance by Cochran’s Q heterogeneity test. See Table 1 for deﬁnitions. data set (PsA-BSTOP) and the validation data set (UK Biobank ICD-10), with C statistics of <0.6 by external validation (Figure 2). Each model was characterized by high sensitivity but low speciﬁc- ity, indicating a high rate of false positives (see Supplementary Table 7, available at http://onlinelibrary.wiley.com/doi/10.1002/ art.42154). In addition, calibration and clinical utility were found to be poor for both the random forest model and the conditional inference forest model (see Supplementary Figures 5 and 6). The best performing model based on accuracy of discrimination in the validation data set was the random forest model, where the C statistic was found to be 0.61 (95% conﬁdence interval [95% CI] 0.56–0.76) in internal validation, but which dropped consider- ably to 0.57 (95% CI 0.54–0.61) in external validation. The overall performance of the random forest model as measured by the Brier score was similar for internal and external validation, with Brier scores of 0.22 and 0.30, respectively, suggesting poor agreement in both data sets. RESULTS Both the RF and CF models showed modest performance across the PsA-Biomarkers of Systemic Treatment Outcomes in Psoriasis (PsA-BSTOP) study data set (A) and the UK Biobank Inter- national Statistical Classiﬁcation of Diseases and Related Health Prob- lems, Tenth Revision data set (B). The Concordance statistic for each model was <0.6 by external validation. supporting the notion that psoriasis in patients with PsA is genet- ically and biologically similar to psoriasis in patients with PsC. Interestingly, these pathways for PsA development were reported previously in a study applying the Pi pipeline to a set of 59 SNPs associated with PsA (21). However, of greatest interest is the pri- oritized target pathways that differ between PsA and PsC, which provide insight into the PsA-speciﬁc processes whereby we ﬁnd enrichment in multiple NF-κB signaling annotations and the Wnt signaling pathway. Figure 2. Receiver operating characteristic (ROC) curves showing the sensitivity and speciﬁcity of the random forest (RF) and the condi- tional inference forest (CF) machine learning algorithms in discriminat- ing between psoriatic arthritis (PsA) and cutaneous-only psoriasis (PsC). Both the RF and CF models showed modest performance across the PsA-Biomarkers of Systemic Treatment Outcomes in Psoriasis (PsA-BSTOP) study data set (A) and the UK Biobank Inter- national Statistical Classiﬁcation of Diseases and Related Health Prob- lems, Tenth Revision data set (B). The Concordance statistic for each model was <0.6 by external validation. Figure 2. Receiver operating characteristic (ROC) curves showing the sensitivity and speciﬁcity of the random forest (RF) and the condi- tional inference forest (CF) machine learning algorithms in discriminat- ing between psoriatic arthritis (PsA) and cutaneous-only psoriasis (PsC). Both the RF and CF models showed modest performance across the PsA-Biomarkers of Systemic Treatment Outcomes in Psoriasis (PsA-BSTOP) study data set (A) and the UK Biobank Inter- national Statistical Classiﬁcation of Diseases and Related Health Prob- lems, Tenth Revision data set (B). The Concordance statistic for each model was <0.6 by external validation. Figure 2. Receiver operating characteristic (ROC) curves showing The Wnt signaling pathway plays a key role in bone formation in normal development and in abnormal bone formation in dis- eases such as axial spondyloarthritis and osteoarthritis. The Wnt signaling pathway may also be of particular interest in the setting of PsA, where bone formation in peripheral joints is included in the CASPAR criteria for the classiﬁcation of PsA. RESULTS Other highly ranked genes included UBA52, CNPY2, STAT2, and TYK2. Using the top 1% of ranked genes, we found signiﬁcant enrichment in IFN and interleukin signaling pathways (see Supplementary Table 4, available at http://onlinelibrary.wiley.com/doi/10.1002/ art.42154). These pathways were not found to be enriched when using summary statistics from PsA patients compared to those from PsC patients, suggesting that these pathways are primarily involved in the pathogenesis of psoriasis (see Supplementary Tables 5 and 6, available at http://onlinelibrary.wiley.com/doi/ 10.1002/art.42154). Pathways found to be enriched in the com- parison of PsA to PsC included multiple pathways for NF-κB sig- naling (adjusted P = 1.4 × 10−45) and Wnt signaling (adjusted P = 9.5 × 10−58), which provides compelling evidence that these pathways are potentially involved in the development of PsA. Finally, we used the PsA-BSTOP GWAS data set to develop a PsA risk prediction model using a set of 4,729,872 SNPs and lasso-penalized linear regression (see Supplementary Figure 4B, available at http://onlinelibrary.wiley.com/doi/10.1002/art. 42154). The best model achieved an AUC of 0.66 when assessed using 10-fold cross-validation and consisted of 118 SNPs, 34 of which mapped to the MHC (see Supplementary Figure 7). The SNP weights, P values, and model intercept are reported in Sup- plementary Table 8, available at http://onlinelibrary.wiley.com/doi/ 10.1002/art.42154. Independent validation of this model in the UK Biobank GWAS data set demonstrated an AUC of 0.57. The optimal prediction cutoff value to maximize the true-positive rate and minimize the false-positive rate was 0.3, which resulted in a sensitivity of 0.53 and a speciﬁcity of 0.58. Calibration of this model was found to be poor, with a CITL score of –2.16 (95% CI –2.31, –2.01) (see Supplementary Figure 8), suggesting a general overestimation of risk, and a calibration slope of 1.41 Risk prediction. We assessed the ability of the Michigan classiﬁcation pipeline to discriminate PsA from PsC in our avail- able data sets (see Supplementary Figure 4A). The 2 reported sta- tistical approaches (the random forest model and the conditional inference forest model) performed poorly across both the training COMPARATIVE GENETIC ANALYSIS OF PsA AND PSORIASIS 1541 Figure 2. Receiver operating characteristic (ROC) curves showing the sensitivity and speciﬁcity of the random forest (RF) and the condi- tional inference forest (CF) machine learning algorithms in discriminat- ing between psoriatic arthritis (PsA) and cutaneous-only psoriasis (PsC). DISCUSSION Using a large integrated data set of PsA patients, PsC patients, and controls, we have been able to provide accurate heritability estimates, identify a novel susceptibility locus, explore key biologic pathways associated with the development of PsA, and explore the utility of prediction models for classifying PsA risk. While the individual SNP analysis showed large overlap between PsC and PsA, pathway analysis revealed important differences, including enrichment of PsA-signiﬁcant SNPs found in key path- ways such as NF-κB and Wnt signaling. A previous study by Aterido et al investigated pathways associated with PsA susceptibility and reported signiﬁcant asso- ciation with the glycosaminoglycan metabolism pathway (Reactome R-HSA-1630316) (25). However, no association between PsA susceptibility and the glycosaminoglycan metabo- lism pathway was observed in our data, as none of the highly pri- oritized genes overlapped with genes in this pathway annotation. These differing results could be attributed to differences in the methods used for mapping SNPs to genes, as in the study by Aterido et al SNPs were assigned to genes based solely on prox- imity. It is now well recognized that causal genes are not always those that are closest to the GWAS hit, and the causal SNP may exert its regulatory effect on distant genes. The Pi pipeline addresses this limitation by including gene expression data and chromatin conﬁrmation data in order to capture evidence for SNP–gene physical interactions in addition to proximity informa- tion (19). Aterido et al also reported that the SNP rs10865331 at the B3GNT2 locus was associated with the risk of developing PsA but not PsC (P = 0.029). While we found signiﬁcant associa- tion with this SNP when comparing PsA patients to controls (P = 2.05 × 10−7), we found no evidence that this association is PsA-speciﬁc based on our stratiﬁed analyses comparing PsA to PsC using PsA-BSTOP data (P = 0.41) or on the larger meta- analysis using UK Biobank and Immunochip data (P = 0.31). The SNP-based heritability estimates reported herein sup- port recent ﬁndings by Li et al (7) and show comparable heritability of PsA and PsC. Our results do not support the previous family and population estimates that suggest a substantially larger heri- table component for PsA above that of psoriasis alone (4–6). Sev- enteen genome-wide associations were identiﬁed, including rs5754467 (P = 1.61 × 10−9) which maps to chromosome 22q11 and is near the genes UBE2L3, YDJC, and CCDC116. RESULTS Blocking of Dkk-1 (an inhibitor of Wnt signaling) in mice has been shown to halt progressive and erosive joint destruction in inﬂammatory arthritis by encouraging bone formation (22). Interestingly, a previ- ous study on PsA demonstrated that PsA patients had lower levels of Dkk-1 compared to healthy controls, and treatment with secukinumab increased these levels over a period of 6 months to normal Dkk-1 serum levels (23). In contrast, another study reported no signiﬁcant difference in levels of Dkk-1 in patients with PsA without radiographic axial disease compared to healthy con- trols (24). Therefore, further work is required to understand the role of Wnt signaling in PsA. (95% CI 0.95–1.86), suggesting that the predictions were too moderate and showing limited variation in the predicted probabilities. DISCUSSION This SNP has not been previously reported in the setting of PsA, but is highly correlated (r2 >0.8 based on SNP data from a north- ern European population) with a previously identiﬁed psoriasis SNP. This correlation does not represent a PsA-speciﬁc genetic effect (12), but further supports the genetic similarity of psoriasis in both patients with PsA and patients with PsC. We used the Pi pipeline to identify key genes and pathways involved in PsA susceptibility. In analyses of PsA patients com- pared to controls, we replicated the previously reported ﬁndings of prioritized genes (ICAM1, IRF1, STAT2, and TYK2) and target pathways (IFN and interleukin signaling) for psoriasis, further Our prediction models showed only modest ability to dis- criminate PsA from PsC in the available data sets, which was 1542 SOOMRO ET AL consistent with the ﬁndings of a recently published study by Smith et al (26). For the ﬁrst approach, we attempted to reproduce a previously published classiﬁcation pipeline by following the description and published model parameters, which allowed us to reproduce the workﬂows (13). Following the author recommen- dations, we used the 2 sets of genetic features that were selected using a well-phenotyped cohort and estimated the model param- eters in our data to capture cohort-speciﬁc effects and optimize performance. Second, we attempted to develop a model for our existing data sets through external validation. However, neither of these approaches achieved satisfactory discrimination either with internal or external validation. Given that the predictive perfor- mance of a model using the same data on which it was developed (often referred to as apparent performance) will tend to give an optimistic estimate of the model’s performance, it is not uncom- mon for a prediction model to achieve lower performance results when applied to an external population. as external validation tests the transportability of models to plausi- bly related populations (29). While polygenic risk scores capture the heritable component of disease susceptibility, they fail to cap- ture the more dynamic risk factors that can modulate susceptibil- ity, such as environmental and lifestyle risk factors. In addition, studies have shown that genetic risk factors can be independent of known clinical risk factors (30). ACKNOWLEDGMENTS The lack of discrimination observed in our data sets could be due to the differences in demographic and clinical characteristics of participants in our data sets compared to those of the partici- pants in the original study. PsA is clinically a heterogeneous dis- ease and differing proportions of patients with oligoarticular or polyarticular arthritis mutilans and axial disease (each with poten- tially differing genetic risk factors) could have contributed to the decreased performance of the model. This potential issue was recognized by the authors of the original study and, although we followed the author recommendations by modeling the effects of these markers in our data to learn these cohort-speciﬁc parame- ters, the overall classiﬁcation performance remained low (13). We are grateful for the assistance given by The University of Man- chester IT Services and for the use of the Computational Shared Facility. We thank all of the patient participants and acknowledge the enthusias- tic collaboration of all clinicians and research teams in the UK and the Republic of Ireland who recruited for this study. We gratefully acknowl- edge the substantial contribution to administration of this project by the following members of the Data Monitoring Committee (DMC) of the BADBIR Study Group: Dr. Robert Chalmers, Dr. Carsten Flohr (Chair), Dr. Karen Watson, and David Prieto-Merino. We also thank the following members of the BADBIR Steering Committee: Oras Alabas, Professor Jonathan Barker, Gabrielle Becher, Anthony Bewley, David Burden, Simon Morrison, Professor Phil Laws (Chair), Mr. Ian Evans, Professor Christopher Grifﬁths, Shehnaz Ahmed, Dr. Brian Kirby, Elise Kleyn, Ms. Linda Lawson, Teena Mackenzie, Tess McPherson, Dr. Kathleen McElhone, Dr. Ruth Murphy, Professor Anthony Ormerod, Dr. Caroline Owen, Professor Nick Reynolds, Amir Rashid, Professor Catherine Smith, and Dr. Richard Warren. We are grateful to the following members of the BSTOP Steering Committee for their valuable role in the oversight of the study delivery: Professor David Burden (Chair), Professor Cather- ine Smith, Professor Stefan Siebert, Professor Sara Brown, Helen McA- teer, Dr. Julia Schoﬁeld, and Dr. Nick Dand. Finally, we acknowledge the enthusiastic collaboration of all the dermatologists and specialist nurses in the UK and the Republic of Ireland who provided the BADBIR and BSTOP data. The principal investigators at the participating sites can be found at the following website: http://www.badbir.org/ Clinicians/. Open access funding was enabled and organized by Projekt DEAL. ACKNOWLEDGMENTS An important limitation of our study was the potential impact of poor phenotype speciﬁcity in the PsC cohorts where the exis- tence of undiagnosed PsA could have confounded the perfor- mance of any classiﬁcation models. Although the participants in the BSTOP study were not screened for the absence of PsA by a rheumatologist, they were routinely followed-up with an average of 8 consultations and had a psoriasis disease duration of 27 years without a recorded diagnosis of PsA. Additionally, restricting analyses to a subgroup of PsC patients with psoriasis for a duration of ≥10 (to minimize the risk of undiagnosed PsA) did not improve the performance of the predictive model (data not shown). However, given the extent of undiagnosed PsA in dermatology clinics, we cannot exclude the possibility of undiag- nosed PsA in this group, which would have impacted both feature selection and model performance (27). Furthermore, given that patients in the PsC group were treated with biologic drugs that are also effective in the treatment of PsA, it is possible that PsA development was prevented in susceptible individuals, thus limit- ing the power of the models to discriminate between groups (28). DISCUSSION This suggests that future research on PsA susceptibility in patients with psoriasis should move toward combining clinical data and genetics from data col- lected longitudinally, using a prospective study design in patients with clinically well-deﬁned PsC before treatment with biologic drugs, to create an integrated risk score. Therefore, these future efforts should also investigate the integration of more dynamic biomarkers, such as the host microbiome and immunophenotyp- ing, into the development of PsA risk prediction models. AUTHOR CONTRIBUTIONS All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the ﬁnal ver- sion to be published. Dr. Barton had full access to all of the data in the study and takes responsibility for the integrity of the data and the accu- racy of the data analysis. Study conception and design. Soomro, Stadler, Dand, Barker, Smith, Bowes, Barton. Acquisition of data. Dand, Jadon, Duckworth, Ho, Marzo-Ortega, Helliwell, Ryan, Kane, Korendowych, Simpson, Packham, McManus, Gabay, Lamacchia, Nissen, Brown, Verstappen, Barker, Smith, FitzGer- ald, McHugh, Warren, Bowes, Barton. Analysis and interpretation of data. Soomro, Stadler, Dand, Bluett, Jalali-najafabadi, Duckworth, Van Staa, Barker, Smith, Bowes, Barton. REFERENCES 17. Bowes J, Budu-Aggrey A, Huffmeier U, Uebe S, Steel K, Hebert HL, et al. Dense genotyping of immune-related susceptibility loci reveals new insights into the genetics of psoriatic arthritis. Nat Commun 2015;6:6046. 1. Ritchlin CT, Colbert RA, Gladman DD. Psoriatic Arthritis. N Engl J Med 2017;376:957–70. 2. Ibrahim G, Waxman R, Helliwell PS. The prevalence of psoriatic arthritis in people with psoriasis. Arthritis Rheum 2009;61:1373–8. 18. Magi R, Morris AP. GWAMA: software for genome-wide association meta-analysis. BMC Bioinformatics 2010;11:288. 3. Alinaghi F, Calov M, Kristensen LE, Gladman DD, Coates LC, Jullien D, et al. Prevalence of psoriatic arthritis in patients with psoria- sis: a systematic review and meta-analysis of observational and clinical studies. J Am Acad Dermatol 2019;80:251–65. 19. Fang H, De Wolf H, Knezevic B, Burnham KL, Osgood J, Sanniti A, et al. A genetics-led approach deﬁnes the drug target landscape of 30 immune-related traits. Nat Genet 2019;51:1082–91. 4. Chandran V, Schentag CT, Brockbank JE, Pellett FJ, Shanmugarajah S, Toloza SM, et al. Familial aggregation of psoriatic arthritis. Ann Rheum Dis 2009;68:664–7. 20. Abraham G, Kowalczyk A, Zobel J, Inouye M. SparSNP: fast and memory-efﬁcient analysis of all SNPs for phenotype prediction. BMC Bioinformatics 2012;13:88. 5. Moll JM, Wright V. Familial occurrence of psoriatic arthritis. Ann Rheum Dis 1973;32:181–201. 21. Bui A, Liu J, Hong J, Hadeler E, Mosca M, Brownstone N, et al. Iden- tifying novel psoriatic disease drug targets using a genetics-based pri- ority index pipeline. J Psoriasis Psoriatic Arthritis 2021;6:185–97. 6. Brandrup F, Holm N, Grunnet N, Henningsen K, Hansen HE. Psoriasis in monozygotic twins: variations in expression in individuals with identical genetic constitution. Acta Derm Venereol 1982;62:229–36. 22. Diarra D, Stolina M, Polzer K, Zwerina J, Ominsky MS, Dwyer D, et al. Dickkopf-1 is a master regulator of joint remodeling. Nat Med 2007; 13:156–63. 7. Li Q, Chandran V, Tsoi L, O’Rielly D, Nair RP, Gladman D, et al. Quan- tifying differences in heritability among psoriatic arthritis (PsA), cutane- ous psoriasis (PsC) and psoriasis vulgaris (PsV). Sci Rep 2020;10: 4925. 23. Fassio A, Gatti D, Rossini M, Idolazzi L, Giollo A, Adami G, et al. Secu- kinumab produces a quick increase in WNT signalling antagonists in patients with psoriatic arthritis. Clin Exp Rheumatol 2019;37:133–6. 8. Bowes J, Budu-Aggrey A, Huffmeier U, Uebe S, Steel K, Hebert HL, et al. Dense genotyping of immune-related susceptibility loci reveals new insights into the genetics of psoriatic arthritis. REFERENCES Nat Commun 2015;6:6046. 24. Jadon DR, Sengupta R, Nightingale A, Lu H, Dunphy J, Green A, et al. Serum bone-turnover biomarkers are associated with the occurrence of peripheral and axial arthritis in psoriatic disease: a prospective cross-sectional comparative study. Arthritis Res Ther 2017;19:210. 9. Bowes J, Ashcroft J, Dand N, Jalali-Najafabadi F, Bellou E, Ho P, et al. Cross-phenotype association mapping of the MHC identiﬁes genetic variants that differentiate psoriatic arthritis from psoriasis. Ann Rheum Dis 2017;76:1774–9. 25. Aterido A, Cañete JD, Tornero J, Ferrandiz C, Pinto JA, Gratacos J, et al. Genetic variation at the glycosaminoglycan metabolism pathway contributes to the risk of psoriatic arthritis but not psoriasis. Ann Rheum Dis 2019;78. 10. Budu-Aggrey A, Bowes J, Loehr S, Uebe S, Zervou MI, Helliwell P, et al. Replication of a distinct psoriatic arthritis risk variant at the IL23R locus. Ann Rheum Dis 2016;75:1417–8. 26. Smith MP, Ly K, Thibodeaux Q, Beck K, Yang E, Sanchez I, et al. Eval- uation of a genetic risk score for diagnosis of psoriatic arthritis. J Psoriasis Psoriatic Arthritis 2020;5:61–7. 11. Okada Y, Han B, Tsoi LC, Stuart PE, Ellinghaus E, Tejasvi T, et al. Fine mapping major histocompatibility complex associations in psoriasis and its clinical subtypes. Am J Hum Genet 2014;95:162–72. 27. Mease PJ, Gladman DD, Papp KA, Khraishi MM, Thaçi D, Behrens F, et al. Prevalence of rheumatologist-diagnosed psoriatic arthritis in patients with psoriasis in European/North American dermatology clinics. J Am Acad Dermatol 2013;69:729–35. 12. Tsoi LC, Spain SL, Knight J, Ellinghaus E, Stuart PE, Capon F, et al. Identiﬁcation of 15 new psoriasis susceptibility loci highlights the role of innate immunity. Nat Genet 2012;44:1341–8. 28. Rosenthal YS, Schwartz N, Sagy I, Pavlovsky L. Incidence of psoriatic arthritis among patients receiving biologic treatments for psoriasis: a nested case–control study. Arthritis Rheumatol 2022;74:237–43. 13. Patrick MT, Stuart PE, Raja K, Gudjonsson JE, Tejasvi T, Yang J, et al. Genetic signature to provide robust risk assessment of psoriatic arthritis development in psoriasis patients. Nat Commun 2018;9: 4178. 29. Steyerberg EW, Vergouwe Y. Towards better clinical prediction mod- els: seven steps for development and an ABCD for validation. Eur Heart J 2014;35:1925–31. 14. Taylor W, Gladman D, Helliwell P, Marchesoni A, Mease P, Mielants H, and the CASPAR Study Group. Classiﬁcation criteria for psoriatic arthritis: development of new criteria from a large international study. Arthritis Rheum 2006;54:2665–73. 30. Study conception and design. Soomro, Stadler, Dand, Barker, Smith, Bowes, Barton. Study conception and design. Soomro, Stadler, Dand, Barker, Smith, Bowes, Barton. Study conception and design. Soomro, Stadler, Dand, Barker, Smith, Bowes, Barton. Acquisition of data. Dand, Jadon, Duckworth, Ho, Marzo-Ortega, Helliwell, Ryan, Kane, Korendowych, Simpson, Packham, McManus, Gabay, Lamacchia, Nissen, Brown, Verstappen, Barker, Smith, FitzGer- ald, McHugh, Warren, Bowes, Barton. Acquisition of data. Dand, Jadon, Duckworth, Ho, Marzo-Ortega, Helliwell, Ryan, Kane, Korendowych, Simpson, Packham, McManus, Gabay, Lamacchia, Nissen, Brown, Verstappen, Barker, Smith, FitzGer- ald, McHugh, Warren, Bowes, Barton. In conclusion, predicting the risk of PsA development in patients with psoriasis remains an important research question, and external validation in addition to statistical validation is an important step in the clinical translation of PsA prediction models, Analysis and interpretation of data. Soomro, Stadler, Dand, Bluett, Jalali-najafabadi, Duckworth, Van Staa, Barker, Smith, Bowes, Barton. Analysis and interpretation of data. Soomro, Stadler, Dand, Bluett, Jalali-najafabadi, Duckworth, Van Staa, Barker, Smith, Bowes, Barton. COMPARATIVE GENETIC ANALYSIS OF PsA AND PSORIASIS 1543 ADDITIONAL DISCLOSURES 15. Dand N, Duckworth M, Baudry D, Russell A, Curtis CJ, Lee SH, et al. HLA-C*06:02 genotype is a predictive biomarker of biologic treat- ment response in psoriasis. J Allergy Clin Immunol 2019;143: 2120–30. Author Ryan is an employee of Genuity Science Dublin. Author Brown is an employee of Genomics England. 16. Bycroft C, Freeman C, Petkova D, Band G, Elliott LT, Sharp K, et al. The UK Biobank resource with deep phenotyping and genomic data. Nature 2018;562:203–9. REFERENCES Khera A V, Emdin CA, Drake I, Natarajan P, Bick AG, Cook NR, et al. Genetic risk, adherence to a healthy lifestyle, and coronary disease. New Engl J Med 2016;375:2349–58.
W2312257440.txt	https://europepmc.org/articles/pmc3928315?pdf=render	en		SYSTEMATIC REVIEW OF BIOMARKERS FOR DISEASE PROGRESSION IN ALZHEIMER'S DISEASE	Journal of neurology, neurosurgery and psychiatry	2,013	cc-by	7,178	A Systematic Review of Biomarkers for Disease Progression in Alzheimer’s Disease David J. M. McGhee1, Craig W. Ritchie2, Paul A. Thompson3, David E. Wright3, John P. Zajicek4, Carl E. Counsell1 1 Division of Applied Health Sciences, University of Aberdeen, Aberdeen, Scotland, United Kingdom, 2 Centre for Mental Health, Imperial College London, London, England, United Kingdom, 3 Centre for Health and Environmental Statistics, Plymouth University, Plymouth, England, United Kingdom, 4 Clinical Neurology Research Group, Plymouth University, Plymouth, England, United Kingdom Abstract Background: Using surrogate biomarkers for disease progression as endpoints in neuroprotective clinical trials may help differentiate symptomatic effects of potential neuroprotective agents from true slowing of the neurodegenerative process. A systematic review was undertaken to determine what biomarkers for disease progression in Alzheimer’s disease exist and how well they perform. Methods: MEDLINE and Embase (1950–2011) were searched using five search strategies. Abstracts were assessed to identify papers meriting review in full. Studies of participants with probable Alzheimer’s disease diagnosed by formal criteria were included. We made no restriction on age, disease duration, or drug treatment. We only included studies with a longitudinal design, in which the putative biomarker and clinical measure were both measured at least twice, as this is the only appropriate study design to use when developing a disease progression biomarker. We included studies which attempted to draw associations between the changes over time in the biomarker used to investigate disease progression and a clinical measure of disease progression. Results: Fifty-nine studies were finally included. The commonest biomarker modality examined was brain MRI (17/59, 29% of included studies). Median follow-up in included studies was only 1.0 (IQR 0.8–1.7) year and most studies only measured the putative biomarker and clinical measure twice. Included studies were generally of poor quality with small numbers of participants (median 31 (IQR 17 to 64)), applied excessively restrictive study entry criteria, had flawed methodologies and conducted overly simplistic statistical analyses without adjusting for confounding factors. Conclusions: We found insufficient evidence to recommend the use of any biomarker as an outcome measure for disease progression in Alzheimer’s disease trials. However, further investigation into the efficacy of using MRI measurements of ventricular volume and whole brain volume appeared to be merited. A provisional ‘roadmap’ to improve the quality of future disease progression biomarker studies is presented. Citation: McGhee DJM, Ritchie CW, Thompson PA, Wright DE, Zajicek JP, et al. (2014) A Systematic Review of Biomarkers for Disease Progression in Alzheimer’s Disease. PLoS ONE 9(2): e88854. doi:10.1371/journal.pone.0088854 Editor: Malú G. Tansey, Emory University, United States of America Received December 4, 2013; Accepted January 10, 2014; Published February 18, 2014 Copyright: ß 2014 McGhee et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This article presents independent research funded by the National Institute for Health Research (NIHR) under its Programme Grants for Applied Research Programme (Grant Reference Number RP-PG-0707-10124). The views expressed in this article are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health. The funders had no role in study design, data collection, data analysis, data interpretation, writing of the report or for the decision to submit for publication. Competing Interests: The authors have declared that no competing interests exist. E-mail: david.mcghee@abdn.ac.uk currently not possible to directly measure the number of remaining cortical neurons in vivo and, therefore, alternative approaches are required. Clinical assessments in Alzheimer’s disease using scales to measure cognitive impairment, disability, quality of life, or global disease severity are affected by symptomatic effects of therapy and are unable to differentiate this effect from diseasemodification, at least in the short-term. Various clinical trial designs have been developed to try to adjust for symptomatic effects of putative neurodegenerative agents and, therefore, allow clinical rating scales to be used as endpoints. These include long-term follow up studies of placebotreated and active-agent treated patients looking for sustained divergence, measuring outcomes following a wash-out period, and Introduction The rising prevalence of Alzheimer’s disease, the commonest neurodegenerative disorder, and the associated financial and social costs this brings, presents a major challenge to governments of countries with an ageing population [1]. Given that only symptomatic treatments for Alzheimer’s disease currently exist, much research has focused on the development of drugs which slow or even halt neurodegeneration and, therefore, clinical progression. However, clinical trials in neurodegenerative disorders have struggled to separate out symptomatic effects of potential therapeutic agents (e.g. due to increased synaptic acetylcholine) from true disease-modifying effects. In Alzheimer’s disease, it is PLOS ONE \| www.plosone.org 1 February 2014 \| Volume 9 \| Issue 2 \| e88854 Biomarkers for Disease Progression in AD Given the methodological and statistical weaknesses we identified in studies of biomarkers for disease progression in Parkinson’s disease (PD) in a previous systematic review [8], we aimed to determine whether the same problems were prevalent in Alzheimer’s disease research. We, therefore, aimed to critique data from identified disease progression biomarker studies relating to study design, participant characteristics, and statistical analyses undertaken, in order to produce guidelines for future studies. delayed start trial designs [2]. However, analytic and logistical problems with these trial designs have as yet restricted their use [3]. An alternative approach, the focus of much primary research, is the use of a surrogate outcome biomarker as an endpoint in neuroprotective clinical trials. Surrogate outcome biomarkers are objectively measured characteristics of a disease, which act as indicators of the underlying pathogenic process responsible for disease progression, including the change in that process following a therapeutic intervention [4,5]. To allow their use in clinical trials surrogate outcome biomarkers must have a strong association with a clinical endpoint or outcome known to measure the effect of a therapeutic intervention on disease progression, for which the biomarker can act as a substitute. Surrogate biomarkers for disease progression in Alzheimer’s disease could shorten the duration of phase III trials and thereby reduce the cost and time required to get a drug to market. Unfortunately at present there is not a single accepted surrogate outcome biomarker for any neurodegenerative disorder. Much has been written about the features that a biomarker for disease progression in neurodegenerative disorders, such as Alzheimer’s disease, should possess [6,7]. The ideal surrogate biomarker should: Methods Following the development of a review protocol, literature searches were conducted in the databases MEDLINE (1950 to November 2011) and Embase (1980 to November 2011), using the OVID search interface. Five separate search strategies, based on previous searches developed by an experienced information scientist, were run in each database. The first four were based on free-text words identified through background reading of relevant review articles. These searches included potential (1) blood, (2) urine or cerebrospinal fluid (CSF), (3) imaging and (4) neurophysiological biomarkers. A fifth search using generic terms for biomarkers based on index headings was also run in both databases. For details of the search strategy please see document S1. The searches were limited to human studies. Only English language articles were included, due to lack of resources for translation. Reference lists of included articles and relevant review articles were checked to identify any studies which the electronic search strategy may have missed. 1. Change with neurodegeneration (i.e. degeneration of cortical neurons); 2. Show an association with the clinical phenotype arising secondary to this degenerative process; 3. Have a direct association with disease progression, without intermediate variables; 4. Have robust longitudinal data linking it to disease progression; 5. Not be influenced by symptomatic treatment, but only by a true change in the neurodegenerative process; 6. Predict long-term changes in disease progression by short-term changes in the biomarker; 7. Be generalisable to people with differing characteristics (e.g. age, gender, race); 8. Be continually variable (ideally linearly for simplicity); 9. Be sensitive, reflecting small changes in disease progression; 10. Be quick and cheap to measure, and amenable to blinded assessment; 11. Be suitable for measurement reliably across different centres; 12. Be suitable for repeated measurement in the same patient; 13. Be safe and tolerable to the patient. Validation of the electronic search strategy The electronic search strategy was validated by hand searching five years of the two journals from which most of the included articles came: Neurology (1995–1999) and Archives of Neurology (2001–2005). The number of relevant and irrelevant articles identified by hand searching and by the electronic search, was used to calculate the sensitivity and specificity for the electronic search strategy. Study selection A single reviewer examined abstracts retrieved by the electronic search to identify articles meriting review in full. Full length articles were then reviewed before data were extracted from relevant papers. In both stages the inclusion and exclusion criteria detailed below were applied. Only studies of participants with probable Alzheimer’s disease diagnosed by formal criteria [9–13] were included. Studies which included participants with prodromal Alzheimer’s disease or mild cognitive impairment (MCI) were only included if progression to Alzheimer’s disease was confirmed in all participants by clinical follow-up. No restriction was made on the grounds of participant’s age, disease duration, or drug treatment. As emphasised in our previous systematic review of biomarkers for disease progression in PD [8], a cross-sectional study design, in which an association between a biomarker and a clinical measure of disease progression is examined at a single time point in a group of patients with differing disease severity, is not suitable to examine for a relationship between the change in a clinical measure and the change in a biomarker over time within individuals with a neurodegenerative disorder. We, therefore, limited this review to studies with a longitudinal design, where the biomarker and clinical measure were recorded at least twice. Studies which investigated the efficacy of using a biomarker, including (but not restricted to) imaging, blood tests, tests of CSF As Alzheimer’s disease is a complex neurodegenerative disorder in which many different pathophysiological processes have been implicated (e.g. tau and amyloid deposition, abnormalities of cholesterol metabolism, inflammation, oxidative damage and lysosomal dysfunction) it is not surprising that many different candidate biomarkers for disease progression in Alzheimer’s disease have been studied. However, the literature in this area has never been brought together systematically. We, therefore, aimed to undertake a systematic review to assess what potential surrogate biomarkers for disease progression in Alzheimer’s disease exist, whether any meet the criteria for use in clinical trials, and if not which looks most promising. We did not aim to review the literature for diagnostic biomarkers (i.e. those designed to aid early diagnosis in the pre-motor or motor phase) or prognostic biomarkers (i.e. those aimed at identifying patients who progress at different rates). PLOS ONE \| www.plosone.org 2 February 2014 \| Volume 9 \| Issue 2 \| e88854 Biomarkers for Disease Progression in AD Table 1. Quality criteria to assess studies examining surrogate biomarkers for disease progression[8]. Question Yes No (1) Was the primary aim of the study to validate a biomarker for disease progression? 32 54 (2) Did the study detail a scientifically valid reason for choosing the given biomarker for investigation? 59 100 (3) Has the reproducibility of measuring the biomarker in the same centre by different trained personnel, and between centres, been evaluated? 2 3 (4) Has an assessment of the effect of likely confounding factors (e.g. age, gender, smoking status, and being on symptomatic treatment) on the measurement of the biomarker been made? 1 2 (5) Has an assessment of the validity and reliability of the criterion (e.g. clinical rating scale) used been made? 54 92 (6a) Was a power calculation undertaken to determine the required number of participants? 3 5 (6b) If a power calculation was undertaken, was the number of participants included appropriate? 1 2 (7) Was the study longitudinal? 59 100 (8) Was the study prospective? 49 83 (9) Was there a sufficient period of follow-up? 26 44 (10) Were the biomarker and clinical measures of disease severity measured on $3 occasions? 7 12 (11) Was measurement of the biomarker blind to participant characteristics? 25 42 (12) Did$75% of participants entering the study complete the full follow-up period? 42 71 (13) Were cases unselected/unbiased (no exclusion criteria)? 16 27 (14) Were associations between the biomarker and clinical measures of disease severity examined for using appropriate statistical modelling (e.g. linear mixed modelling) with adjustment for confounding factors, rather than simply correlation analysis? 7 12 (15) Were results of statistical analyses reported in sufficient detail to allow the inclusion of the study results in a meta-analysis? 14 24 doi:10.1371/journal.pone.0088854.t001 and neurophysiological tests, to investigate disease progression in Alzheimer’s disease were included. To qualify for inclusion there must have been an attempt to assess an association between the change in a biomarker and the change in a clinical measure of disease progression over time. Acceptable clinical measures included measures of cognitive impairment, disability, handicap, quality of life, and global clinical assessments. Only studies exploring associations between a biomarker and the total score from a clinical rating scale, rather than its subsections, were included. The subsections of most clinical measures would not be acceptable outcome measures for neuroprotective trials and, therefore, developing surrogate biomarkers for them was not felt to be relevant. However, exceptions were made for the following clinical rating scale subsections, which may be acceptable outcome measures for disease-modification trials: Alzheimer’s disease assessment scale cognitive (ADAS-cog) and non-cognitive (ADAS-noncog) subsections [14]; Blessed dementia scale change in performance of everyday activities subsection (Blessed ADL) [15]; CAMCOG memory subsection [16]. Similarly, only studies examining for associations between putative biomarkers and global measures of cognition, rather than individual neuropsychological tests were included. Furthermore, studies solely examining for associations between biomarkers and measures of neuropsychiatric impairment were not included, as depression and behavioural disturbance are not clearly associated with disease progression in Alzheimer’s disease [17]. Studies examining the relationship between a biomarker and treatment status, the presence or severity of complications related to therapy, or duration of illness were excluded. We also excluded studies which examined for associations between symptomatic improvement, as measuring by clinical rating scales, and the change in the level or activity of cholinesterase enzymes in the blood or CSF following commencement of a cholinesterase inhibitor. As we wished to develop a biomarker for disease PLOS ONE \| www.plosone.org progression rather than a way of measuring the response to symptomatic therapy, these studies were not felt to be relevant. Data extraction Study methods and results were extracted by a single reviewer, and to check for accuracy this was performed twice. Data were extracted, using a data extraction sheet (document S2) relating to the following: (1) study design including restrictiveness of criteria for entry into the study; (2) setting; (3) study population, including number of participants, gender ratio, disease duration at baseline, baseline measures of disease severity and baseline treatment status; (4) specific biomarkers investigated; (5) statistical analyses performed; (6) results of statistical analyses of the associations between the biomarkers and clinical measures of disease severity; (7) analysis of the effect of drug treatment on the biomarker; (8) economic analysis of using the biomarker; (9) measures of suitability and acceptability of the test to patients. The restrictiveness of the inclusion and exclusion criteria applied to each study was graded as: none, explicit statement that only criteria to exclude other causes of dementia were applied; mild #3 criteria applied (except those described under moderate); moderate, 4–5 criteria applied or evidence of an attempt to limit by age, gender, cognitive state, drug therapy for Alzheimer’s disease (e.g. all de-novo); severe$6 criteria applied; not detailed, no mention of whether criteria were applied. Methodological quality No validated tool to measure the quality of studies investigating surrogate biomarkers as outcome measures exists. An attempt was, therefore, made to assess study quality using a quality questionnaire developed in our previous systematic review of biomarkers for disease progression in PD (table 1). 3 February 2014 \| Volume 9 \| Issue 2 \| e88854 Biomarkers for Disease Progression in AD Most articles did not provide information pertinent to question five (‘has an assessment of the validity and reliability of the criterion used been made?’), perhaps because it was assumed that readers would be aware of the psychometric properties of the criterion used. We, therefore, scored papers favourably for question five if they used a criterion examined in the review of outcome measures in clinical trials in Alzheimer’s disease from the Canadian Coordinating Office for Health Technology Assessment (CCOHTA) [18]. Whilst the examination of the properties of a given clinical outcome measure in this review neither implies adequate or favourable psychometric assessment, it does at least indicate that some degree of psychometric assessment has occurred. Where more than one clinical rating scale was used to draw associations with a biomarker in a single paper, question five was marked favourably as long as at least one of the clinical measures was in the aforementioned review. With regards to question nine (‘was there a sufficient period of follow-up?’) we denoted a sufficient period of follow-up in this review as longer than one year. Although this may be an insufficient period of follow-up to detect significant disease progression in Alzheimer’s disease, we hoped this cut-off would at least help differentiate very short studies from those with longer periods of follow-up. participants, confirmed using neuropathological diagnostic criteria [19,20]. As illustrated in table 2, almost half of the included studies did not describe their setting, but the vast majority of those who did were based in outpatient departments. Similarly, almost a third of studies failed to mention whether inclusion and exclusion criteria were applied. Of those providing this information more than three quarters applied moderately to severely restrictive study entry criteria. All of the included studies used an impairment or disability scale as the clinical measure of disease progression used to test for an association with a biomarker. None of the studies used measures of quality of life or handicap as a clinical outcome measure. Characteristics of study participants As illustrated in table 2, the median number of study participants was low at 31 (interquartile range (IQR) 17 to 64). The mean age of those included was fairly young at 73.0 (standard deviation (SD) 4.0) years of age, particularly considering that the median duration of disease at study entry was 3.6 (IQR 2.9 to 4.3) years. The majority of participants were not on a cognitive enhancer at baseline and had mild dementia, as assessed by the MMSE. Unfortunately, insufficient numbers of studies quoted participants’ baseline scores on other widely used cognitive rating scales to allow meaningful descriptive statistics relating to these measures to be calculated. Data analysis and synthesis Given the likelihood that included studies would examine the relationship of multiple different putative biomarkers with multiple different clinical measures of disease severity, we were aware that any data synthesis would be qualitative in nature. Quality criteria The median total score produced by applying the quality questionnaire to each of the included studies was 7.0 (IQR 6.0 to 7.0) out of a possible 16 (table 1). There was no evidence to suggest that the quality scores achieved for recently published studies were better than for those published in the past. In just over half of the included studies the primary aim was to develop a biomarker for disease progression. Whilst all studies were rated as having given a valid reason for choosing the biomarker in question for investigation, this question was difficult to score for studies whose primary aim was not to develop a biomarker for disease progression. In those cases credit was given for a reasonable explanation of why the studies true aims were scientifically credible. The vast majority of studies did not describe the reproducibility of measuring the biomarker, even in a single centre, and in most cases no details of the effects of confounding factors on the biomarker under investigation were described. The majority of studies did, however, use at least one clinical rating scale examined in the CCOHTA review. Only three studies undertook a power calculation to determine the number of participants, and only one of these recruited the required number of participants. The median length of follow-up was only 1.0 (IQR 0.8 to 1.7) years, and most studies only measured the putative biomarker and clinical measure of disease severity twice (mean number of timepoints 2.3 (SD 0.9)). Unfortunately in a few studies it was impossible to ascertain exactly how long participants were followed-up, or how many measurements were taken. Over half of the included studies also failed to state whether measurement of the biomarker was undertaken by an operator blind to the participants’ characteristics. In most studies over 75% of those entering the study at baseline completed the follow-up period. However, in many cases it appeared that analyses were restricted to a select cohort of patients, drawn from a larger unspecified cohort, who had Results As shown in Figure 1, the electronic searches identified 8234 records. After removing duplicates, 5416 unique records identified by the electronic search were screened, in addition to a further 22 records identified while performing the hand search or on reviewing reference lists of relevant review articles and included articles. The full-text articles of 308 records were then assessed for eligibility, and of those 249 articles were excluded. Finally data were extracted from a total of 59 articles. Hand searching Hand searching to validate the electronic search strategy revealed a sensitivity of 60.0% (95% CI 17.0–92.7) and a specificity of 99.1% (95% CI 98.8–99.3). The number of included articles identified by the electronic search in both journals within the chosen time period was small (Archives of Neurology n = 2, Neurology n = 1). The low sensitivity related to the finding of one additional article in each journal on hand searching. However, both these articles had already been found prior to the hand search by searching the reference lists of included articles and review articles. Therefore, the actual sensitivity for the whole search process (electronic search plus review of reference lists) was higher (100.0% (95% CI 46.3–100.0)). Both articles were not identified by the electronic search as they lacked a term in their title relating to the biomarker modality used to examine for an association with a clinical measure of disease progression. Characteristics of included articles All included studies except one made the diagnosis of Alzheimer’s disease using the National Institute of Neurological and Communicative Disorders and Stroke-Alzheimer’s Disease and Related Disorders Association (NINCDS-ADRDA) criteria [9,10]. In two studies the diagnosis was, in at least some PLOS ONE \| www.plosone.org 4 February 2014 \| Volume 9 \| Issue 2 \| e88854 Biomarkers for Disease Progression in AD Figure 1. Flow diagram outlining the selection procedure to identify 59 articles which were included in the systematic review of biomarkers for disease progression in Alzheimer’s disease. Note that of the 20 articles identified by reviewing reference lists, nine were excluded, and 11 were included in the final qualitative synthesis. Both articles identified by hand searching were included in the final qualitative synthesis. doi:10.1371/journal.pone.0088854.g001 of the evidence presented in tables S1 to S9 were measures of ventricular volume (associations with MMSE and ADAS-cog) and whole brain volume (associations with MMSE) made using magnetic resonance imaging (MRI). No studies reported an economic analysis of using the biomarker in question, and nor did any report on the acceptability of the test to individual patients. Six studies did, to some extent, examine the effect of symptomatic drug therapy on the biomarker under examination. completed the study period and, therefore, the true drop-out rate was probably higher. Types of biomarkers investigated The biomarker modalities examined in the 59 included studies are summarised in table 3, along with details of whether or not a significant association between each biomarker modality and a clinical measure of disease progression was demonstrated in each study. Full details of the individual biomarkers examined in each study and their relationship with clinical measures of disease severity are given in tables S1 to S9. Multiple different candidate biomarkers for disease progression have been investigated in Alzheimer’s disease, but the majority of studies examined some form of brain imaging. Those studies which reported a significant relationship were generally single studies examining a specific brain region, electrophysiological feature, blood or CSF constituent, with results which had not been replicated by other groups. In addition they involved small numbers of participants. The only biomarkers which appeared to have sufficient evidence to merit further investigation on the basis PLOS ONE \| www.plosone.org Statistics Correlation analysis, a basic statistical method which can be used to examine for a relationship between two variables, was solely used in 76% (37/49) of the included studies in which a description of the statistical techniques used was provided. Ten of the 59 included studies (17%) failed to report what statistical techniques were employed. Only two studies used a mixed model, despite the advantages this technique conveys in terms of dealing with missing data. Only seven (12%) of the included studies made adjustments for confounding factors. Furthermore, only 14 (24%) 5 February 2014 \| Volume 9 \| Issue 2 \| e88854 Biomarkers for Disease Progression in AD Discussion Table 2. Study characteristics of the 59 included articles and baseline characteristics of participants with Alzheimer’s disease included in those studies. We found insufficient evidence to support the current use of any biomarker to measure disease progression in Alzheimer’s disease clinical trials. Measurements of ventricular volume and whole brain volume made by MRI do, however, appear to merit further investigation. It is possible that the lack of a current biomarker in Alzheimer’s disease is because no suitable biomarker exists or, at least, no single biomarker given the complexity of the disease. However, in keeping with the findings of our previous systematic review in PD [8], at present, it probably also reflects the poor quality of studies which have investigated biomarkers for disease progression. In order to improve future studies we previously developed a provisional ‘roadmap’ for conducting biomarker studies primarily in PD (Figure 2) but this ‘roadmap’ clearly also applies to Alzheimer’s disease and other neurodegenerative diseases. The starting point for any disease progression biomarker study must be a valid reason for selecting a specific biomarker for investigation based on the pathophysiology of the disease in question. Unfortunately, the development of a biomarker was not the primary aim of several studies included in this review; relevant analyses were simply the by-product of studies with an alternative aim (e.g. drug development). The appropriateness of studies with an alternative primary aim undertaking additional analyses to produce information regarding such associations is questionable. As our ‘roadmap’ highlights biomarker studies require careful planning and, therefore, should only run alongside other types of studies (e.g. long-term prognostic studies or clinical trials) when either such planning has taken place or as part of the process of gathering specific preparatory data required for a future formal biomarker study. Whilst this systematic review could be criticised for including studies whose primary aim was not to develop a biomarker for disease progression in Alzheimer’s disease, we did so to ensure our review was as inclusive as possible and to avoid missing any potential biomarkers. Secondly, the reliability of a putative biomarker must be established by demonstrating the reproducibility of its measurement in a single centre by different personnel, and between different centres. With imaging biomarkers characterised by a small change in a small area of the brain reliability of measurement can be a real issue, particularly between different Setting of included studies Outpatient 29 49% Outpatients and inpatients 1 2% Inpatient 1 2% Not detailed 28 47% Inclusion/exclusion criteria applied in included studies None 0 0% Mildly restrictive 5 9% Moderately restrictive 21 36% Severely restrictive 15 26% Not detailed 17 29% Baseline demographics Median number of patients 31 (17 to 64) Mean age (years) 73.0 (4.0) Mean percentage male 42 (14) Median disease duration (years) 3.6 (2.9 to 4.3) Median percentage treated with a cognitive enhancer 0 (0 to 73) 21 (20 to 23) Baseline disease severity Median MMSE The number and percentage of included studies with each study characteristic is presented. Means are presented with standard deviations, and medians with interquartile ranges (IQR). doi:10.1371/journal.pone.0088854.t002 fully reported the outcome of their statistical analyses. Even when basic correlation analyses were conducted, correlation coefficients and significance values were often not reported and in no case were confidence intervals for the correlation coefficient given. Table 3. Comparison of the number of included studies investigating a given biomarker modality with the number reporting a significant association between the biomarker modality and a clinical measure of disease progression. Biomarker modality Number of studies investigating biomarker modality Number of studies reporting a significant association between biomarker modality and a clinical measure of disease progression Brain MRI 17 (29%) 14 CSF 12 (20%) 4 Brain MRS 8 (14%) 8 Serum/plasma/blood 7 (12%) 4 Brain PET 6 (10%) 4 Brain SPECT 5 (9%) 4 Electrophysiology 4 (7%) 3 Brain CT 1 (2%) 1 Ultrasound 1 (2%) 1 Two studies examined for a relationship between two different biomarker modalities (MRI and MRS in one study; MRI and CSF in another study) and a clinical measure of disease progression. (MRI, magnetic resonance imaging; CSF, cerebrospinal fluid; MRS, magnetic resonance spectroscopy; PET, positron emission tomography; SPECT, single-photon emission computed tomography; CT, computed tomography). doi:10.1371/journal.pone.0088854.t003 PLOS ONE \| www.plosone.org 6 February 2014 \| Volume 9 \| Issue 2 \| e88854 Biomarkers for Disease Progression in AD longitudinal studies. However, only three studies in this review performed a power calculation, and only one of these then went on to recruit sufficient participants. Moreover, the small number of participants (median 31 (IQR 17 to 64)) in the studies included in this review is of concern. As studies become smaller it is increasingly likely that potentially significant associations will not be detected, and the number of variables which can be included in multivariate analyses without significantly increasing the risk of spurious findings becomes limited. Whilst we only included longitudinal studies in this review it was clear from filtering the abstracts returned by the electronic search that, as in PD, numerous cross-sectional disease progression biomarker studies have been performed in Alzheimer’s disease. As already discussed, this is not a suitable design to examine for a relationship between a change in a clinical measure and the change in a biomarker over time within individuals with Alzheimer’s disease. The studies included in this review had a median follow-up duration of only 1.0 (IQR 0.8 to 1.7) years, with only 44% of studies following participants up for longer than our chosen discriminator of one year. There is currently no evidence to suggest what the minimum duration of a disease progression biomarker study should be, but it obviously needs to be long enough for a clinically significant change in the criterion, used to draw associations with the putative biomarker, to be observed. However, if a short-term change in a biomarker is to be associated with a long-term change in a clinical outcome measure then clearly a longer period of follow-up is required. In the included studies the biomarker and clinical measures were generally only measured twice (mean 2.3 (SD 0.9) time points). This is clearly insufficient to allow a linear association to be differentiated from a non-linear association. Future studies in this area must be longitudinal and measure the biomarker and clinical measures at several time points (at least three) over a sufficient follow-up period, more likely to be measured in years than months, as only this design will provide sufficient evidence of a biomarkers potential validity. The use of moderately to severely restrictive entry criteria in the majority of studies included in this review will clearly have influenced the participants’ characteristics. In particular, the elderly appear to be underrepresented in the included studies. One American incidence study, for example, found that whilst Alzheimer’s disease had an annual incidence of 280 per 100,000 in those aged 65–69 years of age this rose dramatically to 5610 per 100,000 in those over 90 years of age [22]. Similar results have been reported in other American and European studies [23,24]. This makes the mean age of 73.0 (SD 4.0) years of age of participants in the studies included in this review concerning, particularly as the median duration of disease at study entry was 3.6 (IQR 2.9 to 4.3) years. We would recommend that future studies try to keep their entry criteria as open as possible to maximise the generalisability of their results. Reporting of statistical analyses in the included studies was inadequate. In both correlation and regression analyses, hypothesis testing can be undertaken to determine whether a relationship exists in the population as a whole, and confidence intervals calculated to indicate the strength of that relationship. Whilst all included studies undertook significance testing many failed to report precise significance values, and instead gave results descriptively in the text. Whilst this may reflect pressures of space in published journals, the results should at least be provided as a supplementary online resource. Several studies unfortunately even failed to detail what statistical techniques they used. Without clear reporting of the study methodology, results, and the outcome of Figure 2. Flow diagram outlining a provisional ‘roadmap’ for conducting a study to determine whether a given biomarker is a suitable surrogate for a clinical measure of disease progression. doi:10.1371/journal.pone.0088854.g002 centres which may have different imaging equipment and software. Thirdly, an evaluation of the effect of potential confounding factors on the biomarker (e.g. age, gender, smoking status or cognitive enhancers) should be undertaken. An understanding of the influence of these factors on the biomarker will aid sample size calculations, and allow a rigorous analysis of the final study results by adjusting for these factors. In parallel to this pre-study ‘work-up’ of the biomarker the validity, reliability, and responsiveness, including to clinical change, of the selected criterion against which a biomarker will be examined, must be explored. Extensive work has been undertaken in assessing the validity and reliability of psychometric instruments [21], and a similar approach here would seem sensible. Maximising the scientific rigor of the selected criterion is central to improving the chance of coming to the correct conclusion about the efficacy of a biomarker for disease progression, and will have implications for biomarker study sample size calculations. Following these initial steps it should then be possible to perform a power calculation to determine an appropriate sample size before a biomarker study commences. Sample sizes can be adjusted to accommodate potential losses to follow-up which, as in the studies included in this review, are commonly encountered in PLOS ONE \| www.plosone.org 7 February 2014 \| Volume 9 \| Issue 2 \| e88854 Biomarkers for Disease Progression in AD volume and whole brain volume as biomarkers of disease progression in Alzheimer’s disease does appear to be merited. As in our previous systematic in PD, we found methodological, statistical and reporting flaws in studies examining disease progression in Alzheimer’s disease. Our methodological guidelines should hopefully provide a better chance of making progress in this area, and we would value feedback on them. statistical analyses, investigators devalue their study and risk it being excluded from future systematic reviews or meta-analyses. The statistical techniques applied in the included studies were in several cases inappropriate and, more often than not, too simplistic. There was an overreliance on correlation, which is a limited technique to examine for a relationship between the changes in two variables as it only indicates the strength and direction of a relationship, and does not allow adjustment for confounding factors [25]. There was a tendency in the included articles for multiple individual correlation coefficients and significance values to be calculated after measuring a large number of variables rather than using a multivariate analysis or a higher level of statistical modelling (e.g. a linear mixed model [26]). The majority of studies also failed to adjust for important confounding factors, regardless of what statistical techniques they used. We encountered the same deficiencies in statistical methodologies in the articles included in our previous systematic review of biomarkers for disease progression in PD [8], and in that paper discussed at length potential solutions to these problems. We strongly recommend that future biomarker studies incorporate a range of analyses, rather than simply correlation, in order to explore the validity of more advanced statistical methods. Using appropriate statistical techniques should reduce the chance of type I and type II errors and, thereby, allow sensible conclusions to be drawn about the efficacy of specific biomarkers. Analyses should be planned and conducted by an experienced statistician given the complexities of dealing with repeated measures data. It is pleasing to note that some of the lessons of this systematic review have already begun to be realised by some researchers and put into practice. The longitudinal Alzheimer’s Disease Neuroimaging Initiative (ADNI) [27] aims to measure various putative CSF and imaging biomarkers several times over several years. Their work, like that of the Parkinson’s Progression Markers Initiative (PPMI) [28] and the Parkinson’s Disease Biomarkers Program (PDBP) [29], should mark a major shift in the quality of studies of biomarkers for disease progression, and hopefully lead to advances in this important field. Supporting Information Document S1 Electronic search strategy. (DOCX) Document S2 Data extraction sheet. (DOCX) Table S1 Blood, plasma and serum biomarkers. (DOCX) Table S2 CSF biomarkers. (DOCX) Table S3 Ultrasound biomarkers. (DOCX) Table S4 CT brain biomarkers. (DOCX) Table S5 Brain MRI biomarkers. (DOCX) Table S6 Brain MRS biomarkers. (DOCX) Table S7 Brain SPECT biomarkers. (DOCX) Table S8 Brain PET biomarkers. (DOCX) Table S9 Electrophysiological biomarkers. (DOCX) Checklist S1 PRISMA checklist. (DOC) Conclusions Author Contributions This extensive systematic review found insufficient evidence to recommend the use of any biomarker for measuring disease progression in Alzheimer’s disease clinical trials. However, further examination of the efficacy of MRI measurements of ventricular Conceived and designed the experiments: DJMM CWR JPZ CEC. Performed the experiments: DJMM CEC. Analyzed the data: DJMM CEC. Wrote the paper: DJMM CWR PAT DEW JPZ CEC. Provided statistical expertise: PAT DEW. References 1. Knapp M, Prince M, Albanese E, Banjeree S, Dhanasiri S, et al (2007) A report to the Alzheimer’s Society on the prevalance and economic cost of dementia in the UK produced by King’s College London and the London School of Economics. London: Alzheimer’s Society. 2. Knopman DS (2008) Clinical trial design issues in mild to moderate Alzheimer disease. Cogn Behav Neurol 21: 197–201. 3. 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https://openalex.org/W2766741417	http://www.seer.ufu.br/index.php/biosciencejournal/article/download/35734/20839/	English	null	Aerobic decolorization of azo dye orange g by a new yeast isolate Candida cylindracea SJL6	Bioscience journal	2,017	cc-by	6,486	KEYWORDS: Biodegradation. Fungus. Dyes. KEYWORDS: Biodegradation. Fungus. Dyes. KEYWORDS: Biodegradation. Fungus. Dyes. AEROBIC DECOLORIZATION OF AZO DYE ORANGE G BY A NEW YEAST ISOLATE Candida cylindracea SJL6 ABSTRACT: Biodecolorization of the azo dye Orange G was investigated using a new strain of Candida cylindracea SJL6, isolated from freshwater samples of the Subaé river in Bahia state, Brazil. Strain SJL6 was identified as C. cylindracea on the basis of 26S rDNA region. The various parameters of dye decolorization and cell growth were studied, including the Orange G dye concentration (100 to 500 ppm), temperature (20 to 40 °C), glucose concentration (0 to 5%), and initial pH (3 to 8). Biotoxicity tests were performed using shrimp (Artemia salina) to determine the lethal concentration (LC50) and onion bulbs (Allium cepa) to determine the cytotoxic and genotoxic effects of both Orange G dye and metabolites formed after decolorization. Up to 90% of decolorization of the Orange G dye at 500 ppm was achieved by C. cylindracea SJL6 at 30 °C, pH 3, and 1% glucose. However, the biotoxicity tests showed that there was increased toxicity after decolorization, suggesting partial Orange G dye degradation and production of toxic metabolites. Biosci. J., Uberlândia, v. 33, n. 5, p. 1340-1350, Sept./Oct. 2017 1340 1340 Original Article INTRODUCTION P. et al that of unicellular organisms, in addition to the production of mycelia, which often makes fungi less adaptable to effluent treatment conditions (ASKU; DÖNMEZ, 2003; YU; MEN, 2005). peqSTAR 96× Universal Gradient thermal cycler (PeqLab, Erlangen, Germany) programmed for an initial denaturation at 94 ºC for 3 min, followed by 28 cycles of denaturation (94 °C, 30 s), annealing (58 °C, 1 min), and extension (72 °C, 2 min) and a final extension at 72 °C for 5 min. The amplified products were separated on a 0.8% agarose gel, stained with ethidium bromide, and visualized under ultraviolet light. The PCR fragments were purified using a GFX PCR kit (Amersham Biosciences, Piscataway, NJ, USA), and then the amplicons were sequenced on an automatic sequencer ABI 310 (Applied Biosystems, Foster City, CA, USA) at the Molecular Genetics Laboratory of Federal University of Recôncavo da Bahia. The second round of sequencing was performed using an ABI PRISM 3100 genetic analyzer (Applied Biosystems) by ACTGene Molecular Analysis LTDA (The Rio Grande do Sul, Brazil). The sequences obtained were compared with those available in the GenBank database using the Basic Local Alignment Search Tool (BLAST) at http://www.ncbi.nlm.nih.gov. peqSTAR 96× Universal Gradient thermal cycler (PeqLab, Erlangen, Germany) programmed for an initial denaturation at 94 ºC for 3 min, followed by 28 cycles of denaturation (94 °C, 30 s), annealing (58 °C, 1 min), and extension (72 °C, 2 min) and a final extension at 72 °C for 5 min. The amplified products were separated on a 0.8% agarose gel, stained with ethidium bromide, and visualized under ultraviolet light. The PCR fragments were purified using a GFX PCR kit (Amersham Biosciences, Piscataway, NJ, USA), and then the amplicons were sequenced on an automatic sequencer ABI 310 (Applied Biosystems, Foster City, CA, USA) at the Molecular Genetics Laboratory of Federal University of Recôncavo da Bahia. The second round of sequencing was performed using an ABI PRISM 3100 genetic analyzer (Applied Biosystems) by ACTGene Molecular Analysis LTDA (The Rio Grande do Sul, Brazil). The sequences obtained were compared with those available in the GenBank database using the Basic Local Alignment Search Tool (BLAST) at http://www.ncbi.nlm.nih.gov. Analytical method The decolorization rate was determined by spectrophotometry at 476 nm and calculated using the following equation: Decolorization (%) = (A − B)/A × 100, where A and B represent the initial and final absorbance of the dye, respectively. Decolorization (%) = (A − B)/A × 100, where A and B represent the initial and final absorbance of the dye, respectively. Cellular growth was determined by measuring optical density at 550 nm. INTRODUCTION The use of yeasts presents several biotechnological advantages, not only because of their rapid growth, but also because of their resistance to unfavorable environments and high efficiency in azo dye decolorization (MORTARELL et al, 2012; YANG et al., 2008; LIU et al., 2011; PAJOT et al., 2011; TAN et al., 2014). Therefore, the aim of this work was to evalaute various culture parameters for decolorization of the Orange G azo dye by a new strain of Candida cylindracea SJL6, as well as to evaluate the toxicity of final products of the degradation process. Molecular identification of the yeast Molecular identification of the yeast DNA extraction was performed according to Sampaio et al. (2001) and Almeida (2005). Molecular characterization was performed by sequencing of the D1/D2 region of 26S ribosomal DNA using the NL1 (5′- GCATATCAATAAGCGGAGGAAAAG-3′) and NL4 (5′-GGTCCGTGTTTCAAGACGG-3′) primers (O’DONNEL, 1993). For the polymerase chain reaction (PCR), we used the following reagents and concentrations: 10 ng of DNA, 1× enzyme buffer for Taq DNA polymerase, 3.7 mM MgCl2, 0.6 pmol/µL dNTPs, 0.4 pmol/µL each primer, and 5 U of Taq DNA polymerase, with the final volume adjusted to 50 µL with sterile ultra-pure water. DNA of Saccharomyces cerevisiae (10 ng) was used as a positive control. Amplification was performed in a Decolorization assays y Assays were performed in 250-mL Erlenmeyer flasks containing 50 mL of NDM at varying culture conditions: the dye concentration (100–500 ppm), glucose concentration (0–5%), pH (3–8), and temperature (20–40 ºC). After incubation at 150 rpm for 24 h, 2-mL aliquots were taken from the flasks and centrifuged at 5,000 rpm for 10 min. The supernatant obtained was used to analyze both decolorization rate and toxicity, while the biomass was used to determine cellular growth. All assays were performed in triplicate. As the inoculum, 5 mL of a cellular suspension (optical density at 550 nm = 0.8) was used, which was prepared from a 24-h culture grown in NDM. INTRODUCTION Azo dyes account for 70% of all dyes used in the textile industry (TONY et al., 2009). The success of the wide use of these dyes is related to their easy synthesis, cost-effectiveness, stability, and great structural variety, which enables a wide range of colors and applications. Thus, in addition to the textile industry, these dyes are widely used in the paper, food, leather, cosmetics, and medical industries (CHANG et al., 2004; TELKE et al., 2008). The presence of residues in textile effluents causes significant impacts on the total organic carbon, biochemical oxygen demand (BOD), chemical oxygen demand (COD), suspended solids, salinity, pH, and color, in addition to the recalcitrant nature of azo dyes (FARYAL; HAMEED, 2005; SAVIN; BUTNARU, 2008). The high values of BOD and COD can result in the consumption of dissolved oxygen, causing the death of fish and other aquatic organisms (VON SPERLING, 1996). Furthermore, azo dyes when metabolized by particular enzymes, may generate aromatic amines, which are known for their high toxicity, mutagenicity, and carcinogenicity (SOLÍS et al., 2012). Bioremediation has been considered an environment friendly and cost-competitive approach that produces less sludge and is able to generate end-products of low toxicity or even achieve total mineralization of compounds (RAI et al., 2005). In this context, many microorganisms have been selected every day for the development of new treatments. All this has raised growing concerns and prompted several studies in different fields of knowledge in an attempt to develop effective and low-cost technologies for the removal of dyes from textile effluents. Physical and chemical treatments have been the most widely used; however, they usually require high costs and are not able to completely degrade the molecules, generating significant amounts of chemical sludge, which creates a second pollution problem, waste disposal (ANJANEYULU; CHARY, 2005; SARATALE et al., 2009; BAFANA et al., 2011). Due to their enzymatic apparatus, fungi have a great potential for decolorization of azo dyes (MORTARELL et al., 2012). Through oxidative and ligninolytic non-specific enzyme systems, these microorganisms are able to mineralize various types of synthetic dyes (YANG et al., 2005). However, several factors hamper the use of filamentous fungi in industrial waste treatment, such as stringent conditions for enzyme production, risk of bacterial contamination under non-sterile conditions of wastewater treatment, and slow growth compared to Biosci. J., Uberlândia, v. 33, n. 5, p. 1340-1350, Sept./Oct. 2017 Received: Accepted: 1341 Aerobic decolorization... ALCÂNTARA, T. A. MATERIAL AND METHODS The azo dye used in this study was Orange G (C.I. Name: Acid Orange 10; Sigma–Aldrich, St. Louis, MO, USA). A 10,000 ppm stock solution was prepared by dissolving the dye in distilled water and sterilizing by vacuum filtration (0.22-mm filter, Millipore Corp., Bedford, MA, USA). Culture medium The modified Normal Decolorization Medium (NDM) was used for the experiments, which consisted of (%): yeast extract, 0.25; KH2PO4, 0.5; MgSO4·7H2O, 0.05; CaCl2·2H2O, 0.013; and glucose, 2 (RAMALHO et al., 2004). After sterilization at 121 °C for 20 min, Orange G was added to the medium at different concentrations (100, 200, 300, 400, and 500 ppm). The pH was adjusted to 5.0 with orthophosphoric acid. Biotoxicity test using onion (Allium cepa) The tests using onion bulbs were performed according to Fiskesjö (1985), modified. Non- sprouted onion bulbs of the same origin, approximately 2 cm in size, with external white cataphylls, were purchased at a local market. Before the test, dry external cataphylls were removed without damaging the root meristem, and the bulbs were washed under running water. To avoid dehydration of primary cells, the bulbs were placed in distilled water and kept in the dark for 48 h. Distilled water was changed daily. After two days, bulbs with the size of 1.5 to 2.0 cm were selected. Five bulbs were used for each treatment. The bulbs were placed in test tubes, with their roots in contact with the supernatant (20 mL) obtained after the decolorization process. The Orange G dye solution Biotoxicity tests Acute toxicity test using Artemia salina The acute biotoxicity test was performed with the nauplii of A. salina, according to Matthews (1995), with some modifications. For hatching, 1 g of A. salina cysts was added to 500 mL of artificial Biosci. J., Uberlândia, v. 33, n. 5, p. 1340-1350, Sept./Oct. 2017 1342 ALCÂNTARA, T. A. P. et al Aerobic decolorization... seawater (30 ppt). The cysts were kept at a constant agitation by air pumping for 24 h at room temperature. Then, 10 nauplii of A. salina were transferred to test tubes containing 10 mL of artificial seawater and Orange G dye (positive control) or different concentrations (2.08, 4.2, 8.3, 16.6, 25, 33, 50, and 75%) of the supernatant obtained after the decolorization process and incubated at room temperature for 24 h. Artificial seawater was used as a negative control, and all tests were performed in triplicate. The results were obtained by counting the number of alive A. salina nauplii in each test tube. The calculation of the lethal concentration (LC50) was performed using the Spearman–Karber Trimmed program (AN, 2006). was a positive control, and distilled water was a negative control. After 24 h of exposure, the roots were removed, kept in Carnoy’s fixative solution for 24 h, and then stored in ethanol (70%) under refrigeration until slide preparation. For the slide preparation, roots were removed from ethanol, washed, and hydrolyzed in 1 M HCl at 60 °C for 8 min. Subsequently, a drop of acetic acid (45%) was added to a slide, and the tip was removed from a root, then covered with a coverslip, and crushed. The slide–coverslip assembly was frozen at −80 °C for 5 min, then the coverslip was removed, and the material was stained with 10% Giemsa stain for 2 min. The root material on the blade was observed under an optical microscope at a 400× magnification. About 5,000 cells were analyzed per treatment. The following parameters were observed: (A) a mitotic index (MI) (1,000 cells per bulb); (B) chromosomal aberrations (CAs) in the mitotic cycle (1,000 cells per bulb); and (c) the presence of micronuclei (1,000 cells per bulb). The data were analyzed with the Assistat Software, version 7.7, using analysis of variance, followed by Turkey’s test. Molecular identification A 616-nucleotide sequence of the D1/D2 region of strain SJL6 showed 99% identity with the sequences of the D1/D2 domain of C. cylindracea (Figure 1). gi\|685456613\|Candida quercitrusa isolate MB144 gi\|87298856\|Candida natalensis strain VTT C-04521 gi\|87298851\|Candida anglica gi\|371444538\|Candida fragi strain Kk3 gi\|37933485\| Candida sp. BG01-8-20-001B-2-1 SJL6 gi\|2065486\|Candida cylindracea 98 71 100 56 0.02 Figure 1. Maximum likelihood phylogenetic tree inferred from the alignment of the D1/D2 region sequences the yeast rDNA subunit constructed in the MEGA 6 program. The best model selected by the progra was the Tamura-Nei + Gamma (TN93 + G) model with partial deletion of missing data a confidence level of 1000 bootstrap repeats. gi\|685456613\|Candida quercitrusa isolate MB144 gi\|87298856\|Candida natalensis strain VTT C-04521 gi\|87298851\|Candida anglica gi\|371444538\|Candida fragi strain Kk3 gi\|37933485\| Candida sp. BG01-8-20-001B-2-1 SJL6 gi\|2065486\|Candida cylindracea 98 71 100 56 0.02 Fi 1 M i lik lih d h l ti t i f d f th li t f th D1/D2 i Figure 1. Maximum likelihood phylogenetic tree inferred from the alignment of the D1/D2 region sequences of the yeast rDNA subunit constructed in the MEGA 6 program. The best model selected by the program was the Tamura-Nei + Gamma (TN93 + G) model with partial deletion of missing data and confidence level of 1000 bootstrap repeats. Figure 1. Maximum likelihood phylogenetic tree inferred from the alignment of the D1/D2 region sequences of the yeast rDNA subunit constructed in the MEGA 6 program. The best model selected by the program was the Tamura-Nei + Gamma (TN93 + G) model with partial deletion of missing data and confidence level of 1000 bootstrap repeats. Effects of various parameters on the Orange G decolorization rate and cellular growth of C. cylindracea SJL6 Initial dye concentration y C. cylindracea SJL6 was able to remove more than 90% of the color up to an Orange G concentration of 500 ppm after 24 h, and only a C. cylindracea SJL6 was able to remove more than 90% of the color up to an Orange G concentration of 500 ppm after 24 h, and only a Biosci. J., Uberlândia, v. 33, n. 5, p. 1340-1350, Sept./Oct. 2017 1343 Aerobic decolorization... ALCÂNTARA, T. A. P. et al small biomass reduction occurred with the increasing Orange G concentrations. Thus, it is possible to conclude that C. cylindracea SJL6 proved to be quite tolerant to high concentrations of the dye (Figure 2). Figure 2. Effect of initial concentrations of Orange G on the decolorization rate (■), cell growth (□), and final pH (●) Figure 2. Effect of initial concentrations of Orange G on the decolorization rate (■), cell growth (□), and final pH (●) Results obtained by other authors have shown an inverse relationship between initial concentrations and dye decolorization. Pseudozyma rugulosa Y-48 and Candida krusei G-1 decolored 99% of Reactive Bright Red L-2BP at 200 ppm after 24 h, with the decolorization gradually decreasing as the dye concentrations increased (YU; MEN, 2005). In another study, Candida tropicalis TL-F1 was able to achieve a decolorization rate of more than 97% with the Acid Brilliant Scarlet GR dye in the concentration range from 20 to 100 ppm after 24 h; however, there was a rate reduction to less 80% at higher dye concentrations (120 and 140 ppm) (TAN et al., 2014). Issatchenkia orientalis JKS6 was able to decolorize up to 99% of the Reactive Black 5 dye at 500 ppm after 36 h; however, the decolorization rate decreased to less than 40% at a 1,000 ppm dye concentration (JAFARI et al., 2014). The decrease in the decolorization rate with an increasing dye concentration suggests a toxic action by dye intermediate metabolites produced after cleavage of the chromophore group (WANG et al., 2009). Textile effluents show variations in residual dye concentrations, and therefore, the ability of microorganisms to degrade dyes in a wide range of concentrations becomes an important factor for effective biodegradation (PARSHETTI et al., 2010; AYED et al., 2011). The results showed that C. cylindracea SJL6 was unable to perform decolorization in the absence of an external source of carbon and energy. Initial dye concentration Although there are reports of yeasts that can use a dye as a sole source of carbon and/or nitrogen for dye degradation, this process is highly associated with the cell growth and primary metabolism (LUCAS et al., 2006; YANG et al., 2008; PAJOT et al., 2011; MORTARELL et al., 2012). Therefore, yeasts do not generally grow without glucose or any other readily metabolizable carbon source (SOLÍS et al., 2012). In this work, the increase of glucose concentrations showed no significant effect on both decolorization rate and cellular growth, which remained constant at the glucose concentrations from 1% to 5%. These results are very interesting since low carbon source concentrations reduce the costs of developing biodegradation processes. Moreover, high glucose concentrations in the medium can inhibit dye degradation since microorganisms tend to use an external source of energy instead of a dye (QU et al., 2012). However, no such inhibition was observed in this study up to the maximum glucose concentration studied (5%). For the yeasts Debaryomyces polymorphus and C. tropicalis, Reactive Black 5 dye degradation was highly dependent on the glucose concentration in the medium. Thus, the color removal increased from 76.4% and 92.7%, respectively, after 60 h to 100% between 18 h and 24 h after rising glucose Biosci. J., Uberlândia, v. 33, n. 5, p. 1340-1350, Sept./Oct. 2017 Glucose concentration Figure 3 shows the effects of glucose concentrations on the Orange G dye decolorization. Biosci. J., Uberlândia, v. 33, n. 5, p. 1340-1350, Sept./Oct. 2017 1344 ALCÂNTARA, T. A. P. et al Aerobic decolorization... concentration from 0.5% to 1% (YANG et al., concentration from 0.5% to 1% (YANG et al., concentration from 0.5% to 1% (YANG et al., 2008). Figure 3. Effect of glucose concentrations on the Orange G dye (200 ppm): decolorization rate (■), cell growth (□), and final pH (●). Figure 3. Effect of glucose concentrations on the Orange G dye (200 ppm): decolorization rate (■), cell growth (□), and final pH (●). The optimal glucose concentration was reported to be between 0.5 and 1% for several yeasts such as Pichia sp. TCL upon decolorization of the Acid Red B dye (100 ppm) (QU et al., 2012), I. orientalis JKS2 upon decolorization of the Reactive Black 5 dye (JAFARI et al., 2014), and C. tropicalis TL-F1 upon decolorization of the Acid Bright Scarlet GR dye (100 ppm) (TAN et al., 2014). In this study, an increased glucose concentration favored the cell growth, and a slight increase in biomass was observed, followed by a decrease in pH. The decrease in pH occurred due to glucose metabolism and positively influenced the dye decolorization since yeasts, in general, adapt better to acid environments. ligninolytic enzymes involved in azo dye decolorization are observed at a low pH (SHAH; PATEL, 2014). The same has been described for biodegradation of different dyes by yeasts (JADHAV et al., 2008; LIU et al., 2011; TAN et al., 2014). Enzymes such as manganese peroxidase (MnP), lignin peroxidase (LiP), NADH– dichlorophenol indophenol reductase, and azoreductases are some of the major enzymes involved in dye biodegradation by yeasts (YANG et al., 2005; LUCAS et al., 2006; JADHAV et al., 2008; WAGHMODE et al., 2011). Temperature The maximum Orange G decolorization rate and cell growth were observed in the temperature range from 20 °C to 30 °C. However, decolorization ceased completely at temperatures of 35 °C and above, accompanied by a sharp decrease in cell growth (Figure 5). These results can be explained by a loss of cell viability and/or by denaturation of the enzymes responsible for the dye decolorization (WANG et al., 2009). Biosci. J., Uberlândia, v. 33, n. 5, p. 1340-1350, Sept./Oct. 2017 pH The decolorization rate was above 98% in the pH range between 3 and 6 (Figure 4) and drastically decreased at pH 7 and 8. The cell growth was not affected by the pH variation, although it was slightly better in the pH range between 4 and 5. These results are interesting for the development of biodecolorization processes using C. cylindracea SJL6 since acidic pH inhibits the growth of others microorganisms such as bacteria. It was not possible to correlate the cell growth with the decolorization rate, which suggests that the relationship between pH and decolorization is associated with optimal conditions for the performance and production of enzymes involved in the degradation process. The temperature effect on decolorization does not follow a pattern and varies according to the characteristics inherent to the microorganisms and/or the enzymes involved in the process. For yeasts, the best results are reported at temperatures in the range from 25 °C to 32 °C (ERTUĞRUL et al., 2008; CHARUMATHI; DAS, 2010; GRASSI et al., 2011; LIU et al., 2011; JAFARI et al., 2014). It is generally known that the maximum fungal growth and the maximum activity of Biosci. J., Uberlândia, v. 33, n. 5, p. 1340-1350, Sept./Oct. 2017 1345 ALCÂNTARA, T. A. P. et al Aerobic decolorization... Figure 4. Effect of initial pH on the decolorization rate of Orange G (200 ppm) (■), cell growth (□), and final pH (●). Figure 4. Effect of initial pH on the decolorization rate of Orange G (200 ppm) (■), cell growth (□), and final pH (●). Figure 5. Effect of the temperature on the decolorization rate of Orange G (200 ppm) (■), cell growth (□), and final pH (●). Figure 5. Effect of the temperature on the decolorization rate of Orange G (200 ppm) (■), cell growth (□), and final pH (●). Biosci. J., Uberlândia, v. 33, n. 5, p. 1340-1350, Sept./Oct. 2017 ALCÂNTARA, T. A. P. et al ALCÂNTARA, T. A. P. et al MI tests differed statistically. Regarding the CA index and the presence of micronuclei, there were differences between the treatments and control, but not among the treatments. No significant reduction in MI, caused by the exposure of onion bulbs to the Orange G dye, was observed; on the other hand, a higher reduction of MI occurred after dye decolorization (24 h). mutagenicity, besides providing a cheap, fast, and sensitive method for toxicity monitoring. According to the results (Table 1), cytotoxicity increased after the Orange G decolorization. Cellular aberrations were observed as anaphase bridges, chromosome fragments, sticky chromosomes in the metaphase, nuclear abnormalities, and the presence of micronuclei, which were evaluated separately. All Table 1. Total number of cell in interphase and in division: mitotic index (MI), chromosomal aberrations ra (CA) and micronucleus index (MN) Treatments Interphase cells Cells in division MI (%) CA (%) MN (%) * Control 4133 867 17.34 ±0,5a 0.06 ±0.09b 0 ±0 b Orange G 4572 428 8.56 ±0,62b 0.56 ±0.15a 0.2 ±0.19a Supernatant 4769 231 4.62 ±0,76c 0.46 ±0.21a 0.2 ±0.10a Control: Distilled water; Significant difference from the negative control P<0.05 e *P<0.001; (ANOVA – Tukey Test). Table 1. Total number of cell in interphase and in division: mitotic index (MI), chromosomal (CA) and micronucleus index (MN) number of cell in interphase and in division: mitotic index (MI), chromosomal aberrations rate and micronucleus index (MN) f cell in interphase and in division: mitotic index (MI), chromosomal aberrations rate nucleus index (MN) The increased toxicity after decolorization in both tests can be attributed to the incomplete dye degradation, indicating a biotransformation process. The metabolites produced after dye decolorization can be more toxic in many cases than the original dye molecule, and their toxicity is commonly attributed to aromatic amines, which are often released after reduction of the azo dye linkage (PINHEIRO et al., 2004; BAFANA et al., 2011; SOLÍS et al., 2012). The presence of 4- aminoacetanilide and 5-acetamido-2-amino-1- hydroxy-3,6-naphtalene disulfonic acid was reported to be responsible for the increased toxicity in rats after Violet dye degradation by the bacterium Pseudomonas putida (BEN MANSOUR et al., 2010). The increased toxicity after decolorization in both tests can be attributed to the incomplete dye degradation, indicating a biotransformation process. ALCÂNTARA, T. A. P. et al inhalation routes, in addition to causing skin and eye irritations and being directly involved in hemolytic anemia in vertebrates (SHUKLA et al., 2012). Longer-time fermentation could contribute to increased dye mineralization and reduce or eliminate the metabolite toxicity. In addition, the use of microbial consortia is highly recommended to obtain complete dye degradation. CONCLUSIONS The yeast C. cylindracea SLJ6 was efficient in Orange G dye decolorization, showing good tolerance of high dye concentrations. However, the results suggested incomplete biodegradation, and the metabolites produced after decolorization were more toxic than the original dye molecule, underscoring the need for additional studies to identify the metabolites produced. In addition, new approaches are necessary to reduce the production of potentially toxic metabolites, such as the use of microbial consortia. According to the literature, the Orange G dye is not easily degraded by microorganisms. Fungi such as Thelephora sp. and Pleurotus sajor- caju, which present a recognized potential for the decolorization of different azo dyes, removed only 30% and 50% of the color, respectively (CHAGAS; DURRANT, 2001; SELVAN et al., 2003). Biotoxicity tests increased toxicity after dye decolorization processes. Silva et al. (2012) analyzed the discoloration of the Phthalocyanine Reactive Blue 21 dye by peroxidase of turnip and found that the hydrolysis of the chromophore group increased toxicity after enzyme treatment. Almeida and Corso (2014) also observed that the inhibition of the lettuce (Lactuca sativa) seed germination increased from 5 to 50%, compared with that caused by the untreated dye, after the Procion Red MX–5B dye was decolorized by Aspergillus terreus, indicating incomplete dye degradation and production of toxic intermediates. The toxicity tests were performed using the nauplii of the brine shrimp A. salina as a quick, simple, and inexpensive test, which provides a good indication of the acute toxicity for several compounds and is widely used to determine the toxicity of dyes (PRASAD; RAO, 2013; IQBAL & BHATTI, 2014; PUNZI et al., 2015). The results obtained in this study showed that the decolorization products reached a lethal concentration (LC50) at 17.45%. However, in assays containing only the Orange G dye at different concentrations, no mortality of A. salina was observed. These results suggest that the cleavage of the chromophore group produced toxic intermediates (PUVANESWARI et al., 2006). It is common to see the reports about To confirm the above results, toxicity tests were performed using onions because they are a good indicator of cytotoxicity, genotoxicity, and Biosci. J., Uberlândia, v. 33, n. 5, p. 1340-1350, Sept./Oct. 2017 1346 Aerobic decolorization... ALCÂNTARA, T. A. P. et al ALCÂNTARA, T. A. P. et al Aerobic decolorization... crescimento celular foram: concentração do Alaranjado G (100 a 500 ppm), temperatura (20 a 40 ºC), concentração de glicose (0 a 5%) e pH inicial (3 a 8). Os testes de biotoxicidade foram realizados utilizando o microcrustáceo Artemia salina para determinar a concentração letal (L50) e bulbos de cebola (Alium cepa) para determinar os efeitos citotóxicos e genotóxicos tanto do corante alaranjado G quanto dos metabólitos produzidos após a descoloração. Uma taxa de descoloração acima de 90% foi atingida a 500 ppm por C. cylindracea SJL6 a 30 ºC, pH 3 e 1% de glicose. Entretanto, os testes de biotoxicidade mostraram que ocorreu um aumento da toxicidade após a descoloração, o que sugere uma degradação parcial da molécula do corante Alaranjado G e produção de metabólitos tóxicos. Biosci. J., Uberlândia, v. 33, n. 5, p. 1340-1350, Sept./Oct. 2017 ACKNOWLEDGEMENTS The Orange G decolorization rate by Bacillus megaterium was above 94.5% (TRIPATHI; SRIVASTAVA, 2014). However, the metabolites produced were identified as naphthalene, aldehyde derivative compounds, and phenylhydrazine, which are known for their toxicity by oral, dermal, and The authors are grateful to CAPES and FAPESB for financial support for the realization of this work. RESUMO: A biodescoloração do corante Alaranjado G foi investigada utilizando um novo isolado de Candida cylindracea SJL6, isolado de amostras de água do Rio Subaé, Bahia, Brasil. A linhagem SJL6 foi identificada como Candida cylindracea com base na região 26S do rDNA. Os parâmetros estudados na descoloração do corante e Biosci. J., Uberlândia, v. 33, n. 5, p. 1340-1350, Sept./Oct. 2017 1347 REFERENCES AKSU, Z.; DÖNMEZ, G. A comparative study on the biosorption characteristics of some yeasts for Remazol Blue reactive dye. 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https://openalex.org/W2626319710	https://www.nature.com/articles/s41533-017-0038-6.pdf	English	null	Why do physicians lack engagement with smoking cessation treatment in their COPD patients? A multinational qualitative study	Npj primary care respiratory medicine	2,017	cc-by	6,437	1Department of Family Medicine, Maastricht University Medical Centre, CAPHRI School for Public Health and Primary Care, Maastricht, The Netherlands; 2Department of Community Medicine, General Practice Research Unit, UiT The Arctic University of Tromsø, Tromsø, Norway; 3Centre for Family and Community Medicine, Division of Public Health, Faculty of Health Sciences, Medical University of Lodz, Lodz, Poland; 4Department of Family Medicine, Northern State Medical University, Arkhangelsk, Russia; 5Division of Population Medicine, Institute of Primary Care and Public Health, Cardiff University, Cardiff, UK; 6Department of General Practice, University Medical Centre, Rostock, Germany and 7Institute of General Practice, Addiction Research and Clinical Epidemiology Unit, Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany Correspondence: Eva Anne Marije van Eerd (eva.vaneerd@maastrichtuniversity.nl) ARTICLE OPEN Why do physicians lack engagement with smoking cessation treatment in their COPD patients? A multinational qualitative t d Eva Anne Marije van Eerd1, Mette Bech Risør2, Mark Spigt1,2, Maciek Godycki-Cwirko3, Elena Andreeva4, Nick Francis5, Anja Wollny6, Hasse Melbye2, Onno van Schayck1 and Daniel Kotz1,7 Smoking cessation is the only effective intervention to slow down the accelerated decline in lung function in smokers with chronic obstructive pulmonary disease. Nevertheless, physicians often do not routinely provide evidence-based smoking cessation treatment to their patients. To understand underlying reasons, we explored how physicians engage in smoking cessation treatment in their chronic obstructive pulmonary disease patients. In total, 21 focus group discussions were held with general practitioners and pulmonologists in seven different countries in Europe and Asia. We generated three themes, whereby some of the issues concerned smokers in general: ﬁrst, ‘physicians’ frustration with chronic obstructive pulmonary disease patients who smoke’. These frustrations interfered with the provision of evidence-based treatment and could result in this group of patients being treated unequally. Second: ‘physicians’ limited knowledge of, and negative beliefs about, smoking cessation treatment’. This hindered treating smokers effectively. Third: ‘healthcare organisational factors that inﬂuence the use of smoking cessation treatments’. Money and time issues, as well as the failure to regard smoking as a disease, inﬂuenced how physicians engaged in smoking cessation treatment. Our results indicate that there is a number of barriers to the provision of effective smoking cessation treatment in patients with chronic obstructive pulmonary disease and smokers in general. Introducing an informative smoking cessation programme, including communication skills and ethical issues, in the vocational and postgraduate medical training may help to address these barriers. This is important in order to increase engagement with smoking cessation treatment and to improve quality of chronic obstructive pulmonary disease care. npj Primary Care Respiratory Medicine (2017) 27:41 ; doi:10.1038/s41533-017-0038-6 www.nature.com/npjpcrm Received: 1 November 2016 Revised: 4 May 2017 Accepted: 10 May 2017 RESULTS Physicians’ frustration with COPD patients who smoke Many physicians shared negative feelings towards patients with COPD who continued smoking. This seems to hinder them in providing evidence-based treatment, including smoking cessation treatment. Physicians’ limited knowledge of, and negative beliefs about, smoking cessation treatment Physicians seemed to feel responsible for motivating patients to quit smoking. Many of the physicians felt they lacked experience and conﬁdence in helping patients to stop smoking. Some also questioned the effectiveness of smoking cessation treatments. In the discussion of this, the physicians seemed inclined to switch from COPD-speciﬁc topics to topics regarding the general smoking population. ‘Yes, make them, make them to wish to stop smoking (laughs a bit) is what we should do.’ [FGD 1, Norway, GP] Several discussions focused on how exhausting it is for physicians to spend their valuable time on COPD patients and to prescribe expensive medication to relieve symptoms. Never- theless, many physicians seemed to conﬂate the importance of supporting patients to quit smoking with a personal desire to try and make them stop. They reported frustration regarding patients who demanded treatment but refused to accept responsibility for the treatment themselves. Even though all the physicians emphasised that smoking cessation is the most essential treatment for smokers with COPD, some of them questioned whether this also applied to particular groups of patients, e.g., late stages of COPD. ‘No, it has been proven that if you do not smoke you are better off; if you quit at a young age you are better off… but those people who are at a certain stage of COPD …those data are not convincing.’ [FGD 3, Netherlands, pulmonologist] (There are studies that show that also smokers in severe and very severe stages of COPD beneﬁt from quitting smoking in terms of their quality of life and lung function17, 18) ‘They [COPD patients that smoke] are a very difﬁcult patient group, especially those that are just on the cusp of needing long term oxygen and you’re trying to convince them that you’re not going to give it to them if they don’t stop smoking … it’s very frustrating if they still persist…’ [FGD 3, Wales, Pulmonologist] Physicians from the Netherlands, Germany and Norway expressed the view that there were COPD patients who did not necessarily feel better after quitting smoking. INTRODUCTION patients still continue smoking even though quitting is their best treatment option. Chronic obstructive pulmonary disease (COPD) is a major public and individual health problem. Experts think that, by 2020, COPD will rank ﬁfth worldwide in terms of burden of disease and third in terms of mortality.1 Higher mortality rates and an accelerated decline in lung function are seen in patients with COPD who continue smoking.1 Smoking cessation is therefore the most important treatment for patients with COPD, as it is the only evidence-based intervention which has been proven to slow down the accelerated decline in lung function.1 Physicians play a very important role in providing effective smoking cessation treatment for these patients. However, literature shows that they do not routinely deal with smoking cessation during their consultations with smokers.6–8 According to a large European review, several factors seem to inﬂuence physicians’ engagement in smoking cessation: patient character- istics; physician-characteristics, including attitude and cessation- speciﬁc knowledge and skills; structural factors, including reim- bursement and time required.8 A different review found that some physicians had negative thoughts about smoking cessation treatment as they considered it time-consuming and ineffective, and they lacked conﬁdence in their ability to discuss smoking cessation with their patients.9 To promote smoking cessation, international COPD guidelines include recommendations for effective smoking cessation inter- ventions.1, 2 They state, in accordance with the Cochrane review on smoking cessation in patients with COPD, that a combination of behavioural and pharmacological interventions is superior to any other treatment.3 Nevertheless, the prevalence of smoking in patients with COPD is still high and there is evidence that it exceeds the rate of smoking in the general population.4 There is only limited evidence that patients get motivated to quit on receiving a diagnosis of COPD.5 This implies that many COPD To our knowledge, no attention has been given in the literature speciﬁcally to physicians’ views on smoking cessation treatment for smokers with COPD. Whereas, for this group of smokers, it is more urgent that they quit smoking, while it is even more difﬁcult due to their higher addiction levels and higher susceptibility to developing depressive symptoms.10–12 There are studies exploring Published in partnership with Primary Care Respiratory Society UK Physicians’ engagement in smoking cessation treatment EAM van Eerd et al. 2 ‘I switched to ‘light’ cigarettes’ and, ‘Doctor, I’ve quit! I have not smoked for three days!’ He smoked for 50 years before’. INTRODUCTION [FGD 2, Russia, pulmonologists] experiences with COPD care in general, but these studies do not speciﬁcally address smoking cessation.13–16 From these studies, it is apparent that engagement in COPD care is inﬂuenced by physician-related factors such as perceived low self-efﬁcacy in the provision of treatment and lack of conﬁdence in the effects of treatments.13, 16 Also, patient-related factors such as being non- compliant and not taking responsibility for their condition were barriers for carrying out COPD care.15 Furthermore, awareness of available treatments and support for patients with COPD, as well as the time required for such treatments seemed to create organisational barriers.14 It is not clear whether these factors also hinder smoking cessation care in patients with COPD or if there are different issues in this speciﬁc population. Eventually, all these frustrations resulted in a general feeling of dissatisfaction with regards to supporting COPD patients with quitting smoking. ‘…it is almost like tilting against windmills.’ [FGD 3, Poland, pulmonologist] These negative experiences with smokers with COPD seemed to contribute to physicians being prejudiced against smokers with COPD. Some physicians discussed the issue that these prejudge- ments provoke the risk of treating smokers with COPD differently. Some said that they found smokers with COPD ‘nonsensical’ and it was their own fault they had COPD, and were more reluctant to prescribe expensive medication. Another physician said that if a COPD patient had quit smoking ‘he has a plus in our view and we should take care of him’ [FGD 1, Poland, GP]. The aim of this study was therefore to explore how physicians engage in smoking cessation treatment for their COPD patients, by exploring their attitudes, reported practice and experienced problems in this regard. Once researchers and physicians understand speciﬁc needs and concerns regarding utilising cessation treatments for this particular group of smokers, they may address these problems in order to increase smoking cessation in COPD patients. In conclusion, these physicians felt responsible for ‘making’ COPD patients quit smoking. However, they experienced many patient-related barriers which frustrated them. These frustrations provoked the risk of developing negative feelings about this group of patients, and subsequently treating them unequally and potentially depriving them of the most effective treatments. Healthcare organisational factors that inﬂuence the use of smoking cessation treatments Healthcare organisational factors that inﬂuence the use of smoking cessation treatments Interpretation of ﬁndings in relation to previously published work Physicians experienced frustration when working with smokers with COPD, as these patients were perceived as not taking responsibility for their disease. Present societal norms emphasise personal responsibility for one’s health21 and physicians expect their patients to act in the interest of successful health maintenance.22 Smokers with COPD fall short of these expecta- tions as their disease has been caused by their own smoking behaviour, and many do not act to stop the progression of their disease by quitting. In this societal context, smokers with COPD feel stigmatised and they might be hesitant to contact their physicians because they fear criticism21 and become irritated by their advice to stop smoking.23 However, research shows the vast majority of smokers are happy for their physician to raise the topic of smoking, if the advice is given in an empathetic and patient- centred manner.23–25 In order to create an environment with less frustration, it is important that physicians contribute to reducing the negative effect of stigma,21 as this interferes with the physician-patient relationship. In our view, some physicians need to develop a less paternalistic style, and create room for the smokers’ own views.12, 21 In this theme, the inﬂuence of health care organisational factors on smoking cessation treatment is discussed. In these discussions, the physicians broadened their views to include issues related to smoking cessation in general. Smoking cessation treatment, in most countries, did not appear to be common practice in COPD care. Physicians from the Netherlands, Wales, Germany and Hong Kong reported lack of time and ﬁnancial reimbursement as important obstacles for providing smoking cessation treatment. ‘It [quitting smoking] is part of the questionnaire, it is one of the items on the list you need to cover… but it is so time- consuming.’ [FGD 1, Netherlands, GP] The physicians in the Netherlands felt there was not sufﬁcient time in the COPD check-up appointment to discuss smoking cessation. In Germany, they grumbled about the price/quality ratio of the existing smoking cessation treatments. ‘It’s not economical but you shouldn’t lose your enthusiasm there.’ [FGD 3, Germany, pulmonologist] Many physicians reported limited knowledge, conﬁdence and skills regarding evidence-based smoking cessation treatments. RESULTS It was difﬁcult for them to explain the beneﬁts of quitting to their patients without the prospect of a positive result. Other frustrations that hindered physicians in working with smokers with COPD were difﬁculties in communication. Physicians reported that some patients no longer attended surgery because they do not like hearing about smoking cessation or feel guilty about their smoking behaviour. Furthermore, several physicians stated that they were inexper- ienced in providing smoking cessation treatment. They reported ﬁnding it hard to talk to patients about smoking habits, regarding it as a delicate subject. They would also like to have tools for the promotion of smoking cessation, such as tools for communication and patient education. This was one of the topics of discussion about smoking cessation in general and not only about smokers with COPD. ‘It’s difﬁcult to treat and handle this group of patients, right? You need to strongly motivate them in order to [make them stop smoking] … if you press them too much you will reject them; if you press them too hard to make them stop smoking you will make them your enemies: that’s the ﬁne line.’ [FGD 1, Norway, GP] ‘We need resources on creating an atmosphere and educating patients…in order to increase their motivation to quit smoking and increase the success rate…’ [FGD 3, Hong Kong, GP] Some also believed patients were ‘lying’ about their smoking behaviour. Others reported that COPD patients, when questioned about their smoking behaviour, consciously or unconsciously, trivialized their smoking behaviour. Besides Hong-Kong, Norway and Russia in particular brought up this issue. In Russia, they also discussed the effectiveness of Published in partnership with Primary Care Respiratory Society UK npj Primary Care Respiratory Medicine (2017) 41 Physicians’ engagement in smoking cessation treatment EAM van Eerd et al. Physicians’ engagement in smoking cessation treatment EAM van Eerd et al. 3 ‘Every alcoholic receives a detox, but a nicotine patient has to pay for his addiction himself. And this isn’t fair as both are partly recognised as addictions.’ [FGD 3, Germany, GP] pharmacotherapy in the general smoking population. In Germany, there seemed to be a lack of trust in nicotine replacement therapy (NRT). Physicians in both Russia and Germany discussed the view that these aids would only delay quitting and not cure these patients (NRT has been proven to improve long-term abstinence from tobacco19). RESULTS In addition, smoking cessation medication was said to be very expensive and therefore not widely used. In Germany, the physicians concluded that the most important element in smoking cessation is the communication with the patient. Apparently, in some countries, insurance companies and other important institutions in healthcare organisation consider smok- ing addiction to be different from other addictions. They do not provide the same facilities for smokers as for other addicted patients, such as alcoholics, whilst smoking is ofﬁcially recognised as a drug-related illness by addiction treatment care. ‘And the communicative intervention, so to say, is more successful [in motivating quitting] than, for instance, a nicotine patch.’ [FGD 3, Germany, pulmonologist] Published in partnership with Primary Care Respiratory Society UK npj Primary Care Respiratory Medicine (2017) 41 Main ﬁndings In the focus group discussions (FGDs), the physicians mentioned patient-related, physician-related and healthcare organisational factors that hindered the routine provision of evidence-based smoking cessation treatment to patients with COPD. Some of the issues were not speciﬁc for smokers with COPD, but concerned smokers in general. Firstly, physicians experienced great frustra- tion regarding smokers with COPD. In their view, smokers with COPD tended not to accept responsibility for their treatment; often were not honest about their smoking habits; trivialized the consequences of smoking and avoided health care visits. These frustrations provoked the risk of developing negative feelings about this group of patients, and treating them unequally by depriving them of effective treatments. Secondly, physicians lacked experience with different smoking cessation treatments. Thirdly, money and time issues, but also the failure to regard smoking as a disease, inﬂuenced physicians’ treatment of smokers with COPD and also smokers in general. Besides these general discussions, there were some conversa- tions that focussed more on smokers with COPD. The tenor of these discussions was speculative about communication, educa- tional and social tools for motivating COPD patients to quit. For example, some physicians thought it might help to dramatise and scare the patient and they would like to have tools to illustrate the harm smoking does to your lungs (Confronting smokers with undetected COPD in a randomised controlled trial did not increase the rate of smoking cessation20; however, in a recent cohort study, a new diagnosis of COPD seemed to be linked to smoking cessation5). Furthermore, social inﬂuences were discussed as motivational tool: for example, a story was told about a grand- father who quit smoking when his granddaughter said he was not allowed to die yet. Data collection We used FGDs to explore how physicians comment on their engagement in smoking cessation treatment for COPD patients. A FGD design was found suitable as FGDs serve to create data within a group setting, and we wished to take advantage of the different inputs from physicians and their co-construction of meaning, rather than relying on individual interview data. Further, FGDs have the potential to bring forth and discuss different spontaneous viewpoints in relatively understudied domains, such as this domain.36, 37 We performed a secondary analysis together with researchers who had held the FGDs for the primary research. The FGDs were held in seven different countries in 2011–2012: Wales, the Netherlands, Poland, Russia, Germany, Norway and Hong Kong. These countries were selected due to earlier research collaboration on respiratory diseases. The FGDs were originally designed to explore general practitioners’ (GPs) and pulmonologists’ views and concerns on the management of COPD exacerbations in general.15, 38 Strengths and limitations of this study The primary dataset that we used was extensive, therefore the strength of this study is to be found in the participation of several countries in order to ﬁnd common concerns regarding smoking cessation in different healthcare settings. We did not aim for a comparative study as a strict comparison between countries could not be made due to the plethora of information and scattered quotes on smoking. Furthermore, both GPs and pulmonologists attended the FGDs. This gave a fuller picture of physicians’ concerns regarding smoking cessation. In theme three, we brieﬂy mention the importance of the different healthcare contexts for the interpretation and analysis of the information. A weakness in this study is that we were not able to specify exactly what role the different health contexts played for the construction of the concerns. Analysis of the data was done together with two researchers that participated in the primary data collection and analysis, and was proof read by all representatives from the different countries that participated. One other limitation is that the FGDs were originally designed to explore GPs’ and pulmonol- ogists’ views and concerns on the management of COPD exacerbations in general. Smoking cessation is, of course, very important herein, but was not the main focus of the FGDs. On the other hand, this concomitantly resulted in important gut reactions on smoking related issues. Healthcare organisational factors that inﬂuence the use of smoking cessation treatments A Europe-wide literature review demonstrated that the proportion of GPs that offer intensive smoking cessation interventions is low and that one of the reasons for this was limited knowledge and skills.8 This is conﬁrmed by studies examining risk factor management and COPD management in general.14, 26 In national and international guidelines, there are overviews of the different effective smoking cessation options,27, 28 but literature has shown that physicians are not always familiar with these guidelines.13 Several factors can inﬂuence physicians’ adherence to guidelines, such as physicians’ view of their role in COPD care and how complex certain care is.14 In this study, we have seen that many physicians do not have faith in the guideline recommendations. The time spent on smoking cessation treatments is not recovered, as only a few people eventually quit smoking. In Russia and Germany, they discussed the importance of how smokers are ‘labelled’. This was not a COPD-speciﬁc discussion but concerned all smokers. ‘I consider that for some people, smoking is a disease, the same as alcohol dependency, for example.’ [FGD 3, Russia, GP] In line with this, they discussed the availability and reimburse- ment of facilities for smoking cessation and compared it to facilities for other addictions in Germany. Published in partnership with Primary Care Respiratory Society UK Physicians’ engagement in smoking cessation treatment EAM van Eerd et al. 4 Besides, the physicians mentioned that they thought commu- nication was more important than prescribing pharmacotherapy and that it is difﬁcult to familiarise oneself with communicative tools via theory. In order to increase skills and knowledge, it seems important to provide a smoking cessation programme in vocational training and postgraduate smoking cessation training for GPs. In this training, physicians should be taught evidence- based smoking cessation treatment and practise communication, besides reﬂection on personal norms and values. Implications for future research, policy and practice Implications for future research, policy and practice To promote physicians’ engagement in smoking cessation treatment, we advise introducing an informative smoking cessa- tion programme, including communication and ethic, into the vocational training and postgraduate medical training. The attachment of stigma should be considered in this training and how this interferes with giving professional smoking cessation support. In addition evidence-based smoking cessation treatment should be taught and communication should be practiced. Healthcare organisational factors that inﬂuence the use of smoking cessation treatments For future research, it would be interesting to take a more detailed look into the hypothesis generating results of this analysis including the inﬂuence of different healthcare systems on how smoking cessation treatment is performed. This will inform us about which elements are essential for performing effective smoking cessation treatment. p Healthcare organisational factors inﬂuence how physicians engage in COPD care and smoking cessation. Each of the seven countries involved in this study organises COPD care in a different way. The Netherlands,29 Germany,30 Wales31 and Norway32 have the most structured COPD care. In Poland,33 Russia and Hong- Kong, the disease management programs seem to be less established. Even with structured care, however, there are several issues that complicate providing evidence-based smoking cessa- tion treatment. Lack of time is often mentioned in the literature as a barrier to giving smoking cessation treatment.8, 9 In this study, the physicians further complained that the ﬁnancial reward is insufﬁcient. To our knowledge, there are no studies on the differences in outcomes when comparing the different healthcare systems. We can, however, see that in the Netherlands, Germany, Wales, Norway and Poland, the smoking rate is between 20–26%.34 In Russia, this number is much higher: 37.3% of people over 15 years of age smoke.34 In China, almost 50% of men smoke, but almost no women.34 It would be interesting to know if the way the healthcare system is organised inﬂuences smoking cessation, especially for smokers with COPD who, even at the best of times, experience difﬁculties in quitting.35 METHODS p q g As smoking is ofﬁcially recognised as an illness in addiction treatment care, it is interesting to note that in none of the countries rehabilitation programs for smoking are offered in the same way as rehabilitation from other drugs. This shows that there is still a stigma attached to smoking that considers it to be different from other addictions. If smoking is seen as an addiction such as with other drug addictions, then structured nicotine dishabituation programmes might be established and reimbursed. CONCLUSIONS Our results show physicians’ barriers to providing effective smoking cessation treatment in patients with COPD and smokers in general. This is inﬂuenced by general issues and attitudes regarding smoking cessation treatment and the management of patients with COPD. We saw a lot of frustration, inexperience and stigmatisation in physicians regarding smoking cessation in their COPD patients and in the general smoking population. Also, money and time issues hindered the physicians. It is important to increase engagement with smoking cessation treatment and thereby improve quality of COPD care. Published in partnership with Primary Care Respiratory Society UK npj Primary Care Respiratory Medicine (2017) 41 Analysis 4. Shahab, L., Jarvis, M. J., Britton, J. & West, R. Prevalence, diagnosis and relation to tobacco dependence of chronic obstructive pulmonary disease in a nationally representative population sample. Thorax 61, 1043–1047 (2006). The available data were then analysed using an inductive thematic analysis40 and we coded the text without initially relating it to the aim of the study. As there were no predeﬁned categories, this enabled the evolution of different categories through the coding process. E.E. formulated codes for all text that was linked to smoking and smoking cessation. Codes were then discussed in detail and sorted into categories and subcategories (E.E., M.B.R.). For example, ‘patients not open to physicians’ smoking cessation advice’ was a subcategory of ‘patients who do not quit’. The codes were then discussed within the research team (M.B. R., M.S., D.K.) and the essential contents of the codes were divided into three candidate themes related to the overall aim. Simultaneously, the ﬁnal aim of the study was discussed within the team (O.V.S., M.B.R.) and the themes could then be ﬁnalised. This process resulted in three themes: (1) ‘physicians’ frustration with COPD patients who smoke’; (2) ‘physicians’ limited knowledge of, and negative beliefs about, smoking cessation treatment’; (3) ‘healthcare organisational factors that inﬂuence the use of smoking cessation treatments’. E.E. checked to ensure that the resulting thematic map accurately reﬂected the meaning of units evident in the dataset as a whole.40 5. Danielsen, S. E., Lochen, M. L., Medbo, A., Vold, M. L. & Melbye, H. A new diagnosis of asthma or COPD is linked to smoking cessation - the Tromso study. Int. J. Chron. Obstruct. Pulmon. Dis. 11, 1453–1458 (2016). 6. Coleman, T., Wynn, A., Barrett, S. & Wilson, A. Discussion of NRT and other anti- smoking interventions in UK general practitioners’ routine consultations. Nicotine Tob. Res. 5, 163–168 (2003). 7. Ellerbeck, E. F., Ahluwalia, J. S., Jolicoeur, D. G., Gladden, J. & Mosier, M. C. Direct observation of smoking cessation activities in primary care practice. J. Fam. Pract. 50, 688–693 (2001). 8. Stead, M., Angus, K., Holme, I., Cohen, D. & Tait, G. Factors inﬂuencing European GPs’ engagement in smoking cessation: a multi-country literature review. Br. J. Gen. Pract. 59, 682–690 (2009). 9. Vogt, F., Hall, S. & Marteau, T. M. General practitioners’ and family physicians’ negative beliefs and attitudes towards discussing smoking cessation with patients: a systematic review. Addiction 100, 1423–1431 (2005). 10. REFERENCES 1. Global Initiative for Chronic Obstructive Lung Disease (GOLD). Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease. http://www.goldcopd.org/. Accessed 20 April 2016. 2. National Clinical Guideline Centre. Chronic obstructive pulmonary disease: management of chronic obstructive pulmonary disease in adult in primary and secondary care. http://guidance.nice.org.uk/CG101/Guidance/pdf/English. Accessed 20 April 2016. 3. van Eerd E. A., van der Meer R. M., van Schayck O. C., Kotz D. Smoking cessation for people with chronic obstructive pulmonary disease. Cochrane Database Syst. Rev. (8), CD010744 (2016). Analysis Tonnesen, P. et al. Smoking cessation in patients with respiratory diseases: a high priority, integral component of therapy. Eur. Respir. J. 29, 390–417 (2007). 11. van Eerd, E. A. et al. Do we need tailored smoking cessation interventions for smokers with COPD? A comparative study of smokers with and without COPD regarding factors associated with tobacco smoking. Respiration 90, 211–219 (2015). Data collection This means that the results are reﬂections of most discussed concerns and we see them as hypothesis generating for future in-depth research in this ﬁeld. g Each country performed three FGDs with 4–7 participants considering that the chosen participants and the number of FGDs would provide sufﬁcient information to answer the original study questions. The number of participants in each FGD should ideally be between 6–8 as often recommended,39 but due to cancellations the number differed in the end. For each FGD new participants were recruited: FGD 1 with only GPs; FGD 2 with only pulmonologists; FGD 3 with a mix of GPs and pulmonologists. In FGD 1 and 2, GPs and pulmonologists got the chance to discuss issues from the viewpoint of their discipline without being interfered by the other discipline. In FGD 3 these issues could then be combined in an interdisciplinary discussion. The GPs and pulmonologists were sampled in order to cover both rural and urban GP practices, as well as private- based and hospital-based pulmonologists.15, 38 All researchers who were responsible for conducting the FGDs participated in a 3-day workshop in order to streamline the methods across countries. All countries used the same interview guide containing topics based on known issues of concern to GPs. The interview guide used in FGD 1 was revised before FGD 2 due to intermediary analysis and this revised version with speciﬁc sub questions was used in FGD 2. The pulmonologists were asked to discuss the routines in general practice as they were known to them, but inviting them to be open about their own views and concerns. Between FGD 2 and 3 another revision of the interview guide was made in order to make FGD 3 focus especially on topics that were still unclear but also on solutions to identiﬁed problems. Published in partnership with Primary Care Respiratory Society UK npj Primary Care Respiratory Medicine (2017) 41 Physicians’ engagement in smoking cessation treatment EAM van Eerd et al. Physicians’ engagement in smoking cessation treatment EAM van Eerd et al. 5 Ethical considerations Methods were performed in accordance with relevant regulations and guidelines. Relevant ethical approval was obtained from the appropriate bodies in the participating countries; South East Wales Research Ethics Committee Panel (Wales), Medical Ethical Review Committee of University Hospital Maastricht/Maastricht University (The Netherlands), Ethics Com- mittee of Northern State Medical University (Russia). Participants provided written informed consent. Identiﬁers were removed from the transcriptions locally prior to distribution among the network. 12. van Eerd E. A. M., Risor M. B., van Rossem C. R., van Schayck O. C. P., Kotz D. Experiences of tobacco smoking and quitting in smokers with and without chronic obstructive pulmonary disease-a qualitative analysis. BMC Fam. Pract. 16, 164 (2015). 13. Salinas, G. D. et al. Barriers to adherence to chronic obstructive pulmonary dis- ease guidelines by primary care physicians. Int. J. Chron. Obstruct. Pulmon. Dis. 6, 171–179 (2011). 14. Johnston, K. N., Young, M., Grimmer-Somers, K. A., Antic, R. & Frith, P. A. Why are some evidence-based care recommendations in chronic obstructive pulmonary disease better implemented than others? Perspectives of medical practitioners. Int. J. Chron. Obstruct. Pulmon. Dis. 6, 659–667 (2011). Data availability The data that support the ﬁndings of this study are available from the corresponding author upon reasonable request and with permission of the lead investigator (HM). Int. J. Chron. Obstruct. Pulmon. Dis. 6, 659–667 (2011). 15. Risør M. B., Spigt M., Iversen R. et al. The complexity of managing COPD exacerbations: a grounded theory study of European general practice. BMJ Open 3, e003861 (2013). 16. Perez, X., Wisnivesky, J. P., Lurslurchachai, L., Kleinman, L. C. & Kronish, I. M. Barriers to adherence to COPD guidelines among primary care providers. Respir. Med. 106, 374–381 (2012). Data assessment To address the additional research question for this secondary analysis, we assessed the quality of the available dataset and discussed whether the existing dataset had the potential to inform us about physicians’ engagement in smoking cessation. All transcripts were reviewed by E.E. in order to discover whether they were relevant for this secondary analysis. This was then discussed within the research team (M.B.R., M.S., D.K.) and it was agreed that the dataset as a whole offered rich data on physicians’ engagement in smoking cessation. Most of the data for the secondary analysis arose from questions in all FGDs about difﬁcult COPD patients. FGD 1 and 2 included a question asking, ‘Could you describe speciﬁcally challenging or difﬁcult clinical situations with COPD-patients in general? ’ FGD 3 included a question asking, ‘Who are the difﬁcult patients and how do you reach them? What do you think are the key issues/bottlenecks regarding the difﬁcult patients? ’ These questions led to discussions about smoking and smoking cessation in COPD patients. Furthermore, we found valuable information in discussions on challenges when coping with patients with COPD; how to improve care and own views and concerns. Also, sections in which exacerbation management, self-management and guidelines were discussed provided interesting information. Information from FGD 3 contributed the most to this secondary analysis. AUTHOR CONTRIBUTIONS All FGDs were transcribed verbatim from audio recording by the local researchers and translated into English by a skilled translator. However, some inconsistencies in English language occurred and we show adjusted quotes in the results section with the original quotes in the attachment. For this analysis, the focus group transcripts were imported into Nvivo 10. E.A.M.V.E.: study conception, data assessment, data analysis, data interpretation and lead writing. M.B.R.: data collection, study conception, data interpretation and writing. M.S.: data collection, study conception, data interpretation, writing. M.G-C., E.A., N.F., A.W.: data collection and writing. H.M.: study conception of the original study and writing. O.V.S.: study conception, writing. D.K.: study conception, writing. ADDITIONAL INFORMATION Supplementary Information accompanies the paper on the npj Primary Care Respiratory Medicine website (doi:10.1038/s41533-017-0038-6). Supplementary Information accompanies the paper on the npj Primary Care Respiratory Medicine website (doi:10.1038/s41533-017-0038-6). Competing interests: The authors declare no competing ﬁnancial interests. Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Published in partnership with Primary Care Respiratory Society UK ACKNOWLEDGEMENTS This study is published on behalf of the PEXACO research team. Thanks to all those who arranged and participated in the focus groups. We thank Carol Herman for language checking. PEXACO was funded by the Norwegian Research Council, grant number 202650//H10. O.S. received institutional funds from Pﬁzer. 17. Chen, J. et al. Effectiveness of individual counseling for smoking cessation in smokers with chronic obstructive pulmonary disease and asymptomatic smokers. Exp. Ther. Med. 7, 716–720 (2014). npj Primary Care Respiratory Medicine (2017) 41 npj Primary Care Respiratory Medicine (2017) 41 Published in partnership with Primary Care Respiratory Society UK Physicians’ engagement in smoking cessation treatment EAM van Eerd et al. 6 33. Godycki-Cwirko, M. et al. 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The implementation of health care aimed at the cessation of tobacco use, treatment of tobacco dependence and consequences of tobacco consumption in the Russian Federation. Ration. Pharmacother. Cardiol 12, 403–413 (2016). 21. Halding, A. G., Wahl, A. & Heggdal, K. ‘Belonging’. ‘Patients’ experiences of social relationships during pulmonary rehabilitation. Disabil. Rehabil. 32, 1272–1280 (2010). consumption in the Russian Federation. Ration. Pharmacother. Cardiol 12, 403–413 (2016). 36. Kvale, S. B. S. Interviews: learning the craft of qualitative research interviewing. (SAGE Publications, 2009). 22. Dieterich, A. The modern patient-threat or promise? Physicians’ perspectives on patients’ changing attributes. Patient Educ. Couns. 67, 279–285 (2007). 37. Bourgeault I., Dingwall R., & de Vries R. The SAGE handbook of qualitative methods in health research. (SAGE Publications Ltd, 2010). 23. Butler, C. C., Pill, R. & Stott, N. C. 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https://openalex.org/W4293239185	https://ee.openlibhums.org/article/id/3331/download/pdf/	English	null	Expensive Health: Health-seeking Behaviours in Diversified Medical Markets	Ethnologia Europaea	2,022	cc-by	7,613	Tanja Bukovčan 2022: Expensive Health: Health-seeking Behaviours in Diversified Medical Markets. Ethnologia Europaea 52(2): 1–18. DOI: https://doi.org/10.16995/ee.3331 1 For Russia, see Temkina (2020); Borozdina & Novkunskaya (2022); Rivkin-Fish (2005); for Ukraine see Bazylevych (2009); for Poland see Węgrzynowska (2021); for Hungary see Rubashkin et al. (2021); for Lithuania see Praspali auskiene (2016); and for Serbia, see Pantović (2016). Expensive Health Expensive Health Health-seeking Behaviours in Diversified Medical Markets Tanja Bukovčan, University of Zagreb, Croatia, tbukovcan@ffzg.hr Tanja Bukovčan, University of Zagreb, Croatia, tbukovcan@ffzg.hr This article analyses how the metamorphosis of a state-funded healthcare system into a market- oriented system in Croatia since the 1990s has influenced the health-seeking behaviour of patients. Through in-depth interviews, patients were asked to identify their satisfaction with various health services and providers. Their answers reveal a complex narrative setup in which the possibility to select another healthcare therapy or provider was linked to their “willingness to pay”. The interview responses uncovered inequalities in the context of healthcare, as well as the politics and powers behind allocating and negotiating value in health-seeking. Ethnologia Europaea is a peer-reviewed open access journal published by the Open Library of Humanities. © 2022 The Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. See http://creativecommons.org/licenses/by/4.0/. OPEN ACCESS Ethnologia Europaea is a peer-reviewed open access journal published by the Open Library of Humanities. © 2022 The Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. See http://creativecommons.org/licenses/by/4.0/. OPEN ACCESS Ethnologia Europaea is a peer-reviewed open access journal published by the Open Library of Humanities. © 2022 The Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. See http://creativecommons.org/licenses/by/4.0/. OPEN ACCESS Ethnologia Europaea is a peer-reviewed open access journal published by the Open Library of Humanities. © 2022 The Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. See http://creativecommons.org/licenses/by/4.0/. OPEN ACCESS 2 “I could have died by then…” The difference? You ask me about the difference? (…) I can just call and get an appointment tomorrow, if I pay. But when I called the hospital, they told me they have an opening in April next year [laughter]. I could easily have died by then… A middle-aged woman in the waiting area of a private endocrinology clinic in the city of Zagreb explained the situation for getting healthcare. The clinic was situated on the outskirts of the city in a brand-new building, the waiting room was shining white and looked like an Ikea showroom. The woman in question had problems “with her thyroid, severe, cancer maybe” and I asked about her reasons for choosing private over public healthcare as part of my eleven-year-long research into medical pluralism and medical markets in Croatia. Based on patients’ narratives, this article analyses health-seeking behaviours in diversified medical markets in transition. In Croatia after the 1990s, these markets have followed the metamorphosis of the state-funded healthcare system into a market-oriented system. Given the joint historico-political moment of transition to post-socialism, processes of healthcare transformation in the context of shifting market conditions have been similar across many East European countries (Džakula et al. 2014; Faith 2007; Figureas et al. 2004; Silverman et al. 2019; Stepurko et al. 2014, 2015).1 Two authors doing research comparable to mine in former countries of the Soviet Union point out that “[o]ver the last three decades, free health care has shifted to a mix of public, private, and semi-private services influenced by neoliberal ideology” (Temkina & Rivkin-Fish 2020: 340). Common aspects across Eastern Europe include: the transformation of patients into consumers (Temkina 2020); the mix of private and public services (Węgrzynowska 2021); questions of shifting rights and responsibilities in healthcare with accompanying changes in power and trust (Temkina & Rivkin-Fish 2020; Bazylevych 2009); and problems linked to the existence of traditions of informal payments and corruption (Rivkin-Fish 2005; Praspaliauskiene 2016; Rubashkin et al. 2021). This article tackles related questions, but draws its analytical power from the viewpoints of the patients themselves, and seeks to follow their pathway selection processes in healthcare decision-making (Khan, Pintelon & Martin 2022). The article will contribute to the wider discussion on the aspects and implications of patient-centred healthcare that Ethnologia Europaea 3 has been present in research on health and healthcare since the 2000s (cf. Anderson 2002; Reynolds 2009; Zandbelt et al. 2007). g 3 My broader study started in 2010 and included 112 individuals in total. Related publications: Tanja Bukovčan 2010: Nije za one slabih živaca – pitanja etičnog i moralnog relativizma u medicinskim etnografijama [Only for those with nerves out of steel – the issue of ethical and moral relativism in medical ethnographies]. Etnološka tribina: Godišnjak Hrvatskog etnološkog društva 40(33): 45–56; Tanja Bukovčan 2007: Advocacy and Activism in Complementary and Alternative Medicine Research – a Croatian Example. Anthropology in Action 15(1): 35–42; Tanja Bukovčan 2013: Through an Hourglass: Re-thinking Ethnography of the Relationship between Biomedicine and Complementary and Alternative Medicine in Croatia. Etnološka tribina: Godišnjak Hrvatskog etnološkog društva 43(36): 90–98. “I could have died by then…” Newly available possibilities to make individual healthcare choices require both financial and treatment rationales on the part of patients. The article asks: almost two hundred years after the birth of public health and the development of equity of access to healthcare into a supposedly universal human right2, what are the factors and specific cultural-political contexts that have made patients “willing to pay” (Donaldson 2001), and accept a “fee-for-service” system (Bazylevych 2009)? Since post-socialism is still widely used as a common term for a period, an experience, and a context, this article remains duly cautious in reaching broader conclusions in terms of post-socialist healthcare transformations. In particular, the Yugoslav healthcare system, initially set up by one of the founders of the World Health Organization, Andrija Štampar, was specific, innovative, and modernistic in its approach not only in regional, but also in global terms (cf. Dugac 2005; Brown & Fee 2006). Štampar’s incentive to make science and medicine accessible to all (Dugac 2005) started as a state project in as early as the late 1920s and over the following decades resulted in a broad and effective network of day clinics, and healthcare reachable in many rural and remote regions of former Yugoslavia. It should be noted that narratives and attitudes about socialism in Croatia are highly diverse. Although the shared cultural-political context of post-socialism is well known and aspects of the processes of post-socialist healthcare transformation may be comparable, many researchers warn against overgeneralization of the whole concept of post-socialism (Velikonja 2009; Pehe 2014; Jelača, Kolanović & Lugarić 2017; Bailyn, Jelača & Lugarić 2018). 2 Oliver & Mossialos (2004); Greenhalgh (2018); Topp et al. (2018). Methodology, Research Sites and Participants The data presented here were analysed from in-depth interviews and the life histories of 37 individuals – 17 men and 20 women, with two young men in their 20s, five women in their 20s to 30s and others in their late 40s or older – who formed a segment of my broader research on medical pluralism in Zagreb, the capital of Croatia (with a population of approximately 1 million).3 Final interviews for this article were conducted Ethnologia Europaea 4 just before 2020, the year of the Covid-19 breakout, and hence it does not cover shifts in care-seeking behaviours caused by the pandemic. For the purpose of writing this article, I selected those 37 individuals from the larger research group who, in their search for a suitable therapy and cure for their mainly chronic diseases, shifted between public or private biomedicine providers on the one hand and complementary and alternative medicine on the other. My initial field sites were the waiting rooms of general practitioners, public hospitals, private clinics, and practitioners of complementary and alternative medicine (CAM). In-depth interviews were individual and conducted in a separate room if available on site, or at another location later. I interviewed some persons twice for additional clarity and content. Computer-assisted qualitative data analysis was done using the software program NVivo. Ethnologia Europaea Medical Markets and Healthcare Alternatives Before the 1990s, public, universal, and state-funded healthcare in Croatia was easily accessible and available. The first state-funded health insurance was institutionalized in 1947, and the first day clinic was opened in 1953 (Letica, Popović & Škrbić 1981). Universal health insurance coverage rose to a staggering 85 percent of the entire population in 1979 (Letica, Popović & Škrbić 1981). During the late 1970s and 1980s, budget commitment was relatively high, at 370 euro per capita for health insurance, with biomedicine then being several times cheaper than it is now, since “the fast development of biomedical technology significantly raised the costs borne by healthcare systems” (OECD 2015; cf. Sorenson, Drummond & Bhuiyan Khan 2013). The private healthcare sector prior to the 1990s was virtually non-existent. After that decade, however, healthcare shifted from being exclusively public to being semi-private and private (Temkina & Rivkin- Fish 2020). Today the total budget commitment for healthcare in Croatia has risen to 1,241 euro per capita, yet this is still under the EU average and amounts to 7.4 percent of the GDP as compared to the European average of 9.9 percent (Stašević, Derk & Ropac 2019; OECD & WHO 2017). Even more telling are the figures showing that over the last ten years in Croatia the percentage of the healthcare costs covered by public resources fell from 86 percent to 77 percent, while the percentage covered by private resources rose from 14 percent to 23 percent (Stašević, Derk & Ropac 2019). Of the percentage covered by private resources, 15 percent came from direct payments and 8 percent from private health insurance other than the mandatory insurance (Zrinščak 2007; OECD & WHO 2017). With the growing number of private practices and the burgeoning burden of public healthcare costs for the state, it would be safe to assume that this tendency will continue. In addition to private practices, another segment that was absent from Croatian pre-1990s healthcare was healthcare alternatives. Healthcare alternatives to Ethnologia Europaea Ethnologia Europaea 5 biomedicine were restricted to only two registered homeopathic practitioners actively working in pre-1990s’ Zagreb. However, by 2020, there were dozens of new therapies, providers, and clinics. In October of 2018 a new Clinic of Ayurvedic Medicine was opened in the centre of Zagreb, after a few already existing centres, and people wearing Balenciaga bags, very expensive brand items, showed up at the Clinic’s red-carpet grand opening. Medical Markets and Healthcare Alternatives Even though some south-eastern European countries since the 1990s have been trying to impose state control over the upsurge of the private health sector (for Greece see Kondilis et al. 2007), medical markets have continued to proliferate in Croatia. Hence, in the short span of thirty years, the decision-making processes of patients related to their health-seeking have changed significantly. As in Russia, neoliberal ideology has also played a role in affecting the patients’ attitudes (Temkina & Rivkin- Fish 2020). The notion of free choice was applied to medical markets after the 1990s. In the market economy, choosing therapies and treatments meant that patients had to estimate their curative potential and assess their value. “I want to get what I want for my money”, said one of the patients who was constantly switching between public and private biomedicine and CAM: “you can’t measure one hundred euros’ worth of health, of course, but you can surely try to help yourself and that’s exactly what I’m doing”. Emphasized in this account was the “I”, the notion that the choices made by patients to ensure better health were their own personal responsibility, something Artvinli Faith (2007) recognized in his analysis of health policy in Turkey: “the neo- liberal approach transforms basic concepts, defining access to health care as an issue of personal responsibility, not a public responsibility” (Faith 2007: 195). Neoliberalism does not, of course, cover only the health sector. However, when applied to individual health, to the health of a particular “I”, [t]his logic of choice turns the patient into a customer who is expected to choose the best way to take care of themselves. Staying healthy is then a choice, a clearly defined transaction in which something is exchanged, a product against a price. Medical Markets and Healthcare Alternatives (Alftberg & Hansson 2012: 417) One of the patients explained to me what could be described as a “formula” for cost- effectiveness over time that she used when assessing, calculating, and negotiating value in her health-seeking strategies: “This one [provider] is more expensive (…), but she might turn out cheaper in the end, if this helps (…).” Therefore, instead of the free public services of the past for which there were no alternatives, there has now appeared a plethora of choices which, as “clearly defined transactions” (Alftberg & One of the patients explained to me what could be described as a “formula” for cost- effectiveness over time that she used when assessing, calculating, and negotiating value in her health-seeking strategies: “This one [provider] is more expensive (…), but she might turn out cheaper in the end, if this helps (…).” Therefore, instead of the free public services of the past for which there were no alternatives, there has now appeared a plethora of choices which, as “clearly defined transactions” (Alftberg & Ethnologia Europaea Ethnologia Europaea 6 Hansson 2012), are rightfully payable. For most of the persons I interviewed, the fee- for-service system (Bazylevych 2009) has meant that good service is strongly implied in the fee. A Different Kind of Inequality The third point to be discussed here regarding shifts in the health-seeking behaviours of patients in the newly diversified medical market is the new distribution of inequality throughout the healthcare sector. Unsurprisingly, “willingness to pay (WTP) is associated with ability to pay” (Donaldson 2001: 186). The persons with whom I spoke were regularly using different healthcare therapies, providers, institutions and systems according to their own pathway selection processes in healthcare decision-making. Their high socioeconomic status was the precondition for having the possibility to exhibit diverse health-seeking behaviours and sift through cares and cures, goods and services. The cost of private healthcare is by no means low, given that the average monthly net income per capita in Croatia is around 900 euro.4 One visit to a private paediatrician in Croatia in 2020 could cost from 20 to 50 euro, a gynaecologist visit from 50 to 100, and a visit to a specialist, for example an endocrinologist, from 70 to 150 euro. General physical examinations in private clinics are also highly popular and can cost from 500 to 1,000 euro. In case of chronic illnesses or prolonged need for medical care, for example in case of pregnancy, the costs can rise significantly. Like Pantović’s research in Serbia showed (Pantović 2016), my research in Croatia found that some women also paid for private check-ups and treatments with a certain obstetrician/gynaecologist throughout their pregnancy. This specialist would be present at their delivery in the public hospital in which she or he was also working. Considering that the monthly costs of regular pregnancy check-ups could, on average, amount to 100–150 euro (significantly more in cases of high-risk pregnancies), it was evident that only new mothers with an extra 1,000 euro or more in their budget could afford such privileged treatment. Public healthcare was also, of course, paid through mandatory health insurance, but this came directly from one’s gross salary and people did not perceive it as payment. The very rare criticisms of paying mandatory health insurance in my data were ambiguous: “I also pay additionally [a] private insurer, but that’s my problem, I think it’s safer that way. But of course, I pay the mandatory… they take it from my salary every month.” Only 4 percent of the insured population, the employed, pay the full mandatory health insurance contribution of 15 percent (Vončina et al. 4 https://www.ceicdata.com/en/indicator/croatia/monthly-earnings (accessed October 15, 2021). Consumers beyond Health The second point I address in this article is the onset of consumer-like behaviour among patients (Temkina & Rivkin-Fish 2020), which has led to changes in their expectations and new logics behind their “willingness to pay” (Donaldson 2001). Positive aspects of private healthcare that the patients I spoke with primarily listed were “easy” and “quick access”. Instead of waiting “months” for diagnostic tests or check-up appointments at public hospitals, access to healthcare in the private sector costs money, of course, but was immediate and guaranteed. Many of those interviewed also positively reported the ways in which private practitioners and providers paid more attention and spent more time with them, as expressed by an elderly woman: You get there, report at the desk and you don’t even have time to take off your coat, they call your name. He [the doctor] spends all the time with you, doesn’t rush in and out, doesn’t talk on the phone, doesn’t yell to the nurse about other patients and things that have nothing to do with you … Many patients reported that the whole process and experience of going to private health providers was much cosier, “more human” or “more dignified”, with no cramped waiting rooms or long waiting hours. At the beginning of our interviews, patients would rarely mention medical advantages, but rather spoke about access, assets, convenience, accessibility, appearance, care, attention, time, comfort, or the quality and time-management of the service. From their explanations could be discerned a perceived association between convenience and better service on the one hand, and better medical care on the other. This finding is reminiscent of Cam Donaldson’s (2001) research in the field of health economics regarding the eliciting of patients’ values through the concept of “willingness to pay”: Some treatment alternatives may not differ in terms of health gain, but patients may still have preferences for one over the other based precisely on the need for something which “goes beyond health”. Examples of “going beyond health” are the preferences patients might have for location of care, the process of care (i.e. non-health-generating aspects of health-care delivery) and autonomy in care. (Donaldson 2001: 183) Ethnologia Europaea Ethnologia Europaea 7 The perceptions of expertise and efficient care among the patients I interviewed were similarly dependent on the factors and conditions which “went beyond health”. As consumers, they talked about choosing goods and services and paying for them. A Different Kind of Inequality 2018), which affects the quality and efficiency of healthcare system and poses Ethnologia Europaea Ethnologia Europaea 8 8 problems for its financial sustainability (ibid.). Hence, policy makers themselves are aware of the problems in terms of patients being given adequate quality in return for the regular monthly reductions from their salaries (ibid.). 5 https://zdravstvo.gov.hr/vijesti/ministarstvo-zdravlja-predstavilo-projekt-smanjenja-listi-cekanja-u-hrvatskim-bol nicama/92 (accessed April 14, 2021). Nostalgia for Imagined Equality Nostalgia and post-socialist legacies come together in broader conceptualizations of how experiences and practices of post-socialism work. However, just as with the concept of post-socialism itself, researchers have warned against an over-generalizing interpretation of the concept of post-socialist nostalgia (Pehe 2014), and against viewing it as yet another “umbrella term to describe a number of disparate and at times contradictory phenomena” (Pehe 2014: 6). Therefore, I have restricted the usage of the term nostalgia to Svetlana Boym’s (2001) interpretation. She sees nostalgia as having a utopian dimension, since its aim is neither directed towards the future, nor strictly towards the past (Boym 2001: 13). Nostalgia, therefore, continually offers new re-readings of both past and present (Boym 2001: 14). The need for new readings is expected in times of massive sociopolitical upheaval, which is why nostalgia is common in all post-socialist settings (Velikonja 2009; Mihelj 2017; Condrache 2020). Boym nonetheless insists that socialism is not the only historical period for which nostalgia has become a kind of coping mechanism (Boym 2001). In the context of healthcare settings in Croatia, the people I interviewed frequently told nostalgic narratives about the universally free healthcare of the past. The narratives were mostly told by members of older generations, that is, to those who were born before the 1990s and had personal experiences of life in Yugoslavia. As one man in his 50s explained: “Before, we could just enter a hospital and would get everything we needed. Today we can’t even get an appointment.” His comment refers to the long hospital waiting lists that are still identified as a pressing problem in the current “strategic plan” of the relevant Ministry of Health of the Republic of Croatia (MZRH, 2020–2022). In 2012 the Ministry initiated a pilot project to analyse the reasons for the long waiting lists and concluded that the main contributing factors were “poor organization of [the] healthcare system in general, [and] poorly defined responsibilities shared between the Ministry, Croatian Institute of Public Health and the hospital management”.5 However, the current “strategic plan” still outlines them as an unsolved issue. Problems such as these in public healthcare create opportunities for the private sector, which is now an integral part of the post-transition system of healthcare. 5 https://zdravstvo.gov.hr/vijesti/ministarstvo-zdravlja-predstavilo-projekt-smanjenja-listi-cekanja-u-hrvatskim-bol nicama/92 (accessed April 14, 2021). Ethnologia Europaea 9 9 Another frequently repeated nostalgic theme was a revisionist call for the status I have named equality-in-lesser-quality (EILQ). Nostalgia for Imagined Equality Equality-in-lesser-quality refers to the frequently expressed attitudes by patients that the general standard in hospitals was rather poor in pre-transition Croatia, as compared to today. These same patients, however, still praised the healthcare of the past as the better one. Hospitals were frequently described by patients as “shabby, overcrowded, in old Austro-Hungarian buildings, with bathing and toilet facilities on one floor”. Yet healthcare in this period was deemed efficient, accessible, and equally available to everyone. The idyllic narrative of all-encompassing health equity in the past is evident from statements like “even the homeless could simply walk into the hospital and have a surgery”. The old system of healthcare was usually praised by my interviewees, precisely because of the existing popular memory of equality and equity. According to one public health expert working at the Andrija Štampar School of Public Health: [The] Croatian healthcare system, although reformed, has retained some of its char acteristics from the period of self-management. Its legacy [of] self-management includes the powerful voice of the system’s users and employees, and the strong belief by the public that medical services should be free at the point of use with little regard for the cost of care provided. (Džakula et al. 2007: 119)6 Thus, it seems that the desirable perception of equality-in-lesser-quality was centred on a paradoxical nostalgia aimed at a past (Boym 2001: 14) that was reportedly “worse” in most aspects but carried within itself a utopian promise of a better future that is inaccessible today (Boym 2001: 14). Thus, it seems that the desirable perception of equality-in-lesser-quality was centred on a paradoxical nostalgia aimed at a past (Boym 2001: 14) that was reportedly “worse” in most aspects but carried within itself a utopian promise of a better future that is inaccessible today (Boym 2001: 14). 7 See Rivkin-Fish (2005); Praspaliauskiene (2016); Rubashkin et al. (2021); Stepurko et al. (2014); Stepurko et al. (2015). 6 Author’s translation. Informal Payments and Other Shady Things There were other areas of course, in which the past was less than idyllic in many respects (see Koleva 2016). On the margins of the proclaimed universal and free healthcare during socialism, other informal costs were readily present in many Central and Eastern European countries as well as in the former member states of the Soviet Union.7 Many societal contexts, from big business to medicine, recognize the distinction between gifts and bribes (Sherry 1983: 160). The distinction is rarely straightforward however, with one easily being mistaken for the other, and “the nature of this ‘mistake’ [ranges] from earnest to contrived” (Sherry 1983: 160). i e Rivkin-Fish (2005); Praspaliauskiene (2016); Rubashkin et al. (2021); Stepurko et al. (2014); Stepurko et al. (2015). Ethnologia Europaea 10 My analysis suggests that phenomena related to corruption and informal payments in healthcare can be divided into three categories: (small) gifts, direct corruption in the form of illegal payments, and “personal connections”. From the accounts of my patients, small gifts in pre-transition times would be brought equally either before or after the service had been provided: “It was not a big deal, but it looked good” since “it was the right thing to do.” Admittedly, the gifts were rather small (Stepurko et al. 2014) and tended to be consumable foods associated with leisure or celebrations: “I would bring him a bottle of brandy”, or “chocolates, better quality”, or “wine, not cheap”, also “coffee, of course, you know, the red brick….” (a popular vacuum-packed coffee by the company Franck). Some gifts were deliberately intended for more than one healthcare worker: “cookies, big packages, so they could share”, or were homegrown goods: “tangerines, three kilos, organic, I grow them myself”. This type of patient behaviour was mostly directed towards hospital doctors and staff, and much less towards general practitioners. Informal payments of small gifts like flowers, chocolate, or wine by patients to healthcare workers have been documented in many countries (Stepurko et al. 2014: 3). Stepurko and colleagues (2014: 3) emphasize that “such gifts are not typically expected by providers and the amount and quality of the treatment patients receive is not dependent on it”. In my research data, gifts had not been typically expected, since they could easily be given after the provision of the service, just out of gratitude, appreciation, or acknowledgement of healthcare workers’ efforts. Ethnologia Europaea 8 Unfortunately, there are no clear-cut historical explanations as to why the envelopes in question in pre-transition Croatia were blue. The leading historico-cultural assumption is that the workers received their monthly salaries in those blue envelopes and were just re-using them for a similar purpose (Jelača, Kolanović & Lugarić 2017). However, there is no evidence to corroborate that claim. Informal Payments and Other Shady Things Whether or not gift- giving as part of patients’ health-seeking behaviour is seen as acceptable depends highly on the setup of the entire system. These practices tend to exist exclusively inside the context “of strong norms and regulations which shape staff behaviour” (Stepurko et al. 2014: 3) and “it is recognized that they have to be regulated and monitored” (Stepurko et al. 2014: 3), even though it remains unclear by whom and how. Furthermore, such regulation might be incorporated into the system and might thus influence the behaviour of healthcare workers, but this regulation does not apply to the patients themselves or to their placing an added value on the doctor–patient exchange strengthened by gift-giving. The topics of gift-giving and gift-receiving behaviours are classic anthropological themes that highlight how much of societal behaviour revolves around the practices of exchange and reciprocity. “The expectation that there will be something in return presupposes the establishment of an enduring relationship”, Benson and Carter point out (2008: 3), since “by accepting the gift, the recipient is obliged to re-enter the cycle as a giver” (Benson & Carter 2008: 2). In the context of providing medical services, doctors are the expected givers of (good) service and through this, they re-enter the cycle of exchange. Ethnologia Europaea 11 The second category of informal payments mentioned in my data were illegal cash payments. The older the people with whom I talked, the more common, even acceptable, this practice seemed to them. “We had to pay for the hip operation… in our family, a cousin, we gave [the doctor] the blue envelope, it’s not like he asked for it, I guess, but he accepted it.” The phenomenon of giving “envelopes” has been analysed in detail by Rima Praspaliauskiene (2016). In her research, she encountered the custom of giving white envelopes and saw them “as a practice of health and care” (Praspaliauskiene 2016: 582): An envelope of money given to a doctor transcends the material patient–doc tor transaction and emerges as a productive force for coping with illness, medical encounters, and misfortunes. (Praspaliauskiene 2016: 582) Praspaliauskiene (2016) notes that the procedure was comparatively similar to what I heard in Croatia, but the colour of envelopes was white in Lithuania in contrast to blue envelopes in pre-transition Croatia.8 I posit that bribing doctors was not only a sporadic practice, a sociocultural phenomenon, but also a widely accepted cultural narrative. Informal Payments and Other Shady Things Older patients with whom I spoke would readily mention “the blue envelopes” as something that was common, but would rarely name a single instance of ever seeing it themselves or experiencing it in any other context than merely as a story they had heard. Shame, embarrassment, and the need for discretion were affectively linked to this practice, and in the account mentioned above, the blue envelope was hidden in the wine bag and given by one member of the family to the doctor when he was alone in his office. The gift and monetary bribe thus went together and could not be distinguished from each other. The transaction was never mentioned again and never verbally acknowledged as ever taking place. The practice of cash payments seems to be even rarer today, according to my interviewees. However, related research by Stepurko and colleagues (2015) suggests that the practice might have been modified, but nevertheless still occurs due to “insufficient resources (low income of physicians) and inadequate governance (poor political-regulatory context)” in addition to earlier socialcultural customs (Stepurko et al. 2015). The third category of value-driven health-seeking behaviours in the informal realm was the search for “private connections” or “acquaintance relations” (Bazylevych Ethnologia Europaea Ethnologia Europaea 12 2009). This phenomenon is known as “guanxi in China (…), wasta in the Arab world (…), jeitinho in Brazil (…), pulling strings in English speaking countries (…), blat in post- Soviet spaces (…), vrski in The Republic of North Macedonia (…), veze in Serbia, Croatia, and Bosnia and Herzegovina, and as vruzki in Bulgaria” (Williams & Yang 2017: 59). Older respondents readily explained that in pre-transition times one would need a “personal connection” or would feel compelled to seek one in order to access better care. A common phrase in connection-seeking would be, as many reported, “do you have anyone in [for example] Vinogradska?” The person asked could be anyone, a friend, a neighbour, a colleague – anyone who could have acquaintances working in a hospital. The question itself meant that the seeker was searching for a doctor working at that specific hospital, Vinogradska, who would admit, take care of, and perhaps operate on the person himself/herself or a member of his/her family. In such cases assumed efficacy of care and success of the cure depended on the pre-evaluated benefit of having a “connection”. Ethnologia Europaea Informal Payments and Other Shady Things Finding a connection did not automatically mean that the doctor in question would require or should be offered payment in the aforementioned blue envelope. However, since this instance of connected care was deemed more valuable, a payment could be part of it. Strategies of building and retaining complicated and complex relationships between doctors and patients required knowing the rules of the game so one could play it, either as a patient or a healthcare worker. Therefore, strategies of allocating and negotiating the value of caring and curing existed as informal practices in the previous era of nominally unpaid healthcare. Some of these older behaviours have been retained in a somewhat modified form. Research participants confirmed that small gifts are still exchanged in public hospitals and with the same logic both before and after the service has been rendered. These gifts are interpreted as symbolic and as gifts of gratitude and appreciation. Public opinion and attitudes towards the giving of envelopes has obviously changed to the extent that respondents unanimously claimed that envelope-giving does not exist anymore. One of the important findings of my study is that neither gifts nor envelopes are exchanged in the private health sector today: “it did not even cross my mind to bring anything”, emphasized one of the interviewees. Unambiguously mone tary transactions have replaced the culturally evaluated transactions of the past. The third category of value-driven health-seeking behaviours, personal connections, still exists, but with a significant shift, giving rise to the phenomenon of semi-private healthcare (Temkina & Rivkin-Fish 2020). In this form of healthcare, patients move from private to public healthcare (see Pantović 2016 for Serbia) and continue their treatment in the public health sector with the same doctors, avoiding the waiting lists 13 or getting some other benefit that would not otherwise have been accessible to them. According to my interview participants, this was a deliberate strategy used by some patients. Conclusions The central premise of this article is that both the willingness to pay (Donaldson 2001) and adhering to a fee-for-service system (Bazylevych 2009) form a constitutive part of the medical market in Croatia today, compared to the pre-transition period. This premise was corroborated by patients’ accounts: health-seeking behaviours have become dependent on parallel payable healthcare and healthcare alternatives, and on the consumer-like expectations of patients which “go beyond health” (Donaldson 2001). They are also centred around patients assessing the general quality of their experience with healthcare providers. At the beginning of this article, I asked: what has made some people in Croatia willing to give up relatively low-cost universal public healthcare in favour of paying more for private treatment? In the context of changing medical markets which modify and redefine the way we conceptualize and commodify our health and wellbeing, the patients interviewed for this article associated their ability to select between different therapies and providers with a curative potential. Patients drew direct attention to the fact that a higher level of quality is now accessible – if one has the money. At the same time, some patients’ narratives evoked an imagined health equity in which all citizens had equal access to the less biotechnologically advanced healthcare offered by the pre- transition state. In my research I discovered 1) nostalgic narratives that had emerged and refer to past access to free healthcare and call for equality-in-lesser-quality; and 2) the paradoxical fact that older health-seeking behaviours came with hidden – but widely known – costs in the form of informal gifts or the seeking and sustaining of personal connections. These costs were in no way significantly lower than the costs of private medical care today. Interestingly, patients indicated that their selection of care and cures did not involve a meticulous, detailed analysis of available medical treatments. Instead, their decisions were situational, contextual, individual, changeable, affective, and “intuitive”, and they stemmed from personal experiences and perceptions. Patients liked having choices and negotiated politics and power from the basis of their own mindful bodies. Contributing to this is the fact that when entering the market, medical clinics behaved as would any other commercial enterprise. Popular culture, gala events organized by large private clinics for their opening, and the successful public relations campaigns of specific clinics, therapies and practices seemed to be an increasingly important factor Ethnologia Europaea Ethnologia Europaea 14 in patients’ decisions. Ethnologia Europaea Competing Interests The author has no competing interests to declare. Conclusions Market-based healthcare and its accompanying inequalities have become socially legitimized through patients’ attitudes that were based less on a careful evaluation of the pros and cons, options and costs, and more on the fact that patients enjoyed following their bodily intuitions at any given moment. Market-based healthcare allowed them to do this rather than go through treatment regimes decided upon by institutions, the state, or political decision-making. This attitude is likely to be one reason why patients do not resist or protest against the obvious inequalities throughout the present-day healthcare sector. At the time of its emergence, the term medical pluralism was applied to problems surrounding the hegemony of biomedicine, but today this definition seems inadequate (cf. Raffaetà et al. 2017). The increasing complexity of medical pluralism today should alert scholars to the problems of financing, state budgets, migration, solidarity, neoliberalism, inclusion, exclusion, and the near-utopian aspirations towards a universal right to health. Patients’ expectations stem from a complex mix of past, present, and more hopeful future, but for the time being, these expectations are not being met. When the selection of health pathways is directly dependent on an individual’s socioeconomic status, health equity is a distant reality. 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DOI: https://doi.org/10.1515/jeb-2017-0006 Zandbelt, Linda C., Ellen M.A. Smets, Frans J. Oort, Mieke H. Godfried & Hanneke C. de Haes 2007: Medical Specialists’ Patient-centered Communication and Patient-reported Outcomes. Medical Care 45(4): 330–339. DOI: https://doi.org/10.1097/01.mlr.0000250482.07970.5f Zrinščak, Siniša 2007: Zdravstvena politika Hrvatske: U vrtlogu reformi i suvremenih društvenih izazova [The Croatian Health Policy: In the Whirlpool of Reforms and Contemporary Social Challenges]. Revija za socijalnu politiku 14(2): 193–220. DOI: https://doi.org/10.3935/rsp.v14i2.697 Tanja Bukovčan, PhD, Ass. Professor, works at the Department of Ethnology and Cultural Anthropology, Faculty of Humanities and Social Sciences, University of Zagreb, Croatia, and teaches courses in medical and visual anthropology. Her research interests include medical pluralism and medical markets, ethnography in clinical practice, cultural competence in healthcare and visual ethnography. Tanja Bukovčan, PhD, Ass. Professor, works at the Department of Ethnology and Cultural Anthropology, Faculty of Humanities and Social Sciences, University of Zagreb, Croatia, and teaches courses in medical and visual anthropology. Her research interests include medical pluralism and medical markets, ethnography in clinical practice, cultural competence in healthcare and visual ethnography. (tbukovcan@ffzg.hr)
https://openalex.org/W73371683	https://hal.inria.fr/hal-01054675/document	English	null	Teaching Practice from the Perspective of ICT Student Teachers at the Faculty of Education, Charles University in Prague	IFIP advances in information and communication technology	2,010	cc-by	5,442	To cite this version: Miroslava Černochová. Teaching practice from the perspective of ICT student teachers at the Faculty of Education, Charles University in Prague. IFIP TC 3 International Conference on Key Competencies in the Knowledge Society (KCKS) / Held as Part of World Computer Congress (WCC), Sep 2010, Brisbane, Australia. pp.44-55, 10.1007/978-3-642-15378-5_5. hal-01054675 Distributed under a Creative Commons Attribution 4.0 International License HAL Id: hal-01054675 https://inria.hal.science/hal-01054675v1 Submitted on 8 Aug 2014 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License Teaching practice from the perspective of ICT student teachers at the Faculty of Education, Charles University in Prague Miroslava Černochová Charles University in Prague, Faculty of Education, Dpt. of Information Technology and Technical Education, M.Rettigové 4, 116 39 Praha 1, Czech Republic, miroslava.cernochova@pedf.cuni.cz Abstract. The author worked for some years as a coordinator, methodology consultant and supervisor of teaching practice of ICT student teachers at the Faculty of Education in Prague. In her paper she summarizes experiences and data collected by questionnaires during years 2004-2008 by student teachers within their teaching practice on subjects related to Computer Science, Informatics and ICT Education in Czech Basic and Secondary Schools. The results came out of evaluative questionnaires and comments published in on- line support. A video-record will form a part of the paper presentation. Keywords: Teaching practice, ICT teacher, information education, Informatics, computer technology, teacher education, lesson plan, Primary/Secondary school, Moodle. 1 Introduction The paper is dedicated to a teaching practice of student teachers implemented into a study program of a Master’s Degree study of Information and Technical Education at the Faculty of Education in Prague. During five years of their study ICT student teachers have to successfully get through two periods of teaching practice in basic or secondary schools in subjects dedicated to ICT, Informatics or Computer Science. After graduation they are fully qualified basic or secondary school teachers. They would work as primary or secondary school teachers of ICT, Informatics or Computer Science. They would work at schools also as a computer nets supervisor and they might be employed as ICT coordinators and consultants. They would provide advice how to apply ICT in school practice to teachers or head teachers. 1.1 Teaching practice of ICT student teachers in primary or secondary schools Generally, the teaching practice of student teachers of the Faculty of Education in Prague is organized in the Year 4 and Year 5. Each of them takes four weeks. Fig. 1. A position of teaching practice in a study program of student teachers at the Faculty of Education In Year 4: 4 week of teaching practice In Year 5: 4 weeks of teaching practice Fig. 1. A position of teaching practice in a study program of student teachers at the Faculty of Education Within each of four weeks of an obligatory teaching practice the ICT student teachers assist or stand-by teachers in classrooms. During this period they design their own lesson plans and teach a minimum of 12 lessons in the classroom environment. (I.e. during the university study, students teach at least 24 lessons in Basic or Secondary schools). They analyze and evaluate their teaching experience together with their mentors who work in a close collaboration with a didactic consultant from the Faculty of Education. For each lesson ICT student teachers design their lesson plans while using an identical template (see Appendix 1). They can ask a mentor for advice to tailor lesson plans or they can consult with a didactics consultant or their peers from the Faculty of Education on-line. Via the on-line support organized in Moodle, ICT student teachers deliver a set of lesson plans, a questionnaire, reports and news about their teaching practice. Together they can discuss some technological problems or pedagogical situations in forums then. ICT teacher students can complete their four-week teaching practice in Basic or Secondary schools not only in Prague, but also whenever in the Czech Republic. Some students prefer conducting their teaching practice at schools they attended as a child. Their former teachers can become their mentors and colleagues and can monitor their first professional achievements. In such cases ICT student teachers find good relationships with their mentors as very important for their teaching practice. In 2002 we introduced in Moodle on-line support in order to improve coordination and management of teaching practice for our ICT student teachers. The on-line support is very useful and helpful for all, student teachers and mentors, who should visit, monitor and supervise each student teacher in their schools. From 2007 onwards we also started recording our student teachers by a digital camera in schools. 1 In 2004 - 2007 only 13% ICT teachers in Primary or Secondary schools used text-books in their teaching with children. 1.1 Teaching practice of ICT student teachers in primary or secondary schools After teaching practice in schools all video-recordings are analyzed in a seminar together with all ICT student teachers. Video-records allow them to see the real situation in the classroom of various schools, the equipment of their computer-labs, the interactions between ICT students and pupils etc. Some ICT student teachers, during their university study, are employed in basic or secondary schools as non-qualified teachers of informatics or computer science subjects. In such cases their work is validated as teaching practice; it means they complete their teaching practice in the schools where they work. The ICT students who work as non-qualified teachers, unlike their student colleagues who are not employed in schools, have been prepared not only to teach children, but also in other fields and responsibilities of the teacher profession, primarily in: • assessment of pupils learning outcomes, • assessment of pupils learning outcomes, • a legal agenda related to schooling, • communication with parents, etc. • communication with parents, etc. These ICT student teachers who already work in schools are very active in seminars at the Faculty of Education. They ask questions that distinguish them from their university school-mates, exploit their experience from teaching in schools and present interesting ideas on how to implement ICT into education. Additionally, they usually design high-quality teaching or study materials. 1.2 How ICT student teachers develop lesson plans During a teaching practice in schools ICT student teachers must develop lesson plans in identical templates (see Appendix 1). The items of the template don’t correspond with a structure of lesson plans that (teachers) students usually prepare. The template serves not only to describe a scenario of their teaching lesson, but also as a didactic exercise to develop some of student teachers’ skills and to support their own thinking about teaching and learning from different perspectives: most of the student teachers have a problem formulating clever questions or suggesting an interesting problem to be solved. Most of student teachers don’t consider a terminology and lexis of their subjects. And it is difficult for them to develop a concept structure of ICT subjects in minds of their pupils. Most of the ICT student teachers (66%) design their teaching without any help of their mentors. The question of how to design a lesson is more complicated. The reason is that in most schools1 ICT teachers do not use any textbook. Most of teachers (about 79%) develop their own study materials for pupils. For making lesson plans ICT students mainly use the Internet (90% of students – see Fig.2). In the lesson planning process the ICT student teachers pay great attention to these aspects: p • Time table of teachers and pupils activities and a structure of lesson • Educational content of lesson • Proposal of activities and assignments for pupils. The ICT students found out that designing timetables for lessons and motivating pupils to take active participation (see Fig. 3) are the biggest problems. Sometimes activities they prepare for pupils are either too easy or not stimulating tasks without any respect to personal priorities, interests or preferences of their students. Another problem that ICT student teachers met with was how to motivate children for ICT activities. Fig 2. Types of resources used by ICT student teachers for lesson plan development Fig 2. Types of resources used by ICT student teachers for lesson plan development Among Czech schools there are a lot of differences in approaches to teaching ICT subjects. Most of them concentrate on development of fundamental ICT skills of pupils. They mainly focus on training in MS Office software. In some schools children produce animations or multimedia applications using digital cameras and special SW. Rarely the Czech Basic or Secondary schools implement basis of programming into compulsory ICT subjects (see Fig. 2 Imagine and Baltik have become a favorite SW for pupils’ development of programming thinking and algorithm skills in Czech primary schools. 1.2 How ICT student teachers develop lesson plans 4). Some Czech schools organize for children facultative courses in programming. ICT student teachers found out that in basic schools some teachers2 are dedicated to programming in Karel and in secondary schools mainly in HTML or Pascal. Most of ICT student teachers would prefer to teach programming than to train fundamental ICT skills with editors or spreadsheets. From September 2007 Czech schools apply a new curriculum concept defined by the Framework Educational Program where ICT is in a totally new position. According to this curriculum document, ICT has become a new compulsory educational domain with a similar position as Math, Languages, Art or Science Education traditionally have. Lessons about ICT are integrated into Primary Education in Grades 4 and 5 (for children of age 9- 11). Therefore some ICT teacher students during their practice also taught courses with children of age 11-12. There they could experience how children of that age like activities with computers. Fig. 3 The main problems the ICT student teachers have met during their teaching practice (% student teachers) Fig. 3 The main problems the ICT student teachers have met during their teaching practice (% student teachers) Fig. 3 The main problems the ICT student teachers have met during their teaching practice (% student teachers) Fig 4. Teaching of programming in Czech Basic and Secondary schools Fig 4. Teaching of programming in Czech Basic and Secondary schools During their teaching practice ICT student teachers could see how schools have been applying new curriculum policy, how ICT has been integrated into project works at schools, which topics of Informatics are being taught to children in Grade 4, etc. 1.3 A situation in schools where ICT student teachers complete teaching practice Most of the ICT student teachers did their practice in schools involved in the “Governmental Information Policy in Education” (GIPE). In 2002-2006 the GIPE equipped Czech schools with computer technology and SW to integrate ICT into education, to train teachers in ICT skills and to organize educational projects within ICT. But since 2007 the Czech Republic hasn’t had any financial support of that sort and further ICT development at schools doesn’t exist. The ICT student teachers could see the impact of this fact and experience how schools differ in ICT equipment, SW and services for teachers and pupils. Nevertheless, step-by-step most Czech schools have succeeded in getting interactive whiteboards that caused a boom in motivation of Czech teachers to work with computers and Internet. In the last two years most of the ICT student teachers could use interactive whiteboard in schools, too. Czech teachers to work with computers and Internet. In the last two years most of the ICT student teachers could use interactive whiteboard in schools, too. In 2006 some schools, that facilitate teaching practice to our ICT student teachers, have participated in many EU Projects (eTwinning, CALIBRATE, etc.). Some ICT student teachers could participate in these EU projects activities. “I was surprised by a strong-willed effort by my supervisor teacher who was not only an ICT and Art Education teacher, but also a principal of the school. His pupils master graphic Most of the ICT student teachers appreciate: a) high-tech equipment at schools (data-projector, interactive whiteboard, wireless mouse and keyboards, etc.). Computer labs in schools are arranged as multifunctional classrooms with very modern furniture, where they could be given not only Informatics lessons, but also lessons of Music or Art Education. In the last two years student teachers had the opportunity to use an interactive whiteboard in most schools. “I really liked the computer labs; there are three computer labs in the school that are all arranged in non-traditional ways. A teacher in my school designed the furniture. I also liked the Internet cafe and library in the school.” b) a good “climate” and partnership among teachers in schools. Their open approaches to pupils enable them to find more flexible solutions for unknown problems. Teachers teach pupils skills of teamwork. From ICT student teachers’ point of view there are a lot of kind people at schools. p p p c) teachers’ effort to upgrade HW and SW in schools. ICT student teachers were surprised by how much effort teachers had to make if they wanted to upgrade the equipment and SW applications at the schools. During their university study, the ICT student teachers had no reason to think about it because the Faculty offered them the use of professional SW and high-quality HW. However, at schools today ICT teachers can’t only teach their subjects, but they also have to take care of SW and HW. In many schools there is also a lot of “old fashion” technology; schools don’t have enough money to buy new technology. For maintaining old computers it is necessary to have enough spares and components. ICT student teachers were surprised by “teachers’ active attempts to get needed components and spare parts for old fashioned computers and hardware”. ICT student teachers could get experience with some SW that has not been applied at the Faculty of Education – for example Master Eye that helps teachers to organize work on PC with children and that could be very useful in computer labs without data-projects. d) a chance to use an interactive whiteboard. Till 2007 ICT student teachers haven’t been able to use an interactive whiteboard at the Faculty of Education. However, most of the schools where they do teaching practice are equipped with them. ICT student teachers appreciated the possibility to work with this new type of whiteboard. SCHOOLS FROM THE ICT STUDENT TEACHERS’ PERSPECTIVE Most of the ICT student teachers appreciate: “In my school non suitable user data security could protect against abuse or their complete discarding by other pupils.” … “For teachers who don’t teach ICT subjects it was very difficult to use a computer lab. The lab could be used only for ICT lessons.” … “I didn’t like the school had two computer labs, but only one of them was connected to the b) and to approaches to teaching of ICT subjects: b) and to approaches to teaching of ICT subjects: “In my school, children prefer to play computer games what isn´t so difficult and boring according them therefore they regard ICT lessons as not very important. …Teachers didn’t give special professional attention to gifted pupils who knew a lot about technology and more than their teachers. In the school there dominates a practical orientation of ICT education to develop only fundamental user’s skills and to achieve a general “computer literacy”…. “I don’t agree children cannot learn programming and haven’t any textbooks.” Most of the ICT student teachers appreciate: e) ways how in some primary schools children from age 7 to 10 are being encouraged to work with ICT. These schools decided to find a space in their school timetable to allow teachers to work with young children in computer labs. techniques and Art thinking – you can see the pupils’ pictures and presentations in school halls, classrooms and teachers rooms – the school looks like a Gallery of Art.” , f) the high interest of children in computers , f) the high interest of children in computers g) a fact that in few schools all teachers could use ICT in all classrooms. In some schools ICT student teachers could see how schools utilize Moodle. Some ICT student teachers had some critical comments to: a) conditions for ICT Education g) a fact that in few schools all teachers could use ICT in all classrooms. “In my school there were limited technical conditions for ICT education. It is a pity that children couldn’t learn more about WWW.”... “In the computer lab there was clutter and very often some teachers came in to speak with my supervisor teacher. I expected that it would be possible to use a data projector for my teaching as I was accustomed at the Faculty of Education, but the school didn’t have it. It threw me into confusion. Therefore I decided to create my Web pages where I could publish all study and teaching materials and instructions for my pupils (www.vyukarudna.wz.cz).” WHAT SHOULD BE IMPROVED AND CHANGED IN SCHOOLS? ICT student teachers were asked to propose what could be improved in a school where they did their teaching practice. Most of their recommendations on how to change a situation in their school related to a technological improvement of schools: “I recommend to install a bigger monitor for teachers (in labs for Biology Education and Foreign Language Education) and for students, too.” … “I would reduce a path to a common directory on a server where children can save their work. It was a problem for children.” “I would install Total Commander that could be for children more practical and understandable.” … “I would move the teacher workstation to another place in the computer lab.” … “I would change how the computers are arranged in a net; I would change the administration of user’s profile.” “I would support more the Internet activities of students, but it would be necessary also to have better security for the computer network.” … “I would like to recommend to a computer-net administrator to equip schools with advanced technology” ... “and to upgrade SW.” “It would be very useful for teachers to install dataprojectors in all classrooms with computers.” “I would prefer to install in all computer labs of school the same operating system (for example Windows XP).” “I would prefer to install in all computer labs of school the same operating system (for example Windows XP).” “I would like to install more educational SW.” “I would like to install more educational SW.” “In my school in a computer lab where I had lessons with children there wasn’t a dataprojector, so I always had to borrow it from another classroom and after my lesson I had to put it back. It was not very practical. The security setting did not allow me to open an e-mail account on a public portal and children didn’t have any e-mail accounts in the school server either. No existing e-mail accounts forced the school to break collaboration with other schools.” In 2005 the ICT student teachers recommended TECHNOLOGICAL CONDITIONS IN SCHOOLS Teaching practice in schools is a good opportunity for ICT student teachers to see a real situation in schools with all problems that schools have with ICT services and ICT in education: “In my school where I did teaching practice there were three supervisors for administration of computer labs. But I know some schools without any qualified supervisors where supervision of the computer lab is done by a teacher without any qualification in ICT.” … “My supervisor- ICT teacher was very busy; he is the only specialist for ICT subjects in the school and zároveň a supervisor of computer classrooms. He also helps his colleagues with their computers to solve their user problems – this hectic schedule sometimes influences the quality of his teaching.” According to comments by ICT student teachers in 2005, student teachers were faced with teachers who had very low computer literacy. Therefore teachers who mastered work with ICT were very busy and overloaded by activities with ICT. “In my school some teachers even rejected installing computers in their working room.” Some ICT student teachers struggled with a lot of technical problems. In 2007 some of them had to use very old computer machines without any data projectors and with low speed connectivity to the Internet. For instance, in 2007 one basic school didn’t have a computer-net, there were only 10 isolated computers in a classroom. Generally, some schools have no data projector or have very low speed connectivity to the Internet. Other problems that influenced the teaching practice of ICT student teachers were a lack of scanners, printers or multimedia workstations. Some problems originated in a bad conception of network administration and in setting users´ rights. Internet.” … “Computers were situated in the computer lab in a manner that pupils were sitting with their backs to a teacher and to a data-projector.” In 2005 the ICT student teachers recommended to school ICT administrators and ICT coordinators: To get rid of blackboard and to install a new one in a better place in the classroom. To re-arrange workstations in a classroom. To buy a dataprojector or to install Master Eye. To have computer labs accessible to all teachers and students. To integrate Moodle into school work. To buy more computers. To install other e-mail system (not GroupWise). To use also another graphic editor that could be used also at home by students. To employ more ICT administrators and ICT coordinators. to ICT teachers: To introduce textbooks for students. To teach programming. to head teachers: to ICT teachers: To introduce textbooks for students. To teach programming. to head teachers: To recruit qualified ICT teachers for ICT subjects. To recruit qualified ICT teachers for ICT subjects. In 2006 ICT student teachers recommended to school ICT administrators and ICT coordinators: To allow pupils to work in computer labs after their school activities and during breaks. To darken computer labs. To install another computer lab. To buy another SW – also for a teaching basis of database and programming. To change arrangement of computers and furniture in computer labs. to teachers: To extend ICT courses to others grades. To involve pupils (school) into an international or national project activity. To motivate pupils to apply ICT in practice and in life. In 2007 ICT student teachers advised to school ICT coordinators: To use Vision studio or similar SW for monitoring student’s individual activities. To install a camera-control system (to protect technology equipment against losses). To install dataprojectors in all computer labs. To allow teachers to conncet a notebook. To install an interactive whiteboard In 2007 ICT student teachers advised to school ICT coordinators: To use Vision studio or similar SW for monitoring student’s individual activities. To install a camera-control system (to protect technology equipment against losses). To install dataprojectors in all computer labs. To allow teachers to conncet a notebook. To install an interactive whiteboard 1.4 Student teacher professional development from a student teacher perspective After teaching practice ICT student teachers have to fill evaluative questionnaires where they have to answer some questions related to conditions for information education in schools and to the approach of teachers and school management to a new curriculum concept of ICT education. All lesson plans, video-recordings and documents developed by ICT students in their teaching practice form a part of a student portfolio that is applied in some courses oriented on methodology and didactic aspects of information and ICT education. QUESTION: WHAT HAVE ICT STUDENT TEACHERS LEARNT IN SCHOOLS DURING THEIR TEACHING PRACTICE? In 2004: “I have learned very much. As a teacher I met so many problems (SW, computer net, HW, connectivity) that I didn’t expect that I had to think about how to solve them.” … “It was my first experience with young students as my pupils.” … “Teaching practice was super. You could see that theory and practice are two totally different things! It seems a lot of my teachers at the Faculty of Education forgot this fact!” … “I was surprised how easy it was for me to teach!” … “At first I was slightly afraid to teach children a subject that I couldn’t learn personally in my basic school. But at the end it was a fantastic experience for me!” … “I was teaching at a basic school – it was nice practice for me. I am curious about how it would be to teach at a secondary school.” “I completed my practice at a secondary school. I was curious about how my female colleagues would be able to teach teenagers like I had to teach.” … “I designed learning activities for children by my own concept to be interesting also for me personally. I exploited my personal experiences with ICT.” … “I was kindly surprised how teachers, school management and my supervisor-teacher accepted me. I was very happy to be and to teach there. It influenced me to become a teacher!” “The teaching practice is very short, but for student teachers it is very exacting. During four weeks we have to get experience with teaching in two specializations. Fortunately my practice was successful – I am employed in this schools and my teaching practice was realized with my pupils. There was only one problem – my supervisor-teacher is a student of the Technical University, who is an expert in ICT and Computer Science, but he has no pedagogical background like me. He could be more convenient as a supervisor-teacher for ICT subjects with talented children.” “I liked my teaching practice. It was my first experience with being a teacher. At first I was afraid but after my first lesson I discovered that it is a fantastic and creative job to be a teacher. I was glad to see how my pupils collaborate and work. Step by step I have managed to motivate all pupils to do given assignments. QUESTION: WHAT HAVE ICT STUDENT TEACHERS LEARNT IN SCHOOLS DURING THEIR TEACHING PRACTICE? In 2004: Maybe I will succeed in it because I was a new “non-hackneyed element” in a classroom and my pupils did not know what they could expect from me.” QUESTION: WHO HAS INFLUENCE ON TEACHING STYLES OF ICT STUDENT TEACHERS? In Fig 5 you can see who has the greatest impact on the teaching style of the ICT student teachers. Their own basic or secondary school and university teachers have had the most important influence on their approaches to pupils and teaching strategies. Some ICT students are not thinking about professional patterns and they try to tread their own path without any models or patterns. Fig 5 Professional patterns of who has an influence on ICT student teachers Fig. 5 Professional patterns of who has an influence on ICT student teachers THE BIGGEST PROBLEMS FOR ICT STUDENT TEACHERS IN TEACHING PRACTICE For most of ICT student teachers there is a problem organizing their teaching into 45 minute lessons. Another problem that they had relates to a motivation of pupils to be active. ICT student teachers did not see any problem in communication with their pupils (Fig. 3). Some ICT student teachers were faced with teaching both ICT beginners and ICT experienced pupils. They didn’t know how to design lesson in class with a big differences in ICT knowledge and skills of teenager students and whom should they dedicate their teaching to in their 45 minute lessons. “I was not ready to teach students with such big differences in ICT skills and knowledge: I hadn’t time and space to develop the advanced ICT users and at the same time I couldn’t teach beginners in ICT.” 2 A role of Moodle in teaching practice of ICT student teachers A few years ago we decided to use Moodle for coordinating and managing teaching practice of ICT student teachers. In Moodle we publish instructions for students on what to do and documents (lesson plan template, templates for time-table, etc.). In Moodle ICT student teachers can ask colleagues and didactics specialists and inform them about the situation in their schools. We endeavor to answer students immediately. “I would like to appreciate a clear structure and function of Moodle support. Thanks to very clear instructions I could see how teaching practice proceeds in other schools of my colleagues, what I have to do. Our teachers from the Faculty of Education answered us immediately. Each important question was answered.” ... “I would appreciate the Moodle support which helped me very quickly understand a system on how teaching practice is organized. It is a pity, that other departments of the faculty do not use similar support.” 3 Conclusions The teaching practice has a key role in university teacher education of ICT student teachers. It is a pity at the Faculty of Education in Prague student teachers can experience their teacher profession with pupils only during 8 weeks. It is a too short time period to prepare student teachers for their profession and to demonstrate mastering key professional competences [6]. Some students who study in a full-time study program at the Faculty of Education and at the same time work at schools as part-time non-qualified ICT teachers, achieve in didactic courses and in their diploma thesis significantly better results and apply more professional approaches to given tasks and problems than students who only study in full-time study programs. 2. Van der Linde Diane, van der Hoeff Aike, Pronk Hans, van AST Michel ICT Knowledge base. Version 1.0. (2009) References 1. Moodle. Http://moodle.pedf.cuni.cz. 1. Moodle. Http://moodle.pedf.cuni.cz. 2. Van der Linde Diane, van der Hoeff Aike, Pronk Hans, van AST Michel ICT Knowledge base. Version 1.0. (2009) 3. JOMITE Group SpriTE Teaching Practice Assessment Checklist. (2009) 3. JOMITE Group SpriTE Teaching Practice Assessmen 4. Vlastní hodnocení škol. ČŠI, Praha. 4. Vlastní hodnocení škol. ČŠI, Praha. 5. Janík, Tomáš Didaktické znalosti obsahu a jejich význam pro oborové didaktiky, tvorbu kurikula a učitelské vzdělávání. Brno. (2009). 6. Hodnocení studentů studijních programů učitelství při plnění souvislé pedagogické praxe z oboru it (IKT) KITTV, PedF UK, Praha. (2010). Appendix 1 Lesson Plan Grade: Subject: Lesson plan author: p Educational program (curriculum document) applied by the school: p Educational program (curriculum document) applied by the school: 1. Preliminary requirements for pupil’s knowledge: Give a list of pupil’s knowledge and skills to be able to understand a topic for a lesson. For example • Pupils are able to..., Pupils understand..., Pupils master in ...) p 2. Main aim of the lesson: • Particular goals: 2. Main aim of the lesson: • ICT competencies (related to the document of the Framework Educational Program) Give a list of competencies that you will contribute to develop in your lesson. y 3. Text-books, study materials that would be used by pupils for your lesson. 4. Aids, tools that would be used in a lesson. 5. A list of technology equipment and HW that would be used in your lesson by a teacher and by pupils. For each tool explain for which activity it will be used. 6 A list of SW that would be used in your lesson and to prepare for your lesson 7. Draw a scheme of classroom arrangement.) 7. Draw a scheme of classroom arrangement.) 8. Teaching methods and strategies with the explanation how you will apply teaching methods and which teaching strategies you will prefer for your teaching. 9. Procedure: Describe instructions on how to order lesson procedures. Your description ought to be formulated as a manual on how to realize your lesson plan. 10. Questions that pupils should be able to answer at the end of the lesson. 11. Assignments and problems to be solved by students. Give also their solution and numeral account). 12. Homework. Formulate problems and activities for homework. p 13. Evaluation: Explain criteria how student outcomes will be evaluated. 14. Addition and extra topics, assignments for talented pupils: Propose how you will organize a lesson with a classroom where you have found some talented pupils. 15. WWW: Give a list of WWW pages used in your teaching. 15. WWW: Give a list of WWW pages used in your teaching. 17. Vocabulary of terms from ICT and Informatics related to the topic: Give a list of terms with explanations understandable and dedicated to your pupils. 18. Term and its explanation: ….. 2. What didn’t you manage? TEACHER SELF-EVALUATION AND AUTO-REFLECTION 1. How did you succeed your lesson? 2. What didn’t you manage?
https://openalex.org/W2761292554	https://revistas.pucsp.br/index.php/dic/article/download/32072/23758	Portuguese	null	Intervenção fonoaudiológica precoce em afasia infantil decorrente de um acidente vascular cerebral: relato de caso	Distúrbios da Comunicação	2,017	cc-by	3,611	ARTIGOS ARTIGOS http://dx.doi.org/10.23925/2176-2724.2017v29i3p480-486 Intervención fonoaudiológica precoz en afasia infantil debido a un accidente vascular cerebral: reporte de un caso Natalia Caroline Favoretto* Natalia Gutierrez Carleto* Paula Grandini Cunha* Vanessa Clivelaro Bertassi Panes* Adriano Yacubian Fernandes* Dionísia Aparecida Cusin Lamônica* Magali de Lourdes Caldana* Intervenção fonoaudiológica precoce em afasia infantil decorrente de um acidente vascular cerebral: relato de caso Early speech-language intervention in childhood aphasia after a stroke: case report Intervención fonoaudiológica precoz en afasia infantil debido a un accidente vascular cerebral: reporte de un caso Resumo O Acidente Vascular Cerebral é menos frequente em crianças do que em adultos, porém possui um poder devastador para o futuro destes indivíduos, mesmo com indícios de melhor recuperação das alterações. O objetivo deste estudo é descrever os achados fonoaudiológicos de um caso de Acidente Vascular Cerebral infantil, evidenciando a evolução após 12 meses de terapia fonoaudiológica, iniciada * Faculdade de Odontologia de Bauru, Universidade de São Paulo, Bauru, SP, Brasil * Faculdade de Odontologia de Bauru, Universidade de São Paulo, Bauru, SP, Brasil Contribuição dos autores: NCF: avaliação do sujeito e elaboração do planejamento terapêutico fonoaudiológico do caso clínico, e elaboração do artigo; NGC: avaliação do sujeito e elaboração do planejamento terapêutico fonoaudiológico do caso clínico, e elaboração do artigo; PGC: avaliação do sujeito e elaboração do planejamento terapêutico fonoaudiológico do caso clínico, e elaboração do artigo; VCBP: discussões interdisciplinares; AYF: análise de neuroimagens; elaboração do artigo; DACL: co-orientação do estudo; MLC: supervisão dos atendimentos fonoaudiológicos; elaboração do artigo. E-mail para correspondência: Natalia Caroline Favoretto - natalia.favoretto@yahoo.com Recebido: 07/03/2017 Aprovado: 29/08/2017 Contribuição dos autores: NCF: avaliação do sujeito e elaboração do planejamento terapêutico fonoaudiológico do caso clínico, e elaboração do artigo; NGC: avaliação do sujeito e elaboração do planejamento terapêutico fonoaudiológico do caso clínico, e elaboração do artigo; PGC: avaliação do sujeito e elaboração do planejamento terapêutico fonoaudiológico do caso clínico, e elaboração do artigo; VCBP: discussões interdisciplinares; AYF: análise de neuroimagens; elaboração do artigo; DACL: co-orientação do estudo; MLC: supervisão dos atendimentos fonoaudiológicos; elaboração do artigo. E-mail para correspondência: Natalia Caroline Favoretto - natalia.favoretto@yahoo.com Recebido: 07/03/2017 Aprovado: 29/08/2017 E-mail para correspondência: Natalia Caroline Favoretto - natalia.favoretto@yahoo.com R bid 07/03/2017 480 Distúrb Comun, São Paulo, 29(3): 480-486, setembro, 2017 ARTIGOS Intervenção fonoaudiológica precoce em afasia infantil decorrente de um acidente vascular cerebral: relato de caso precocemente, após lesão neurológica adquirida. Trata-se de um indivíduo de 11 anos, gênero masculino, com normalidade de desenvolvimento neuropsicomotor e independência para as atividades funcionais durante a primeira infância. Em março de 2015 sentiu um mal súbito, foi encaminhado para o hospital de urgência e diagnosticado com Acidente Vascular Cerebral, do tipo hemorrágico, em região fronto- parietotemporal esquerda. Foram realizados procedimentos cirúrgicos, e a internação durou 25 dias. No momento da alta hospitalar houve a orientação sobre a necessidade de atendimento fonoaudiológico. A primeira avaliação de linguagem evidenciou afasia adquirida do tipo emissiva, conforme as classificações propostas para crianças. Resumo Foram realizadas 91 sessões de terapia fonoaudiológica, com duração de 50 minutos, pelo período de 12 meses. Ao analisar as habilidades formais de linguagem que permaneceram alteradas, observa-se que as relacionadas à leitura e escrita são as de maior significância, interferindo no desempenho escolar e desenvolvimento comunicativo. Assim, além do atendimento fonoaudiológico até que seja possível uma comunicação funcional, é importante que haja o apoio escolar. Palavras-chave: Fonoaudiologia; Linguagem; Acidente Vascular Cerebral; Crianças. Abstract Stroke is less common in children than in adults, but it has a devastating power for the future of these individuals, even with signs of better recovery from the changes. The aim of this study is to describe the speech therapy findings of a case of stroke child, showing the progress after 12 months of speech therapy initiated early after acquired neurological injury. This is an individual of 11 years old, male, with neurodevelopment normality and independence to the functional activities during early childhood. In March of 2015, he suffered a sudden illness, was referred to the emergency hospital and diagnosed with hemorrhagic stroke, in the left frontoparetotemporal region. Surgical procedures were performed, and hospitalization lasted 25 days. At the time of hospital discharge, there was guidance about the need for speech therapy care. In the first evaluation of language, there was evidence of acquired aphasia of the emissive type, according to the classifications proposed for children. A total of 91 speech therapy sessions lasting 50 minutes were performed for 12 months. When analyzing formal language skills that have remained altered, it is observed that those related to reading and writing are those of greater significance, interfering in school performance and communicative development. Thus, in addition to speech and language therapy until functional communication is possible, it is important that there be school support. Keywords: Speech, Language and Hearing Sciences; Language; Stroke; Child. Keywords: Speech, Language and Hearing Sciences; Language; Stroke; Ch Introdução Para as crianças, a afasia pode ser subdivida em desenvolvimental e adquirida. Se ocorrer an tes dos dois anos de idade a afasia é considerada desenvolvimental, se ocorrer após, adquirida. A idade de dois anos é um marcador de tempo devido ao fato de que, nesta faixa etária, a criança deve ter desenvolvido aspectos mínimos da linguagem, como alguns fonemas e a capacidade de produzir sentenças simples7. A classificação dos tipos de afasia na infância também pode se diferenciar de acordo com as manifestações linguísticas, de forma mais abrangente, dividindo-se em afasia emissiva e receptiva, considerando os parâmetros de expressão e compreensão, respectivamente8. O Acidente Vascular Cerebral (AVC) pode ser definido como o rápido desenvolvimento de sinais clínicos decorrentes de distúrbios focais ou globais da função cerebral, com sintomas com duração superior a 24 horas, de origem vascular, resultando alterações cognitivas e sensório-motoras, de acordo com a área e a extensão da lesão1. Estima-se que, uma em cada cinco mulheres e, um em cada quatro homens poderão sofrer um AVC até os 85 anos de idade, e este risco aumenta após os 55 anos2. Quando se trata de crianças, a variável é de dois a oito casos a cada 100.000 crianças até a idade de 14 anos, sendo que são acontecimentos raros e isolados, de caráter importante pela gravida de de suas complicações e variedade de etiologias. Algumas das principais causas de AVC em crianças são: cardiopatia congênita, doença hematológica (anemia falciforme, policitemia, púrpura alo -imune do recém-nascido), doença auto-imune, coagulopatias, vasculite, anomalias vasculares, in fartos venosos, doenças metabólicas, vasoespasmo, traumas, desidratação, neoplasias, entre outras3,2. O AVC é menos frequente em crianças do que em adultos, porém quando ocorre, possui um poder devastador para o futuro destes indivíduos, mesmo com indícios de melhor recuperação das alterações por esta população4. Estima-se que, uma em cada cinco mulheres e, um em cada quatro homens poderão sofrer um AVC até os 85 anos de idade, e este risco aumenta após os 55 anos2. Quando se trata de crianças, a variável é de dois a oito casos a cada 100.000 crianças até a idade de 14 anos, sendo que são acontecimentos raros e isolados, de caráter importante pela gravida de de suas complicações e variedade de etiologias. Resumen El accidente cerebrovascular es menos común en niños que en adultos, pero tiene un poder devastador para el futuro de estes individuos aun que se tenga indicios de mejor recuperación de las alteraciones. El objetivo de este estudio es describir los hallazgos fonoauiológicos de un caso de Accidente Vascular Cerebral Infantil, que muestra el progreso después de 12 meses de terapia fonoaudiológica iniciada precozmente, en un caso de lesión neurológica adquirida. Se trata de un individuo de 11 años, de sexo masculino, con normalidad el desarrollo neuropsicomotor y independencia de las actividades funcionales durante la primera infancia. En marzo el año 2015 sintió un mal súbito, fue remitido al hospital de emergencia y diagnosticado como Accidente Vascular Cerebral, de tipo hemorrágico en la región frontoparietotemporal izquierda. Se realizaron procedimientos quirúrgicos y la hospitalización duró 25 días. En el momento del alta hospitalaria hubo orientación sobre la necesidad de tratamiento fonoaudiológico. La primera evaluación del lenguaje mostró afasia adquirida de tipo emisivo, de acuerdo con las clasificaciones propuestas para los niños. Se realizaron 91 sesiones de terapia fonoaudiológica, con una duración de 50 minutos, el período de 12 meses. Mediante el análisis de las habilidades lingüísticas formales que permanecieron cambiada, se observa que las relacionadas con la lectura y la escritura son las más significativas, porque interfieren con el desempeño escolar y desarrollo comunicativo. Por lo tanto, además de la terapia fonoaudiologoca hasta que una comunicación funcional sea posible, es importante el apoyo escolar. Palabras clave: Fonoaudiologia; Lenguaje; Acidente Vascular Cerebral; Niño. Palabras clave: Fonoaudiologia; Lenguaje; Acidente Vascular Cerebral; Niño 481 Distúrb Comun, São Paulo, 29(3): 480-486, setembro, 2017 ARTIGOS Natalia C. Favoretto, Natalia G. Carleto, Paula G. Cunha, Vanessa C. B. Panes, Adriano Y. Fernandes, Dionísia A. C. Lamônica, Magali de L. Caldana ARTIGOS Introdução O tipo e a gravidade da afasia estão diretamente relacionados ao local e extensão da lesão neurológi ca adquirida, sendo a relação entre áreas cerebrais e a linguagem alvo de estudos há anos, mostrando principalmente a importância do hemisfério es querdo, mais especificamente as áreas de Broca e Wernicke9. Outro fato conhecido é que regiões extras influenciam diretamente e indiretamente nas habilidades linguísticas, bem como o hemisfério direito, responsável pelos aspectos paralinguísticos da comunicação10. Considerando os aspectos citados, ressalta-se que, em todos os casos de AVC, é de suma impor tância considerar a etiologia, história clínica, local e extensão da lesão, relacionando tais fatores com as alterações apresentadas pelos sujeitos acometidos, principalmente naqueles em que houver a presença da afasia, pois cada caso é único e possui diferentes manifestações que podem atingir todos os níveis formais da linguagem, dentre eles a fonologia, mor fologia, sintaxe, semântica, pragmática e o léxico. O AVC pode ser dividido em dois tipos: isquê mico e hemorrágico. O AVC isquêmico (AVCI) é caracterizado pela obstrução de um vaso sanguíneo que bloqueia o fluxo de sangue para as células cere brais, não ocorrendo o recebimento necessário dos nutrientes indispensáveis ao metabolismo de suas células1,2. Enquanto o AVC hemorrágico (AVCH) consiste no rompimento de um vaso sanguíneo, gerando o extravasamento de sangue e aumento da pressão intracraniana1. O tipo de AVC mais comum, em qualquer faixa etária, é o AVCI5. O objetivo do presente estudo é descrever os achados fonoaudiológicos de um caso de AVC infantil, evidenciando a evolução após 12 meses de terapia fonoaudiológica, iniciada precocemente, após lesão neurológica adquirida e, dessa forma, reforçar a importância da intervenção precoce, a fim de proporcionar um prognóstico favorável à pessoa com afasia. Após o acometimento, independente do tipo de AVC, um dos danos mais comuns ao sujeito é o distúrbio adquirido de linguagem, denominado afasia, que é caracterizada por alterações linguísti cas de origem discursiva e articulatória, produzida por lesão focal no sistema nervoso central, em áreas responsáveis pela linguagem, podendo ou não estar associada a outras alterações cognitivas, tornando o indivíduo dependente de recursos auxiliares para a produção e/ou interpretação da linguagem6. Apresentação do caso clínico Avaliações pré e pós-intervenção fonoaudiológica Habilidades Resultados pré-intervenção fonoaudiológica Resultados pós-intervenção fonoaudiológica Fluência Inalterado Inalterado Leitura Alterado Alterado Escrita Alterado Alterado Nomeação Alterado Inalterado Repetição Alterado Inalterado Evocação Alterado Inalterado Compreensão da LO Inalterado Inalterado Compreensão da LE Alterado Alterado Legenda: LO: linguagem oral. LE: linguagem escrita Tabela 1. Avaliações pré e pós-intervenção fonoaudiológica Habilidades Resultados pré-intervenção fonoaudiológica Resultados pós-intervenção fonoaudiológica Fluência Inalterado Inalterado Leitura Alterado Alterado Escrita Alterado Alterado Nomeação Alterado Inalterado Repetição Alterado Inalterado Evocação Alterado Inalterado Compreensão da LO Inalterado Inalterado Compreensão da LE Alterado Alterado Legenda: LO: linguagem oral. LE: linguagem escrita Tabela 1. Avaliações pré e pós-intervenção fonoaudiológica Legenda: LO: linguagem oral. LE: linguagem escrita a malformação arteriovenosa, caracterizada por fístulas arteriovenosas que possuem um epicentro denominado ninho, composto por artérias que nutrem e aumentam o volume das veias, podendo estar presentes no nascimento ou se evidenciarem durante o desenvolvimento do indivíduo, sendo que elas não diminuem ou regridem de forma espontâ nea. As mudanças hormonais para a puberdade ou traumas, podem mobilizar o crescimento da malfor mação arteriovenosa, pois as veias de drenagem se tornam mais evidenciadas, tortuosas e distendidas, podendo ocasionar uma hemorragia14. Alguns Apresentação do caso clínico O trabalho fundamentou-se em um estudo de caso clínico de um paciente atendido na Clínica de Fonoaudiologia da instituição de origem. O estudo não foi enviado ao Comitê de Ética em Pesquisa com Seres Humanos (CEP), pois ao iniciar o trata 482 Distúrb Comun, São Paulo, 29(3): 480-486, setembro, 2017 ARTIGOS Intervenção fonoaudiológica precoce em afasia infantil decorrente de um acidente vascular cerebral: relato de caso mento fonoaudiológico, o responsável pelo pacien te assinou o Termo de Autorização para Utilização dos Dados, como previsto e concedido pela própria instituição, dispensando a aprovação pelo CEP. panhado de edema cerebral, com a necessidade de outro procedimento cirúrgico para descompressão intra-craniana. A alta hospitalar aconteceu 25 dias após o acometimento, com orientações sobre a necessidade de atendimento fonoaudiológico. ç p p ç p O paciente compareceu à Clínica de Fonoau diologia acompanhado dos pais, que participaram da anamnese e auxiliaram no relato da seguinte história clínica: indivíduo de 11 anos de idade, gênero masculino, com normalidade de desenvol vimento neuropsicomotor e independência para as atividades funcionais durante a primeira infância. Em março de 2015 sentiu mal estar seguido de sín cope, foi socorrido em um breve período de tempo, aproximadamente 10 minutos, e encaminhado para o hospital, onde foi realizada avaliação neurológica por meio de tomografia computadorizada de crânio, com cortes axiais, utilizando o aparelho Multi Slice, que fornece reconstruções multiplanares, sem a infusão intravenosa do meio de contraste iodado. Imediatamente, foi diagnosticado AVCH em região fronto-parietotemporal esquerda e realizada neuro cirurgia para conter a hemorragia, sendo que, após quatro dias, houve novo foco hemorrágico acom A primeira avaliação de linguagem ocorreu em abril de 2015, por meio do Teste de Reabilitação das Afasias – versão adaptada11 e do Token Test – versão adaptada para crianças12, evidenciando a classifica ção de afasia adquirida do tipo emissiva, conforme as classificações propostas para crianças7,8. Foram realizadas 91 sessões de terapia fo noaudiológica, com duração de 50 minutos, pelo período de 12 meses. A intervenção foi realizada por três terapeutas distintas, com revezamento de terapeutas, respeitando o ciclo da grade curricular do curso de graduação em Fonoaudiologia, sendo todas as sessões supervisionadas por uma única docente especialista na área. Os planejamentos terapêuticos foram abrangentes para atender todas as alterações fonoaudiológicas encontradas na avaliação (tabela 1). Tabela 1. Referências Em relação à reabilitação precoce, quando o paciente é submetido a terapia desde a fase aguda do AVC, a recuperação pode ser até duas vezes maior quando comparada a recuperação espontâ nea. Além disso, se a terapia fonoaudiológica for iniciada após a fase aguda, os resultados podem ser menos expressivos, ainda que significativos18. Levando em consideração que o tratamento fono audiológico inciou-se precocemente, em torno de um mês após o acometimento cerebral, foi possível observar melhora significativa nas habilidades co municativas, visto que, ao comparar as avaliações pré e pós-intervenção fonoaudiológica, nota-se que, de seis habilidades alteradas, restaram apenas três que não apresentaram melhora expressiva. 1. World Health Organization (WHO). Neurological disorders: public health challenges [Acesso em 20 out de 2016]. Disponíel em: http://www.who.int/mental_health/neurology/neurological_ disorders_report_web.pdf. 2. Liégio LM, Borges DM, Faria PHC, Pacheco LP, Martins PP. Atendimento ao acidente vascular encefálico infantil: perspectiva de neuropediatras. Enfermaria Global 2011; 4(22); 1-14. 3. Mekitarian Filho E, de Carvalho WB. Stroke in children. J Pediatr (Rio J). 2009; 85(6): 469-79. 4. Avila, LN. A linguagem em crianças e adolescentes acometidos por acidente vascular cerebral infantil. Porto Alegre. 4. Avila, LN. A linguagem em crianças e adolescentes acometidos por acidente vascular cerebral infantil. Porto Alegre. Dissertação [mestrado] – Universidade Federal Rio Grande do Sul. Faculdade de Medicina; 2009. Dissertação [mestrado] – Universidade Federal Rio Grande do Sul. Faculdade de Medicina; 2009. 5. Rotta NT & Ranzan J. Acidente vascular cerebral. In: Rotta NT, Ohlweiler L, Riesgo RS. Rotinas em Neuropediatria. Porto Alegre: Artmed; 2005.p.123-130. Ao analisar as habilidades formais de lingua gem que permaneceram alteradas após 12 meses de intervenção, observa-se que as relacionadas à leitura e escrita são as de maior significância, interferindo no desempenho escolar e desenvolvi mento comunicativo, sendo, portanto, necessário o acompanhamento fonoaudiológico unido ao apoio escolar. Dessa forma, por mais que a afasia na infância tenha um melhor prognóstico, essa criança precisará de acompanhamento por um período maior, compreendendo este por toda sua infância e até a aquisição total da linguagem em seu período escolar, devido às possíveis dificuldades 8, o que reforça a importância e a necessidade de comunicação e parceria com toda a equipe escolar, promovendo qualidade no processo de reabilitação. 6. Coudry MIH. Diário de Narciso: discurso e afasia: análise discursiva de interlocuções com afásicos. 2. ed. São Paulo: Martins Fontes; 1996. 7. Van Hout A. Acquired afasia in childhooh. In Segalowits SJ, Rapin I. Discussão Dentre os tipos de AVC, o isquêmico é o mais comum independente da faixa etária, relacionando -se com fatores de risco de ordens hereditárias. Em contrapartida, o AVCH está mais relacionado a pro blemas de malformação vascular, estando presentes ou ausentes outros fatores de risco adquiridos ou hereditários13. Algumas das anomalias vasculares são as malformações arteriais, arteriovenosas e fístulas arteriovenosas. Dentre estas, merece destaque 483 Distúrb Comun, São Paulo, 29(3): 480-486, setembro, 2017 ARTIGOS Natalia C. Favoretto, Natalia G. Carleto, Paula G. Cunha, Vanessa C. B. Panes, Adriano Y. Fernandes, Dionísia A. C. Lamônica, Magali de L. Caldana Figura 1. Controle pós-craniectomia na região fronto-parietotemporal esquerda Figura 1. Controle pós-craniectomia na região fronto-parietotemporal esquerda Figura 2. Tomografia Computadorizada de Crânio, após craniectomia, destacando área hipoatenuante de aspecto sequelar na região fronto-parietotemporal esquerda Figura 2. Tomografia Computadorizada de Crânio, após craniectomia, destacando área hipoatenuante de aspecto sequelar na região fronto-parietotemporal esquerda este tipo de AVC na infância, o que corrobora com os achados da literatura. critérios estabelecidos na década de 60 ainda são utilizados e consideram o início da puberdade aos oito anos para as meninas e em nove anos para os meninos15. Apesar de o tipo mais comum de AVC ser o isquêmico, o caso clínico abordado relata um AVC do tipo hemorrágico ocorrido durante o período de puberdade e resultante de malformação arteriovenosa, que é o principal fator de risco para A relação entre tipo, local e extensão da lesão e manifestações na comunicação do indivíduo merecem atenção, visto que, os lobos temporal, parietal e região pré-frontal do hemisfério esquer do atuam de forma direta nos processos léxico -semânticos16, havendo forte correlação entre as 484 Distúrb Comun, São Paulo, 29(3): 480-486, setembro, 2017 ARTIGOS Intervenção fonoaudiológica precoce em afasia infantil decorrente de um acidente vascular cerebral: relato de caso Conclusão áreas têmporo-parietais e as tarefas de discurso, repetição, nomeação, compreensão da escrita e da leitura9. A lesão cerebral do paciente compreende as áreas supracitadas, exceto região pré-frontal, o que reforça a concordância entre manifestações linguísticas e locais de lesões neurológicas adqui ridas do caso estudado. Um acometimento cerebral infantil não é algo comum e casual. Ao longo dos anos, a variável de crianças atingidas vem aumentando exponen cialmente, por isso, é necessário que haja mais estudos, uma vez que sua etiologia, diagnóstico e prognóstico são desafiadores. É importante que este indivíduo seja assistido de forma precoce, refor çando a importância da intervenção em período de maior neuroplasticidade, e que seja acompanhado até atingir uma comunicação funcional. Além disso, destaca-se a importância da associação entre os achados fonoaudiológicos e neurológicos para que ocorra maior sucesso nos processos de reabilitação da linguagem. O efeito da lesão cerebral em crianças é dife rente do efeito em adultos17. Caso uma criança seja acometida por uma lesão cerebral que comprometa completamente seu hemisfério dominante, poderá apresentar afasia, mas com maior chance de recupe ração da linguagem, pois a reorganização cerebral, decorrente da neuroplasticidade, encontra-se em maior potencial comparado à individuos adultos e idosos, fato que possivelmente colaborou no pro cesso de reabilitação neste relato de caso, visto que o paciente iniciou a intervenção fonoaudiológica aos 11 anos de idade. Distúrb Comun, São Paulo, 29(3): 480-486, setembro, 2017 12. Di Simoni F. The Token Test for children. Boston: Teaching Resources Corporation, 1978. Referências Hanbook of Neuropsychology, vol 8. 2. ed, Amsterdam: Elsevier, 2003. 8. Ortiz, KZ. Distúrbios neurológicos adquiridos – linguagem e cognição. Barueri: Manole; 2005.p.60-61, 9. Vieira ACC, Roazzi A, Queiroga BM, Asfora R, Valença MM. Afasias e áreas cerebrais: argumentos prós e contras à perspectiva localizacionista. Psicol. Reflex e Crit. 2011; 24(3): 588-96. 10. Fonseca RP, Wagner GP, Rinaldi J, Parente MAMP. O envelhecimento influencia as habilidades pragmáticas, léxico-semânticas e prosódicas do hemisfério direito? Estudos interdisciplinares sobre o envelhecimento 2007; 12:53-79. 11. Jakubovicz R. Teste de Reabilitação das Afasias. Rio de Janeiro: Revinter, 2005. 485 Distúrb Comun, São Paulo, 29(3): 480-486, setembro, 2017 ARTIGOS Natalia C. Favoretto, Natalia G. Carleto, Paula G. Cunha, Vanessa C. B. Panes, Adriano Y. Fernandes, Dionísia A. C. Lamônica, Magali de L. Caldana 12. Di Simoni F. The Token Test for children. Boston: Teaching Resources Corporation, 1978. 16. Pereira JR, Reis AM, Magalhães Z. Neuroanatomia funcional: Anatomia das áreas activáveis nos usuais paradigmas em ressonância magnética funcional. Acta Med Port 2003; 16:p.107-16. 13. Gandra JD, Bastos MAR. Fatores de risco para ocorrência de acidente vascular encefálico em indivíduos de 0 a 20 anos. Enferm Rev 2014 jan/abr; 17(1): p.61-78. 17. Thompson RF. O cérebro – uma introdução à neurociência. 3.ed. Santos: Santos; 2005. p.444. 14. Gontijo B, Pereira LB, Silva CMR. Malformações vasculares. Na Bras Dermatol 2004 jan/fev;79(1):p.7-25. 18. Robey RR. The efficacy of treatment for aphasic persons: a meta-analysis. Brain and Language.1994; 47(4): p.582-608. 15. Marshall WA, Tanner JM. Variations in the pattern of pubertal changes in girls. Arch Dis Child 1969; 44:p.291-303. 486
https://openalex.org/W2187121830	https://zenodo.org/records/8042321/files/ijpbs%201.pdf	English	null	EFFICACY OF 2% BLEACHING POWDER IN CONTROLLING SECONDARY CONTAMINATION OF PEBRINE DISEASE INTASAR SILKWORM ANTHEREAE MYLITTA DRURY (DABA T.V)	Zenodo (CERN European Organization for Nuclear Research)	2,013	cc-by	3,563	LAKSHMI VELIDE Department of Biotechnology, GokarajuRangaraju Institute of Engineering and Technology, Bachupally, Kukatpally,Hyderabad, Andhra Pradesh, India-500090 Department of Biotechnology, GokarajuRangaraju Institute of Engineering and Technology, Bachupally, Kukatpally,Hyderabad, Andhra Pradesh, India-500090 Int J Pharm Bio Sci 2013 Oct; 4(4): (B) 1228 - 1233 Int J Pharm Bio Sci 2013 Oct; 4(4): (B) 1228 - 1233 Research Article Biotechnology ABSTRACT Pebrinedisease found to be highly virulent and harm the larval and cocoon c tropical tasar silkworm Antheraeamylitta Drury (Daba TV).Therefore an been made to evaluate the effect of 2% bleaching powder solution in co pebrine disease attained through secondary contamination by studying cocoon characters. In comparison with the healthy control (T1 batch larva untreated plantation),the results reveal a significant decrease span,larvalweight,number of cocoons harvested, effective rearing,cocoonweight,shellweight,shellratio,filament length, reelability, silkreeled, length of shell, width of shell, shell thickness, peduncle thickne length, peduncle weight, peduncle diameter in T2 batch (infected larva untreated Terminaliaarjuna plants) but a slight decrease was observed (healthy and infected larvae reared combined on 2% bleaching powder sol plantation).The results also show that maximum mortality in T1 batch a larvae was due to viral and bacterial infections and other factors rather t disease whereas in case of T2 batch nearly 50% of larval mortality pebrinedisease.Based on the results obtained from present study 2% bleac solution is found efficient in controlling pebrine disease attained throug contamination in Daba TV. KEYWORDS: pebrine, secondary contamination, 2%Bleaching powder solution, Da Corresponding author LAKSHMI VELIDE Department of Biotechnology, GokarajuRangaraju Institute of Engineering Bachupally, Kukatpally,Hyderabad, Andhra Pradesh, India-50 Pebrinedisease found to be highly virulent and harm the larval and cocoon characters of tropical tasar silkworm Antheraeamylitta Drury (Daba TV).Therefore an attempt has been made to evaluate the effect of 2% bleaching powder solution in controlling the pebrine disease attained through secondary contamination by studying larval and cocoon characters. In comparison with the healthy control (T1 batch larvae reared on untreated plantation),the results reveal a significant decrease in larval span,larvalweight,number of cocoons harvested, effective rate of rearing,cocoonweight,shellweight,shellratio,filament length, reelability, weight of silkreeled, length of shell, width of shell, shell thickness, peduncle thickness, peduncle length, peduncle weight, peduncle diameter in T2 batch (infected larvae reared on untreated Terminaliaarjuna plants) but a slight decrease was observed in T3 batch (healthy and infected larvae reared combined on 2% bleaching powder solution treated plantation).The results also show that maximum mortality in T1 batch and T3 batch larvae was due to viral and bacterial infections and other factors rather than pebrine disease whereas in case of T2 batch nearly 50% of larval mortality was due to pebrinedisease.Based on the results obtained from present study 2% bleaching powder solution is found efficient in controlling pebrine disease attained through secondary contamination in Daba TV. EFFICACY OF 2% BLEACHING POWDER IN CONTROLLING SECONDARY CONTAMINATION OF PEBRINE DISEASE INTASAR SILKWORM ANTHEREAE MYLITTA DRURY (DABA T.V) LAKSHMI VELIDE INTRODUCTION STATISTICAL ANALYSIS Each assay was replicated 3 times. Values were expressed as mean ± SE ofreplicationand Student’s t-test was applied to locate significant (P≤ 0.05) differences between treated and untreated larvae. INTRODUCTION cocoons were preserved in the cages made up of wire mesh of size 2ft x 2ft x 2ft under temperature of 29±1ºC and humidity 70±1%. The emerged moths were tested for pebrine by a method derived from that used in sericulture 11. In this method, the abdomen of moth is severed with scissors, placed in a small mortar, mixed with water and crushed with pestle. A drop of the smear is placed on a clean slide and examined under a microscope of 600X magnification for Nosema sp., spores. (19).The eggs laid by healthy and infected moths were collected and incubated for further research.The hatched larvae were reared in three different fields to prevent secondary contamination.Two days before brushing of larvae and for every four days of interval throughout the rearing period,one of the rearing field along with the trunks of Terminaliaarjuna plants was sprayed with 2% Bleaching powder solution which is prepared from the commercially available bleaching powder. The first instar larvae hatched from the eggs laid by the healthy moths were kept as T1batch and reared on untreated plantation.The first instar larvae hatched from the eggs laid by the pebrine infected moths were kept as T2 batch and reared on untreated plantation.The first instar larvae hatched from the eggs laid by the healthy and pebrinised moths were kept combined as T3batch and reared on 2% bleaching powder solution treated plantation. Each batch had three replications of 50 larvae and was reared till cocooning following standard procedure.Larvae that were died because of pebrinewere examined for the presence of spores under light microscope everyday till spinning and included for data analysis. Anthereaemylitta Drury a lepidopteran insect of the Saturniidae family produces tasar silk of commercial importance.This species is endemic and distributed in different geographical regions of India in the form of 44 ecological races.The varied voltinism and higher interference of environment on silkworm rearing performance, heavy mortality of larvae due to pests, predators, parasites attack, reduced the crop yield and made the silk industry unreliable1,2. Pebrineis one of the dreadful disease seen in Anthereaemylitta Drury (Daba TV), caused by intracellular parasite Nosemaspecies.Pebrine can be acquired from the mother moth (primary infection) or through the excreta of pebrinised larvae and the contaminated environment (secondary infection). The pathogen is causing considerable yield loss upto 40% in combination with other pathogens3. INTRODUCTION Black pepper like spots on the integument of infected larvae are the infected hypodermal cells which become enlarged and vacuolated get blackened due to the formation of melanin 4.The infected larvae of Bombyxmori and Anthereaemylittadrury show significant changes in the cocoon weight, shell weight, denier, reelability etc.,5,6. Three Nosema sp. from three non-mulberry silkworms as Nosemamylitta from Antheraeamylitta, Nosema.ricini from Philosamiaricini and Nosemaassamensis from Antheraeaassamensis7.No silkworm race reported to be completely immune to pebrine. Effectiveness of fungicides (Carbendazim) and antimalarial drugs (Chloroquine) to control the disease has been investigated by several workers 8,9,10. The present work includes the study of larval and cocoon characters of Daba TV to understand the effect of 2% bleaching powder solution in controlling pebrine disease attained through secondary contamination. cocoons were preserved in the cages made up of wire mesh of size 2ft x 2ft x 2ft under temperature of 29±1ºC and humidity 70±1%. The emerged moths were tested for pebrine by a method derived from that used in sericulture 11. In this method, the abdomen of moth is severed with scissors, placed in a small mortar, mixed with water and crushed with pestle. A drop of the smear is placed on a clean slide and examined under a microscope of 600X magnification for Nosema sp., spores. (19).The eggs laid by healthy and infected moths were collected and incubated for further research.The hatched larvae were reared in three different fields to prevent secondary contamination.Two days before brushing of larvae and for every four days of interval throughout the rearing period,one of the rearing field along with the trunks of Terminaliaarjuna plants was sprayed with 2% Bleaching powder solution which is prepared from the commercially available bleaching powder. The first instar larvae hatched from the eggs laid by the healthy moths were kept as T1batch and reared on untreated plantation.The first instar larvae hatched from the eggs laid by the pebrine infected moths were kept as T2 batch and reared on untreated plantation.The first instar larvae hatched from the eggs laid by the healthy and pebrinised moths were kept combined as T3batch and reared on 2% bleaching powder solution treated plantation. Each batch had three replications of 50 larvae and was reared till cocooning following standard procedure.Larvae that were died because of pebrinewere examined for the presence of spores under light microscope everyday till spinning and included for data analysis. This article can be downloaded from www.ijpbs.net B - 1228 KEYWORDS: pebrine, secondary contamination, 2%Bleaching powder solution, Daba TV. Corresponding author LAKSHMI VELIDE Department of Biotechnology, GokarajuRangaraju Institute of Engineering and Technology, Bachupally, Kukatpally,Hyderabad, Andhra Pradesh, India-500090 This article can be downloaded from www.ijpbs.net B - 1228 KEYWORDS: pebrine, secondary contamination, 2%Bleaching powder solution, Daba TV. Corresponding author LAKSHMI VELIDE Department of Biotechnology, GokarajuRangaraju Institute of Engineering and Technology, Bachupally, Kukatpally,Hyderabad, Andhra Pradesh, India-500090 EYWORDS: pebrine, secondary contamination, 2%Bleaching powder solution, Daba TV. LAKSHMI VELIDE Department of Biotechnology, GokarajuRangaraju Institute of Engineering and Technology, Bachupally, Kukatpally,Hyderabad, Andhra Pradesh, India-500090 This article can be downloaded from www.ijpbs.net B - 1228 Int J Pharm Bio Sci 2013 Oct; 4(4): (B) 1228 - 1233 MATERIALS AND METHODS Daba TV cocoons were collected as per the standard norms such as weight, colour,size of cocoons and length of the peduncle from the field plantation of Central silk board,Chennor, AdilabadDistrict,Andhra Pradesh, India. The This article can be downloaded from www.ijpbs.net B - 1229 This article can be downloaded from www.ijpbs.net B - 1229 B - 1229 Int J Pharm Bio Sci 2013 Oct; 4(4): (B) 1228 - 1233 RESULTS AND DISCUSSION A.mylitta infected with Nosema sp. reduced the relative growth rate of the larvae12. The administration of certain chemicals like prostaglandins increases the larval lifecycle in silkworm13.Present results also show that out of 150 larvae brushed the number of cocoons formed were significantly low in case of T2 batch which in turn reduced the ERR%. Much variation was not observed between T1 and T2 batches in case of number of cocoons formed and ERR%. Present studies shows that the pebrine virulence was high and had much impact on various characters of the ecorace.Table 1 show that T2 and T3 batch larvae have shown 21% and 4.7% reduction in their larval span in comparison with T1.In case of T2 batch the larval weight was 22% less than T1 batch.The decrease in food consumption, digestion, relative consumption rate, efficiency of conversion of ingested food in fifth instar of Table 1 Effect of 2% bleaching on rearing performance of Daba TV Ecorace Table 1 Effect of 2% bleaching on rearing performance of Daba TV Ecorace Larval characters Healthy Control Larvae reared on untreated plants (T1) Infected Control larvae reared on untreated plants (T2) Larvae rearedon2%Bleaching Powder Solutiontreated plants(T3) No.of larvae brushed 150 150 150 Larval span(days) 43 34(-20.9) 41(-4.7) Larval weight(g) 36 28(-22.2) 35(-2.8) No.of cocoons Harvested 135 76(-43.7) 118(-12.6) Effective rate of rearing by number (ERR) (%) 90 50.6(-43.8) 79(-12.2) Table 2 explains the mortality rate of Daba TV larvae .In case of T1 batch the mortality rate was maximum due to infections and other attacks rather than pebrine disease. Nearly 50% mortality rate in T2 batch can be attributed to pebrine disease. In case of T3 batch maximum loss was found to be due to micro-organisms attack and other factors. Each value represents the mean of 3 different observations. Each value represents the mean of 3 different observations. mylittaand increases the cocoon weight and shell weight 10.The administration of certain neurohumoral factors, vertebrate harmones, chemicals like prostaglandins increases the cocoon weight, shell weight in silkworm13,15,16.It is evident from the results that the SR% of T1 and T3 batches did not vary much whereas T2 batch has shown a significant reduction. The results also show that filament length of T2 and T3 batch cocoons have decreased by 60 and 10% Table 3 indicate the rearing performance of 2% bleaching powder solution in controlling pebrine disease and the influence on cocoon characters.In comparison with the healthy control cocoon weight of T2 and T3 batches found decrease by 19 and 6% respectively. It is observed that high shell weight was recorded in T3 batch cocoons compared to T2 which may be due to the resistance attained against pebrine disease.6working on pebrine disease in Andhra local ecorace,Anthereaemylittadrury have recorded low cocoon weight and shell weight in the cocoons of both trasovarial, secondarily infected larvae rather than the healthy control. A.mylitta larvae infected with Nosema species have shown decrease in shell weight 14. 0.005% carbendazim treatment of larval stages during rearing has a definite effect in suppressing the development of Nosemasp. in A. mylittaand increases the cocoon weight and shell weight 10.The administration of certain neurohumoral factors, vertebrate harmones, chemicals like prostaglandins increases the cocoon weight, shell weight in silkworm13,15,16.It is evident from the results that the SR% of T1 and T3 batches did not vary much whereas T2 batch has shown a significant reduction. The results also show that filament length of T2 and T3 batch cocoons have decreased by 60 and 10% respectively when compared to that of healthy control.Pebrine disease in Andhra local ecorace will seriously affect the filament length 6.It is found that the B1, B2, B3 and B4 batch cocoons have decreased in reel ability by 1.17, 3.11, 2.33 and 3.11% respectively than that of healthy cocoons. In case of reel ability there is no much variation between T1 and T3 batches whereas a significant reduction was noticed in T2 batch cocoons.Pebrine disease will significantly affect the reel ability in tasar cocoons 6. When compared with the healthy control the weight of the silk reeled of T2 batch cocoon was very much low by 32%, whereas in T3 batch cocoons it was low by 8%.The reduced silk gland weight in A.mylitta larvae infected with Nosema sp. Each value represents the mean of 3 different observations. This article can be downloaded from www.ijpbs.net B 1230 Each value represents the mean of 3 different observations. Table 2 Effect of 2% bleaching on mortality of Daba TV during larval stages No.of larvae lost due to Healthy Control larvae reared on untreated plants (T1) Infected Control larvae reared on untreated plants (T2) Larvaerearedon2%Bleaching Powder Solutiontreated plants(T3) Pebrine 0 35 3 Viral 3 12 9 Bacterial 5 11 10 Others 7 16 10 Total 15 74 32 Each value represents the mean of 3 different observations. Table 3 Effect of 2% bleaching on Commercial characters of Daba TV Ecorace Cocoon characters Healthy Control larvae reared on untreated plants (T1) Infected Control larvae reared on untreated plants (T2) Larvaerearedon2%Bleaching Powder Solution treated plants(T3) Cocoon weight(g) 9.68±1.12 7.86±0.06(-18.8) 9.07±1.02(-6.3) Shell weight(g) 0.97±0.21 0.68±0.02(-29.9) * 0.88±0.03(-9.3) Shell ratio (%) 10±0.08 8.65±0.05(-13.5) 9.71±0.05(-2.9) * Filament length(m) 354.82±2.87 143.5±1.65(-59.6) 320.45±0.07(-9.7) * Reelability(%) 5.16±0.25 4.32±0.04(-16.3) 5.1±0.13(-1.2) * Reeledsilkweight(g) 0.5±0.08 0.34±0.05(-32.0) 0.46±0.05(-8) * Denier 12.68±0.15 21.32±0.25(+68.13) 12.92±0.25(+1.9) * The values presented into the parentheses indicate the percent increase (+) or decrease (-) over control. Significantly different at P ≤0.05 (Students’ t-test). All the values are the mean values of five replications Table 2 Effect of 2% bleaching on mortality of Daba TV during larval stages Each value represents the mean of 3 different observations. This article can be downloaded from www.ijpbs.net B - 1230 Int J Pharm Bio Sci 2013 Oct; 4(4): (B) 1228 - 1233 Table 3 indicate the rearing performance of 2% bleaching powder solution in controlling pebrine disease and the influence on cocoon characters.In comparison with the healthy control cocoon weight of T2 and T3 batches found decrease by 19 and 6% respectively. It is observed that high shell weight was recorded in T3 batch cocoons compared to T2 which may be due to the resistance attained against pebrine disease.6working on pebrine disease in Andhra local ecorace,Anthereaemylittadrury have recorded low cocoon weight and shell weight in the cocoons of both trasovarial, secondarily infected larvae rather than the healthy control. A.mylitta larvae infected with Nosema species have shown decrease in shell weight 14. 0.005% carbendazim treatment of larval stages during rearing has a definite effect in suppressing the development of Nosemasp. in A. Each value represents the mean of 3 different observations. will finally reduces the silk production14. Lowest denier value can be attributed to healthy control cocoons next comes the T3 batch cocoons. A significant increase (68%) in the denier values of T2 batch cocoons was noticed which can be attributed to the serious impact of pebrine infection and reduction in silk quality.6working on pebrine disease in Andhra local ecorace, A. mylittadrury have reported a significant variation between the denier values of nosema infected cocoons and healthy control.Silk produced from the cocoons of pebrine infected larvae is usually much inferior 17. Table 4 Effect of 2% bleaching on Shell and peduncle characters of Daba T.V ecorace Cocoon characters Healthy Control larvae reared on untreated plants (T1) Infected Control larvae reared on untreated plants (T2) Larvaerearedon2%Bleaching PowderSolutiontreated Plants (T3) Length of shell(cm) 4.03±0.15 2.05±0.03(-49.1) 3.98±0.12(-1.3) Width of shell(cm) 2.56±0.17 1.02±0.05(-60.2) * 2.39±0.16(-6.7) Shell thickness(mm) 0.53±0.05 0.14±0.02(-73.6) * 0.39±0.04(-26.5) Peduncle thickness (mm) 2.43±0.17 1.08±0.04(-55.5) 2.21±0.14(-9.1) * Peduncle length(mm) 4.15±0.19 1.18±0.08(-71.5) 3.54±0.18(-14.7) * Peduncle weight(g) 0.13±0.05 0.03±0.01(-76.9) * 0.09±0.02(-30.7) Peduncle diameter (cm) 1.18±0.04 0.06±0.02(-94.9) * 1.08±0.06(-8.5) The values presented in the parentheses indicate the percent increase (+) or decrease (-) over control. *Significantly different at P ≤0.05 (Students’ t-test). All the values are the mean values of five replications Table 4 Effect of 2% bleaching on Shell and peduncle characters of Daba T.V ecora Table 4 shows the results of shell and peduncle characters. In comparison with T1 batch cocoons, the shell length of T2 batch cocoons was 49% low but T3 batch cocoons did not show much variation.In case of T2 batch, the cocoons have shown significant decrease in shell width than T3 batch when compared with T1.A noticeable decrease of 73% was identified in the shell thickness of T2 batch cocoons when compared to T1 batch.T3 batch cocoons have shown 26% less shell thickness than T1 batch cocoons.Thick shell of Daba T.V. denotes the successful adaptatation of the ecorace18.When compared with T1 This article can be downloaded from www.ijpbs.net B - 1231 This article can be downloaded from www.ijpbs.net B - 1231 Int J Pharm Bio Sci 2013 Oct; 4(4): (B) 1228 - 1233 batch,the peduncle thickness value recorded for T2 batch cocoons was 55% low whereas T3 batch cocoons have shown almost the similar peduncle thickness. Each value represents the mean of 3 different observations. The Peduncle length of T2 batch cocoon was found decreased significantly in relation with T1 batch but T3 batch cocoons have shown a slight decrease.The peduncle weight of T2 batch and T3 batch cocoons was 76.9% and 30.7% less than T1 batch cocoons. An enormous reduction in peduncle diameter was recorded in T2 batch cocoons in comparisonwithT1batch cocoons.Peduncle diameter in T3 batch cocoons did not show much variation from the healthy control.Thus in conclusion 2% bleaching powder solution is efficient in controlling secondary contamination of pebrine disease and the treatment improves the larval characters, cocoon characters of infected Daba TV. ACKNOWLEDGEMENT The authors would like to thank The TasarKhadi Silk Production and Sales Co- operative Society Ltd.Pothreddypally, for providing the reeling equipment and Central silk board,Chennor,for the permission given to carry out our investigation . REFERENCES 1. 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https://openalex.org/W3108597374	https://aprp.msal.ru/jour/article/download/2582/1574	Russian	null	Ensuring the Reliability of Information on the Internet: Modern Legal Framework and Legal Practice	Aktualʹnye problemy rossijskogo prava	2,020	cc-by	5,091	© Симонова С. В., 2020 * Симонова Снежана Владимировна, кандидат юридических наук, старший преподаватель кафедры со- циального и семейного законодательства Ярославского государственного университета имени П. Г. Де- мидова, эксперт аппарата Уполномоченного по правам человека в Ярославской области Советская ул., д. 14, г. Ярославль, Россия, 150003 1 Исследование выполнено при финансовой поддержке РФФИ и ЭИСИ в рамках научного проекта № 20-011-31346 © Симонова С. В., 2020 DOI: 10.17803/1994-1471.2020.120.11.172-179 С. В. Симонова* snezh-simonova@yandex.ru ПРАВОВОЕ РЕГУЛИРОВАНИЕ В ИНФОРМАЦИОННОЙ СФЕРЕ DOI: 10.17803/1994-1471.2020.120.11.172-179 Обеспечение достоверности информации в сети «Интернет»: современные правовые основы и юридическая практика1 Аннотация. В работе представлен анализ российской юридической практики по вопросу распространения в сети «Интернет» недостоверной общественно значимой информации. На основе исследования норма- тивных, правоприменительных и лингвистических источников автор дает юридическую оценку свойству информационной достоверности, отграничивает данное свойство от смежных явлений, а также формули- рует условия реализации законного интереса по получению достоверной информации. Особое внимание уделяется анализу материалов практики привлечения граждан к административной ответственности за распространение заведомо недостоверной общественно значимой информации в сети «Интернет». Ав- тор приходит к выводу о широком распространении в судебной практике презумпций недостоверности цифровых сообщений и их угрозы общественному порядку и безопасности. Отмечается, что недостовер- ная общественно значимая информация интерпретируется в практике через призму не соответствующих действительности сведений, а бремя доказывания истинности публикуемых в сети «Интернет» сообщений возлагается, как правило, на их распространителей. Ключевые слова: информация; достоверность; распространение информации; Интернет; киберпространство; административная ответственность; правонарушение; судебная практика; доказывание; злоупотребление свободой массовой информации. Для цитирования: Симонова С. В. Обеспечение достоверности информации в сети «Интернет»: современ- ные правовые основы и юридическая практика // Актуальные проблемы российского права. — 2020. — Т. 15. — № 11. — С. 172–179. — DOI: 10.17803/1994-1471.2020.120.11.172-179. 1 Исследование выполнено при финансовой поддержке РФФИ и ЭИСИ в рамках научного проекта № 20-011-31346 Актуальные проблемы российского права. 2020. Т. 15. № 11 (120) ноябрь 172 Симонова С. В. Симонова С. В. Обеспечение достоверности информации в сети «Интернет»: современные правовые основы и юридическая практика1 Обеспечение достоверности информации в сети «Интернет»: современные правовые основы и юридическая практика1 2 The reported study was funded by RFBR and Expert Institute for Social Research (EISI) according to the research project № 20-011-31346. 3 СЗ РФ. 2019. № 12. Ст. 1221. 4 СЗ РФ. 2019. № 12. Ст. 1217. Snezhana V. Simonova, Cand. Sci. (Law), Senior Lecturer of the Department of Social and Family Legislation, P. G. Demidov Yaroslavl State University, Expert of the Administrative Office of the Commissioner for Human Rights in the Yaroslavl Region ul. Sovetskaya, d. 14, Yaroslavl, Russia, 150003 snezh-simonova@yandex.ru Abstract. The paper presents an analysis of Russian legal practice on dissemination of unreliable socially significant information on the Internet. Based on the study of regulatory, law enforcement and linguistic sources, the author provides a legal assessment to information reliability characteristic, delimits it from related phenomena, and formulates the conditions for the realization of a legitimate interest in obtaining reliable information. Particular attention is given to an analysis of cases of bringing citizens to administrative responsibility for the dissemination of deliberately unreliable socially significant information on the Internet. The author concludes that the presumptions of unreliability of digital messages and their threat to public order and safety are widely spread in judicial practice. It is noted that unreliable socially significant information is interpreted in practice through the prism of information that does not correspond to reality, and the burden of proving the truth of messages published on the Internet is, as a rule, on their distributors. Keywords: information; reliability; spread of information; Internet; cyberspace; administrative responsibility; offense; arbitrage practice; proof; abuse of freedom of the media. Cite as: Simonova SV. Obespechenie dostovernosti informatsii v seti «Internet»: sovremennye pravovye osnovy i yuridicheskaya praktika [Ensuring the Reliability of Information on the Internet: Modern Legal Framework and Legal Practice]. Aktualnye problemy rossiyskogo prava 2020;15(11):172-179. DOI: 10.17803/1994-1471.2020.120.11.172- 179. (In Russ., abstract in Eng.). течение 2019 г. представляют интерес с точки зрения обобщения применяемых механизмов принудительного обеспечения достоверности информационных потоков в киберпространстве. Как представляется, одним из ключевых вопросов, важных для оценки действующего правового регулирования отношений в области распространения информации в сети «Интер- нет», является вопрос о качественных характе- ристиках информации и о возможности обеспе- чения данного качества при помощи правовых механизмов. Должна ли информация, распро- страняемая в цифровой среде, стремиться к достоверности? В 2020 г. исполняется год с момента при- нятия в России «пакета законов», направ- ленных на ограничение и деликтизацию распространения в сети «Интернет» недосто- верной общественно значимой информации, — Федерального закона от 18.03.2019 № 31‑ФЗ «О внесении изменений в статью 15.3 Феде- рального закона “Об информации, информаци- онных технологиях и о защите информации”»3 и Федерального закона от 18.03.2019 № 27‑ФЗ «О внесении изменений в Кодекс Российской Федерации об административных правонару- шениях»4. С одной стороны, реформа привела к актуализации вопроса о содержании и юриди- ческих пределах права на информацию в циф- ровой среде. С другой стороны, промежуточные итоги реализации указанных нововведений в течение 2019 г. Ensuring the Reliability of Information on the Internet: Modern Legal Framework and Legal Practice2 Snezhana V. Simonova, Cand. Sci. (Law), Senior Lecturer of the Department of Social and Family Legislation, P. G. Demidov Yaroslavl State University, Expert of the Administrative Office of the Commissioner for Human Rights in the Yaroslavl Region ul. Sovetskaya, d. 14, Yaroslavl, Russia, 150003 snezh-simonova@yandex.ru Snezhana V. Simonova, Cand. Sci. (Law), Senior Lecturer of the Department of Social and Family Legislation, P. G. Demidov Yaroslavl State University, Expert of the Administrative Office of the Commissioner for Human Rights in the Yaroslavl Region ul. Sovetskaya, d. 14, Yaroslavl, Russia, 150003 snezh-simonova@yandex.ru Snezhana V. Simonova, Cand. Sci. (Law), Senior Lecturer of the Department of Social and Family Legislation, P. G. Demidov Yaroslavl State University, Expert of the Administrative Office of the Commissioner for Human Rights in the Yaroslavl Region ul. Sovetskaya, d. 14, Yaroslavl, Russia, 150003 snezh-simonova@yandex.ru представляют интерес с точки зрения обобщения применяемых механизмов принудительного обеспечения достоверности информационных потоков в киберпространстве. В Как представляется, одним из ключевых вопросов, важных для оценки действующего правового регулирования отношений в области распространения информации в сети «Интер- нет», является вопрос о качественных характе- ристиках информации и о возможности обеспе- чения данного качества при помощи правовых механизмов. Должна ли информация, распро- страняемая в цифровой среде, стремиться к достоверности? Вне зависимости от того, где именно — в сети «Интернет» или за ее пределами — рас- пространяются сведения, свойство достоверно- Актуальные проблемы российского права. 2020. Т. 15. № 11 (120) ноябрь 173 Правовое регулирование в информационной сфере В поисках ответа на вышеуказанные вопро- сы целесообразно обратиться к существую- щему правовому регулированию, практике и лингвистическим источникам. Конституция РФ, гарантируя в ст. 29 свободу поиска, получения, производства и распространения информации, не содержит требований, которым должны со- ответствовать распространяемые сведения. Ис- ключение составляют лишь конституционные нормы о праве граждан на получение досто- верной информации о состоянии окружающей среды. Обратившись к развивающему поло- жения Конституции РФ нормативному регули- рованию, можно установить неоднозначность юридической оценки информационной досто- верности. сти информации следует рассматривать через призму блага5. Безусловно, особое значение данное благо будет иметь для отношений в сети «Интернет», где риски негативных последствий оборота недостоверной информации крайне высоки. Вместе с тем не любой принудитель- ный механизм обеспечения информационной достоверности отвечает личным, общественным и государственным интересам. сти информации следует рассматривать через призму блага5. Безусловно, особое значение данное благо будет иметь для отношений в сети «Интернет», где риски негативных последствий оборота недостоверной информации крайне высоки. Вместе с тем не любой принудитель- ный механизм обеспечения информационной достоверности отвечает личным, общественным и государственным интересам. Например, мониторинг сети «Интернет» со- ставляет важнейшее направление деятельности большинства правозащитных институтов. Ин- тернет-источники служат одним из основных каналов получения властными органами све- дений о нарушениях или об угрозах нарушения прав и законных интересов граждан. Обнару- жение подобных информационных поводов в ряде случаев предопределяет дальнейшие активные действия по содействию восстановле- нию и защите нарушенных прав. С учетом этого запрет под угрозой наказания распространять недостоверные сведения может обернуться потерей из поля зрения государства фактов реального нарушения прав, что не отвечает не только общественным, но и государственным интересам. Так, в Федеральном законе от 27.07.2006 № 149‑ФЗ «Об информации, информацион- ных технологиях и о защите информации»6 достоверность определяется через призму принципа правового регулирования отноше- ний в сфере информации (ст. 5 По точному замечанию В. В. Невинского, «обязанность человека выходить в информационное поле с достоверной информацией объективно обусловлена» (см.: Невинский В. В. «Цифровые права» человека: сущность, система, значение // Конституционное и муниципальное право. 2019. № 10. С. 26–32). 6 СЗ РФ. 2006. № 31 (1 ч.). Ст. 3448. 7 СЗ РФ. 2017. №20. Ст. 2901. 7 СЗ РФ. 2017. № 20. Ст. 2901. у 6 СЗ РФ. 2006. № 31 (1 ч.). Ст. 344 Snezhana V. Simonova, Cand. Sci. (Law), Senior Lecturer of the Department of Social and Family Legislation, P. G. Demidov Yaroslavl State University, Expert of the Administrative Office of the Commissioner for Human Rights in the Yaroslavl Region ul. Sovetskaya, d. 14, Yaroslavl, Russia, 150003 snezh-simonova@yandex.ru 3), а в Стратегии развития информационного общества в РФ на 2017–2030 годы7 — как потребность граждан и общества. В большинстве же правовых актов достоверная информация рассматривается как объект права, реализуемого в строго опреде- ленной системе отношений. Как представляется, правовые основы обес- печения достоверности информации должны оформляться с учетом ответов на следующие вопросы: Интересно, что федеральный законодатель и до принятия анализируемых Федеральных законов № 31‑ФЗ и № 27‑ФЗ уже выделял круг сведений, к которым предъявлялось требование достоверности: это и информация о деятельно- сти государственных органов, органов местно- го самоуправления, и рекламная информация, и информация об изготовителе (исполнителе, продавце) и о реализуемых им товарах (работах, услугах) и пр. Содержание правовых норм, за- крепляющих указанные требования, позволяет установить, что реализация гарантированного законом права получать достоверные сведе- ния по соответствующему вопросу служит целям 1) вопрос о содержании понятия достоверности информации; 2) вопрос о возможности включения в содер- жание права на информацию правомочия получения достоверных сведений; 3) вопрос о возможности выведения юридиче- ского значения достоверности информации с позиции категории права и (или) законного интереса; 4) вопрос об условиях наделения субъекта ин- формационных отношений обязанностью распространять достоверные сведения. Актуальные проблемы российского права. 2020. Т. 15. № 11 (120) ноябрь 174 Симонова С. В. Симонова С. В. Обеспечение достоверности информации в сети «Интернет»: современные правовые основы и юридическая практика1 Симонова С. В. Обеспечение достоверности информации в сети «Интернет»: современные правовые основы и юридическая практика1 2014 г. и впоследствии отмененные нормы об обязанности блогеров обеспечивать достовер- ность распространяемой информации8. обеспечения иных законных интересов (напри- мер, интересов потребителя в области компе- тентного выбора товара, интересов в области экологической безопасности и взаимодействия с органами власти). Кроме того, учитывая, что распространение в сети «Интернет» недостоверной общественно значимой информации под видом достоверных сообщений охватывается составами админи- стративных правонарушений, предусмотренных частями 9–11 ст. 13.15 Кодекса РФ об админи- стративных правонарушениях, сомнения вызы- вает возможность полноценного доказывания качеств недостоверности распространяемой информации в рамках производства по делам об административных правонарушениях. Кроме того, право граждан на достоверную информацию реализуется в системе отноше- ний, где управомоченным субъектом выступает гражданин (группа или объединение граждан), а обязанным субъектом — компетентный субъект. Основу такой компетентности состав- ляет осведомленность относительно деятельно- сти, информацию о которой требуется раскрыть (продавец, государственный орган, орган мест- ного самоуправления), либо профессиональная квалификация в области оборота информации (СМИ, журналист). К примеру, практике известны случаи рас- пространения непроверенной информации, содержащей утверждения о нарушении прав. 8 См.: Федеральный закон от 05.05.2014 № 97‑ФЗ «О внесении изменений в Федеральный закон “Об информации, информационных технологиях и о защите информации” и отдельные законодательные акты Российской Федерации по вопросам упорядочения обмена информацией с использованием ин- формационно-телекоммуникационных сетей» // СЗ РФ 2014 №19 Ст 2302 9 См.: Закон РФ от 07.02.1992 № 2300-1 (ред. от 18.07.2019) «О защите прав потребителей». П. 1 ст. 10 // СЗ РФ. 1996. № 3. Ст. 140 ; постановление Пленума ВАС РФ от 08.10.2012 № 58 «О некоторых вопросах практики применения арбитражными судами Федерального закона “О рекламе”». П. 29 // Вестник ВАС РФ. 2012. № 12. Актуальные проблемы российского права. 2020. Т. 15. № 11 (120) ноябрь 8 См.: Федеральный закон от 05.05.2014 № 97‑ФЗ «О внесении изменений в Федеральный закон “Об информации, информационных технологиях и о защите информации” и отдельные законодательные акты Российской Федерации по вопросам упорядочения обмена информацией с использованием ин- формационно-телекоммуникационных сетей» // СЗ РФ. 2014. № 19. Ст. 2302. 9 См.: Закон РФ от 07.02.1992 № 2300-1 (ред. от 18.07.2019) «О защите прав потребителей». П. 1 ст. 10 // СЗ РФ. 1996. № 3. Ст. 140 ; постановление Пленума ВАС РФ от 08.10.2012 № 58 «О некоторых вопросах практики применения арбитражными судами Федерального закона “О рекламе”». П. 29 // Вестник ВАС РФ. 2012. № 12. Snezhana V. Simonova, Cand. Sci. (Law), Senior Lecturer of the Department of Social and Family Legislation, P. G. Demidov Yaroslavl State University, Expert of the Administrative Office of the Commissioner for Human Rights in the Yaroslavl Region ul. Sovetskaya, d. 14, Yaroslavl, Russia, 150003 snezh-simonova@yandex.ru Зачастую вывод об отсутствии или наличии в решениях, действиях или бездействии субъек- тов права нарушений закона может быть сделан лишь на основе специальных знаний и по ито- гам проверочных мероприятий. В связи с новы- ми законами возникает вопрос о возможности привлечения к ответственности лиц, распро- странивших не подтвердившиеся сведения, ибо очевидно, что информация о нарушении прав является общественно значимой. Очевидно, что содержание Федеральных за- конов № 31‑ФЗ и № 27‑ФЗ в некотором смысле «ломает» сложившийся подход к определению системы отношений, для которой свойство до- стоверности информации имеет юридическое значение. Так, обновленным регулированием обязанность воздерживаться от сообщения недостоверных сведений возлагается на не ограниченный по признаку компетентности круг субъектов. В условиях отсутствия в зако- нодательстве понятия общественно значимой информации и гарантированного права на до- стоверную общественно значимую информа- цию невозможно говорить о том, что реализа- ция данной обязанности обеспечивает защиту какого-либо законного интереса. Между тем российский опыт правотворческой деятельности подтверждает нежизнеспособность норм, воз- лагающих обязанности в области информаци- онной достоверности на некомпетентный круг субъектов. Достаточно вспомнить принятые в Анализ российской юридической практики позволяет установить, что в зависимости от си- стемы общественных отношений в понятие «до- стоверность» может вкладываться различный смысл. Так, достоверность информации в сфере рекламы оценивается не только с позиции со- ответствия действительности сообщаемых сведений, но и через призму их полноты и до- статочности для формирования истинного впечатления о рекламируемом объекте9, по- скольку, по мысли судов, умолчание определен- 9 См.: Закон РФ от 07.02.1992 № 2300-1 (ред. от 18.07.2019) «О защите прав потребителей». П. 1 ст. 10 // СЗ РФ. 1996. № 3. Ст. 140 ; постановление Пленума ВАС РФ от 08.10.2012 № 58 «О некоторых вопросах практики применения арбитражными судами Федерального закона “О рекламе”». П. 29 // Вестник ВАС РФ. 2012. № 12. Актуальные проблемы российского права. 2020. Т. 15. № 11 (120) ноябрь 175 Правовое регулирование в информационной сфере ных (в том числе негативных) сведений может искажать смысл информации10. Во-первых, категория недостоверной обще- ственно значимой информации рассматрива- ется в правоприменительной практике через призму не соответствующих действительности сведений. Как правило, суды исходят из того, что указанное несоответствие может содержать в себе признаки как полной, так и частичной «фальсификации»12. В последнем случае име- ется в виду искажение отдельных элементов содержания распространяемых сообщений. В то же время в системе отношений, возни- кающих в связи с защитой чести, достоинства и деловой репутации, недостоверные сведе- ния оцениваются на предмет их порочащего характера. 10 См.: Трофимов В. Н. Подборка судебных решений за 2018 год: статья 5 «Общие требования к рекламе» Федерального закона от 13.03.2006 № 38‑ФЗ «О рекламе» // СПС «КонсультантПлюс». 11 См., например: апелляционное определение Московского городского суда от 06.08.2018 № 33- 34054/2018 // СПС «КонсультантПлюс». 12 См.: постановление Якутского городского суда Республики Саха (Якутия) от 25.07.2019 по делу № 5-1442/2019 // URL: http://jakutsky.jak.sudrf.ru/ (дата обращения: 15.01.2020). 13 См.: Экспертное заключение Совета при Президенте РФ по развитию гражданского общества и правам человека на принятые Государственной Думой закон «О внесении изменений в статью 15.3 Федерального закона “Об информации, информационных технологиях и о защите информации”» и на закон «О внесении изменений в статью 13.15 Кодекса Российской Федерации об административных правонарушениях» от 11.03.2019. П. 5 // URL: http://president-sovet.ru/documents/read/639/ (дата обращения: 10.01.2020). 13 См.: Экспертное заключение Совета при Президенте РФ по развитию гражданского общества и правам человека на принятые Государственной Думой закон «О внесении изменений в статью 15.3 Федерального закона “Об информации, информационных технологиях и о защите информации”» и на закон «О внесении изменений в статью 13.15 Кодекса Российской Федерации об административных правонарушениях» от 11.03.2019. П. 5 // URL: http://president-sovet.ru/documents/read/639/ (дата обращения: 10.01.2020). 10 См.: Трофимов В. Н. Подборка судебных решений за 2018 год: статья 5 «Общие требования к рекламе» Федерального закона от 13.03.2006 № 38‑ФЗ «О рекламе» // СПС «КонсультантПлюс». Snezhana V. Simonova, Cand. Sci. (Law), Senior Lecturer of the Department of Social and Family Legislation, P. G. Demidov Yaroslavl State University, Expert of the Administrative Office of the Commissioner for Human Rights in the Yaroslavl Region ul. Sovetskaya, d. 14, Yaroslavl, Russia, 150003 snezh-simonova@yandex.ru При этом наибольшие трудности возникают в практике по вопросам разграни- чения утверждений о фактах (информацион- ных сведений) и оценочных суждений, а также оценки распространяемых сведений с позиции умаления ими чести, достоинства и деловой ре- путации. Данный тезис подтверждается боль- шим числом отмененных судебных решений, а также противоположными заключениями линг- вистических экспертиз, в которых одним и те же заявлениям даются различные по характеру оценки11. Вместе с тем, обратившись к толковым сло- варям русского языка, можно установить, что слово «недостоверный» определяется с по- зиции качества, отражающего сомнения в до- стоверности чего-либо. Понятие же достовер- ности, в свою очередь, отличается богатством смыслового значения и интерпретируется как с позиции явления, не вызывающего сомнений, так и с позиции точности, подлинности и реаль- ности определенных утверждений, источников, сведений, событий и пр. Очевидно, что с точки зрения первой интерпретации отождествление свойства недостоверности и свойства несоответ- ствия действительности информации едва ли справедливо признать корректным. Вместе с тем с 2019 г. оценка достоверного характера распространяемой в сети «Интернет» информации составляет неотъемлемую часть процесса по делам об административных пра- вонарушениях. От результатов данной оценки будет во многом зависеть решение вопроса о привлечении граждан к административной от- ветственности. Практика применения ч. 9–11 ст. 13.15 КоАП РФ в настоящее время находится на стадии становления. При этом обращает на себя внимание то, что многие из вынесенных в 2019 г. судебных решений о привлечении граж- дан к ответственности за распространение недо- стоверной общественно значимой информации были отменены в апелляционном порядке. Ана- лиз принятых к настоящему времени судебных актов позволяет прийти к следующим основным выводам. Обозначенные выше тонкости толкования единиц русского языка имеют ключевое значе- ние для понимания позиции отдельных россий- ских правозащитных институтов, призывающих к разграничению недостоверной информации и информации, не соответствующей действитель- ности. Так, согласно экспертному мнению Со- вета при Президенте РФ по развитию граждан- ского общества и правам человека13 (далее — СПЧ), не соответствующая действительности информация указывает на факты или события, 10 См.: Трофимов В. Н. Подборка судебных решений за 2018 год: статья 5 «Общие требования к рекламе» Федерального закона от 13.03.2006 № 38‑ФЗ «О рекламе» // СПС «КонсультантПлюс». Актуальные проблемы российского права. 2020. Т. 15. № 11 (120) ноябрь 176 Симонова С. В. Симонова С. В. Обеспечение достоверности информации в сети «Интернет»: современные правовые основы и юридическая практика1 Симонова С. В. Обеспечение достоверности информации в сети «Интернет»: современные правовые основы и юридическая практика1 ровать и на судебной стадии производства по делам об административных правонарушениях. Актуальные проблемы российского права. 2020. Т. 15. № 11 (120) ноябрь Актуальные проблемы российского права. 2020. Т. 15. № 11 (120) ноябрь 14 См.: определение Октябрьского районного суда города Архангельска от 06.06.2019 по делу № 5-309/2019 // URL: http://oktsud.arh.sudrf.ru/ (дата обращения: 15.01.2020). 15 См.: постановление Якутского городского суда Республики Саха (Якутия) от 25.07.2019 по делу № 5-1442/2019. 16 См.: постановление Селемджинского районного суда Амурской области от 26.09.2019 по делу № 5-45/2019 // URL: http://selemdginskiy.amr.sudrf.ru/ (дата обращения: 15.01.2020). 17 См.: постановление Селемджинского районного суда Амурской области от 26.09.2019 по делу № 5-44/2019. 18 В Экспертном заключении СПЧ от 11.03.2019 справедливо отмечается, что у лица, не являющегося журналистом, существенно меньше возможностей для проверки распространяемой информации. От- ветственность же за распространение слухов под видом достоверных сообщений предусмотрена для представителей журналистского сообщества. Snezhana V. Simonova, Cand. Sci. (Law), Senior Lecturer of the Department of Social and Family Legislation, P. G. Demidov Yaroslavl State University, Expert of the Administrative Office of the Commissioner for Human Rights in the Yaroslavl Region ul. Sovetskaya, d. 14, Yaroslavl, Russia, 150003 snezh-simonova@yandex.ru не имевшие места в реальности, и в силу этого относится к объективной истине, в то время как недостоверная информация отражает уровень доверия к источнику информации. Из данной трактовки следует вывод о возможности оцен- ки уровня достоверности информации исклю- чительно на основе субъективной шкалы веры человека в истинность распространяемых им сведений. Так, при рассмотрении анализируемой ка- тегории дел отдельные суды возлагают бремя доказывания достоверности распространенных сведений на лиц, в отношении которых возбуж- дено дело об административном правонару- шении15. В ряде случаев выводы о том, что рас- пространяемые сообщения не соответствуют действительности, опираются на экспертные заключения, заключения по результатам ла- бораторных исследований и иные подобные специальные документы16. Однако тот факт, что проверка достоверности отдельных сообщений возможна исключительно на основе специаль- ных знаний, не расценивается в практике в ка- честве обстоятельства, исключающего вывод об осознании гражданами недостоверности опубликованных ими сообщений17. Соответ- ственно, риски распространения сообщений без проведения дополнительных проверочных мероприятий возлагаются на граждан. По мнению СПЧ, при рассмотрении дел об административных правонарушениях, пред- усмотренных ч. 9–11 ст. 13.15 КоАП РФ, суду недопустимо «вторгаться в такие далекие от права вопросы, как вера и доверие». Однако анализ уже сложившейся правоприменитель- ной практики показывает, что суды оценивают достоверность информации всего лишь с пози- ции ее соответствия действительности и не рас- сматривают вопрос о вере граждан в истинность распространяемых сообщений. Во-вторых, исследование материалов пра- воприменительной практики показывает, что вопрос достоверности распространенной ин- формации зачастую не становится предметом всесторонней оценки должностных лиц органов внутренних дел, возбуждающих дела об адми- нистративных правонарушениях, предусмотрен- ных частями 9–11 ст. 13.15 КоАП РФ. Отсутствие в протоколах об административных правонару- шениях доводов в пользу недостоверности опу- бликованной в сети «Интернет» информации в некоторых случаях становится основанием их возвращения судьями для устранения недо- статков14. Однако презумпция недостоверности информации в сети «Интернет» может домини- Вместе с тем установление истинного пси- хического отношения граждан к достоверности записей, публикуемых ими в сети «Интернет», имеет ключевое значение для решения вопро- са о наличии в их действиях предусмотренных частями 9–11 ст. 13.15 КоАП РФ составов адми- нистративных правонарушений. Пренебреже- ние данным правилом фактически приводит к запрету распространения слухов, правдивость которых не очевидна и требует организации специальных проверочных мероприятий18. Обозначенное указывает на то, что в со- временном процессе по делам о привлечении постановление Селемджинского районного суда Амурской области от 26.09.2019 по делу 45/2019 // URL: http://selemdginskiy.amr.sudrf.ru/ (дата обращения: 15.01.2020). постановление Селемджинского районного суда Амурской области от 26.09.2019 по делу 4/2019. 14 См.: определение Октябрьского районного суда города Архангельска от 06.06.2019 по делу № 5-309/2019 // URL: http://oktsud.arh.sudrf.ru/ (дата обращения: 15.01.2020). 15 С Я Р б С (Я ) 25 07 2019 18 В Экспертном заключении СПЧ от 11.03.2019 справедливо отмечается, что у лица, не являющегося журналистом, существенно меньше возможностей для проверки распространяемой информации. От- ветственность же за распространение слухов под видом достоверных сообщений предусмотрена для представителей журналистского сообщества. Snezhana V. Simonova, Cand. Sci. (Law), Senior Lecturer of the Department of Social and Family Legislation, P. G. Demidov Yaroslavl State University, Expert of the Administrative Office of the Commissioner for Human Rights in the Yaroslavl Region ul. Sovetskaya, d. 14, Yaroslavl, Russia, 150003 snezh-simonova@yandex.ru 18 В Экспертном заключении СПЧ от 11.03.2019 справедливо отмечается, что у лица, не являющегося журналистом, существенно меньше возможностей для проверки распространяемой информации. От- ветственность же за распространение слухов под видом достоверных сообщений предусмотрена для представителей журналистского сообщества. 18 В Экспертном заключении СПЧ от 11.03.2019 справедливо отмечается, что у лица, не являющегося журналистом, существенно меньше возможностей для проверки распространяемой информации. От- ветственность же за распространение слухов под видом достоверных сообщений предусмотрена для представителей журналистского сообщества. Актуальные проблемы российского права. 2020. Т. 15. № 11 (120) ноябрь 177 Правовое регулирование в информационной сфере граждан к административной ответственности за злоупотребление свободой массовой инфор- мации (в том состоянии, в котором этот процесс существует на настоящий момент) менее отчет- ливо выражены присущие гражданскому судо- производству состязательность и равноправие сторон. Между тем доказывание достоверности той или иной информации представляет весь- ма трудную задачу для граждан в случае, если они это будут делать самостоятельно. Достаточ- но вспомнить «закон о праве на забвение»19, предписания которого так и не стали реально работающими и эффективными. Как представ- ляется, причины этого во многом связаны с тем, что на лиц, требующих от операторов поисковых систем прекращения выдачи ссылок на опре- деленные страницы сайтов, стало возлагаться бремя доказывания недостоверности и неак- туальности распространяемой о них на данных страницах информации20. Анализируя материалы правоприменитель- ной практики по делам о привлечении граждан к ответственности за правонарушения, пред- усмотренные частями 9–11 ст. 13.15 КоАП РФ, можно установить отсутствие единого подхода к определению негативных последствий распро- странения в сети «Интернет» недостоверной ин- формации. Так, в отдельных протоколах об ад- министративных правонарушениях и решениях судов первой инстанции в качестве угрожающих общественному порядку рассматриваются такие гипотетические последствия распространения недостоверной общественно значимой инфор- мации, как, например, усиление политических настроений граждан в условиях чрезвычайной ситуации, формирование у граждан убежде- ния в противоправном характере деятельно- сти правоохранительных органов, возмущение граждан и митинги и пр. В ряде случаев вопрос о содержательных характеристиках и реально- сти угрозы массового нарушения общественно- го порядка может не исследоваться органами внутренних дел в принципе, в то время как в судах апелляционной инстанции обращается внимание на необходимость всестороннего до- казывания данных обстоятельств21. В-третьих, необходимым условием при- влечения граждан к ответственности за рас- пространение недостоверной общественно значимой информации в сети «Интернет» по смыслу ч. 9–11 ст. 13.15 КоАП РФ является угроза или реальное наступление определенных зако- нодателем негативных последствий. В ряде смежных составов КоАП РФ, например связан- ных с нарушением порядка организации либо проведения публичных мероприятий (ч. 19 См.: Федеральный закон от 13.07.2015 № 264‑ФЗ «О внесении изменений в Федеральный закон “Об информации, информационных технологиях и о защите информации” и статьи 29 и 402 Гражданского процессуального кодекса Российской Федерации» // СЗ РФ. 2015. № 29 (ч. I). Ст. 4392 21 См.: решение Верховного Суда Республики Саха (Якутия) от 21.05.2019 по делу № 7/1-137/2019 // URL: http://vs.jak.sudrf.ru/ (дата обращения: 15.01.2020). 19 См.: Федеральный закон от 13.07.2015 № 264‑ФЗ «О внесении изменений в Федеральный закон “Об информации, информационных технологиях и о защите информации” и статьи 29 и 402 Гражданского процессуального кодекса Российской Федерации» // СЗ РФ. 2015. № 29 (ч. I). Ст. 4392 20 См., например: апелляционное определение Московского городского суда от 06.06.2017 № 33- 19040/2017 // СПС «КонсультантПлюс» ; апелляционное определение Московского городского суда от 12.09.2017 № 33-334821/2017 // СПС «КонсультантПлюс». 21 См.: решение Верховного Суда Республики Саха (Якутия) от 21.05.2019 по делу № 7/1-137/2019 // URL: http://vs.jak.sudrf.ru/ (дата обращения: 15.01.2020). информации, информационных технологиях и о защите информации и статьи 29 и 402 Гражданского процессуального кодекса Российской Федерации» // СЗ РФ. 2015. № 29 (ч. I). Ст. 4392 20 См., например: апелляционное определение Московского городского суда от 06.06.2017 № 33- 19040/2017 // СПС «КонсультантПлюс» ; апелляционное определение Московского городского суда от 12.09.2017 № 33-334821/2017 // СПС «КонсультантПлюс». 22 См.: Application no. 33846/07, Węgrzynowski and Smolczewski v. Poland, Judgment of 16 July 2013, § 65 // URL: http://hudoc.echr.coe.int/eng?i=001-122365 (дата обращения: 15.01.2020). Snezhana V. Simonova, Cand. Sci. (Law), Senior Lecturer of the Department of Social and Family Legislation, P. G. Demidov Yaroslavl State University, Expert of the Administrative Office of the Commissioner for Human Rights in the Yaroslavl Region ul. Sovetskaya, d. 14, Yaroslavl, Russia, 150003 snezh-simonova@yandex.ru 3, 4, 6, 6.1 ст. 20.2), аналогичные последствия также выступают в качестве необходимого элемента объективной стороны состава правонарушения. Однако как показывает практика применения данных норм, угроза причинения вреда охра- няемым законом ценностям зачастую конста- тируется судами со слов сотрудников полиции и не является, как правило, самостоятельным предметом доказывания. Между тем едва ли отмеченные обстоятель- ства можно расценить в качестве реальных угроз общественному порядку и обществен- ной безопасности. Связь между информацией, размещенной в сети «Интернет», и реальными событиями всегда будет косвенной ввиду боль- шого числа внешних факторов, которые вмеши- ваются в эту причинную связь. Подтвердить же доказательствами объективную связь между вбросом сведений в Интернет и событием в ре- альной жизни невероятно сложно. С учетом изложенного очевидно, что при- чины сегодняшних проблем правопримени- 19 См.: Федеральный закон от 13.07.2015 № 264‑ФЗ «О внесении изменений в Федеральный закон “Об информации, информационных технологиях и о защите информации” и статьи 29 и 402 Гражданского процессуального кодекса Российской Федерации» // СЗ РФ. 2015. № 29 (ч. I). Ст. 4392 21 См.: решение Верховного Суда Республики Саха (Якутия) от 21.05.2019 по делу № 7/1-137/2019 // URL: http://vs.jak.sudrf.ru/ (дата обращения: 15.01.2020). Актуальные проблемы российского права. 2020. Т. 15. № 11 (120) ноябрь 178 Симонова С. В. Симонова С. В. Обеспечение достоверности информации в сети «Интернет»: современные правовые основы и юридическая практика1 Симонова С. В. Обеспечение достоверности информации в сети «Интернет»: современные правовые основы и юридическая практика1 жиновски и Смолчевски против Польши» нега- тивно оценил практику удаления недостовер- ной информации из сети «Интернет» исходя из правила о недопустимости «переписывания истории»22. По мысли Суда, однажды размещен- ные в сети «Интернет» сведения могут в даль- нейшем использоваться в образовательных и исследовательских целях. В случае же удаления информации ее повторное размещение может оказаться невозможным. Аналогично возникнут сложности и с восстановлением прав лиц, не- обоснованно привлеченных к ответственности за распространение недостоверной обществен- но значимой информации. тельной практики, связанной с юридической оценкой фактов распространения недосто- верной общественно значимой информации, заложены в содержании самих запрещающих норм. Многие из отмеченных противоречий предопределены особенностями формули- рования нормативных предписаний, а также конструкций составов административных пра- вонарушений, предусмотренных частями 9–11 ст. 13.15 КоАП РФ. Как представляется, при применении ана- лизируемых положений крайне важно иметь в виду позицию Европейского Суда по правам человека, который в решении по делу «Вегр- БИБЛИОГРАФИЯ 1. Невинский В. В. «Цифровые права» человека: сущность, система, значение // Конституционное и муниципальное право. — 2019. — № 10. — С. 26–32. 1. Невинский В. В. «Цифровые права» человека: сущность, система, значение // Конституционное и муниципальное право. — 2019. — № 10. — С. 26–32. 2. Трофимов В. Н. Подборка судебных решений за 2018 год: статья 5 «Общие требования к рекламе» Федерального закона от 13.03.2006 № 38‑ФЗ «О рекламе» // СПС «КонсультантПлюс». 2. Трофимов В. Н. Подборка судебных решений за 2018 год: статья 5 «Общие требования к рекламе» Федерального закона от 13.03.2006 № 38‑ФЗ «О рекламе» // СПС «КонсультантПлюс». Материал поступил в редакцию 22 января 2020 г. Материал поступил в редакцию 22 января 2020 г. Актуальные проблемы российского права. 2020. Т. 15. № 11 (120) ноябрь REFERENCES (TRANSLITERATION) 1. Nevinskij V. V. «Cifrovye prava» cheloveka: sushchnost’, sistema, znachenie // Konstitucionnoe i municipal’noe pravo. — 2019. — № 10. — S. 26–32. 1. Nevinskij V. V. «Cifrovye prava» cheloveka: sushchnost’, sistema, znachenie // Konstitucionnoe i municipal’noe pravo. — 2019. — № 10. — S. 26–32. 2. Trofimov V. N. Podborka sudebnyh reshenij za 2018 god: stat’ya 5 «Obshchie trebovaniya k reklame» Federal’nogo zakona ot 13.03.2006 № 38-FZ «O reklame» // SPS «Konsul’tantPlyus». 2. Trofimov V. N. Podborka sudebnyh reshenij za 2018 god: stat’ya 5 «Obshchie trebovaniya k reklame» Federal’nogo zakona ot 13.03.2006 № 38-FZ «O reklame» // SPS «Konsul’tantPlyus». 179 179
https://openalex.org/W3015261173	https://iris.uniroma1.it/bitstream/11573/1388531/1/Luccarini_Stability_2020.pdf	English	null	Stability of the Meat Protein Type I Collagen: Influence of pH, Ionic Strength, and Phenolic Antioxidant	Foods	2,020	cc-by	7,239	Received: 5 March 2020; Accepted: 8 April 2020; Published: 11 April 2020 Abstract: The water-holding capacity (WHC) is among the key factors in determining the quality of meat and its value, which is strongly inﬂuenced by the content and quality of the connective tissue proteins like collagen. Therefore, the factors that inﬂuence the proteins’ stability, e.g., pH, ionic strength, and the antioxidants which are used to increase the meat shelf-life, also aﬀect the WHC. The interaction of collagen, whose structure is strongly inﬂuenced by the interaction with water molecules, can be studied following the behavior of water diﬀusion by low-resolution 1H NMR experiments. The present study is addressed to study the collagen stability as a function of pH, ionic strength, and phenolic antioxidants like catechin. The experimental study demonstrated how the 1H NMR time domain (TD) experiments are able to evaluate the hydration properties of collagen, not only as a function of ionic strength and pH, but also in determining the ability of catechin to interact both on the surface of the collagen ﬁbrils and inside the ﬁbrillar domain. Keywords: meat; collagen; pH; ionic strength; catechin; 1H NMR; T2 relaxation Stability of the Meat Protein Type I Collagen: Inﬂuence of pH, Ionic Strength, and Phenolic Antioxidant Massimo Lucarini 1,, Alessandra Durazzo 1 , Fabio Sciubba 2, Maria Enrica Di Cocco 2, Raﬀaella Gianferri 2, Mosè Alise 3 , Antonello Santini 3 , Maurizio Delﬁni 2 and Ginevra Lombardi-Boccia 1 Massimo Lucarini 1,, Alessandra Durazzo 1 , Fabio Sciubba 2, Maria Enrica Di Cocco 2, Raﬀaella Gianferri 2, Mosè Alise 3 , Antonello Santini 3 , Maurizio Delﬁni 2 and Ginevra Lombardi-Boccia 1 1 CREA-Research Centre for Food and Nutrition, Via Ardeatina 546, 00178 Rome, Italy; alessandra.durazzo@crea.gov.it (A.D.); g.lombardiboccia@crea.gov.it (G.L.-B.) 1 CREA-Research Centre for Food and Nutrition, Via Ardeatina 546, 00178 Rome, Italy; alessandra.durazzo@crea.gov.it (A.D.); g.lombardiboccia@crea.gov.it (G.L.-B.) g g g 2 Department of Chemistry, “Sapienza” University of Rome, Piazzale Aldo Moro 5, 00185 Roma, Italy; Fabio.sciubba@uniroma1.it (F.S.); mariaenrica.dicocco@uniroma1.it (M.E.D.C.); raﬀaella.gianferri@uniroma1.it (R.G.); maurizio.delﬁni@uniroma1.it (M.D.) g 3 Department of Pharmacy, University of Napoli Federico II, Via D. Montesano 49, 80131 Napoli, Ital alisemose@gmail.com (M.A.); asantini@unina.it (A.S.) g * Correspondence: massimo.lucarini@crea.gov.it; Tel.: +39-06-51494446 foods foods Foods 2020, 9, 480; doi:10.3390/foods9040480 foods foods 1. Introduction The water content of meat, approximately 75%, and the water-holding capacity (WHC) are both key factors in determining the technological quality of meat and that of meat products [1,2]. Among the constituents of meat, proteins play a fundamental role in retaining water, [3] and, consequently, the factors inﬂuencing the protein stability also aﬀect the WHC [1]. The main proteins responsible for the WHC in meat are myoﬁbrils and the collagen of connective tissue. Myoﬁbrillar proteins are the most studied, since they are responsible for most of the water retained in the muscles. Collagen is ubiquitous in all vertebrates, and its structure is stabilized by extensive hydrogen bonds where water molecules are a relevant part of the ordered hydrogen-bonding existing network [4]. Among the factors to be considered in the technological processes of the conversion of muscle into meat, there are the pH, the ionic strength, and the antioxidant molecules, which are used to slow down the oxidative process onset. Changes in the pH around the isoelectric point of the meat proteins causes a change in the net charge (positive or negative) of proteins, determining a greater electrostatic repulsive force among Foods 2020, 9, 480; doi:10.3390/foods9040480 www.mdpi.com/journal/foods 2 of 10 Foods 2020, 9, 480 the protein chains, which leads to an increased swelling of the protein system with an increase in the WHC. Similarly, the meat proteins’ electronic charge is also aﬀected by the ionic strength that is able to move the isoelectric point of the proteins, causing a diﬀerent mobility of the water inside the protein domain system. Oxidation of meat has a relevant eﬀect on quality parameters, and for this reason, antioxidant molecules are used to slow down this process. The use of natural antioxidants to prevent and slow down the oxidation process in biological matrices is relevant. Proteins in biological matrices are, together with lipids, one of the main targets of the oxidant compounds for both their reaction rate and abundance [5,6]. Recently, a mechanism for the antioxidant action of catechin, a ﬂavan-3-ol, namely (2R, 3S)-2-(3,4-dihydroxyphenyl)-3,4-dihydro-2H-chromene-3,5,7-triol, towards collagen, has been proposed [7]. Madhan et al. [8], by using circular dichroism and Fourier transform infrared spectroscopy (FTIR), reported how hydrogen bonding and hydrophobic interactions represent the main forces in collagen stabilization due to the interaction with vegetal origin polyphenols. 2. Materials and Methods Collagen type I ﬁbers from bovine Achilles tendon (BAT), (±) catechin and all reagents were purchased from Sigma (Sigma Chemicals Co., St. Louise, MI, USA) and were used without further puriﬁcation for the experimental study. 1. Introduction The interaction of catechin with the collagen could inﬂuence the WHC of the macromolecule; in fact, the molecular size of catechin ﬁts the ones of collagen pentaﬁbrils. These last can then stabilize the collagen structure through inter and intramolecular hydrogen bonds [8]. Collagen molecules can diﬀer, not only in their molecular and supramolecular structures, but also in their distributions in the tissues and their functions [9]. In this study, attention has been mainly addressed to type I collagen, since it is ubiquitous in all vertebrates, and it represents at least 90% of total collagen types [10]. Nuclear magnetic resonance time domain (NMR TD) is a nondestructive method that allows the characterization of water mobility in addition to its distribution [11]. Many studies published have correlated low-ﬁeld 1H NMR measurements with the WHC. The transverse relaxation time (T2) has been used to get information on the meat structure, with a high correlation with meat proteins’ properties [12]. Low-resolution 1H NMR has been used to characterize foods by determining both the decay rate and the amplitude of the 1H NMR signal. The T2 relaxation curve in heterogeneous systems is represented by a multiexponential decay curve that can be attributed to the presence in the sample of elements with structural diﬀerences [13]. The decay rate of the 1H NMR signal is lower in these systems compared to the one corresponding to bulk water and can be quantitatively rationalized. In this study, the dynamical properties of the water molecules present in the hydration network of collagen by a systematic approach based on 1H NMR signal relaxation determinations have been assessed. The behaviors of water dynamics under diﬀerent experimental conditions that can occur in meat products have been investigated by evaluating the water proton relaxation rates. Physiological changes in the ionic strength and pH have been considered, as well as the eﬀects of the interactions of collagen with a small antioxidant molecule like catechin. Further information about collagen and water dynamic behaviors have been obtained by transverse 1H NMR relaxation times and self-diﬀusion measurements. To the authors’ knowledge, no other study has been up to today reported on the use of 1H NMR TD to assess the functional properties of collagen. 2.1. Study of Collagen Stability as Function of pH and Ionic Strength Three diﬀerent solutions were prepared at pH 5.4, 6.2, and 7.0 and each of them with three diﬀerent ionic strengths (0.29, 0.45, and 0.71 M), for a total of nine buﬀer solutions. The range of pH and ionic strengths have been chosen and considered in order to represent the variation of these parameters in the meat [14]. The buﬀer solutions were prepared using 50 mM citric acid monohydrate (10.507 g/L) and trisodium dehydrate (14.707 g/L). pH was adjusted with 4 M NaOH Foods 2020, 9, 480 3 of 10 or 4 M HCl, and ionic strength (I) was adjusted by the addition of NaCl according to the following Equations (1): 1 or 4 M HCl, and ionic strength (I) was adjusted by the addition of NaCl according to the following Equations (1): 1 I = 1 2 X CiZi2 (1) (1) where Ci stands for the concentration, and Zi stands for the ion charge. Considering our experim he ion strength (I) was calculated by using the following Equation (2): where Ci stands for the concentration, and Zi stands for the ion charge. Considering our experiment, the ion strength (I) was calculated by using the following Equation (2): I = 1 2([H2A]1 + h HA−2i 22 + h A−3i 32 + 2[NaCl]) (2) (2) Collagen was hydrated by adding a buﬀer solution to dried samples and left for two days at 4 ◦C before 1H NMR experimental determinations. Collagen was hydrated by adding a buﬀer solution to dried samples and left for two days at 4 ◦C before 1H NMR experimental determinations. 2.2. Catechin-Treated Collagen Collagen (2.5 mg/mL) was treated with catechin (0.001 M) for 24 h at room temperature (25 ◦C) in the dark and without any shaking, as described in the supplementary material of a previously published work by Lucarini et al. [7]. Catechin solutions were spectrophotometrically titrated at 280 nm using a Cary 60 UV–Vis spectrophotometer (Agilent Technologies, Santa Clara, CA, USA) before and after the collagen addition to estimate the catechin amount in the complex. Collagen-catechin complex sample was dialyzed for 24 h at 4 ◦C against distilled water. Dialysis bag, manufactured by Spectrum Medical Industries and with a molecular weight cut-oﬀless than 14KD, were purchased from Fischer Scientiﬁc (Waltham, MA, USA). At the end of dialysis, the collagen-treated samples were freeze-dried. After catechin treatment, the collagen color became slightly pink. 2.3. 1H NMR Time Domain (TD) Samples were hydrated by an addition of deionized distilled water or one of the nine buﬀer solutions and left at 4 ◦C for two days to allow collagen hydration. The ratio buﬀer and dry collagen matter was 3 (g/g). The hydrated samples were placed in 1H NMR tubes and immediately sealed. Measurements were performed with a Minispec mq 20-pulsed 1H NMR spectrometer (Bruker Spectrospin Company, Silberstreifen, Germany) with an operating frequency of 20 MHz for protons (magnetic ﬁeld strength: 0.47 T). The NMR spectrometer was equipped with an external thermostat (Julabo F25, Julabo Labortechnik GmbH, Seelbach, Germany) in order to maintain the selected temperature conditions (t = 25 ◦C). Before the NMR measurements, the tube was placed into the 1H NMR probe as long as needed for thermal equilibration (t = 15 min). Longitudinal relaxation measurements (T1) were performed by an inversion recovery (IR) se Transverse relaxation measurements (T2) were performed by a Carr-Purcell-Meiboom-Gill (CPMG) sequence [15]. For each sample, 49 scans were acquired, with a recycle delay of 40 s. The decay of the transverse magnetization was triexponential; the amplitudes and relaxation rates of the three components were calculated by a nonlinear least-squares data ﬁtting with a proprietary self-made computer software based on the Marquardt algorithm [16]. Self-diﬀusion measurements were carried out using a standard Stejskal-Tanner (PFG spin-echo) sequence [17]. 3. Results 3.1. 1H NMR Characterization of Type I Collagen 3.1.1. Time Domain Measurements 3.1.1. Time Domain Measurements The water properties of collagen recovered from the bovine Achilles tendon system were studied by 1H NMR T2 relaxation measurements. 4 of 10 Foods 2020, 9, 480 Foods 2020, 9, 480 The CPMG decay data found for hydrated collagen samples were multicomponent, as generally reported in compartmentalized or heterogeneous systems [18,19]. The sum of three exponentials gave the best ﬁt. As shown in Table 1, the three T2 values were 6 ± 1, 41 ± 7, and 447 ± 15 ms, with relative fractions of 56 ± 5, 36 ± 4, and 8 ± 2%, respectively. Table 1. Transverse relaxation time (T2i), population (ﬁ), and diﬀusion coeﬃcient (Di) values (mean ± standard deviation) related to each water fraction i obtained for hydrated collagen. Table 1. Transverse relaxation time (T2i), population (ﬁ), and diﬀusion coeﬃcient (Di) values (mean ± standard deviation) related to each water fraction i obtained for hydrated collagen. Fraction i 1 2 3 T2i (ms) 6 ± 1 41 ± 7 447 ± 15 ﬁ(%) 56 ± 5 36 ± 4 8 ± 2 Di (10−5cm2s−1) 0.71 1.22 2.31 Table 1 shows the three values of Di associated with the three water fractions. The Di values were D1 = 0.71 × 10−5 cm2 s−1 for the fast-relaxing fraction 1, D2 = 1.22 × 10−5 cm2 s−1 for the intermediate fraction 2, and D3 = 2.17 × 10−5 cm2 s−1 for the slow-relaxing fraction 3. We observed from relaxation measurements in hydrated collagen that multiple water species were detectable (the water relaxation curve was three exponential), with one component having the same relaxation time as pure water and the others two characterized by a reduced T2. The three water fraction localizations were assigned based on available literature data [18–20]. Considering the work by Kopp et al. [21], they describe a protein solution which can be viewed as made by three diﬀerent water environments: the buried water molecules, the water hydration shell around the protein, and the bulk water [21]. In particular, the structural bonded water corresponds to unfreezable water molecules in line with the collagen hydration models proposed [21,22]. They are linked by triple hydrogen bonds involving hydroxyproline or by double hydrogen bonds in the available sites inside the triple helix [22]. 3. Results It has been supposed, assuming non-interacting bonds, that four hydrogen bonds are involved in the linkage of water at the protein–water interface, as it has been reported by Privalov [4]. A cross-relaxation and transverse relaxation 1H NMR study of partially hydrated collagen (h < 0.3), at diﬀerent temperatures and water activities, showed that T2 values of water protons were less than 300 ms [23]. In the experimental conditions of this research (CPMG echo time of 100 ms), the 1H NMR signal cannot be assigned to this interstitial water fraction, whereas the water molecules localized in the micro ﬁbrils (between the triple helix of tropocollagen) mainly contribute to the shortest transverse relaxation time (T21 = 6 ms). The intermediate T2 (T22 = 41 ms) was assigned to the water fraction placed in the interﬁbrillar space. A part of this water fraction is related to the collagen structure and provides water bridges between the collagen microﬁbrils [24]. The molecules of water present in fraction 2 are more mobile than those of fraction 1 but are aﬀected by the macromolecule, as conﬁrmed by the small diﬀerences in the related diﬀusion coeﬃcients (D1 = 0.71 × 10−5 cm2/s and D2 = 1.22 × 10−5 cm2/s). These two slow-relaxing fractions are the hydration water of the tropocollagen. The phenomenon of restriction explains their D1,2 values, whereas the observed decrease is due to the so-called direct hydration eﬀect [25]. For the third fraction, a long T2 (T23 = 447 ms) is reported, as well as a diﬀusion coeﬃcient (D3 = 2.31 × 10−5 cm2/s) close to the value for pure water, and corresponds to the “free” or “bulk” water fraction. The best ﬁt obtained for the longitudinal relaxation time T1 was monoexponential, with a value of 950 ms. The ratio T1/T2 was found >1.6. This result indicated that the motion of water molecules was anisotropic and characterized by a distribution of correlation times [26]. That T1 was monoexponential is not anomalous, since in heterogeneous macromolecular systems, T2 is almost exclusively considered. This is related to some experimental aspects in the measurement of relaxation times: while T2 can be carefully and quickly determined by the CPMG sequence, an accurate T1 determination can be challenging in heterogeneous systems, where, generally, T1 >> T2. 3. Results Since 5 of 10 Foods 2020, 9, 480 measuring the recovery curve of the longitudinal magnetization using the inversion recovery sequence requires an adequate series of recovery delays and long repetition times (≥5 T1), to ensure complete longitudinal relaxation, a number has often been measured low of the experimental points for T1 determination, and it has not always been possible, by deconvolution of the curve, to calculate the same number of T1 components as those obtained from T2 measurements. Anyhow, the correspondence of the T1 values with the T2 values and the ratio T1/T2 can be considered suﬃcient for the data interpretation, since this ratio is potentially useful information for the assessment of the anisotropy existing in heterogeneous systems [13]. Foods 2019, 8, x FOR PEER REVIEW 5 of 10 Anyhow, the correspondence of the T1 values with the T2 values and the ratio T1/T2 can be considered sufficient for the data interpretation, since this ratio is potentially useful information for the Several works reported for water molecules not undergoing proton chemicals exchange a linear relationship of the proton spin-spin relaxation time with the reciprocal of the temperature [27,28]. Indeed, a T2 minimum is expected if the chemical exchange mechanism is the main contribution to relaxation. In a temperature range from 277 to 323 K, such a behavior has been displayed by the T23 component, as reported in Figure 1. assessment of the anisotropy existing in heterogeneous systems [13]. Several works reported for water molecules not undergoing proton chemicals exchange a linear relationship of the proton spin-spin relaxation time with the reciprocal of the temperature [27,28]. Indeed, a T2 minimum is expected if the chemical exchange mechanism is the main contribution to relaxation. In a temperature range from 277 to 323 K, such a behavior has been displayed by the T23 t t d i Fi 1 nt, as reported in Figure 1. Figure 1. Temperature dependence of the T23 values of the collagen sample. Figure 1. Temperature dependence of the T23 values of the collagen sample. Figure 1. Temperature dependence of the T23 values of the collagen sample. Figure 1. Temperature dependence of the T23 values of the collagen sample. 3. Results The presence of a T23 minimum, as function of temperature, indicates that proton relaxation measurements of water in such a system can be interpreted based on an ongoing chemical exchange process between water protons and hydrogen present on the hydroxyl groups of the polymer unit side chains. The presence of a T23 minimum, as function of temperature, indicates that proton relaxation measurements of water in such a system can be interpreted based on an ongoing chemical exchange process between water protons and hydrogen present on the hydroxyl groups of the polymer unit side chains. 3.1.2. Collagen Fibers as Affected by pH and Ionic Strength 3.1.2. Collagen Fibers as Aﬀected by pH and Ionic Strength Nine buffer solutions at three pH values, 5.4, 6.2, and 7.0, were prepared at three different ionic strengths: 0.29, 0.45, 0.71 M, as indicated in the Materials and Methods section. Prior to the 1H NMR measurements, the collagen fibers were hydrated by adding the buffer to the dried sample and left for two days at 4 °C. T2 relaxation times of hydrated collagen showed a triexponential decay with all buffer systems studied. T21, T22, and T23 relaxation times at the three ionic strengths as a function of pH are shown in Figures 2, 3 and 4, respectively. Both the ionic strength and pH affects significantly the T2 relaxation times. Such behavior was displayed by each of the three ionic strengths studied Nine buﬀer solutions at three pH values, 5.4, 6.2, and 7.0, were prepared at three diﬀerent ionic strengths: 0.29, 0.45, 0.71 M, as indicated in the Materials and Methods section. Prior to the 1H NMR measurements, the collagen ﬁbers were hydrated by adding the buﬀer to the dried sample and left for two days at 4 ◦C. T2 relaxation times of hydrated collagen showed a triexponential decay with all buﬀer systems studied. T21, T22, and T23 relaxation times at the three ionic strengths as a function of pH are shown in Figures 2–4, respectively. Both the ionic strength and pH aﬀects signiﬁcantly the T2 relaxation times. Such behavior was displayed by each of the three ionic strengths studied solutions. 6 of 10 6 of 10 Foods 2020, 9, 480 Foods 2019, 8, x FOR ds 2019, 8, x FOR PEER REVIEW 6 o Figure 2. Relationship between the pH and T21 relaxation time in collagen at different ionic strengths. Figure 3. Relationship between the pH and T22 relaxation time in collagen at different ionic strengths. Figure 2. Relationship between the pH and T21 relaxation time in collagen at diﬀerent ionic strengths. Figure 2. Relationship between the pH and T21 relaxation time in collagen at different ionic strengths. Figure 3. Relationship between the pH and T22 relaxation time in collagen at different ionic strengths. Figure 3. Relationship between the pH and T22 relaxation time in collagen at diﬀerent ionic strengths. Figure 2. Relationship between the pH and T21 relaxation time in collagen at different ionic strengths. Figure 2. Relationship between the pH and T21 relaxation time in collagen at diﬀerent ionic strengths. Figure 2. 3.1.2. Collagen Fibers as Affected by pH and Ionic Strength 3.1.2. Collagen Fibers as Aﬀected by pH and Ionic Strength A strong eﬀect of both the ionic strength and the pH on the distribution of T2 relaxation times was observed as reported in Figures 2–4. g g the pH on the distribution of T2 relaxation times was observed as reported in Figures 2, 3, and 4. Significant increases in the T23 relaxation times, which correspond to the “free” or “bulk” water fraction, with the increase of the ionic strength and pH were reported. Previous works, by investigating the effect of increasing the ionic strength in myofibrils by means of 1H NMR T2 relaxation measurements, showed how T2 relaxation times increase in accordance with the ionic strength [14,33,34]. The swelling of the collagen lattice with the increase of pH and ionic strength can Signiﬁcant increases in the T23 relaxation times, which correspond to the “free” or “bulk” water fraction, with the increase of the ionic strength and pH were reported. Previous works, by investigating the eﬀect of increasing the ionic strength in myoﬁbrils by means of 1H NMR T2 relaxation measurements, showed how T2 relaxation times increase in accordance with the ionic strength [14,33,34]. The swelling of the collagen lattice with the increase of pH and ionic strength can well explain the obtained data [14,33,35]. well explain the obtained data [14,33,35]. These results confirm that the influence of pH changes on the fibrillar proteins’ net charge, and consequently, filament spacing is largest around the pK—that is, around pH~5 for the myofibrillar proteins; this support that the changes in relaxation time are related with the collagen lattice spacing, as underlined in the work by Offer and Trinick [30]. Several studies reported how T2 relaxation measured in muscle-based foods correlates with the total water content [36,37] and how T2 relaxation times are influenced by the ionic strength and pH. Noticeably, the effect of increasing pH from 5.4 to 6.2 had higher impact on the T23 relaxation than of increasing pH from 6.2 to 7.0 (figure 4) when ionic strength was 0.71 M. An evident effect of the ionic strength on T23 relaxation times was observed at pH 6.2, when the ionic strength varies from 0.46 to 0.71 M. This indicates a correlation between ionic strength and the degree of fibrillar swelling. 3.1.2. Collagen Fibers as Affected by pH and Ionic Strength 3.1.2. Collagen Fibers as Aﬀected by pH and Ionic Strength Relationship between the pH and T21 relaxation time in collagen at different ionic strengths. Figure 2. Relationship between the pH and T21 relaxation time in collagen at different ionic strengths. Figure 2. Relationship between the pH and T21 relaxation time in collagen at diﬀerent ionic strengths. Figure 2. Relationship between the pH and T21 relaxation time in collagen at different ionic strengths. Figure 2. Relationship between the pH and T21 relaxation time in collagen at different ionic strengths. Figure 2. Relationship between the pH and T21 relaxation time in collagen at diﬀerent ionic strengths. strengths. g p p g strengths. Figure 3. Relationship between the pH and T22 relaxation time in collagen at different ionic Figure 2. Relationship between the pH and T21 relaxation time in collagen at diﬀerent ionic strengths. Figure 3. Relationship between the pH and T22 relaxation time in collagen at different ionic strengths. Figure 3. Relationship between the pH and T22 relaxation time in collagen at diﬀerent ionic strengths. g Fi 3 R l ti hi b t th H d T l ti ti i ll t diff t i i Figure 3. Relationship between the pH and T22 relaxation time in collagen at different ionic strengths Figure 3. Relationship between the pH and T22 relaxation time in collagen at diﬀerent ionic strengths. 7 of 10 7 of 10 Foods 2020, 9, 480 Figure 4. Relationship between the pH and T23 relaxation time in collagen at different ionic h Figure 4. Relationship between the pH and T23 relaxation time in collagen at diﬀerent ionic strengths. Figure 4. Relationship between the pH and T23 relaxation time in collagen at different ionic Figure 4. Relationship between the pH and T23 relaxation time in collagen at diﬀerent ionic strengths. strengths. Both the ionic strength and pH are reported to influence the water-holding capacity [29–31]. This behavior has been attributed to the effect of the pH on the net charge of the proteins, which determines the amount of water in these structures [32]. A strong effect of both the ionic strength and Both the ionic strength and pH are reported to inﬂuence the water-holding capacity [29–31]. This behavior has been attributed to the eﬀect of the pH on the net charge of the proteins, which determines the amount of water in these structures [32]. 3.1.2. Collagen Fibers as Affected by pH and Ionic Strength 3.1.2. Collagen Fibers as Aﬀected by pH and Ionic Strength Indeed, a major change in the T2 relaxation times at pH 7.0 was observed when the ionic strength was respectively 0.29 and 0.46 M respect to the increase These results conﬁrm that the inﬂuence of pH changes on the ﬁbrillar proteins’ net charge, and consequently, ﬁlament spacing is largest around the pK—that is, around pH~5 for the myoﬁbrillar proteins; this support that the changes in relaxation time are related with the collagen lattice spacing, as underlined in the work by Oﬀer and Trinick [30]. Several studies reported how T2 relaxation measured in muscle-based foods correlates with the total water content [36,37] and how T2 relaxation times are inﬂuenced by the ionic strength and pH. Noticeably, the eﬀect of increasing pH from 5.4 to 6.2 had higher impact on the T23 relaxation than of increasing pH from 6.2 to 7.0 (Figure 4) when ionic strength was 0.71 M. An evident eﬀect of the ionic strength on T23 relaxation times was observed at pH 6.2, when the ionic strength varies from 0.46 to 0.71 M. This indicates a correlation between ionic strength and the degree of ﬁbrillar swelling. Indeed, a major change in the T2 relaxation times at pH 7.0 was observed when the ionic strength was respectively 0.29 and 0.46 M respect to the increase showed at 0.71 M (Figure 4). g p y p wed at 0.71 M (figure 4). The myofibrillar swelling resulting from salting could be described hence by two main The myoﬁbrillar swelling resulting from salting could be described hence by two main mechanisms ndicated by Oﬀer and Trinick [30]; in particular: y g g g y hanisms, as indicated by Offer and Trinick [30]; in particular: (i) the bond of negatively charged ions and the increase of electrostatic repulsion between the (i) the bond of negatively charged ions and the increase of electrostatic repulsion between the ments, and ( ) g y g p filaments, and (ii) the removal of one or more transverse structural constraints in the structure, allowing the (ii) the removal of one or more transverse structural constraints in the structure, allowing the ﬁlament lattice to expand. g y g p ments, and (ii) the removal of one or more transverse structural constraints in the structure, allowing the (ii) the removal of one or more transverse structural constraints in the structure, allowing the ment lattice to expand. filament lattice to expand. 3.2. Characterization of Type I Collagen Modiﬁed with Catechin. ( g ) 3 2 Ch t i ti f T I C ll M difi d ith C t hi The water properties of collagen ﬁber systems modiﬁed with catechin were characterized using 1H NMR T2 relaxation measurements. 3.2. Characterization of Type I Collagen Modified with Catechin. The water properties of collagen fiber systems modified with catechin were characterized using 1H NMR T l i The CPMG decay of data from hydrated-modiﬁed collagen samples were multicomponent, as expected. Additionally, in this case, the sum of three exponentials led to the best ﬁt. The three T2 values thus obtained were compared with those found for the collagen ﬁbers (see Table 1) and are shown in Figure 5. 1H NMR T2 relaxation measurements. The CPMG decay of data from hydrated-modified collagen samples were multicomponent, as expected. Additionally, in this case, the sum of three exponentials led to the best fit. The three T2 values thus obtained were compared with those found for the collagen fibers (see Table 1) and are shown in Figure 5 Figure 5. A comparison among the three transverse relation time (T2) components of collagen (blue columns) with those of collagen modified with catechin 0.01 M (green columns). Figure 5. A comparison among the three transverse relation time (T2) components of collagen (blue columns) with those of collagen modiﬁed with catechin 0.01 M (green columns). Figure 5. A comparison among the three transverse relation time (T2) components of collagen (blue columns) with those of collagen modified with catechin 0.01 M (green columns). Figure 5. A comparison among the three transverse relation time (T2) components of collagen (blue columns) with those of collagen modiﬁed with catechin 0.01 M (green columns). Catechin strongly affects the T22 and T23 water relaxation times of collagen. As mentioned before, the intermediate T22 was attributed to the water fraction in the interfibrillar space, and the third fraction (T23) corresponded to the free water fraction. These results indicate that catechin has been able to interact with collagen both on the fibrillar surface and inside the interfibrillar space. Water T23 relaxation value increases when the catechin is bound to the surface of collagen. This result could indicate that water interacts less with the surface of the polymer due to the formation of a complex between the catechin and collagen. Catechin strongly aﬀects the T22 and T23 water relaxation times of collagen. 3.2. Characterization of Type I Collagen Modiﬁed with Catechin. ( g ) 3 2 Ch t i ti f T I C ll M difi d ith C t hi As mentioned before, the intermediate T22 was attributed to the water fraction in the interﬁbrillar space, and the third fraction (T23) corresponded to the free water fraction. These results indicate that catechin has been able to interact with collagen both on the ﬁbrillar surface and inside the interﬁbrillar space. Water T23 relaxation value increases when the catechin is bound to the surface of collagen. This result could indicate that water interacts less with the surface of the polymer due to the formation of a complex between the catechin and collagen. 3.1.2. Collagen Fibers as Affected by pH and Ionic Strength 3.1.2. Collagen Fibers as Aﬀected by pH and Ionic Strength Since the mechanism involving the removal of structural constraints in the collagen structure is a Since the mechanism involving the removal of structural constraints in the collagen structure is a signiﬁcative element for the swelling, it is possible to hypothesize that an upper level for the degree of filament lattice to expand. Since the mechanism involving the removal of structural constraints in the collagen structure is a Since the mechanism involving the removal of structural constraints in the collagen structure is a signiﬁcative element for the swelling, it is possible to hypothesize that an upper level for the degree of Foods 2020, 9, 480 8 of 10 swelling exists and that “saturation” is achieved in the ionic strength range between 0.46 and 0.71 M (Figure 4). , , of swelling exists and that “saturation” is achieved in the ionic strength range between 0.46 and 0.71 M (figure 4) 3.2. Characterization of Type I Collagen Modiﬁed with Catechin. ( igu e ) 3 2 Ch t i ti f T I C ll M difi d ith C t hi 4. Conclusions 4. Conclusions The present study demonstrates that 1H NMR TD can be used to study the water dynamics of collagen thanks to its own longitudinal relaxation processes (T1 or spin-lattice) and transversal relaxation (T2 or spin-spin). Therefore, this approach could improve the understanding of how meat- processing factors can affect collagen swelling [38]. The spin-spin relaxation times reflect the molecular mobility of the collagen chain in accordance with changes in its own chemical structure also affecting meat quality traits related to the pH and ionic strength. Furthermore, the spin-spin relaxation time component T23 can be correlated to the WHC of the collagen-hydrated molecule. In fact, the composition and distribution of T23 fully represents the WHC: the higher the free water percentage, the lower the water retained inside the polymer and, consequently, also the WHC. NMR relaxometry also allowed to investigate the interaction between collagen and catechin, an The present study demonstrates that 1H NMR TD can be used to study the water dynamics of collagen thanks to its own longitudinal relaxation processes (T1 or spin-lattice) and transversal relaxation (T2 or spin-spin). Therefore, this approach could improve the understanding of how meat-processing factors can aﬀect collagen swelling [38]. The spin-spin relaxation times reﬂect the molecular mobility of the collagen chain in accordance with changes in its own chemical structure also aﬀecting meat quality traits related to the pH and ionic strength. Furthermore, the spin-spin relaxation time component T23 can be correlated to the WHC of the collagen-hydrated molecule. In fact, the composition and distribution of T23 fully represents the WHC: the higher the free water percentage, the lower the water retained inside the polymer and, consequently, also the WHC. Foods 2020, 9, 480 9 of 10 NMR relaxometry also allowed to investigate the interaction between collagen and catechin, an antioxidant molecule used in meat processing. In a previous work [7], it has been shown, from mono- and bi-dimensional proton NMR and 13C cross polarization magic angle spectroscopy experiments, that collagen-catechin interactions preferentially occur between the catechin B ring and the collagen proline and hydroxyproline amino acids. The results reported in this study can give further information on the interaction between catechin and collagen, indicating that catechin is able to bind collagen both on the ﬁbrillar surface and inside the interﬁbrillar spaces of the polymer, giving a deeper insight in the understanding of the interaction between catechin and collagen. References 1. Cheng, Q.; Sun, D.-W. Factors Aﬀecting the Water Holding Capacity of Red Meat Products: A Review of Recent Research Advances. Crit. Rev. Food Sci. Nutr. 2008, 48, 137–159. [CrossRef] [PubMed] 1. Cheng, Q.; Sun, D.-W. Factors Aﬀecting the Water Holding Capacity of Red Meat Products: A Review of Recent Research Advances. Crit. Rev. Food Sci. 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https://openalex.org/W1488885587	http://jurnal.pei-pusat.org/index.php/jei/article/download/94/pdf1	English	null	Keanekaragaman dan Kelimpahan Belalang dan Kerabatnya (Orthoptera) pada Dua Ekosistem Pegunungan di Taman Nasional Gunung Halimun-Salak	Jurnal Entomologi Indonesia	2,015	cc-by	5,337	ABSTRACT Diversity and Abundance of Grasshopper and Its Relatives (Orthoptera) on Two Mountainous Ecosystems of Gunung Halimun- Salak National Park. A study on diversity and abundance of grasshopper and its relatives (Orthoptera) was conducted at two mountainous rainforest ecosystems (Mounts Kendeng and Botol) of Gunung Halimun-Salak National Park. A hundred meters of a line transect was used to sample and set up several insect traps (yellow pan, malaise, pit fall, bait pit fall, sweep net, and light traps), and insect sweepings as well. The light traps were set up at about fifty meters distance from the end of the sampling sites. A total individual collected by traps was combined on every comparable sampling site. Total individuals of the Orthoptera captured were 414; consisted of 25 species of 9 families. Both species diversity and number of families were higher at Mount Kendeng rather than Mount Botol. Number of species of each family usually similar except on family of Grillidae was much higher at Mount Kendeng. Species belong to Phasmidae was not recorded at Mount Kendeng, while species belong to both families of Gryllotalpidae and Tettigonidae were not captured at Mount Botol as well. Overal there was a difference in the species richness at each between. Shannon Diversity Index (H’) and evenness (E) were higher at Mount Kendeng (2.44 and 0.81) rather than Mount Botol (1.80 and 0.66). Similarity Index of Jaccard (Cj) and Sorenson (Cn) of both localities were similar (0.40 and 0.32). Herbivores were most dominant at both localities (Phasmidae, Tetrigidae, Acrididae, Gryllidae, dan Gryllotalpidae), followed by omnivores (Blattidae), scavenger (Gryllacrididae), and predator (Mantidae). KEY WORDS: diversity, grasshopper, mountainous ecosystems, Gunung Halimun-Salak National Park misalnya belalang, jangkrik, dan kecoa. Nama belalang sudah sangat terkenal dalam sejarah kuno sebagai makanan manusia dan penghancur tanaman pertanian (LAI 2007), dan makanan bagi satwa liar (Kahono & Amir 2003). Jenis belalang yang terkenal di Nusa Tenggara Timur, KEY WORDS: diversity, grasshopper, mountainous ecosystems, Gunung Halimun-Salak National Park J. Entomol. Indon., September 2010, Vol. 7, No. 2, 100-115 J. Entomol. Indon., September 2010, Vol. 7, No. 2, 100-115 Perhimpunan Entomologi Indonesia PENDAHULUAN Belalang dan kerabatnya ordo Orthoptera merupakan salah satu anggota dari kelompok serangga (kelas Insecta). Jenis-jenisnya mudah dikenal karena memiliki bentuk yang khusus 100 Nety Virgo Erawati dan Sih Kahono: Keanekaragaman dan Kelimpahan Belalang Lampung (Sumatera Selatan) dan beberapa daerah lainnya di Indonesia adalah Locusta migratoria atau belalang kembara, memiliki ke- mampuan melakukan peledakan popu- lasi (outbreak) yang dapat meng- hancurkan ribuan hektar tanaman pertanian terutama padi dan jagung. Jenis-jenis belalang lainnya yang dikenal di Indonesia adalah belalang kayu (Valanga nigricornis), belalang ranting (Phobaeticus chani), belalang daun (Phyllium fulchrifolium), bela- lang sembah (Hierodula vitrea), kecoa (Periplaneta americana), dan jangkrik (Gryllus mitratus) (Koleksi MZB). naik-turunnya populasi dan outbreak. Untuk memantau keanekaragaman ha- yati perlu dilengkapi informasi jumlah individu (kelimpahan) dan fungsi atau peranannya pada suatu habitat dan ekosistem (Primack et al. 1988; Oliver & Beatti 1992, 1996). Kelimpahan jenis serangga sangat ditentukan oleh aktifitas reproduksinya yang didukung oleh lingkungan yang cocok dan ter- cukupinya kebutuhan sumber makan- annya. Kelimpahan dan aktifitas repro- duksi serangga di daerah tropik sangat dipengaruhi oleh musim (Wolda & Wong 1988), karena musim ber- pengaruh kepada ketersediaan sumber pakan dan kemampuan hidup serangga yang secara langsung mempengaruhi kelimpahan. Belalang dan kerabatnya hidup di berbagai tipe lingkungan atau eko- sistem antara lain hutan, semak/ belukar, lingkungan perumahan, lahan pertanian, dan sebagainya (Kalshoven 1981; Meyer 2001; Erniwati 2003). Di alam, belalang berperan sebagai pemangsa, pemakan bangkai, pengurai material organik nabati dan hewani, pemakan bagian tumbuhan hidup dan mati, dan musuh alami dari berbagai jenis serangga lainnya (Borror et al. 1992; Gwynne et al. 1996; Meyer 2001; Kahono & Amir 2003). Ekosistem merupakan lingkungan biologi yang berisi organisme hidup, non-biotik, dan komponen fisik yang saling berinteraksi (Cambell & Neil 2009). Perbedaan struktur dan komposisi penyusun suatu ekosistem menyebabkan perbedaan karakter ekosistem yang mempengaruhi ke- anekaragaman dan kelimpahan biota yang tinggal di dalamnya. Dataran tinggi biasanya mempunyai keane- karagaman dan kelimpahan yang lebih rendah jika dibandingkan dengan data- ran rendah (Wolda 1983). Di daerah padang rumput, kelimpahan dan bio- masa belalang berkurang pada musim semi (Porter & Redak 1996). Setiap kelompok serangga mempunyai respon yang berbeda terhadap perubahan Permasalahan pada penelitian bela- lang dan kerabatnya ordo Orthoptera adalah rendahnya pengetahuan keane- karagaman, sebaran, populasi dan as- pek biologi dasar lainnya. Penelitian ekologi populasi termasuk monitoring fluktuasinya secara sistematis akan dapat meramalkan terjadinya regulasi 101 J. Entomol. Indon., September 2010, Vol. 7, No. 2, 100-115 musim dan iklim (Wolda 1978, 1983; Kahono 2006). PENDAHULUAN Belalang dan kerabat- nya ordo Orthoptera sangat penting peranan dan fungsinya dalam menjaga keseimbangan ekosistem hutan (Gwy- nne et al. 1996; Erniwati 2003). BAHAN DAN METODE Taman Nasional Gunung Halimun- Salak (TNGH-S) secara geografis terletak antara 106o21”-106o38”BT dan 6o37”-6o51”LS (Djuwarsah 1997). Rata-rata curah hujan mencapai 4,181 mm per tahun (Kahono & Noerdjito 2002) dan kelembaban rata-rata 80%. Musim kering terjadi sekitar bulan Juni-Agustus (Manikam 1998). Rata- rata suhu maksimum bervariasi antara 31o-34,5oC dan suhu minimum antara 18,3oC-23,4oC, sedangkan variasi suhu hariannya berkisar antara 24,7oC- 26,6oC (Djuwarsah 1997). Hutan di kawasan Taman Nasional Gunung Halimun-Salak (TNGH-S) merupakan salah satu hutan tropik basah yang terdiri atas gugusan bukit dan pegunungan. Sampai saat ini, penelitian tentang kelompok belalang dan kerabatnya ordo Orthoptera di Indonesia masih terbatas jumlah dan aspek penelitiannya (Kalshoven 1981; Erniwati 2003). Penelitian keaneka- ragaman dan kelimpahan Orthoptera dilakukan di dua ekosistem pegu- nungan yang berbeda yaitu Gunung Kedeng (GK) dan Gunung Botol (GB), dengan maksud untuk mengetahui keanekaragaman dan kelimpahan jenis belalang dan kerabatnya ordo Orthop- tera yang menempati dua ekosistem tersebut. Kajian terhadap peran setiap jenis belalang dan kerabatnya dapat memberikan pengetahuan tentang fungsi kelompok ini di lingkungannya. Hasil penelitian ini akan bermanfaat untuk penelitian monitoring terhadap perubahan iklim dan ekosistem di waktu yang akan datang, yang juga akan berguna bagi penyusunan ber- bagai kebijakan perlindungan dan pemanfaatan yang lestari sumber daya hayati khususnya jenis-jenis Orthop- tera di TNGH-S. Pengambilan sampel Orthoptera dilakukan pada Januari, Februari, dan Maret 2003 di Gunung Kendeng (GK) (1.050-1.400 m dpl.) dan Gunung Botol (GB) (1.500-1.800 m dpl.). Pengambilan sampel dilakukan dengan membuat garis transek utama dengan panjang seratus (100) meter mengikuti jalur jalan setapak. Setiap sepuluh (10) meter pada transek utama dibuat transek sekunder tegak lurus sepanjang lima (5) meter ke kanan dan kiri. Pada titik-titik yang telah dibuat pada tran- sek utama maupun transek sekunder dipasang secara acak perangkap se- rangga. Khusus pemasangan light trap dilakukan dengan jarak kira-kira lima puluh (50) meter dari ujung transek utama, untuk mengurangi pengaruhnya terhadap perangkap lainnya. BAHAN DAN METODE Pengumpulan spesimen dilakukan dengan menggunakan enam alat pe- nangkap serangga, yaitu jaring se- 102 Nety Virgo Erawati dan Sih Kahono: Keanekaragaman dan Kelimpahan Belalang rangga (sweep net) yang diayun sebanyak 15 ayunan, dengan ulangan sebanyak 5 kali; yellow pan trap sebanyak 10 buah yang dipasang selama 24 jam, malaise trap sebanyak 2 buah yang dipasang selama 2 hari, pitfall trap sebanyak 10 buah yang dipasang selama 2 hari, bait pitfall trap dengan umpan ayam busuk sebanyak 10 buah yang dipasang selama 24 jam, dan perangkap cahaya 100 watt (light trap) sebanyak 1 buah yang dipasang dari jam 18:30-21:30 WIB. Beberapa metode pengambilan sampel tersebut digunakan dalam penelitian ini agar kesempatan menangkap menjadi lebih tinggi. Beberapa penelitian memodifi- kasi beberapa metode yang sudah ada agar hasil tangkapan dapat diperoleh secara optimal dan dapat dibandingkan dan dianalisis secara kuantitatif (Toda & Kitcing 1999; Borror et al. 1992). Identifikasi spesimen dilakukan secara morfospecies, dibandingkan dengan spesimen ilmiah yang telah teridentifi- kasi di Laboratorium Entomologi, Pusat Penelitian Biologi-LIPI dan me- nggunakan beberapa referensi ilmiah. Untuk mempermudah dalam analisis data maka hasil tangkapan dikelom- pokkan menurut waktu (Januari, Februari, dan Maret) serta tempat (GK dan GB). Keanekaragaman hayati (biodiver- ekosistem dan bagaimana kemerataan jumlah individu yang tersebar di antara jenis tersebut (evenness) (Magurran 1987). Pada penelitian keaneka- ragaman hayati ini menampilkan daftar jenis dan informasi lainnya, misalnya jumlah individu, fungsi, dan habitat tempat hidupnya. Dipilih cara meng- ukur keanekaragaman dengan meng- gunakan Indeks Keanekaragaman Sha- nnon (Indeks Keanekaragaman Sha- nnon-Wienner) memakai jumlah jenis, kelimpahan atau jumlah individu setiap jenis, dan menggabungkan keduanya. Nilai keanekaragaman bervariasi, se- makin tinggi nilainya berarti keaneka- ragaman jenis semakin tinggi. Sebaran keanekaragaman (evenness) merupa- kan perbandingan antara nilai keaneka- ragaman yang diperoleh dengan nilai keanekaragaman maksimum. Nilai evenness berkisar antara 0 dan 1. Nilai 1 apabila antar species mempunyai kelimpahan sama atau seragam. Untuk mengetahui kesamaan jumlah species yang ditemukan pada dua lokasi, atau dua bulan yang berbeda pada lokasi yang sama, atau bulan yang sama pada lokasi yang berbeda menggunakan Indeks Kesamaan Sorenson, nilainya 0 sampai dengan 1, nilai 1 berarti jumlah species yang ditemukan di dua lokasi adalah sama dan nilai 0 berarti jumlah species yang ditemukan di dua lokasi adalah berbeda sama sekali. bN = jumlah individu pada lokasi b dapat dihitung dengan menggunakan Indeks Kesamaan Jaccard, nilainya 0 sampai dengan 1, nilai 1 atau 100% berarti species yang ditemukan di dua lokasi memiliki kesamaan/kemiripan sempurna, dan nilai 0 atau 0% berarti tidak ada kemiripan sama sekali. BAHAN DAN METODE Per- hitungan lain, kesamaan/kemiripan komposisi spesies antar lokasi (pro- porsi species yang sama antar lokasi) Keanekaragaman hayati (biodiver- sity) sebagai kegiatan yang meng- ungkapkan jumlah jenis (kekayaan jenis atau species richness) yang ditemukan pada suatu komunitas/ 103 J. Entomol. Indon., September 2010, Vol. 7, No. 2, 100-115 bN = jumlah individu pada lokasi b Keanekaragaman Dari data penelitian diperoleh 414 individu Orthoptera yang termasuk dalam 9 famili dan 25 jenis. Jumlah individu di GB lebih tinggi daripada GK, tetapi jumlah famili dan jenis lebih tinggi di GK daripada GB. Di GK diperoleh 136 individu yang termasuk dalam 8 famili dan 20 jenis dan GB diperoleh 278 individu yang termasuk dalam 7 famili dan 15 jenis. Jumlah individu, famili, dan jenis di GB lebih banyak pada Februari dibandingkan dengan Januari dan Maret. Di GK, jumlah individu lebih banyak pada Februari (53 individu) tetapi jumlah famili dan jenis lebih banyak pada Maret daripada bulan lainnya (Tabel 1). Sampel yang diperoleh dihitung jumlah individu (N), jumlah famili (F), dan jumlah jenisnya (S). Keaneka- ragaman Orthoptera dihitung berdasar- kan indeks keanekaragaman Shannon (H’), sebaran keanekaragaman Sha- nnon (E), indeks kesamaan Jaccard (Cj), indeks kesamaan Sorenson (Cn), serta kelimpahan relatif (KR) (Magurran 1987). Persamaan dalam perhitungan indeks tersebut adalah sebagai berikut: H’ = -∑ni/N ln ni/N E = H’ ln S Cj = j/(a+b-j) Cn = 2jN/(aN+bN) KR = ni/N x 100% Keterangan: H’ = -∑ni/N ln ni/N E = H’ ln S Cj = j/(a+b-j) Cn = 2jN/(aN+bN) KR = ni/N x 100% Keterangan: Walaupun dalam penelitian ini memiliki jumlah jenis Orthoptera lebih sedikit dibandingkan yang dilakukan oleh Erniwati (2003), namun mem- punyai informasi baru tentang per- bedaan keanekaragaman dan kelimpa- an Orthoptera pada dua ekosistem yang berbeda di TNGH-S, antara GK dan GB. Keanekaragaman Orthoptera yang tertinggi di GK ditunjukkan oleh banyaknya jumlah jenis yang ditemu- kan. Jumlah jenis Orthoptera di GK (20 jenis) lebih tinggi dibandingkan dengan GB (15 jenis). Keterangan: ni = jumlah individu pada i jenis j = jumlah jenis yang ditemukan pada lokasi a dan b a = jumlah jenis yang ditemukan pada lokasi a b = jumlah jenis yang ditemukan pada lokasi b jN = jumlah kelimpahan terendah yang terdapat pada lokasi a dan lokasi b aN = jumlah individu pada lokasi a 104 Nety Virgo Erawati dan Sih Kahono: Keanekaragaman dan Kelimpahan Belalang Tabel 1. Keanekaragaman Jumlah individu (N), famili (F), jenis (S), indeks keanekaragaman Shannon (H’), dan sebaran keanekaragaman Shannon (E) jenis Orthoptera di GK dan GB, TNGH-S Lokasi Januari Februari Maret Subtotal Total GK N 34 53 49 136 F 5 6 8 8 S 10 12 14 20 H’ 2,09 1,99 2,08 2,44 E 0,91 0,80 0,79 0,81 GB N 78 117 83 278 F 5 6 5 7 S 10 12 10 15 H’ 1,62 1,77 1,47 1,80 E 0,70 0,71 0,64 0,66 Subtotal N 112 170 132 414 F 6 6 9 9 S 14 18 19 25 H’ 1,99 2,09 2,10 2,72 E 0,76 0,72 0,71 0,71 Keterangan : GK = Gunung Kendeng; GB = Gunung Botol Indeks keanekaragaman Shannon dan sebaran keanekaragaman Shannon (evenness) di GK (H’ = 2,44 dan E = 0,81) lebih tinggi dibandingkan di GB (H’ = 1,80 dan E = 0,66). Ada korelasi positif antara hasil penelitian Ortho- ptera dan kerabatnya ini dengan penelitian Atmowidi (2000), Suantara (2000), dan Utomo (2001) yang menyatakan bahwa keanekaragaman dan sebaran keanekaragaman serangga (Hymenoptera, Lepidoptera, dan Dip- tera) di GK lebih tinggi dibandingkan di GB. Lebih rendahnya keanekaragaman Orthoptera di GB belum diketahui secara jelas. Tidak diukur curah hujan di kedua Gunung tersebut, hanya dari perkebunan teh terdekat (Malasari) curah hujan tidak menunjukkan perbedaan selama pengamatan. Curah hujan bulanan selalu di atas 100 mm mengindikasikan rendahnya musim kering (Whitmore 1984). Dari data vegetasi menunjukkan bahwa keanekaragaman flora di GK lebih tinggi daripada GB (Manikam 1998; Atmowidi 2000; Suantara 2000). Literatur umum mengatakan bahwa 105 J. Entomol. Indon., September 2010, Vol. 7, No. 2, 100-115 keanekaragaman serangga berkorelasi positif dengan tingkat kompleksitas lingkungannya. Hutan yang lebih komplek (jenis tumbuhannya, iklim, ekosistemnya, dan landscape) biasanya memiliki keanekaragaman serangga yang lebih tinggi. Sejak tahun 2000 hutan di GB telah mengalami berbagai kemunduran karena penebangan dan alih fungsi hutan yang memicu tumbuhnya tumbuhan sekunder (semak, rumput, dan belukar) yang mengundang berbagai jenis serangga pendatang setelah suatu lingkungan baru terjadi. Jadi, lingkungan GB sebagai lingkungan hutan primer plus lingkungan baru. Keanekaragaman Orthoptera di GK (2,44) lebih tinggi dari pada di GB (1,80) yang berbeda dengan keanekaragaman kumbang di GB sedikit lebih tinggi daripada di GK, GB (3,59) dan GK (3,55) (Maulinda 2003). Fenomena keaneka- ragaman yang demikian banyak terjadi pada lingkungan tropis (Wolda 1978). turut pada Februari dan Maret. Keanekaragaman Belum cukup data yang dapat menerangkan secara jelas mengapa keanekaragaman pada Januari lebih tinggi daripada di bulan lainnya. Hal ini dapat disebab- kan karena Januari lebih ‘favorable’ dilihat dari lebih banyaknya daun-daun muda (Apud, komunikasi pribadi) yang disukai oleh Orthoptera pada umumnya. Kesamaan Kesamaan jenis Orthoptera ber- dasarkan indeks kesamaan Jaccard antara GK dan GB sebesar 0,40 (Tabel 2). Berdasarkan pada bulan peng- ambilan sampel, kesamaan jenis Orthoptera pada Januari, Februari, dan Maret di GK berkisar antara 0,29-0,40, sedangkan di GB berkisar antara 0,47- 0,82. Indeks kesamaan jenis Orthop- tera antar lokasi (GK dan GB) pada ketiga bulan tersebut berkisar antara 0,22-0,44. Kesamaan jenis berdasarkan indeks kesamaan Sorenson di GK dan GB sebesar 0,32 (Tabel 3). Berdasarkan bulan pengambilan sampel, kesamaan jenis pada Januari, Februari, dan Maret di GK berkisar antara 0,30-0,53; sedangkan di GB berkisar antara 0,59- 0,71. Indeks kesamaan Sorensen Orthoptera antar lokasi (GK dan GB) pada ketiga bulan tersebut berkisar antara 0,15-0,36. Kesamaan Sorrensen, menunjukkan bahwa proporsi jumlah spesies Orthoptera yang ditemukan di Berdasarkan waktu pengambilan sampel, keanekaragaman tertinggi ter- jadi pada Maret (H’ = 2,10), disusul Februari (H’ = 2,09), dan Januari (H’ = 1,99). Sebaran indeks keanekaragaman Orthoptera tertinggi terjadi pada Januari (E = 0,76), disusul Februari (E = 0,72), dan Maret (E = 0,71) (Tabel 1). Hal ini menunjukkan bahwa pada Januari sebaran jumlah individu masing-masing jenis adalah tinggi (kelimpahan jumlah individu antar jenis paling tinggi), disusul berturut- 106 Nety Virgo Erawati dan Sih Kahono: Keanekaragaman dan Kelimpahan Belalang Tabel 2. Indeks kesamaan Jaccard (Cj) Orthoptera pada bulan Januari, Februari, dan Maret Lokasi GK Januari GK Februari GK Maret GB Januari GB Februari GB Maret Total GK GK Januari 1 GK Februari 0,29 1 GK Maret 0,50 0,44 1 GB Januari 0,36 0,22 0,33 1 GB Februari 0,38 0,33 0,44 0,47 1 GB Maret 0,33 0,22 0,30 0,82 0,57 1 Total GB 0,40 Keterangan: GB = Gunung Botol; GK = Gunung Kendeng Tabel 2. Indeks kesamaan Jaccard (Cj) Orthoptera pada bulan Januari, Februari, dan Maret Lokasi GK Januari GK Februari GK Maret GB Januari GB Februari GB Maret Total GK GK Januari 1 GK Februari 0,29 1 GK Maret 0,50 0,44 1 GB Januari 0,36 0,22 0,33 1 GB Februari 0,38 0,33 0,44 0,47 1 GB Maret 0,33 0,22 0,30 0,82 0,57 1 Total GB 0,40 Keterangan: GB = Gunung Botol; GK = Gunung Kendeng Tabel 2. Indeks kesamaan Jaccard (Cj) Orthoptera pada bulan Januari, Februari, dan Maret 0,40 0,40 Tabel 3. Kesamaan Indeks kesamaan Sorenson (Cn) Orthoptera pada bulan Januari, Februari, dan Maret Lokasi GK Januari GK Februari GK Maret GB Januari GB Februari GB Maret Total GK GK Januari 1 GK Februari 0,30 1 GK Maret 0,53 0,35 1 GB Januari 0,32 0,15 0,21 1 GB Februari 0,21 0,36 0,19 0,59 1 GB Maret 0,29 0,15 0,18 0,65 0,71 1 Total GB 0,32 Keterangan : GB = Gunung Botol; GK = Gunung Kendeng Tabel 3. Indeks kesamaan Sorenson (Cn) Orthoptera pada bulan Januari, Februari, dan Maret Lokasi GK Januari GK Februari GK Maret GB Januari GB Februari GB Maret Total GK GK Januari 1 GK Februari 0,30 1 GK Maret 0,53 0,35 1 GB Januari 0,32 0,15 0,21 1 GB Februari 0,21 0,36 0,19 0,59 1 GB Maret 0,29 0,15 0,18 0,65 0,71 1 Total GB 0,32 Keterangan : GB = Gunung Botol; GK = Gunung Kendeng Tabel 3. Indeks kesamaan Sorenson (Cn) Orthoptera pada bulan Januari, Februari, dan Maret 0,32 107 107 J. Entomol. Indon., September 2010, Vol. 7, No. 2, 100-115 KR = 16,18%), Rhaphidophora sp. 1 (Gryllacrididae) (16 individu; KR = 11,76%), dan Pycnocelus sp. (Blat- tidae) (15 individu; KR = 11,03%) (Tabel 4 dan Tabel 5). GK dan juga di GB adalah sebanyak 32%. Kesamaan Jaccard, proporsi species Orthoptera yang ditemukan di GK dan juga ditemukan di GB sebanyak 40%. Jenis yang lebih melimpah di GB adalah Nemobius sp. (Gryllidae) (101 individu; KR = 36,33%), kemudian disusul berturut-turut oleh Rhaphido- phora sp. 1 (Gryllacrididae) (87 indi- vidu; KR = 31,29%), Pycnocelus sp. (Blattidae) (20 individu; KR = 7,19%) dan Captotetrix interuptus (Tetrigidae) (17 individu; KR = 6,12%). Empat jenis yang lebih melimpah dari pada yang lainnya di kedua lokasi tersebut adalah Nemobius sp. (106 individu; KR = 25,60%), Rhaphidophora sp. 1 (103 individu; KR = 24,88%), Blatta orientalis (39 individu; KR = 9,42%), dan Pycnocelus sp. (35 individu; KR = 8,45%) (Tabel 4 dan Tabel 5). Kelimpahan Jenis Nilai kelimpahan relatif (KR) di GK yang paling tinggi adalah famili Blattidae (73 individu; KR = 53,68%), kemudian disusul berturut-turut oleh Gryllidae (20 individu; KR = 14,71%), dan Gryllacrididae (19 individu; KR = 13,97%). Sedangkan KR di GB yang paling tinggi adalah famili Gryllidae (101 individu; KR = 36,33%), ke- mudian disusul berturut-turut oleh Gryllacrididae (92 individu; KR = 33,09%), dan Blattidae (52 individu; KR = 18,71%) (Tabel 4 dan Tabel 5). Jenis yang lebih melimpah di GK adalah Blatta orientalis (Blattidae) (28 individu; KR = 20,59%), kemudian disusul berturut-turut oleh Grapto- blatta sp. 1 (Blattidae) (22 individu; Tabel 4. Jumlah individu (N), jenis (S), dan kelimpahan relatif (KR) masing- masing famili Orthoptera di TNGH-S GK GB Famili (F) Jumlah Individu (N) Jumlah Jenis (S) KR (%) Jumlah Individu (N) Jumlah Jenis (S) KR (%) Acrididae 13 3 9,56 8 3 2,88 Tetrigidae 3 1 2,21 17 1 6,12 Gryllacrididae 19 2 13,97 92 2 33,09 Blattidae 73 4 53,68 52 5 18,71 Gryllidae 20 6 14,71 101 1 36,33 Mantidae 6 2 4,41 1 1 0,36 Gryllotalpidae 1 1 0,74 0 0 0 Tettigoniidae 1 1 0,74 0 0 0 Phasmidae 0 0 0 7 2 2,52 Keterangan: GK = Gunung Kendeng; GB = Gunung Botol; KR = Kelimpahan Relatif Tabel 4. Jumlah individu (N), jenis (S), dan kelimpahan relatif (KR) masing- masing famili Orthoptera di TNGH-S 108 Nety Virgo Erawati dan Sih Kahono: Keanekaragaman dan Kelimpahan Belalang Tabel 5. Jumlah individu (N) dan kelimpahan relatif (KR) jenis* Orthoptera di GK dan GB, TNGH-S GK GB Total Famili Jenis Jumlah individu (N) KR (%) Jumlah individu (N) KR (%) Jumlah individu (N) KR (%) Acrididae Phlaeoba sp. 11 8,09 2 0,72 13 3,14 Erucius sp. 1 0,74 1 0,36 2 0,48 Trilophidia sp. 1 0,74 5 1,80 6 1,45 Tetrigidae Captotetrix interuptus 3 2,21 17 6,12 20 4,83 Gryllacrididae Rhaphidophora sp. 1 16 11,76 87 31,29 103 24,88 Rhaphidophora sp. 2 3 2,21 5 1,80 8 1,93 Blattidae Rhicnoda rugosa 8 5,88 0 0 8 1,93 Blatta orientalis 28 20,59 11 3,96 39 9,42 Graptoblatta sp. 1 22 16,18 9 3,24 31 7,49 Graptoblatta sp. 2 0 0 1 0,36 1 0,24 Pycnocelus sp. 15 11,03 20 7,19 35 8,45 Epilampra sp. 0 0 11 3,96 11 2,66 Gryllidae 0 0 0 0 0 0 Trydactilus sp. Kelimpahan Jenis 1 0,74 0 0 1 0,24 Itara microcephala 10 7,335 0 0 10 2,42 Nemobius sp. 5 3,68 101 36,33 106 25,60 Muctibulus sp. 2 1,47 0 0 2 0,48 Cyclopaglum sp. 1 0,74 0 0 1 0,24 Gymnogryllus elegans 1 0,74 0 0 1 0,24 Mantidae Gonypeta punctata 5 3,68 0 0 5 1,21 Ceratocrania macra 1 0,74 0 0 1 0,24 Hierodula vitrea 0 0 1 0,36 1 0,24 Gryllotalpidae Gryllotalpa africana 1 0,74 0 0 1 0,24 Tettigoniidae Paragraecia sp. 1 0,74 0 0 1 0,24 Phasmidae Phasmidae sp. 1 0 0 1 0,36 1 0,24 Phasmidae sp. 2 0 0 6 2,15 6 1,69 Keterangan: * = Penamaan jenis secara morfospecies GK = Gunung Kendeng Tabel 5. Jumlah individu (N) dan kelimpahan relatif (KR) jenis* Orthoptera di GK dan GB, TNGH-S 109 J. Entomol. Indon., September 2010, Vol. 7, No. 2, 100-115 J. Entomol. Indon., September 2010, Vol. 7, No. 2, 100-115 Beberapa jenis yang hanya ditemu- kan di GK adalah Rhicnoda rugosa (Blattidae), Trydactilus sp., Itara microcephala, Muctibulus sp., Cyclo- paglum sp., Gymnogryllus elegans (Gryllidae), Gonypeta punctata, Cera- tocrania macra (Mantidae), Gryl- lotalpa africana (Gryllotalpidae), dan Paragraecia sp. (Tettigoniidae). Jenis yang hanya ditemukan di GB adalah Graptoblatta sp. 2, Epilampra sp. (Blattidae), Hierodula vitrea (Manti- dae), Phasmidae sp. 1, dan Phasmidae sp. 2 (Phasmidae) (Tabel 4 dan Tabel 5). omnivora, predator, dan pemakan bangkai (scavenger). Orthoptera yang berperan sebagai herbivora lebih dominan daripada kelompok lainnya. Orthoptera herbivora terdiri dari famili Acrididae, Tetrigidae, Tettigoniidae, Gryllotalpidae, dan Gryllidae. Orthop- tera omnivora adalah famili Blattidae, scavenger famili Gryllacrididae, dan predator famili Mantidae. Dari 171 individu (14 jenis) Orthoptera herbivora yang tertangkap, walaupun hanya 38 individu terdapat di GK namun keanekaragamannya sangat tinggi yaitu 12 jenis, sebaliknya dari 133 individu yang tertangkap di GB hanya terdiri dari 7 jenis. Dari 125 individu (6 jenis) Orthoptera omnivora yang tertangkap, jumlah individu dan jumlah jenisnya di GK dan GB tidak terlalu berbeda 73 individu (4 jenis) di GK dan 52 individu (5 jenis) di GB. Dari 111 individu (2 jenis) Orthoptera scavenger yang tertangkap, keduanya dijumpai di GK dan GB. Jumlah indi- vidu di GB (92 individu) jauh lebih banyak daripada di GK (19 individu). Dari 7 individu (3 jenis) Orthoptera predator yang tertangkap, dua jenis (6 individu) hanya dijumpai di GK, satu jenis (1 individu) hanya dijumpai di GB (Tabel 6). Kelimpahan Jenis Penelitian komunitas serangga sela- ma setahun lebih oleh Kahono & Woro (2002) menunjukkan adanya sinkroni- sasi antara hujan dan populasi se- rangga, namun dalam penelitian ini hanya dilakukan dalam waktu tiga bulan, maka tidak jelas apakah naik- turunnya populasi selama pengamatan berkaitan dengan naik-turunnya curah hujan. Kelimpahan jumlah individu antar spesies Orthoptera di GK lebih seragam (0,81) dari pada di GB (0,66). Hal yang mirip pada kelimpahan jumlah individu antar spesies kumbang di GK sedikit lebih seragam (0,76) dari pada di GB (0,72) (Maulinda 2003). Belalang dan kerabatnya ordo Orthoptera yang paling banyak ditemukan di TNGH-S (GK dan GB) berperan sebagai herbivora sebesar J. Entomol. Indon., September 2010, Vol. 7, No. 2, 100-115 Peranan Orthoptera Berdasarkan peranannya di alam, jenis-jenis dari ordo Orthoptera di TNGH-S berperan sebagai herbivora, 110 Nety Virgo Erawati dan Sih Kahono: Keanekaragaman dan Kelimpahan Belalang (Mantidae). Orthoptera yang berperan sebagai herbivora mendominasi di TNGH-S (Gambar 1). yang dilakukan oleh Rizali et al. (2002) yang mengemukakan bahwa serangga yang ditemukan di lahan persawahan tepian (41%) yang terdiri dari famili Acridi- dae, Tetrigidae, Tettigoniidae, Gryl- lotalpidae, dan Gryllidae, yang diikuti berturut-turut oleh kelompok omnivora 30% (Blattidae), scavenger 27% (Gryllacrididae), dan predator 2% 111 Tabel 6. Pembagian peranan pada kelompok Orthoptera yang ditemukan di GK dan GB, TNGH-S GK GB Total PERAN Jml. jenis Jml. indiv. Jml. jenis Jml. indiv. Jml. jenis Jml. indiv. Herbivora 12 38 7 133 14 171 Omnivora 4 73 5 52 6 125 Scavenger 2 19 2 92 2 111 Predator 2 6 1 1 3 7 TOTAL 20 136 15 278 25 414 Gambar 1. Persentase dari total jumlah jenis (biru) dan kelimpahan individu (merah) Orthoptera yang dijumpai dalam penelitian berdasarkan peranannya di GK (kiri) dan GB (kanan) 0 60 50 40 30 20 10 28 32,1 20 12,6 8 22,2 0,2 4 Herbivora Omnivora Scavenger Predator % 60 40 20 0 48 9,2 16 17,6 8 4, 8 1, Herbivora Omnivora Scavenger Predator % 4 Tabel 6. Pembagian peranan pada kelompok Orthoptera yang ditemukan di GK dan GB, TNGH-S Gambar 1. Persentase dari total jumlah jenis (biru) dan kelimpahan individu (merah) Orthoptera yang dijumpai dalam penelitian berdasarkan 0 60 50 40 30 20 10 28 32,1 20 12,6 8 22,2 0,2 4 Herbivora Omnivora Scavenger Predator % 60 40 20 0 48 9,2 16 17,6 8 4, 8 1, Herbivora Omnivora Scavenger Predator % 4 Predator Predator Scavenger Gambar 1. Persentase dari total jumlah jenis (biru) dan kelimpahan individu (merah) Orthoptera yang dijumpai dalam penelitian berdasarkan peranannya di GK (kiri) dan GB (kanan) 111 J. Entomol. Indon., September 2010, Vol. 7, No. 2, 100-115 hutan dalam wilayah TNGH-S dido- minasi oleh serangga herbivora. Se- rangga herbivora merupakan pemakan tumbuhan dan dapat menempati ham- pir semua tipe habitat, baik pada kanopi atau tajuk pohon dan belukar. Orthoptera omnivora sebagai pemakan segala jenis makanan, biasanya lebih dikenal sebagai perombak yang me- nempati hampir semua tipe habitat hutan seperti serasah dan material organik tumbuhan. Orthoptera sca- venger merupakan pemakan bangkai, biasanya hidup pada permukaan tanah dan serasah (Meyer 2001). Gunung Kendeng (136 individu). Peranan Orthoptera Jenis yang melimpah di Gunung Kendeng adalah Blatta orientalis, Rhaphido- phora sp 1, Pycnocelus sp., Phlaeoba sp., dan Itara microchepala, sebalik- nya di Gunung Botol adalah Nemobius sp., Rhaphidophora sp 1, dan Pycno- celus sp. Jenis-jenis yang termasuk famili Phasmidae tidak ditemukan di Gunung Kendeng, sebaliknya yang termasuk Gryllotalpidae dan Tetti- gonidae tidak ditemukan di Gunung Botol. Sepuluh jenis (Rhicnoda rugosa, Trydactilus sp., Itara micro- cephala, Muctibulus sp., Cyclopaglum sp., Gymnogryllus elegans, Gonypeta punctata, Ceratocrania macra, Gryllo- talpa africana, and Paragraecia sp.) hanya ditemukan di Gunung Kendeng. Lima jenis (Graptoblatta sp. 2, Epilampra sp., Hierodula vitrea, Phas- midae sp. 1, and Phasmidae sp. 2) hanya ditemukan di Gunung Botol. Kelompok belalang dan kerabatnya yang dominan di Gunung Kendeng dan Botol adalah jenis-jenis yang berperan sebagai herbivora daripada omnivora, scavenger, dan predator. Kecuali scavenger, persentase jenis Orthoptera yang bersifat herbivora, omnivora, dan predator di GK lebih tinggi daripada yang terdapat di GB. Hal demikian juga terjadi pada kelimpahan individu omnivora dan predator. Sebaliknya, kelimpahan indi- vidu herbivora, scavenger di GB lebih tinggi daripada di GK (Gambar 1). Walaupun tidak begitu jelas alasannya namun kondisi ini menunjukkan bahwa lingkungan di GK lebih baik daripada GB (Simbolon & Mirmanto 1997). KESIMPULAN Saudara Sarino (teknisi Labo- ratorium Entomologi, Bisang Zoologi) dan Hendi (Citalahab, Malasari) yang telah membantu pelaksanaan penelitian di lapangan. Dra. Erniwati dan teknisi laboratorium Entomologi, Bidang Zoologi, Pusat Penelitian Biologi-LIPI yang telah membantu sorting, Berdasarkan hasil yang telah diperoleh dapat disimpulkan bahwa. Keanekaragaman belalang dan ke- rabatnya di Gunung Kendeng lebih tinggi (20 jenis dan 8 famili) daripada Gunung Botol (15 jenis dan 7 famili), tetapi kelimpahannya lebih tinggi di Gunung Botol (278 individu) daripada 112 Nety Virgo Erawati dan Sih Kahono: Keanekaragaman dan Kelimpahan Belalang mounting, dan identifikasi. Kepala Balai Taman Nasional Gunung Halimun atas ijin yang telah diberikan. Dr. Tri Atmowidi, M.Si. atas bimbingannya yang telah dilakukan. Kepada mereka semua kami meng- ucapkan banyak terima kasih. Erniwati. 2003. Belalang (Orthoptera) dan kekerabatannya. Di dalam: Amir M, Kahono S (ed.). Serangga Taman Nasional Gunung Halimun Jawa Barat. Biodiversity Conser- vation Project. Hal. 63-76. Gwynne DT, DeSutter L, Flook P, Rowell H. 1996. Orthoptera. Cric- kets, katydids, grasshoppers, etc. Version 01 January 1996. DAFTAR PUSTAKA Atmowidi T. 2000. Keanekaragaman morfospesies Hymenoptera para- sitoid dan senyawa Antiherbivora di Taman Nasional Gunung Hali- mun, Jawa Barat [tesis]. Bogor: Institut Pertanian Bogor. Kahono S. 2006. Respon adaptif kumbang lembing pemakan daun Henosepilachna vigintictopuchtata (Fabricius) (Coleoptera: Coccinel- lidae: Epilachninae) dan tumbuhan inangnya terhadap musim kemarau di daerah beriklim tropis kering Pasuruan dan Malang – Jawa Timur. Berita Biologi 8(3): 193- 200. Borror DJ, Triplehorn CA, Johson NF. 1992. Pengenalan Pelajaran Se- rangga. Partosoedjono S, penerjemah. Yogyakarta: Gadjah Mada University Press. Terjemah- an dari: An Introduction to Study of Insect. Kahono S, Amir M. 2003. Ekosistem dan khasanah serangga Taman Nasional Gunung Halimun. Di dalam: Amir M & Kahono S. (ed.), Serangga Taman Nasional Gunung Halimun Jawa Barat. Biodiversity Conservation Project. Hal. 1-22. Borror DJ, White RE. 1970. Field Guide Insects. New York: Houghton Mifflin Company. Campbell, Neil A. 2009. Biology concepts and connections. 6th (ed.), Benjamin-Cummings Pub Co. Kahono S, Noerdjito WA. 2002. Fluctuation of Rainfall and Insect Community in Gunung Halimun National Park, West Java. Research and Conservation of Biodiversity in Indonesia, Vol. IX, 157-169. Djuwarsah M. 1997. The Soil of Gunung Halimun National Park. Reasearch and Conservstion of Biodiversity in Indonesia. In: The Inventory of Natural Resources in Gunung Halimun National Park. Vol. II. Biodiversity Conservation Project. Kalshoven LGE. 1981. Pests of Crops in Indonesia. Laan PA van der, penerjemah. Jakarta : PT Ichtiar Baru-van Hoeve. Terjemahan dari 113 J. Entomol. Indon., September 2010, Vol. 7, No. 2, 100-115 species: a case of study. Conser- vation Biology 10 (1): 99-109. : De Plagen van de Cultuur- gewassen in Indonesie. LAI. 2007. Holy Bible. New Inter- national Version. Jakarta: Lembaga Alkitab Indonesia.. Primack RB, Supriatna J, Indrawan M, Kramadibrata P. 1988. Biologi Konservasi. Jakarta: Yayasan Obor Indonesia. Magurran AE. 1987. Ecological Diversity and Its Measurement. London: Chapman and Hill. Porter EE, Redak RA. 1996. Short- term recovery of grasshopper communites (Orthoptera: Acridi- dae) of a California native gras- sland after prescribe burning. http://www.ags.uci.edu/eporter/res earch.html. [diakses 3 Februari 2003]. , j , g 2002. Longicorn Beetles from Gunung Halimun National Park, West Java, Indonesia from 1997- 2002 (Coleoptera, Disteniidae and Cerabycidae). Bulletin of Forestry and Forest Products Research Institute 1 (3, 384): 189-223. Rentz DCP. 1996. Grasshopper Country. Australia: CSIRO. Manikam PJ. 1998. Gunung Halimun National Park Plan Book I. Research and Conservation of Biodiversity in Indonesia. Vol III. DAFTAR PUSTAKA Bogor: Information System and Park Management of Gunung Halimun National Park. Rizali A, Buchori D, Triwidodo H. 2002. Keanekaragaman serangga pada lahan persawahan-tepian Hutan: Indikator kesehatan ling- kungan. Hayati 9(2):41-47. Simbolon H & Mirmanto E. 1997. Altitudinal zonation of the forest vegetation in GHNP, West Java. In: Research and Conservation of Biodiversity in Indonesia vol. II. The Inventory of Natural Resources in GHNP. p. 14-35. Maulinda D. 2003. Keragaman Kumbang di Taman Nasional Gu- nung Halimun, Jawa Barat [skrip- si]. Bogor: IPB. Meyer JR. 2001. Orthoptera. http: //www. cals. nscuedu/ course/ ent425/ compendium/ orthop.htmal [diakses 22 Maret 2002]. Suantara IN. 2000. Keragaman kupu- kupu (Lepidoptera) di Taman Nasional Gunung Halimun, Jawa Barat [skripsi]. Bogor: Fakultas Pertanian IPB. Oliver L, Beatti AJ. 1992. A possible method for the rapid assesment of biodiversity. Conservation Biology 7: 562-568. Toda MJ, Kitcing RL. 1999. Forest Ecosystem: the assessment of plant and animal biodiversity in forest ecosystem. International Biodiver- sity Observation Year. Oliver L, Beatti AJ. 1996. Invertebrate morphospecies as surrogates for 114 Nety Virgo Erawati dan Sih Kahono: Keanekaragaman dan Kelimpahan Belalang Wolda H. 1978. Seasonal fluctuation in rainfall, food and abundance of tropical insects. Journal of Animal Ecology 47:369-381. Utomo S. 2001. Keanekaragaman Ordo Diptera (Insecta) di Gunung Kendeng dan Gunung Botol, Taman Nasional Gunung Halimun, Jawa Barat [skripsi]. Bogor: Institut Pertanian Bogor, Fakultas Matematika dan Ilmu Pengetahuan Alam. Wolda H. 1983. Diversity, diversity indices and tropical cockroaches. Oecologia 58:290-298. Wolda H, Wong H. 1988. Tropical insect diversity and seosonality Sweep samples vs Light trap. Prociding Entomology 91(2):203- 216. Whitmore TC. 1984. Tropical rain Forests of the Far East. 2nd ed. Oxford: Clarendon Press. Willemse LPM. 2001. Fauna Ma- lesiana Guide to Pest Orthoptera of Indomalayan Region. Netherlands: Buckhuy Publiser. 115 115
https://openalex.org/W2106740306	https://publications.goettingen-research-online.de/bitstream/2/35706/2/Cereb.%20Cortex-2015-Sch%c3%b6nwiesner-3278-89.pdf	English	null	Parcellation of Human and Monkey Core Auditory Cortex with fMRI Pattern Classification and Objective Detection of Tonotopic Gradient Reversals	Cerebral cortex	2,014	cc-by	13,843	Parcellation of Human and Monkey Core Auditory Cortex with fMRI Pattern Classiﬁcation and Objective Detection of Tonotopic Gradient Reversals Marc Schönwiesner1,2,3, Peter Dechent4, Dirk Voit5, Christopher I. Petkov6 and Katrin Krumbholz7 1Laboratory for Brain, Music and Sound Research (BRAMS), Montreal, Canada, 2Department of Psychology, University of Montreal, Montreal, Canada, 3Montreal Neurological Institute, McGill University, Montreal, Canada, 4Department of Cognitive Neurology, MR-Research in Neurology and Psychiatry, University Medicine Göttingen, Göttingen, Germany, 5Biomedical NMR Research GmbH, Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany, 6Institute of Neuroscience, Newcastle University Medical School, Newcastle upon Tyne, UK and 7MRC Institute of Hearing Research, Nottingham, UK at Niedersaechsische Staats- u. Universitae http://cercor.oxfordjournals.org/ Downloaded from at Niedersaechsische Staats- u. Universitaetsbibliothek Goettingen on October 19, 2015 http://cercor.oxfordjournals.org/ Downloaded from Address correspondence to Marc Schönwiesner, Laboratory for Brain, Music and Sound Research (BRAMS), Pavilion 1420 Mont-Royal, Montreal, Quebec, Canada H3A 2B4. Email: marc.schoenwiesner@umontreal.ca Auditory cortex (AC) contains several primary-like, or “core,” ﬁelds, which receive thalamic input and project to non-primary “belt” ﬁelds. In humans, the organization and layout of core and belt audi- tory ﬁelds are still poorly understood, and most auditory neuroima- ging studies rely on macroanatomical criteria, rather than functional localization of distinct ﬁelds. A myeloarchitectonic method has been suggested recently for distinguishing between core and belt ﬁelds in humans (Dick F, Tierney AT, Lutti A, Josephs O, Sereno MI, Weiskopf N. 2012. In vivo functional and myeloarchitectonic mapping of human primary auditory areas. J Neurosci. 32:16095–16105). We propose a marker for core AC based directly on functional magnetic resonance imaging (fMRI) data and pattern classiﬁcation. We show that a portion of AC in Heschl’s gyrus classiﬁes sound frequency more accurately than other regions in AC. Using fMRI data from macaques, we validate that the region where frequency classiﬁcation perform- ance is signiﬁcantly above chance overlaps core auditory ﬁelds, pre- dominantly A1. Within this region, we measure tonotopic gradients and estimate the locations of the human homologues of the core auditory subﬁelds A1 and R. Our results provide a functional rather than anatomical localizer for core AC. We posit that inter-individual variability in the layout of core AC might explain disagreements between results from previous neuroimaging and cytological studies. functional imaging studies found evidence for 2 primary-like ﬁelds, which were interpreted as the human homologues of A1 and R; rarely has evidence for a third primary-like ﬁeld (RT) been found (see, however, Moerel et al. 2012). Parcellation of Human and Monkey Core Auditory Cortex with fMRI Pattern Classiﬁcation and Objective Detection of Tonotopic Gradient Reversals However, dif- ferent studies have reached conﬂicting conclusions about the ﬁelds’ orientations and tonotopic layouts with respect to macroanatomical landmarks (reviewed in Baumann et al. 2013). All studies have used tonotopic mapping to detect borders between ﬁelds within core AC. The main difference between studies that have reached conﬂicting conclusions is that some studies have used an independent criterion for dis- tinguishing between core and belt auditory ﬁelds. In visual cortex, the boundary between the primary ﬁeld (V1) and non- primary ﬁelds is marked by reversals in the polar-angle com- ponent of the retinotopic gradient (Engel et al. 1994; Sereno et al. 1995). Data from non-human primates and other animals demonstrate that, in AC, the borders between different core ﬁelds run perpendicular to the ﬁelds’ tonotopic gradients and are thus marked by gradient reversals. In contrast, the borders between core and belt ﬁelds run parallel to the tonotopic gradi- ents. This means that these borders are not associated with gra- dient reversals (Morel et al. 1993; Rauschecker et al. 1995; Petkov et al. 2006) and can thus not be detected by tonotopic mapping. Dick et al. (2012) circumvented this problem by using a myeloarchitectonic marker for core AC and then restricting the tonotopic mapping to the core ﬁelds so identi- ﬁed. Building upon earlier ﬁndings of higher myelination in core ﬁelds (Hackett et al. 2001), they improved a previously developed quantitative T1 mapping protocol (Sigalovsky et al. 2006) to estimate myelination across the cortical surface. This method highlighted a region of high myelination on medial HG, consistent with the location of core AC in cytoarchitectonic parcellations in humans (Morosan et al. 2001). The region con- tained 2 tonotopic gradients oriented along the same axis as observed in monkeys (Merzenich and Brugge 1973; Imig et al. 1977; Morel and Kaas 1992; Morel et al. 1993). Moerel and col- leagues found similar results using the independent functional criterion of frequency selectivity, which has been shown to be greater in core than belt ﬁelds (Rauschecker et al. 1995). at Niedersaechsische Staats- u. Universitaetsbibliothek Goettingen on October 19, 2015 journals.org/ Keywords: hearing, tonotopy Keywords: hearing, tonotopy Cerebral Cortex October 2015;25:3278–3289 doi:10.1093/cercor/bhu124 Advance Access publication June 5, 2014 Cerebral Cortex October 2015;25:3278–3289 doi:10.1093/cercor/bhu124 Advance Access publication June 5, 2014 Cerebral Cortex October 2015;25:3278–3289 Cerebral Cortex October 2015;2 doi:10.1093/cercor/bhu124 Cerebral Cortex October 2015;25:3278–3289 doi:10.1093/cercor/bhu124 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/license reuse, distribution, and reproduction in any medium, provided the original work is properly cited. © The Author 2014. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. y buted under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted n in any medium, provided the original work is properly cited. Procedure h h In the human measurements, each trial started with the presentation of a stimulus for 4 s, followed by the functional image acquisition, which took 1 s and then 5 s of silence to yield a 9-s repetition time (TR) in order for any activation due to the scanner noise to die away before the next image acquisition. The stimuli were presented in epochs consist- ing of 2 trials, followed by 2 baseline trials where the stimulus was replaced by 4 s of silence. The experiment contained 20 trials of each of the 8 frequencies (see above). Epochs for different frequencies were presented in pseudorandom order with balanced transition probabil- ities. The experiment consisted of 324 trials in total (20 trials × 8 frequencies + 160 baseline trials + 4 initial dummy trials). It was split evenly into 2 runs of equal duration separated by a short break. To- gether with the structural scans, each session lasted about 60 min. This was a passive listening experiment; in order to stay alert, participants watched a silent subtitled movie of their own choice, which was pre- sented through a projection system with vision-correcting goggles. While it has been shown that task context can have a strong inﬂuence on AC activity (reviewed in Fritz et al. 2007), there is evidence that at- tention has little effect on measures of tonotopic organization (Petkov et al. 2004; Woods et al. 2009). Introduction We use these methods to delineate subdivisions of core AC in several individuals. 1990) across all frequencies. The noise was presented throughout the experiment at a level of 35 dB SPL per ERB, 40 dB below the level of the tones. tones and other narrowband sounds than belt AC (Morel et al. 1993; Rauschecker et al. 1995). The current approach is based on the same general idea but uses multi-voxel pattern classiﬁ- cation and high-resolution fMRI to maximize sensitivity to the functional differences between core and belt AC. We independ- ently validate our method by demonstrating that it correctly identiﬁes core auditory ﬁelds in several macaque monkey fMRI datasets. We then measure tonotopic gradients to delineate the borders of the subdivisions of core AC. Previous studies have assessed tonotopic gradients by manually connecting their low- and high-frequency endpoints, which involves a degree of subjective interpretation. Moreover, some of the endpoints used may have been part of belt AC, which may have skewed the gradient orientations. Here, we restrict the measurement of tonotopic gradients to the region identiﬁed as core by the pattern classiﬁcation procedure, and we use an automated, and thus objective, method for assessing gradient orientations. We use these methods to delineate subdivisions of core AC in several individuals. Two of the 3 monkeys were presented with 6 pure tones, logarith- mically spaced in frequency between 500 Hz and 16 kHz. The third monkey was presented with 2 2-octave-wide noises, centered at 500 Hz and 4 kHz. The stimuli were generated digitally with a 44.1-kHz sampling rate. They had a 50-ms duration (including 8-ms raised- cosine onset and offset ramps) and were presented with a 75-ms inter- stimulus interval and at an intensity of 75 dB SPL. They were presented through MR-compatible headphones modiﬁed to ﬁt the monkeys (MR Confon, for Monkeys 1 and 2, and NordicNeuroLab, for Monkey 3). at Niedersaechsische Staats- u. Universita http://cercor.oxfordjournals.org/ Downloaded from Imaging Protocol Th h g g The human measurements were performed in a horizontal 3-Tesla scanner (Magnetom TIM Trio, Siemens Healthcare) equipped with a 12-channel matrix head-coil (Siemens Healthcare) and using an echo- planar imaging sequence (gradient echo; acquisition time = 1 s, echo time = 36 ms; ﬂip angle = 90°) with sparse sampling (TR = 9 s) to min- imize the effect of scanner noise on the measured activity (Edmister et al. 1999; Hall et al. 1999). Each functional volume comprised 13 slices with an in-plane resolution of 1.5 × 1.5 mm and a thickness of 2.5 mm. The ﬁeld of view was 192 mm. The slices were oriented paral- lel to the average angle of left and right lateral sulci (determined from the structural scan, described later) to fully cover the superior temporal plane (STP) in both hemispheres. As a result, the functional volumes included HG, planum temporale (PT), planum polare, and the superior temporal gyrus and sulcus. A standard whole-brain T1-weighted struc- tural scan (magnetization-prepared rapid gradient echo sequence) with 1-mm3 resolution was also obtained for each participant (acquired before the functional scans). Participants h Seven human participants (4 males, aged between 25 and 35 years) took part in the experiment after having provided informed consent. All were right-handed according to the Edinburgh inventory (Oldﬁeld 1971) and had no history of hearing disorder or neurological disease. The experimental procedures conformed to the World Medical Asso- ciation’s Declaration of Helsinki and were approved by the local ethics committee. echsische Staats- u. Universitaetsbibliothek Goettingen on October 19, 2015 In addition, this paper also presents fMRI datasets from 3 macaque monkeys. One monkey (Monkey 1) was scanned while anesthetized, the other 2 (Monkeys 2 and 3) were awake. Two of the monkey data- sets (Monkeys 1 and 2) were acquired at the Max-Planck-Institute of Biological Cybernetics, Germany (Logothetis group) and have been used in a previous study (Monkeys 1 and 2; Petkov et al. 2006). The third dataset (Monkey 3) is new and was acquired at Newcastle Univer- sity, United Kingdom (Petkov group). Procedures for animal handling, anesthesia, and scanning complied with the guidelines of the Euro- pean Community (EUVD 86/609/EEC) for the care and use of labora- tory animals and were approved by the local authorities. A detailed description of all procedures can be found elsewhere (Logothetis et al. 1999; Petkov et al. 2006). In the monkey measurements, each trial started with the presenta- tion of a train of stimuli for 4–8.5 s, followed by the functional image acquisition, which took 1.5 s to yield a 10-s repetition time (TR; see Petkov et al. 2006 and Petkov et al. 2009 for details). Trials containing sounds were alternated with silent baseline trials. The awaken monkeys were required to maintain visual ﬁxation throughout the auditory stimulation, and so, the number of repetitions per condition, as well as the number of conditions depended on ﬁxation perform- ance. Monkeys 1 and 2 were presented with 12 and 29 repetitions of each of 6 frequencies, respectively, and Monkey 3 was presented with 60 repetitions of the 2 frequency conditions (see above). Introduction Anatomical and neurophysiological studies have established a widely accepted model of the structure of auditory cortex (AC) in non-human primates (Merzenich and Brugge 1973; Imig et al. 1977; Morel and Kaas 1992; Morel et al. 1993; Rauscheck- er et al. 1995; Hackett et al. 1998; Kaas and Hackett 1998, 2000). According to this model, primate AC contains 3 primary- like ﬁelds, collectively referred to as “core,” each with a separ- ate map of sound frequency (tonotopic map). In the macaque, the core ﬁelds are stacked in a posterior-to-anterior direction and surrounded by secondary ﬁelds, referred to as “belt” (reviewed in Petkov et al. 2006; Baumann et al. 2013). Cyto- architectonic data from postmortem human brains are consist- ent with this model of primate AC: they suggest that human AC also contains 3 primary-like ﬁelds, stacked along the long axis of Heschl’s gyrus (HG), which runs in a posteromedial- to-anterolateral direction (Morosan et al. 2001). Functional imaging data, however, have sometimes led to conﬂicting interpretations (reviewed in Baumann et al. 2013). Most In the present study, we propose an independent approach to localize core AC based on functional rather than anatomical properties. Most previous attempts at localizing core AC from functional data (e.g., Wessinger et al. 2001; Petkov et al. 2006; Chevillet et al. 2011; Moerel et al. 2012) exploited the ﬁnding from neurophysiological studies in monkeys that core AC responds better, and with greater frequency speciﬁcity, to pure tones and other narrowband sounds than belt AC (Morel et al. 1993; Rauschecker et al. 1995). The current approach is based on the same general idea but uses multi-voxel pattern classiﬁ- cation and high-resolution fMRI to maximize sensitivity to the functional differences between core and belt AC. We independ- ently validate our method by demonstrating that it correctly identiﬁes core auditory ﬁelds in several macaque monkey fMRI datasets. We then measure tonotopic gradients to delineate the borders of the subdivisions of core AC. Previous studies have assessed tonotopic gradients by manually connecting their low- and high-frequency endpoints, which involves a degree of subjective interpretation. Moreover, some of the endpoints used may have been part of belt AC, which may have skewed the gradient orientations. Here, we restrict the measurement of tonotopic gradients to the region identiﬁed as core by the pattern classiﬁcation procedure, and we use an automated, and thus objective, method for assessing gradient orientations. Data Analysis h f l d The functional data from both the humans and the monkeys were cor- rected for motion artifacts and spatially smoothed with a 2-mm Gauss- ian kernel. Statistical analysis was based on a general linear model (GLM) implemented in BrainVoyagerQX and performed on the origin- al 3D (“volume”) data. Regions of signiﬁcant activation were deter- mined by comparing the responses to the sound conditions with the silent baseline. The GLM was applied both to the individual and ﬁxed-effects group data. For the group analysis and for comparison of individual datasets, the functional volumes were co-registered to the symmetric ICBM152 template. Further analyses were performed in Matlab after importing the data using BrainVoyager’s Matlab toolbox BVQXtools. at Niedersaechsische Staats- u. Universitaetsbibliothek Goettingen on October 19, 2015 s.org/ First, we extracted frequency tuning curves for each voxel by plot- ting the response size (compared with baseline) to the different fre- quency conditions. In previous studies, the voxels’ preferred (or “best”) frequencies have been estimated as the frequency yielding the largest response, or by ﬁtting the voxel frequency tuning curves with a bell-shaped function (e.g., Formisano et al. 2003). The function-ﬁt method has the advantage of utilizing the responses to all frequencies, rather than to just 1 frequency. This reduces susceptibility to noise. However, ﬁtting can become unpredictable when the voxel tuning curves are multi-peaked. Here, we propose a simpler, non-parametric, method, which also utilizes the responses to all frequencies, but without the unpredictability of ﬁtting. Our method involves calculating the centroid of each voxel’s tuning curve according to: Centroid ¼ 1 n X n i¼1 ri n r fi; The monkey data were analyzed in a similar way as the human data, except that we collapsed multiple frequency conditions into a binary, low versus high, classiﬁcation problem (with a chance level of 50%). This allowed us to use the same classiﬁcation problem for all 3 monkeys, 1 of which was presented with only 2 frequency conditions. For Monkey 1, the SVM did not achieve above-chance performance due to the lower number of repetitions relative to the other 2 monkey datasets. For this animal, we used a k-nearest neighbor (with k = 1) classiﬁcation algorithm. The k = 1 nearest neighbor algorithm uses all training data for classiﬁcation, rather than only the data at the decision border, as in the case of the SVM. Data Analysis h f l d For Monkey 2, we conducted both a 2-category (low vs. high) and a 6-category (corresponding to the 6 pre- sented frequencies; see above) classiﬁcation analysis to assess the effect of collapsing different frequencies into a 2-category classiﬁcation problem. where n is the number of presented frequencies (8 in the current ex- periment), fi is the frequency value, and ri is the respective response amplitudes in percent signal change. r is the average response ampli- tude across frequencies. We then estimated the widths of the voxel tuning curves by calculating the spread about their centroids. This in- volved multiplying the normalized responses, ri/ðn rÞ; with the squared distances of the respective frequencies from the centroid, summing across frequencies, and taking the square root. The tuning- curve spreads were then multiplied by 2 ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 2 ln 2 p to derive the full-width-at-half-maximum (FWHM). For comparison, we also ﬁtted the voxel tuning curves with a rounded exponential (roex) function (the roex function is bell-shaped and is commonly used for ﬁtting cochlear ﬁlters from notched-noise data; Rosen and Baker 1994). The ﬁt was constrained so that the maximum of the function fell in the frequency range covered by the stimuli. The best frequencies and tuning-curve widths were taken as the peak and FWHM of the ﬁtted functions. The classiﬁcation accuracies were used to deﬁne “core” AC by thresholding to signiﬁcantly above-chance classiﬁcation performance. In order to validate this classiﬁcation-based method for delineating core AC, the classiﬁcation results for the monkeys were compared with the results from an independent method proposed by Petkov et al. (2006). They identiﬁed macaque core AC as that portion of the STP that (1) responds most strongly to narrowband sounds such as pure tones and (2) contains 3 tonotopic gradients with layouts compatible with those of A1, R, and RT, the locations of which have been veriﬁed in previous anatomical studies (Hackett et al. 2001). As the animals were not sacriﬁced after the fMRI session, no direct cytoarchitectonic parcel- lation of auditory areas was available. The multi-voxel pattern classiﬁcation analysis of the human data was conducted with a type-2 linear-kernel Support Vector Machine (SVM, Cortes and Vapnik 1995), implemented in the PyMVPA Python toolbox (Hanke et al. 2009), and applied to the volume data of each participant separately. Stimuli The training and test phases of the SVM analysis were performed using a 5-fold cross-validation procedure, whereby the dataset is randomly divided into 5 equal parts and each part is used as test data, with the re- maining 4 parts used for training. Classiﬁcation accuracy was estimated by taking the average across the 5 parts. The SVM analysis was per- formed within a 4-mm sphere of all voxels that showed a signiﬁcant re- sponse to at least 1 of the 8 frequency conditions (searchlight procedure; Kriegeskorte et al. 2006). Thus, the classiﬁcation accuracy for each voxel is based on the activity pattern within the voxel’s 34-voxel neighborhood. Group average accuracy was calculated by averaging the co-registered individual classiﬁcation accuracy volumes. The classiﬁcation results were visualized on slices through AC, pitched by ∼30° from the line connecting the anterior and posterior commis- sures to run parallel to the lateral sulcus. In addition, they were also mapped onto the 2-dimensional cortical surface reconstructions for better visualization. Brain surfaces were extracted from the human structural volumes using BrainVoyagerQX (BrainInnovation). Individ- ual brain surfaces were registered to an iterative group average surface (as implemented in BrainVoyager). Surface mapping involves interpol- ation. The classiﬁcation analysis was performed on the original volume, rather than the interpolated surface, data, because interpol- ation introduces correlation between data points, which decreases the power of the classiﬁcation procedure. Classiﬁcation accuracies (percent correct classiﬁcation; the chance level was 12.5%) were trans- formed to z-scores using a binomial distribution and the resulting maps were thresholded at P = 0.01 (uncorrected). A cluster-size thresh- old based on random ﬁeld theory as implemented in FMRIstat (Worsley et al. 2002) was applied to achieve a threshold of P < 0.05, corrected for multiple comparisons. To verify that the classiﬁcation results were stable and not speciﬁc to the particular classiﬁcation algo- rithm used, we repeated the analysis with type-1 SVM and k-nearest neighbor classiﬁers. For each classiﬁer, we performed a basic manual grid search for suitable values of its free parameter (k in case of the k-nearest neighbor classiﬁer and a regularization parameter in case of the SVM). The results were similar between the 2 classiﬁers and robust across a wide range of parameter values. (Monkey 3). Only the right AC was measured with the surface coils (Monkeys 1 and 2), whereas both the left and right ACs were measured with the head coil (Monkey 3). 3280 Parcellation of Core Auditory Cortex • Schönwiesner et al. Stimuli Functional volumes were acquired using a gradient-recalled echo-planar imaging sequence (TE = 16 ms, acquisition time = 1.5 s, TR = 10 s). They consisted of 6–12 slices, or- iented parallel to the relevant lateral sulcus. Each slice consisted of 128 × 128 voxels and was 2 mm thick. The ﬁeld of view was set indi- vidually for each animal and measured between 6.4 × 6.4 and 12.8 × 12.8 cm, corresponding to an in-plane resolution of between 0.5 × 0.5 and 1 × 1 mm. Structural volumes were acquired with a 3-dimensional, modiﬁed driven-equilibrium Fourier transform se- quence (TE = 4 ms, TR = 22 ms, 256 × 256 × 128 voxels, with an FOV of 9.6 × 9.6 × 6.4 cm). (Monkey 3). Only the right AC was measured with the surface coils (Monkeys 1 and 2), whereas both the left and right ACs were measured with the head coil (Monkey 3). Functional volumes were acquired using a gradient-recalled echo-planar imaging sequence (TE = 16 ms, acquisition time = 1.5 s, TR = 10 s). They consisted of 6–12 slices, or- iented parallel to the relevant lateral sulcus. Each slice consisted of 128 × 128 voxels and was 2 mm thick. The ﬁeld of view was set indi- vidually for each animal and measured between 6.4 × 6.4 and 12.8 × 12.8 cm, corresponding to an in-plane resolution of between 0.5 × 0.5 and 1 × 1 mm. Structural volumes were acquired with a 3-dimensional, modiﬁed driven-equilibrium Fourier transform se- quence (TE = 4 ms, TR = 22 ms, 256 × 256 × 128 voxels, with an FOV of 9.6 × 9.6 × 6.4 cm). at Niedersaechsische Staats- u. Universitae http://cercor.oxfordjournals.org/ Downloaded from at Niedersaechsische Staats- u. Univer http://cercor.oxfordjournals.org/ Downloaded from at Niedersaechsische Staats- u. Universitaetsbibliothek Goettingen on October 19, 2015 http://cercor.oxfordjournals.org/ Downloaded from Stimuli For the human measurements, the sound stimuli consisted of bursts of pure tones with frequencies centered around 1 of 8 nominal frequen- cies, logarithmically spaced between 200 and 8000 Hz (200, 338.8, 573.8, 971.9, 1646.2, 2788.4, 4723.1 and 8000 Hz). The bursts were 187.5 ms in duration (including 20-ms squared-cosine onset and offset ramps) and presented once every 250 ms for 4 s. The stimuli were gen- erated digitally (24.4-kHz sampling rate, 24-bit amplitude resolution) using Matlab and Tucker Davis Technologies System 3. In order to min- imize response adaptation, the frequency of the tone bursts was varied randomly according to a uniform distribution of 1 semitone around the nominal frequency. These frequency variations were large enough to be audible, but, at the same time, small enough to be processed within the same cochlear channel (Glasberg and Moore 1990). The stimuli were presented binaurally at a level of 75 dB SPL through MR-compatible high-ﬁdelity headphones (MR Confon). The monkey measurements were performed in vertical primate scanners (Bruker Medical) at 4.7 T (Monkeys 1 and 3) and 7 T (Monkey 2). A primate chair was used for positioning the animals within the magnet bore. Functional and structural volumes were acquired using surface radiofrequency coils of 70- (Monkey 1) or 80-mm (Monkey 2) diameter, positioned over AC of 1 hemisphere, or a whole-head coil In order to minimize the effect of the variation in the normal hearing threshold across frequency, as well as differences in hearing threshold between participants and inhomogeneities in the headphone transfer function, the stimuli were presented in a background of con- tinuous noise with equal energy per cochlear ﬁlter bandwidth (deﬁned as equivalent rectangular bandwidth, or ERB; Glasberg and Moore Cerebral Cortex October 2015, V 25 N 10 3279 (2-category) classiﬁcation problems. Our 8-category frequency classiﬁ- cation problem was solved by partitioning into a complete set of pair- wise binary problems with a majority voting strategy to determine the overall classiﬁcation. This partitioning procedure is part of the PyMVPA toolbox. Prior to the classiﬁcation analysis, the voxel time series were detrended by robust locally weighted least-squares regres- sion (Cleveland and Devlin 1988), mean-corrected, and normalized by their standard deviation. No spatial smoothing was used in this case. Data Analysis h f l d Support vector machines are supervised learn- ing methods that construct 2-category boundaries in sets of training items, each marked as belonging to 1 of the 2 categories. Based on the learned category boundaries, the SVM then predicts whether a new item falls into one category or the other. SVMs only solve binary Delineation of Core Auditory Fields A crucial prerequisite for parcellating core AC is to delineate core from belt AC. The current approach was based on the hy- pothesis that, as neurons in core AC respond with greater ﬁdel- ity and frequency speciﬁcity to narrowband sounds than neurons in the auditory belt and parabelt (Rauschecker et al. 1995), fMRI activity in core AC would be more informative about the presented frequency than activity in non-core AC. We tested this hypothesis using pattern classiﬁcation analysis. Pattern classiﬁcation analysis assesses the relationship between multi-voxel activity patterns and the stimulus input and is, under certain circumstances, sensitive to features of cortical or- ganization that are below the spatial resolution of the fMRI re- cording (Boynton 2005; Kamitani and Tong 2005). Here, at Niedersaechsische Staats- u. Universitaetsbibliothek Goettingen on October 19, 2015 http://cercor.oxfordjournals.org/ Downloaded from 1. Volume slices (A) and surface renderings (B) showing the spatial distribution of classiﬁcation accuracy in AC. Both individual (labeled 1–7) and group average data d Group) are shown. The group data are based on a ﬁxed-effects analysis. The volume slices were oriented to run parallel to the lateral sulcus (see Materials and Methods et schematic brain). The cortical surfaces were extracted from the structural volumes; only the upper surfaces of the left and right temporal lobes are shown with HG, PT, and mporal pole marked in the group average surface (upper left corner in B, viewing direction is indicated by the red arrow in the inset schematic brain). The color overlay on each surface shows the performance accuracy of the classiﬁcation algorithm expressed as proportion of correct classiﬁcations and thresholded at P = 0.01 in the chance al) distribution. Black lines in the group average surfaces indicate the extent of the 10% and 50% probability maps of cytoarchitectonically deﬁned core AC reported by n et al. (2001). The slice and surface representations are provided to facilitate comparisons with previously published results. In both representations, maxima of cation accuracy are visible on or near HG in all participants. at Niedersaechsische Staats- u. Universitaetsbibliothek Goettingen on October 19, 2015 ercor.oxfordjournals.org/ at Niedersaechsische Staats- u. Universitaetsbibliothek Goettingen on October 19, 2015 djournals.org/ Figure 1. Volume slices (A) and surface renderings (B) showing the spatial distribution of classiﬁcation accuracy in AC. Both individual (labeled 1–7) and group average data (labeled Group) are shown. The group data are based on a ﬁxed-effects analysis. Results pattern classiﬁcation analysis was conducted using a machine- learning approach. We trained a machine-learning algorithm (type-2 SVM) to predict the frequency of each presented stimu- lus from the activity within small (4-mm) spherical volumes in AC. Prediction accuracy in human datasets reached up to 50% (i.e., the presented frequency was correctly predicted out of a total of 8 possibilities in 50% of cases), which is well above the 12.5% chance level. A confusion matrix analysis between the predicted and actual frequencies revealed that the majority (68%) of misclassiﬁcations were between neighboring frequen- cies. Figure 1 shows the spatial distribution of the classiﬁcation accuracy on slices through the STP containing HG and on the temporal lobe surfaces. In all 14 hemispheres (7 participants), as well as in the group average data, a contiguous patch of cor- tical surface on or near HG showed classiﬁcation accuracy sig- niﬁcantly above chance. Some participants showed a second, Delineation of Core Auditory Fields Delineation of Core Auditory Fields Delineation of Core Auditory Fields smaller patch of signiﬁcant classiﬁcation accuracy on the PT (6 participants) or planum polare (1 participant). This pattern of results was stable across different classiﬁcation algorithms (we also tested type-1 SVM and k-nearest neighbor algorithms; see Materials and Methods), and a wide range of hyperparameters within each algorithm. Next, we tested whether the classiﬁcation accuracy in the human data correlated with the sharpness of the voxel fre- quency tuning curves or the response amplitude to pure tones, both of which have previously been used as markers for core AC (Wessinger et al. 2001; Petkov et al. 2006; Moerel et al. 2012). We estimated the widths of the voxel frequency tuning curves by measuring the spread around their centroids (see Materials and Methods; Supplementary Fig. 1A). For compari- son, we also estimated the tuning widths by measuring the full-width-at-half-maximum of bell-shaped (roex) functions ﬁtted to the voxel response curves. The 2 measures of tuning width were moderately correlated (Pearson’s r = 0.69, P < 0.0001). The tuning-curve spreads showed a small, but signiﬁ- cant, negative correlation with the classiﬁcation accuracy (r = −0.076, P < 0.0001), indicating that spreads were smaller (i.e., tuning curves were sharper) in voxels with higher classiﬁcation accuracy, as would be expected. The correlation with tuning widths estimated from the ﬁtted roex functions was not signiﬁ- cant (r = −0.015, P = 0.07). To estimate the response amplitude to pure tones, we calculated the maximum of the percentage signal change across the 8 frequencies used in the human mea- surements (Supplementary Fig. 1B), as well as the root-mean- square signal change across all frequencies. Both measures were almost perfectly correlated with each other (r > 0.9, P < 0.0001), and both showed a small, but signiﬁcant, correl- ation with classiﬁcation accuracy (r = 0.08, P < 0.0001), indicat- ing that the pure-tone responses were somewhat larger in voxels with higher classiﬁcation accuracy. g In order to test whether the region of signiﬁcant classiﬁca- tion accuracy coincides with core AC, we applied the classiﬁca- tion method to fMRI datasets from 3 macaque monkeys (4 hemispheres, because only the right hemisphere was mea- sured in Monkeys 1 and 2). In the monkey data, the location of core AC could be veriﬁed using independent functional cri- teria, as well as existing knowledge from previous anatomical and physiological studies (Petkov et al. 2006). Delineation of Core Auditory Fields Figure 2 shows the results from pattern classiﬁcation analysis of the monkey data together with a functional parcellation of each monkey’s AC based on response amplitudes to pure tones and frequency gradient reversals (see Materials and Methods). Similar to the results in humans, classiﬁcation accuracy in a circumscribed region of the monkey AC was greater than that in other regions (the red highlight in Fig. 2B–E shows signiﬁcantly above- chance classiﬁcation accuracy). Importantly, a major portion of this region coincided with the largest core ﬁeld, A1, in all 4 monkey hemispheres. The second-largest core ﬁeld, R, was marked in 2 hemispheres, and belt ﬁelds CM, CL, and AL were each marked in 1 hemisphere. In contrast, the core ﬁeld RT was never marked. The monkey data suggest that signiﬁcant classiﬁcation accuracy is a useful marker for the core auditory ﬁeld A1, and, to a lesser degree, also R. From Monkey 2, sufﬁ- cient data were available to conduct a classiﬁcation analysis for 6 sound frequency conditions (Fig. 2H) in addition to a high- versus low-frequency classiﬁcation (Fig. 2C). Regions of at Niedersaechsische Staats- u. Universitae http://cercor.oxfordjournals.org/ Downloaded from at Niedersaechsische Staats- u. Universitaetsbibliothek Goettingen on October 19, 2015 http://cercor.oxfordjournals.org/ Downloaded from at Niedersaechsische Staats- u. Universitaetsbibliothek Goettingen on October 19, 2015 ournals.org/ We computed a probability map of the region of signiﬁcant classiﬁcation accuracy across our sample of 14 human Figure 2. (A) A slice through a macaque brain (Monkey 1) parallel to the STP and covering AC. The color overlay shows the sound frequency that evoked the strongest response for each voxel. The black lines delineate different functional ﬁelds in AC, identiﬁed based on the response amplitude to pure tones and tonotopic gradient reversals. The core ﬁelds A1 and R as well as belt ﬁelds CM, CL, and AL are labeled. Modiﬁed, with permission, from Petkov et al. (2006). (B–E) The pattern classiﬁcation results from 3 macaque monkeys (4 hemispheres; B, C, E: right AC of Monkeys 1, 2, and 3; D: left AC of Monkey 3; C: the classiﬁcation accuracy when the 6 sound frequency conditions used in Monkey 2 were grouped into high and low frequencies to enable direct comparison with the other monkeys, compare with panel H for results of the 6-frequency classiﬁcation). Delineation of Core Auditory Fields The volume slices were oriented to run parallel to the lateral sulcus (see Materials and Methods and inset schematic brain). The cortical surfaces were extracted from the structural volumes; only the upper surfaces of the left and right temporal lobes are shown with HG, PT, and the temporal pole marked in the group average surface (upper left corner in B, viewing direction is indicated by the red arrow in the inset schematic brain). The color overlay on each slice or surface shows the performance accuracy of the classiﬁcation algorithm expressed as proportion of correct classiﬁcations and thresholded at P = 0.01 in the chance (binomial) distribution. Black lines in the group average surfaces indicate the extent of the 10% and 50% probability maps of cytoarchitectonically deﬁned core AC reported by Morosan et al. (2001). The slice and surface representations are provided to facilitate comparisons with previously published results. In both representations, maxima of classiﬁcation accuracy are visible on or near HG in all participants. Figure 1. Volume slices (A) and surface renderings (B) showing the spatial distribution of classiﬁcation accuracy in AC. Both individual (labeled 1–7) and group average data (labeled Group) are shown. The group data are based on a ﬁxed-effects analysis. The volume slices were oriented to run parallel to the lateral sulcus (see Materials and Methods and inset schematic brain). The cortical surfaces were extracted from the structural volumes; only the upper surfaces of the left and right temporal lobes are shown with HG, PT, and the temporal pole marked in the group average surface (upper left corner in B, viewing direction is indicated by the red arrow in the inset schematic brain). The color overlay on each slice or surface shows the performance accuracy of the classiﬁcation algorithm expressed as proportion of correct classiﬁcations and thresholded at P = 0.01 in the chance (binomial) distribution. Black lines in the group average surfaces indicate the extent of the 10% and 50% probability maps of cytoarchitectonically deﬁned core AC reported by Morosan et al. (2001). The slice and surface representations are provided to facilitate comparisons with previously published results. In both representations, maxima of classiﬁcation accuracy are visible on or near HG in all participants. Cerebral Cortex October 2015, V 25 N 10 3281 signiﬁcant classiﬁcation accuracy were virtually identical in both analyses. 3282 Parcellation of Core Auditory Cortex • Schönwiesner et al. Delineation of Core Auditory Fields 2001; Rademacher et al. 2001). The average overlap of the regions of signiﬁcant classiﬁcation accuracy in any 2 hemispheres was 46%. This is similar to the average overlap in functionally deﬁned core AC ﬁelds in monkeys (∼50%, see Petkov et al. 2006), suggesting that the locations of core AC ﬁelds exhibit a similar degree of inter-individual variability in monkeys and humans. one (Participant 7, left hemisphere). In this participant, the classiﬁcation-based estimate of core AC was located more anteriorly and laterally than expected based on the cytoarchi- tectonic probability map. It is impossible to decide whether the classiﬁcation-based estimate or the cytoarchitectonic prob- ability map is “incorrect” in Participant 7. The fact that the loca- tions of the classiﬁcation-based estimates were consistent across the 2 hemispheres would suggest that they are “correct.” At the same time, however, the classiﬁcation accuracy was gen- erally lower than in other participants, suggesting that the estimates were less reliable. at Niedersaechsische Staats- u. Universita http://cercor.oxfordjournals.org/ Downloaded from Rademacher et al. (2001) reported a maximal distance of about 2 cm between the centroids of any 2 cytoarchitectoni- cally deﬁned core AC regions within 27 individuals. Here, we found a very similar maximal distance of 2.3 cm between the centroids of the regions of signiﬁcant classiﬁcation accuracy in any 2 of our 14 hemispheres. We also quantiﬁed the correspondence between the region of signiﬁcant classiﬁcation performance and a previously reported probability map of core AC based on cytoarchitecton- ic data from 10 postmortem brains (Morosan et al. 2001). For that, we deﬁned 2 regions of interest: one more conservative, corresponding to the 50% probability map of the cytoarchitec- tonically deﬁned core AC (i.e., the region comprising core AC in at least 50% of brains tested), and the other more liberal, cor- responding to the 10% probability map. Outlines of these regions are shown on the group temporal lobe surfaces in Figure 1. We found that the conservative region contained over 90% of the region of signiﬁcant classiﬁcation accuracy in the group average data, indicating that there was a good corres- pondence between the average region of signiﬁcant classi- ﬁcation accuracy and the cytoarchitectonic map of core AC. For individual hemispheres, the correspondence was less good, as would be expected, ranging from as little as 0% to as much as 100%. Estimation of Voxel Best Frequencies f q The results from the previous section indicate that the region of signiﬁcant classiﬁcation accuracy corresponds well with the location of core AC based on cytoarchitectonic criteria in humans and based on independent functional criteria in monkeys. In this section, we describe an automated procedure for measuring tonotopic gradients and ﬁnding their reversals in order to delineate different subﬁelds within core AC. The ﬁrst step in ﬁnding tonotopic gradient reversals is to estimate each voxel’s preferred, or “best,” frequency. Here, the voxel best frequencies were estimated by calculating the centroid of the voxels’ frequency tuning curves (see Materials and Methods). A prerequisite for determining voxel best frequen- cies is that voxel tuning curves are reliable. We tested reliability by computing the distributions across voxels of the coefﬁcients of correlation between the response curves for the ﬁrst and second experimental runs. The response curves were highly correlated between the 2 runs in all participants (the distribu- tions of correlation coefﬁcients were skewed toward high values, with a median value of 0.63, on average). Wilcoxon rank sum tests showed that the distributions were signiﬁcantly different (P < 0.0001) from the respective empirical null distri- butions, estimated by 10 000-fold random sampling (with re- placement) from distributions of correlation coefﬁcients between scrambled voxels. Figure 3. Probability map of estimated core AC. Cortical surfaces of all 14 hemispheres were aligned to the left hemisphere of a target using surface-based alignment of the location of gyri and sulci. The color code indicates the number of hemispheres that contained core AC at a given cortical location. Large overlap of core AC was observed in middle HG. Based on this result, we then computed best-frequency maps using the centroid method (Fig. 4). For comparison, we also derived the best frequencies from the roex functions ﬁtted to the voxel tuning curves (see Materials and Methods). The best-frequency estimates obtained by the 2 methods were highly correlated (Pearson’s r = 0.87, P < 0.0001); both methods generally yielded a gradual variation in best fre- quency across the cortical surface. The best frequencies esti- mated with the centroid method appeared less noisy than those estimated with the function-ﬁt method and thus lent themselves better to extracting best-frequency gradients. In order to parcellate core AC into its subdivisions, we used the best-frequency maps to locate reversals in the tonotopic gradients. Delineation of Core Auditory Fields The red highlight shows the region where the classiﬁcation accuracy was signiﬁcantly above chance (chance levels differed, dependent on the number of frequency conditions). The white lines delineate different AC ﬁelds as in A. F and G show automatically determined borders (dark gray) in core ﬁelds in the region of interest comprising A1 and R as determined by the manual parcellation shown in B and C, respectively. The automatically identiﬁed border between A1 and R coincided exactly with the manual parcellation. The orientation of the slices in A–E and H is depicted in the inset schematic brain. Figure 2. (A) A slice through a macaque brain (Monkey 1) parallel to the STP and covering AC. The color overlay shows the sound frequency that evoked the strongest response for each voxel. The black lines delineate different functional ﬁelds in AC, identiﬁed based on the response amplitude to pure tones and tonotopic gradient reversals. The core ﬁelds A1 and R as well as belt ﬁelds CM, CL, and AL are labeled. Modiﬁed, with permission, from Petkov et al. (2006). (B–E) The pattern classiﬁcation results from 3 macaque monkeys (4 hemispheres; B, C, E: right AC of Monkeys 1, 2, and 3; D: left AC of Monkey 3; C: the classiﬁcation accuracy when the 6 sound frequency conditions used in Monkey 2 were grouped into high and low frequencies to enable direct comparison with the other monkeys, compare with panel H for results of the 6-frequency classiﬁcation). The red highlight shows the region where the classiﬁcation accuracy was signiﬁcantly above chance (chance levels differed, dependent on the number of frequency conditions). The white lines delineate different AC ﬁelds as in A. F and G show automatically determined borders (dark gray) in core ﬁelds in the region of interest comprising A1 and R as determined by the manual parcellation shown in B and C, respectively. The automatically identiﬁed border between A1 and R coincided exactly with the manual parcellation. The orientation of the slices in A–E and H is depicted in the inset schematic brain. 3282 Parcellation of Core Auditory Cortex • Schönwiesner et al. hemispheres (Fig. 3). The right hemispheres were ﬂipped to enable this analysis. We found that the overlap of regions of signiﬁcant classiﬁcation accuracy in 2 or more hemispheres co- incided with the middle part of HG, the same region as identi- ﬁed by cytoarchitectonical parcellations (Morosan et al. Delineation of Core Auditory Fields The liberal region, however, contained 100% of the region of signiﬁcant classiﬁcation accuracy not just in the group average data, but in all individual hemispheres bar Cerebral Cortex October 2015, V 25 N 10 3283 Estimation of Voxel Best Frequencies For instance, if 2 mirror-oriented tonotopic gradients were expected to run parallel to the long (mediolateral) axis of HG, then gradient directions would be determined along that axis of HG. Obvious drawbacks of this method are (1) that it assumes that the gradients adjoining the edge are mirror- symmetric and also (ii) that it requires a prior assumption as to the orientation along which a gradient edge will occur. This is problematic, because the orientations of tonotopic gradients within core AC may vary between participants. A further draw- back is that gradient edges are detected along only one, the preset, orientation, and so, no measure of the reliability of the edge locations can be obtained. Our aim was to reﬁne the gradient-sign method in order to eliminate these problems (Fig. 5). For that, we calculated gradient-sign maps, not just for one, but for all possible orientations between 0 and 179°, in 1°-steps. We then marked the locations of gradient reversals for each orientation using an edge detection ﬁlter (see Fig. 5D for examples). This creates a gradient edge map for each orientation. The edge maps were then summed across all or- ientations to obtain a composite map of gradient edges repre- senting the number of orientations for which an edge was present at a given location. This composite map allowed us to ﬁnd the edges with the greatest number of overlapping rever- sals. Because the orientations were sampled in 1°-steps, the number of reversals represents the angle between the 2 tonoto- pic gradients adjoining at a given edge location. Thus, the edge with the greatest number of overlapping reversals is also the edge with the largest average angle between adjoining gradi- ents and would thus be presumed to represent the most likely border between ﬁelds. In the monkey data, the region of sig- niﬁcant classiﬁcation accuracy overlapped the border between gradient-angle distributions (Langers and van Dijk 2012). Com- putation of gradient-sign maps involves setting an orientation along which to analyze best-frequency gradients, and then de- termining, for each voxel, the direction of the best-frequency change (i.e., rising or falling) along that orientation. Edges between positive and negative gradient directions in such maps mark gradient reversals with respect to the pre-set orien- tation. 3284 Parcellation of Core Auditory Cortex • Schönwiesner et al. Estimation of Voxel Best Frequencies In the majority of previous tonotopic mapping studies, the orientation of tonotopic gradients was assessed by manually connecting their low- and high-frequency endpoints with straight lines. Only a few studies computed gradient-sign maps (Formisano et al. 2003; Petkov et al. 2006) or analyzed Figure 3. Probability map of estimated core AC. Cortical surfaces of all 14 hemispheres were aligned to the left hemisphere of a target using surface-based alignment of the location of gyri and sulci. The color code indicates the number of hemispheres that contained core AC at a given cortical location. Large overlap of core AC was observed in middle HG. Figure 3. Probability map of estimated core AC. Cortical surfaces of all 14 hemispheres were aligned to the left hemisphere of a target using surface-based alignment of the location of gyri and sulci. The color code indicates the number of hemispheres that contained core AC at a given cortical location. Large overlap of core AC was observed in middle HG. Cerebral Cortex October 2015, V 25 N 10 3283 gure 4. Temporal lobe surfaces with renderings of the voxel best frequencies estimated by calculating the centroids of the voxel tuning curves (A), or ﬁtting them with ll-shaped (roex) functions (B). In both panels, the outlines of core AC, estimated by thresholding the classiﬁcation accuracy maps at the signiﬁcance threshold (see Data Analysis), e marked with black lines. at Niedersaechsische Staats- u. Universitaetsbibliothek Goettin http://cercor.oxfordjournals.org/ Downloaded from at Niedersaechsische Staats- u. Universitaetsbibliothek Goettingen on October 19, 2015 http://cercor.oxfordjournals.org/ Downloaded from at Niedersaechsische Staats- u. Universitaetsbibliothek Goettingen on October 19, 2015 ournals.org/ Figure 4. Temporal lobe surfaces with renderings of the voxel best frequencies estimated by calculating the centroids of the voxel tuning curves (A), or ﬁtting them with bell-shaped (roex) functions (B). In both panels, the outlines of core AC, estimated by thresholding the classiﬁcation accuracy maps at the signiﬁcance threshold (see Data Analysis), are marked with black lines. gradient-angle distributions (Langers and van Dijk 2012). Com- putation of gradient-sign maps involves setting an orientation along which to analyze best-frequency gradients, and then de- termining, for each voxel, the direction of the best-frequency change (i.e., rising or falling) along that orientation. Edges between positive and negative gradient directions in such maps mark gradient reversals with respect to the pre-set orien- tation. Estimation of Voxel Best Frequencies For instance, if 2 mirror-oriented tonotopic gradients were expected to run parallel to the long (mediolateral) axis of HG, then gradient directions would be determined along that axis of HG. Obvious drawbacks of this method are (1) that it assumes that the gradients adjoining the edge are mirror- symmetric and also (ii) that it requires a prior assumption as to the orientation along which a gradient edge will occur. This is problematic, because the orientations of tonotopic gradients within core AC may vary between participants. A further draw- back is that gradient edges are detected along only one, the preset, orientation, and so, no measure of the reliability of the edge locations can be obtained. Our aim was to reﬁne the Figure 5. Automatic parcellation of core AC. The extracted individual brain surface (A) is inﬂated (B) and a segment covering STP is cut out. (C) The best-frequency map is masked with the contiguous region of signiﬁcant classiﬁcation performance located closest to HG. This region is taken as estimate of core AC. (D) Gradient reversals are computed for all orientations between 0 and 179°. The reversal maps for different orientations are then summed. The values in the resulting map indicate the angle between the adjoining tonotopic gradients. (E) Stable reversals indicate the location of subﬁeld borders within core AC. at Niedersaechsische Staats- u. Universitaetsbibliothek Goettingen on October 19, 2015 http://cercor.oxfordjournals.org/ Downloaded from Figure 5. Automatic parcellation of core AC. The extracted individual brain surface (A) is inﬂated (B) and a segment covering STP is cut out. (C) The best-frequency map is masked with the contiguous region of signiﬁcant classiﬁcation performance located closest to HG. This region is taken as estimate of core AC. (D) Gradient reversals are computed for all orientations between 0 and 179°. The reversal maps for different orientations are then summed. The values in the resulting map indicate the angle between the adjoining tonotopic gradients. (E) Stable reversals indicate the location of subﬁeld borders within core AC. at Niedersaechsische Staats- u. Universitaetsbibliothek Goettingen on Octo http://cercor.oxfordjournals.org/ aded from Figure 6. Parcellation results and tonotopic gradients for the human data. All individual hemispheres and the group average data are shown. In most hemispheres (11/14), we found 1 tonotopic gradient reversal with a shared low-frequency border, presumably marking the core ﬁelds A1 (red-yellow color gradient, corresponding to low-to-high best frequencies) and R (blue-cyan color gradient). Estimation of Voxel Best Frequencies In 1 hemisphere (Participant 2, right hemisphere), we found an additional high-frequency reversal posterior to A1, which probably marks the border between A1 and either of the belt ﬁelds CM or CL (green-yellow color gradient). In 2 hemispheres (Participants 2 and 6, left hemispheres), no stable gradient reversals were found in the region of signiﬁcant classiﬁcation accuracy. at Niedersaechsische Staats- u. Universitaetsbibliothek Goettingen on October 19, 2015 journals.org/ Figure 6. Parcellation results and tonotopic gradients for the human data. All individual hemispheres and the group average data are shown. In most hemispheres (11/14), we found 1 tonotopic gradient reversal with a shared low-frequency border, presumably marking the core ﬁelds A1 (red-yellow color gradient, corresponding to low-to-high best frequencies) and R (blue-cyan color gradient). In 1 hemisphere (Participant 2, right hemisphere), we found an additional high-frequency reversal posterior to A1, which probably marks the border between A1 and either of the belt ﬁelds CM or CL (green-yellow color gradient). In 2 hemispheres (Participants 2 and 6, left hemispheres), no stable gradient reversals were found in the region of signiﬁcant classiﬁcation accuracy. A1 and R in 2 hemispheres, and between A1 and CM and A1 and CL in 1 hemisphere each (see Fig. 2). The borders between A1 and R and between A1 and CM or CL can be distinguished based on the best frequencies of the adjoining voxels: tonoto- pic gradients share a low-frequency border between A1 and R, but a high-frequency border between A1 and CM or CL. Thus, gradient reversals within regions with low best frequencies likely mark the border between the core ﬁelds A1 and R, whereas gradient reversals within regions with high best fre- quencies likely mark the border between A1 and either of the caudal belt ﬁelds CM or CL. and posterior ﬁelds CM or CL. This is consistent with the region of signiﬁcant pattern classiﬁcation accuracy, which did not include the posterior portions of manually delineated A1. Figure 6 shows the parcellation results for the human data. In 11 of the 14 hemispheres, the region of signiﬁcant classiﬁca- tion accuracy contained 2 tonotopic gradients with a shared low-frequency endpoint, which presumably marks the border between the core ﬁelds A1 and R. One hemisphere showed an additional gradient reversal with a shared high-frequency end- point, located posterior to the low-frequency reversal. Estimation of Voxel Best Frequencies This high-frequency reversal probably marks the border between A1 and either of the caudal belt ﬁelds CM or CL. In the remain- ing 2 hemispheres, the region of signiﬁcant classiﬁcation ac- curacy contained only a single tonotopic gradient and thus no reversals. In these hemispheres, the gradient orientation was consistent with A1. We performed the same automatic detection of area borders in the data from Monkeys 1 and 2 (we could not perform this analysis on Monkey 3 because that dataset contained only 2 sound frequency conditions), using the published delineation of core AC indicated in Figure 2. The automatically determined A1/R borders (Fig. 2F and G) coincided exactly with the pub- lished manual parcellation (Fig. 2B and C, respectively). In addition, we detected a second, more posterior, gradient rever- sal in both cases, which may indicate the border between A1 We measured the direction of the tonotopic gradient with respect to the orientation of the long (mediolateral) axis of HG at each vertex on the ﬂattened cortical surfaces in A1 and R in each participant and the group map (Fig. 7). Mean angles Cerebral Cortex October 2015, V 25 N 10 3285 Figure 7. Circular histograms of tonotopic gradient direction in A1 (black polygons) and R (gray polygons) in left (L) and right (R) hemispheres of the group data and Participants 1–7. Directions are relative to the orientation of HG, whose medial- to-lateral axis corresponds to the horizontal axis in each plot (indicated by the black arrow in plot 2L). Directions were binned every 45° (light-gray circle sections) for plotting. Mean directions and 95% conﬁdence intervals are indicated by small gray triangles. Numbers at the bottom of each graph indicate the upper axis limit of the histogram as number of vertices. Dashed black lines show the orientation of the border between A1 and R, as identiﬁed by the gradient reversal analysis. the tonotopic gradient analysis to the region identiﬁed as core by the pattern classiﬁcation analysis. This avoids the risk of constructing erroneous tonotopic gradients across borders between core and belt ﬁelds (see also Dick et al. 2012; Moerel et al. 2012). We validate our method with fMRI tonotopy data- sets from 3 macaque monkeys. Our results challenge a model of core AC organization in humans proposed by several recent fMRI tonotopy studies (Humphries et al. 2010; Da Costa et al. Location of Core Auditory Fields at Niedersaechsische Staats- u. Universitaetsbibliothek Goettingen on October 19, 2015 .org/ f Frequency classiﬁcation accuracy peaked on or near HG in all of the 14 human hemispheres measured in this study. In the 4 macaque monkey hemispheres, the region of signiﬁcant classi- ﬁcation accuracy consistently overlapped the largest core ﬁeld A1 and, in 2 hemispheres, also the smaller core ﬁeld R. In 2 hemispheres, the signiﬁcant classiﬁcation region also over- lapped the caudal belt ﬁelds CM or CL. This is consistent with previous ﬁndings of tonotopic organization in these areas in primates (Kaas and Hackett 1998; Petkov et al. 2006). Physio- logical studies have shown that frequency selectivity is lower in belt than in core ﬁelds (Rauschecker et al. 1995). This may explain why signiﬁcant classiﬁcation accuracy in the caudal belt was observed in only 2 of our 4 macaque hemispheres. Note, however, that the parcellation of the monkey AC pro- posed by Petkov et al. (2006), against which the current classiﬁcation results were compared, was not veriﬁed by post- mortem anatomical analysis and may thus itself exhibit inac- curacies. That study also reported considerable variability in the tonotopic maps within and between animals. Irrespective, there is a clear convergence of human and macaque results in that the region of signiﬁcant classiﬁcation accuracy corre- sponded roughly to the expected location of core AC and, in the majority of cases, contained 2 tonotopic maps with orienta- tions consistent with the 2 largest core ﬁelds, A1 and R. ranged from almost parallel (1°) to almost perpendicular (88°) with a mean of 34° (interquartile range 30°). The mean angle in the group map was 50°. In individual hemispheres, 19 out of 26 of the mean directions were below 45°. This proportion and larger ones are unlikely to occur under the assumption of an equal distribution of angles within the measured range (under 2% of 100 000 bootstrap samples), suggesting that mean gradients tend to align with the long axis of HG. We also examined the orientation of the border between A1 and R identiﬁed by the gradient reversal analysis. Although these edges are not straight, a representative angle can be computed as the complement to 90° of the direction for which the edge map correlates maximally with the sum of edge maps across all directions (for instance, in Fig. Estimation of Voxel Best Frequencies 2011; Langers and van Dijk 2012) but are consistent with previ- ous cytoarchitectonic (Morosan et al. 2001) and myeloarchitec- tonic (Sigalovsky et al. 2006; Dick et al. 2012) measurements of human core AC and accord with a recently proposed uniﬁed model of the primate primary AC (Baumann et al. 2013). Unlike the myelination measurements, the pattern classiﬁca- tion method involves no specialized imaging sequences or ana- lysis techniques and can also be applied to already existing tonotopy datasets. The current results suggest that, at least at 3 T, where the current human data were acquired, the pattern classiﬁcation approach may be more sensitive than the previ- ously used criterion of sharper frequency selectivity in core AC (Moerel et al. 2012; see below). at Niedersaechsische Staats- u. Universita http://cercor.oxfordjournals.org/ Downloaded from at Niedersaechsische Staats- u. Universitaetsbibliothek Goettingen on October 19, 2015 http://cercor.oxfordjournals.org/ Downloaded from Figure 7. Circular histograms of tonotopic gradient direction in A1 (black polygons) and R (gray polygons) in left (L) and right (R) hemispheres of the group data and Participants 1–7. Directions are relative to the orientation of HG, whose medial- to-lateral axis corresponds to the horizontal axis in each plot (indicated by the black arrow in plot 2L). Directions were binned every 45° (light-gray circle sections) for plotting. Mean directions and 95% conﬁdence intervals are indicated by small gray triangles. Numbers at the bottom of each graph indicate the upper axis limit of the histogram as number of vertices. Dashed black lines show the orientation of the border between A1 and R, as identiﬁed by the gradient reversal analysis. Location of Core Auditory Fields 5D, the gradient reversals at 30° correlate better with the summed map than the ones at 90°). In the group map, the border between A1 and R so computed ran almost parallel to HG (3° on the left and 6° on the right side, Fig. 7). In the majority of the individual hemispheres, these angles were above 70°, that is, almost perpendicular to HG. The mean angle was 67° with an interquartile range of 23° and a range of 15–90°. The classiﬁcation results were robust across different classi- ﬁcation algorithms and parameters, suggesting that the fre- quency classiﬁcation method is a useful in vivo marker of core auditory ﬁeld A1 and, to a lesser extent, R in individual brains. We would expect that the region of signiﬁcant classiﬁcation ac- curacy would also be robust against changes in the stimulation paradigm or parameters. It has been shown that the size of the sustained response (SR) to longer stimulus sequences (>6 s) is affected by the stimulus rate, with faster rates yielding a larger SR in primary ﬁelds, and slower rates yielding a larger SR in nonprimary ﬁelds (Giraud et al. 2000; Harms and Melcher 2002; Seifritz et al. 2002). The current study used relatively short stimulus sequences (4 s), and so, the measured activity would be expected to represent the onset response to the 3286 Parcellation of Core Auditory Cortex • Schönwiesner et al. Discussion This study aimed to map core AC in humans using fMRI. In contrast to previous tonotopy studies, we used multi-voxel pattern classiﬁcation of sound frequency to distinguish core from belt AC, and we assessed tonotopic gradients and gradi- ent reversals using an automated procedure unbiased by prior assumptions to delineate subﬁelds within core AC. We limited sequences, which is relatively little affected by the stimulus rate (e.g., Harms and Melcher 2002). When using longer se- quences, an intermediate stimulus rate should be used in order to create balanced activity across both primary and non- primary ﬁelds. classiﬁcation results and the myelination results by Dick et al. (2012) suggest that, on average across participants, core AC en- compasses only a relatively small, circumscribed region on middle HG. The convergence between independent (functional and anatomical) localizers bolsters conﬁdence in their validity and highlights the potential beneﬁt of combining them in 1 experi- ment. The method proposed by Dick and colleagues and our classiﬁcation method both require thresholding (on the myelin measure, R1, and on classiﬁcation accuracy, respectively) to de- lineate core ﬁelds, and combining information, for instance in a Bayesian framework, may help to reduce potential bias from threshold selection. p y In the human participants, classiﬁcation accuracy correlated weakly with the sharpness of the frequency tuning curves and with the overall response amplitude to pure tones. This was ex- pected because neurons that are more sharply tuned in fre- quency and thus more strongly driven by narrowband sounds such as pure tones should also be more informative about the presented frequency. Sharpness of frequency tuning and re- sponse amplitude to pure tones and other narrowband sounds have been used in previous studies as criterion to delineate core AC in humans (Wessinger et al. 2001; Chevillet et al. 2011). The classiﬁcation method might be expected to be more sensitive than the sharpness-of-tuning criterion, because classi- ﬁcation analysis of multi-voxel activation patterns has been shown to be less susceptible than single-voxel-based analyses to limitations in the measurement spatial resolution (deter- mined by the voxel size and the spatial spread of the blood oxygen level-dependent effect). Multi-voxel pattern analysis can be used to extract information encoded at a somewhat smaller scale than the voxel dimensions and does not require measurable tuning at the voxel level (Boynton 2005; Kamitani and Tong 2005). Discussion The criterion of response amplitude to nar- rowband sounds is premised on the assumption that core and belt AC differ in their frequency tuning properties, but not in their overall sensitivity to sound. Given that response ampli- tudes also depend on stimulus parameters other than frequency composition (e.g., stimulus rate; see above), this assumption cannot be generally valid. Moreover, due to nonlinearities in the blood oxygenation level-dependent effect (Liu et al. 2010), large differences in the neural response amplitude between core and belt AC might translate to only small differences in the ampli- tude of the fMRI signal. at Niedersaechsische Staats- u. Universita http://cercor.oxfordjournals.org/ Downloaded from Orientation of Tonotopic Gradients The orientation of the tonotopic gradients within the core AC region in individual hemispheres varied between 1 and 88° relative to the long axis of HG, with an average of 34° and an interquartile range of 30°. The automatically identiﬁed border between the presumed core ﬁelds A1 and R was oriented almost perpendicular to HG in most hemispheres, with a mean angle of 67°, a range of 15–90°, and an interquartile range of 23°. In the monkey data, the border identiﬁed by our automatic method coincided exactly with the previously published manual delineation. The method proposed here to identify reli- able gradient reversals is robust against variation in gradient orientation, because it is not dependent on any assumption of the orientation of tonotopic gradients with respect to HG. The centroid method for estimating voxel best frequencies helped to reduce noise in the best-frequency maps, which makes the extraction of gradient reversals more reliable. Previous studies have demonstrated good repeatability of tonotopic mapping with fMRI across time and stimulus types (Da Costa et al. 2011; Dick et al. 2012; Moerel et al. 2012). Similarly, the current data showed good repeatability of voxel frequency tuning curves across measurement runs. We therefore think that the observed variability in the tonotopic gradient orientations represent, at least partly, true differences between individuals and hemi- spheres. This is consistent with physiological data: in the earli- est tonotopic mappings of animal AC, Merzenich and colleagues report a signiﬁcant variation in the location of core AC and in the orientation of best-frequency gradients in core AC in macaque monkeys (Merzenich and Brugge 1973), squirrels (Merzenich et al. 1976), and cats (Merzenich et al. 1975). In the description of the results on cat AC, the authors explicitly stated that it was necessary to consider each cat individually in order to arrive at a coherent model of tonotopic organization. Variability in the tonotopic gradients has also been reported in fMRI studies in macaques (Petkov et al. 2006; Baumann et al. 2010; Tanji et al. 2010). Although often de-emphasized, con- siderable inter-individual variation is also evident in human to- notopy studies that show individual data (Schönwiesner et al. 2002; Formisano et al. 2003; Talavage et al. 2004; Humphries et al. 2010; Da Costa et al. 2011; Langers and van Dijk 2012; Dick et al. 2012; Moerel et al. 2012), and in macaque fMRI to- notopic maps (Petkov et al. 2006). References Baumann S, Grifﬁths TD, Rees A, Hunter D, Sun L, Thiele A. 2010. Characterisation of the BOLD response time course at different levels of the auditory pathway in non-human primates. Neuro- image. 50:1099–1108. Baumann S, Petkov CI, Grifﬁths TD. 2013. A uniﬁed framework for the organization of the primate auditory cortex. Front Syst Neurosci. 7:11. In addition to A1 and R, a third core subﬁeld, RT, has been demonstrated in several monkey species. There is growing evi- dence for the existence of RT in humans. Morosan et al. (2001) reported 3 primary-like ﬁelds in human postmortem brains, but the correspondence between these ﬁelds and functionally deﬁned A1, R, and RT, is unclear. While the earlier human to- notopy studies did not ﬁnd a second tonotopic gradient rever- sal (Talavage et al. 2000; Schönwiesner et al. 2002; Formisano et al. 2003; Talavage et al. 2004), some of the more recent studies reported a second reversal, located anterior or lateral to the ﬁrst reversal (Woods et al. 2009; Humphries et al. 2010; Moerel et al. 2012). The second reversal might mark the R–RT border. RT is small and exhibits a less consistent tonotopic or- ganization than A1 and R (Petkov et al. 2006). This may be why RT failed to yield signiﬁcant frequency classiﬁcation ac- curacy in our monkey datasets. Alternatively, the tonotopic map of RT may be incomplete in humans (i.e., only cover a part of the frequency range). An inconsistent or incomplete to- notopic organization of RT may mean that a deﬁnitive marker of the entire core AC may have to comprise a combination of functional, anatomical, and perhaps connectivity criteria. Boynton GM. 2005. Imaging orientation selectivity: decoding con- scious perception in V1. Nat Neurosci. 8:541–542. Chevillet M, Riesenhuber M, Rauschecker JP. 2011. Functional corre- lates of the anterolateral processing hierarchy in human auditory cortex. J Neurosci. 31:9345–9352. Cleveland WS, Devlin SJ. 1988. Locally weighted regression: an ap- proach to regression analysis by local ﬁtting. J Am Stat Assoc. 83:596–610. Cortes C, Vapnik V. 1995. Support-vector networks. Mach Learn. 20:273–297. Da Costa S, van der Zwaag W, Marques JP, Frackowiak RS, Clarke S, Saenz M. 2011. Human primary auditory cortex follows the shape of Heschl’s Gyrus. J Neurosci. 31:14067–14075. Dick F, Tierney AT, Lutti A, Josephs O, Sereno MI, Weiskopf N. 2012. In vivo functional and myeloarchitectonic mapping of human primary auditory areas. J Neurosci. 32:16095–16105. Notes We are thankful to the anonymous reviewers for help in improving earlier versions of this manuscript. Conﬂict of Interest: None declared. Funding This work was supported by the Discipline Bridging Award of the University of Nottingham, UK. MS was supported by the German Academy of Sciences during part of this work, PD was supported by the Volkswagen Foundation, KK was supported by the Medical Research Council (UK), and CP was supported by the Wellcome Trust (UK). Funding to pay the Open Access publication charges for this article was provided by the Medical Research Council (UK). Supplementary Material at Niedersaechsische Staats- u. Universitaetsbibliothek Goettingen on October 19, 2015 http://cercor.oxfordjournals.org/ Downloaded from Supplementary can be found at: http://www.cercor.oxfordjournals. org/. Orientation of Tonotopic Gradients 2011; Langers and van Dijk 2012) proposed a model of human core AC in which the core ﬁelds A1 and R run all the way along the anterior and posterior banks of HG, sharing a border roughly parallel to its long axis (see, for in- stance, Humphries et al. 2010, Fig. 9). The assumption is that the tonotopic gradients in both A1 and R run quasi- perpendicular to HG, connecting the low-frequency region on its anterolateral crown to the high-frequency strips anterior and posterior to HG, respectively. However, our classiﬁcation results suggest that these low- and high-frequency regions do not represent corresponding endpoints of tonotopic gradients. In particular, our results, together with myeloarchitectonic mapping (Dick et al. 2012) and other functional criteria (Moerel et al. 2012), suggest that only the best-frequency pro- gression within a relatively small, circumscribed region around the middle part of HG represents a coherent tonotopic gradi- ent, presumably the gradient of area A1. A quasi-perpendicular layout would also be in direct disagreement with the cytoarchi- tectonic parcellation of human core AC by Morosan et al. (2001). These authors found 3 subﬁelds (which they referred to as Te1.0, Te1.1, and Te1.2), stacked along HG, and with borders running perpendicular, rather than parallel, to its long axis. The best-frequency progression is variable across individ- ual hemispheres but runs approximately parallel to the long axis of HG in the majority of hemispheres tested here (8/14). This is consistent with several earlier human tonotopy studies (Talavage et al. 2000, 2004; Formisano et al. 2003; Upadhyay et al. 2007), which have also found a low- to high-frequency progression from anterolateral-to-posteromedial HG. region of low best frequencies on the anterolateral crown of HG, and a wide collar of high best frequencies around the pos- teromedial end of HG and extending both anteriorly and pos- teriorly along HG. However, different studies have interpreted this pattern differently depending on whether or not they used an independent criterion to delineate core AC. The studies that did not use an independent criterion (Humphries et al. 2010; Da Costa et al. 2011; Langers and van Dijk 2012) proposed a model of human core AC in which the core ﬁelds A1 and R run all the way along the anterior and posterior banks of HG, sharing a border roughly parallel to its long axis (see, for in- stance, Humphries et al. 2010, Fig. 9). Orientation of Tonotopic Gradients The assumption is that the tonotopic gradients in both A1 and R run quasi- perpendicular to HG, connecting the low-frequency region on its anterolateral crown to the high-frequency strips anterior and posterior to HG, respectively. However, our classiﬁcation results suggest that these low- and high-frequency regions do not represent corresponding endpoints of tonotopic gradients. In particular, our results, together with myeloarchitectonic mapping (Dick et al. 2012) and other functional criteria (Moerel et al. 2012), suggest that only the best-frequency pro- gression within a relatively small, circumscribed region around the middle part of HG represents a coherent tonotopic gradi- ent, presumably the gradient of area A1. A quasi-perpendicular layout would also be in direct disagreement with the cytoarchi- tectonic parcellation of human core AC by Morosan et al. (2001). These authors found 3 subﬁelds (which they referred to as Te1.0, Te1.1, and Te1.2), stacked along HG, and with borders running perpendicular, rather than parallel, to its long axis. The best-frequency progression is variable across individ- ual hemispheres but runs approximately parallel to the long axis of HG in the majority of hemispheres tested here (8/14). This is consistent with several earlier human tonotopy studies (Talavage et al. 2000, 2004; Formisano et al. 2003; Upadhyay et al. 2007), which have also found a low- to high-frequency progression from anterolateral-to-posteromedial HG. thus be highly speculative. Moreover, tonotopic gradients would be expected to be less clear outside of core AC. However, at least belt ﬁelds would be expected to exhibit tono- topic organization with best-frequency gradients collinear to those of the adjacent core areas. It may thus be hoped that identiﬁcation of core AC as in the current study will enable de- lineation of at least some of the adjoining ﬁelds. at Niedersaechsische Staats- u. Universitaetsbibliothe http://cercor.oxfordjournals.org/ Downloaded from Supplementary Material Supplementary can be found at: http://www.cercor.oxfordjournals. org/. 3288 Parcellation of Core Auditory Cortex • Schönwiesner et al. Orientation of Tonotopic Gradients at Niedersaechsische Staats- u. Universitaetsbibliothek Goettingen on October 19, 2015 ournals.org/ The results from the classiﬁcation method show a consider- able degree of variability in the location of human core AC across our sample of 14 hemispheres. Much of this variability is due to macroanatomical variability. A probability map of the region of signiﬁcant classiﬁcation accuracy, calculated using surface-based alignment of the temporal lobes across partici- pants and hemispheres, showed a good degree of overlap of core AC around middle HG. The location and variability of the region was consistent with the previous cytoarchitectonic (Morosan et al. 2001; Rademacher et al. 2001) and myelination measurements (Sigalovsky et al. 2006; Dick et al. 2012). The group average region of signiﬁcant classiﬁcation accuracy was for the most part (90%) contained within the cytoarchitectonic 50% probability map of Morosan P et al. (2001). We do not have myelination data for our participants (the data acquisition predated those reports) and thus cannot conduct a direct within-participants comparison of myelination and classiﬁca- tion accuracy. However, at the group level, the region identi- ﬁed by the classiﬁcation method was largely congruent with the region that exhibited high myelination in the study by Dick et al. (2012). Both sets of results suggest that core AC may extend less widely than assumed in several recent human tono- topy studies (Humphries et al. 2010; Da Costa et al. 2011; Langers and van Dijk 2012). These studies assumed that core AC encompasses the entire HG and also some of the regions anterior and posterior to HG. In contrast, both our The general pattern of frequency preference across the STP observed in our group data is consistent with the results found in previous studies (Talavage et al. 2004; Humphries et al. 2010; Da Costa et al. 2011; Langers and van Dijk 2012; Dick et al. 2012; Moerel et al. 2012). All these studies show a broad Cerebral Cortex October 2015, V 25 N 10 3287 region of low best frequencies on the anterolateral crown of HG, and a wide collar of high best frequencies around the pos- teromedial end of HG and extending both anteriorly and pos- teriorly along HG. However, different studies have interpreted this pattern differently depending on whether or not they used an independent criterion to delineate core AC. The studies that did not use an independent criterion (Humphries et al. 2010; Da Costa et al. at Niedersaechsische Staats- u. 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https://openalex.org/W2800582952	https://www.frontiersin.org/articles/10.3389/fgene.2018.00149/pdf	English	null	Complexity of the 5′ Untranslated Region of EIF4A3, a Critical Factor for Craniofacial and Neural Development	Frontiers in genetics	2,018	cc-by	6,852	Complexity of the 5′ Untranslated Region of EIF4A3, a Critical Factor for Craniofacial and Neural Development Gabriella S. P. Hsia1†, Camila M. Musso1†, Lucas Alvizi1, Luciano A. Brito1, Gerson S. Kobayashi1, Rita C. M. Pavanello1, Mayana Zatz1, Alice Gardham2, Emma Wakeling2, Roseli M. Zechi-Ceide3, Debora Bertola1,4 and Maria Rita Passos-Bueno1* 1 Centro de Estudos do Genoma Humano e Células Tronco, Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, São Paulo, Brazil, 2 North East Thames Genetics Service, Great Ormond Street Hospital, London, United Kingdom, 3 Hospital de Reabilitação de Anomalias Craniofaciais, Universidade de São Paulo, São Paulo, Brazil, 4 Instituto da Criança, Hospital das Clínicas da FMUSP, Universidade de São Paulo, São Paulo, Brazil Keywords: acrofacial dysostosis, non-coding region, haplotype, expansion, crossing-over Boles, Center for Neurological and Neurodevelopmental Health, United States *Correspondence: Maria Rita Passos-Bueno passos@ib.usp.br †Co-ﬁrst authors Specialty section: This article was submitted to Genetic Disorders, a section of the journal Frontiers in Genetics Received: 15 February 2018 Accepted: 10 April 2018 Published: 25 April 2018 Citation: Hsia GSP, Musso CM, Alvizi L, Brito LA, Kobayashi GS, Pavanello RCM, Zatz M, Gardham A, Wakeling E, Zechi-Ceide RM, Bertola D and Passos-Bueno MR (2018) Complexity of the 5′ Untranslated Region of EIF4A3, a Critical Factor for Craniofacial and Neural Development. Front. Genet. 9:149. doi: 10.3389/fgene.2018.00149 Specialty section: This article was submitted to Genetic Disorders, a section of the journal Frontiers in Genetics Received: 15 February 2018 Accepted: 10 April 2018 Published: 25 April 2018 Specialty section: This article was submitted to Genetic Disorders, a section of the journal Frontiers in Genetics Received: 15 February 2018 Accepted: 10 April 2018 Published: 25 April 2018 ORIGINAL RESEARCH published: 25 April 2018 doi: 10.3389/fgene.2018.00149 Edited by: Babajan Banganapalli, King Abdulaziz University, Saudi Arabia Repeats in coding and non-coding regions have increasingly been associated with many human genetic disorders, such as Richieri-Costa-Pereira syndrome (RCPS). RCPS, mostly characterized by midline cleft mandible, Robin sequence and limb defects, is an autosomal-recessive acrofacial dysostosis mainly reported in Brazilian patients. This disorder is caused by decreased levels of EIF4A3, mostly due to an increased number of repeats at the EIF4A3 5′UTR. EIF4A3 5′UTR alleles are CG-rich and vary in size and organization of three types of motifs. An exclusive allelic pattern was identiﬁed among affected individuals, in which the CGCA-motif is the most prevalent, herein referred as “disease-associated CGCA-20nt motif.” The origin of the pathogenic alleles containing the disease-associated motif, as well as the functional effects of the 5′UTR motifs on EIF4A3 expression, to date, are entirely unknown. Here, we characterized 43 different EIF4A3 5′UTR alleles in a cohort of 380 unaffected individuals. We identiﬁed eight heterozygous unaffected individuals harboring the disease-associated CGCA-20nt motif and our haplotype analyses indicate that there are more than one haplotype associated with RCPS. The combined analysis of number, motif organization and haplotypic diversity, as well as the observation of two apparently distinct haplotypes associated with the disease-associated CGCA-20nt motif, suggest that the RCPS alleles might have arisen from independent unequal crossing-over events between ancient alleles at least twice. Moreover, we have shown that the number and sequence of motifs in the 5′UTR region is associated with EIF4A3 repression, which is not mediated by CpG methylation. In conclusion, this study has shown that the large number of repeats in EIF4A3 does not represent a dynamic mutation and RCPS can arise in any population harboring alleles with the CGCA-20nt motif. We also provided further evidence that EIF4A3 5′UTR is a regulatory region and the size and sequence type of the repeats at 5′UTR may contribute to clinical variability in RCPS. Reviewed by: Munis Dundar, Erciyes University, Turkey Richard G. 1https://www.ncbi.nlm.nih.gov/tools/primer-blast/ DNA Samples To characterize EIF4A3 5′UTR, 380 DNA samples from unaﬀected individuals unrelated to RCPS families were selected from the biorepository of CEGH-CEL. For haplotype analysis, 13 additional samples were used, 12 are also from CEHG-CEL (four unaﬀected individuals without CGCA-20nt motifs and four with CGCA-20nt motifs; four Brazilian RCPS patients bearing diﬀerent allelic structures) and one sample of a RCPS patient, from the United Kingdom, was sent for diagnosis purposes from North East Thames Genetics Service. In order to evaluate the eﬀects of the motifs on EIF4A3 expression, six DNA samples carrying EIF4A3 alleles with distinct number of motifs were selected from the biorepository of Centro de Estudos do Genoma Humano e Células Tronco (CEGH-CEL) and used for luciferase reporter assay. For methylation assessment, we used DNA samples of RCPS patients (n = 6; homozygous for the 16 repeats allele) from Hospital de Reabilitação de Anomalias Craniofaciais da Universidade de São Paulo (HRAC-USP) and unaﬀected individuals (n = 7; homozygous for the eight repeats allele) from CEGH-CEL. We have shown that an increased number of repeats at 5′UTR of EIF4A3 causes Richieri-Costa-Pereira syndrome (RCPS; OMIM #268305), a rare autosomal-recessive disorder aﬀecting craniofacial and limb development, mainly described in Brazilian patients (Favaro et al., 2011, 2014; Bertola et al., 2017). RCPS individuals show a distinctive allelic pattern, determined not only by the larger number of repeats (>14 as compared to up to 12 repeats in controls), but also by the presence of a unique motif containing G instead of A nucleotide (the ‘disease-associated CGCA-20nt motif’) (Favaro et al., 2014). As the origin of the RCPS disease alleles remains unknown, characterizing the 5′UTR of EIF4A3 in a populational level could give us clues on the mechanisms that originate the EIF4A3 pathogenic alleles (e.g., meiotic instability or unequal crossing-over events), in addition to providing insights on the chance of RCPS arising in other populations. All samples were extracted from peripheral blood using the Gentra Systems Autopure LS (AutoGen) according to the manufacturer’s protocol. EIF4A3 5′UTR Characterization and Haplotype Analysis Sanger sequencing of EIF4A3 5′UTR (NM_014740.3) and ﬁve ﬂanking SNPs (rs11150824, rs2289534, rs3829612, rs10782008, and rs12943620) were performed using BigDye R⃝Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientiﬁc) and the ABI 3730 DNA Analyzer (Applied Biosystems). Sequences were analyzed using Sequencher 5.1 (Gene Codes Corporation) and Mixed Sequences Reader (Chang et al., 2012) software. The last one allowed us to better discriminate the alleles. In order to validate our analysis and obtain more reliability in our data, we randomly selected 30 heterozygous samples with diﬀerent alleles’ structure to sequence each allele separately, using the Illustra GFX PCR DNA and Gel Band Puriﬁcation kit (GE Healthcare) followed by Sanger sequencing. We and others have shown that EIF4A3 downregulation in cellular and animal models leads to defective neural crest cell migration/diﬀerentiation and neural stem cell apoptosis during embryonic development, paralleling RCPS cranioskeletal defects and microcephaly, respectively (Mao et al., 2016; Miller et al., 2017). However, the molecular mechanism responsible for EIF4A3 downregulation remains entirely unknown. Therefore, this work was undertaken to investigate the origin of the pathogenic alleles containing the disease-associated CGCA-20nt motif, as well as to evaluate the functional eﬀects of the 5′UTR motifs on EIF4A3 expression. Insights into the origin and eﬀect of these complex alleles will contribute to a better understanding of regulatory features of 5′UTR regions and their role in craniofacial and neural development. To facilitate results interpretation, we referred SNPs markers according to their genomic relative position on the annotated plus strand (GRCh38/hg38): SNP1 (rs11150824) – SNP2 (rs2289534) – EIF4A3 – SNP3 (rs3829612) – SNP4 (rs10782008) – SNP5 (rs12943620). Primers were designed using Primer-Blast1 (Untergasser et al., 2012) and are described in Supplementary Table S1. Linkage disequilibrium and haplotypes were inferred using Haploview software (Barrett et al., 2005). Citation: Hsia GSP, Musso CM, Alvizi L, Brito LA, Kobayashi GS, Pavanello RCM, Zatz M, Gardham A, Wakeling E, Zechi-Ceide RM, Bertola D and Passos-Bueno MR (2018) Complexity of the 5′ Untranslated Region of EIF4A3, a Critical Factor for Craniofacial and Neural Development. Front. Genet. 9:149. doi: 10.3389/fgene.2018.00149 April 2018 \| Volume 9 \| Article 149 1 Frontiers in Genetics \| www.frontiersin.org Hsia et al. EIF4A3 5′UTR Characterization INTRODUCTION number 1.463.852). All individuals donated biological samples after providing signed informed consent. number 1.463.852). All individuals donated biological samples after providing signed informed consent. Over two-thirds of the human genome is comprised by repetitive elements (de Koning et al., 2011), which have been increasingly associated with functional regulatory roles. Consequently, a variety of human genetic disorders are caused by repeats in coding and non-coding sequences (Cummings and Zoghbi, 2000; Gatchel and Zoghbi, 2005; Mirkin, 2007; La Spada and Taylor, 2010; McMurray, 2010; DeJesus-Hernandez et al., 2011; Renton et al., 2011; Usdin et al., 2015; Haeusler et al., 2016). Most of these diseases are caused by unstable dynamic mutations that usually increase in size during meiotic divisions and have been associated with neurologic disorders (Virtaneva et al., 1997; Cummings and Zoghbi, 2000; Renton et al., 2011; Haeusler et al., 2016). However, poly-A repeats in HOXD13, the causative mechanism of a non- neurological condition, synpolydactyly, represents an exception, in which the most likely mechanism leading to increased poly- A tracts are errors in DNA replication (Muragaki et al., 1996; Warren, 1997; Brown and Brown, 2004). RESULTS The EIF4A3 5′UTR is characterized by the presence of 18- or 20-nucleotide-long motifs diﬀering in the composition of central nucleotides, namely CA-18nt (TCGGCAGCGGCAGCGAGG), CACA-20nt (TCGGCAGCGGCACAGCGAGG), and CGCA- 20nt (TCGGCAGCGGCGCAGCGAGG) (Favaro et al., 2014). Unaﬀected individuals have 3–12 repeats composed mostly by CA-18nt and CACA-20nt motifs, while RCPS patients have 14–16 repeats, with a higher number of the CGCA-20nt motif (Favaro et al., 2014). The motif CGCA-20nt will be herein referred as ‘disease-associated CGCA-20nt motif.’ The EIF4A3 5′UTR is characterized by the presence of 18- or 20-nucleotide-long motifs diﬀering in the composition of central nucleotides, namely CA-18nt (TCGGCAGCGGCAGCGAGG), CACA-20nt (TCGGCAGCGGCACAGCGAGG), and CGCA- 20nt (TCGGCAGCGGCGCAGCGAGG) (Favaro et al., 2014). Unaﬀected individuals have 3–12 repeats composed mostly by CA-18nt and CACA-20nt motifs, while RCPS patients have 14–16 repeats, with a higher number of the CGCA-20nt motif (Favaro et al., 2014). The motif CGCA-20nt will be herein referred as ‘disease-associated CGCA-20nt motif.’ Human embryonic kidney (HEK) 293T cells, cultured in high glucose DMEM supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum (FBS) (all provided by Life Technologies), were plated in 96-well plates 24 h prior to transfection (2 × 104 cells/well). Transient transfections were performed in triplicate by using TurboFectin 8.0 (OriGene) according to the manufacturer’s instruction. Cells were cotransfected with 180 ng of the pGL4.24 constructs and 20 ng of the pRL-SV40 vector (Promega) containing the Renilla luciferase gene, used as a transfection control. The plasmid pLuc generated from pGL3-control template was used as positive control (Soltys et al., 2013). Forty- eight hours after DNA transfection, luciferase activity was measured with the Dual-Glo R⃝Luciferase Assay System in a GloMax Multi 96-microplate Luminometer (Promega). Fireﬂy luminescence results were normalized by Renilla luminescence and the relative luciferase activity was determined. Statistical analyses were performed with one- way ANOVA and Tukey post hoc test. Signiﬁcance was set at p < 0.05. Unaffected Individuals Are Mostly Heterozygous and May Also Present CGCA-20nt Motifs Sanger sequencing of EIF4A3 5′UTR in 380 unaﬀected individuals revealed 43 diﬀerent alleles (Figure 1) in heterozygosis in 85% of individuals. The total number of repeats per allele varied from 2 to 17, and the most common alleles contained 7 (25.46%) or 8 (23.21%) repeats (Supplementary Figure S1). The number and organization of the CA-18nt or CACA-20nt motifs varied between the alleles. We identiﬁed not only alleles containing a single motif, but also complex alleles with multiple organizations of the CA-18nt or CACA-20nt motifs (Figure 1). For example, one allele exclusively constituted by the CA-18nt motif was found. As for the other alleles, the number of the ﬁrst CACA-20nt motifs varied from 2 to 8 with or without diﬀerent combinations of CA-18nt motifs (Figure 1). The most common allele contained seven repeats and was constituted by four repeats of CACA-20nt motif followed by 1 CA-18nt, 1 CACA-20nt, and 1 ﬁnal CA-18nt. We also identiﬁed two atypical alleles comprising 12- or 20-nucleotide-long sequences inserted between motifs, which do not align to any viral sequences or neighboring genes (data not shown). Finally, the disease- associated CGCA-20nt motif, originally found only among RCPS patients, was identiﬁed here in eight heterozygous control Luciferase Assay Although the larger alleles (15 or 16 repeats) have been associated with decreased EIF4A3 expression, the causal Ethics Approval Statement The protocol was approved by the Ethics Committee of Instituto de Biociências at Universidade de São Paulo, Brazil (accession April 2018 \| Volume 9 \| Article 149 Frontiers in Genetics \| www.frontiersin.org 2 EIF4A3 5′UTR Characterization Hsia et al. relationship between number and/or pattern of repeats and gene downregulation is still unknown. Therefore, we investigated the role of the 5′UTR motifs on EIF4A3 expression by luciferase assay. with MethPrimer2 (Li and Dahiya, 2002) and are shown in Supplementary Table S1. Amplicons were checked by agarose electrophoresis and cloned using the TOPO TA Cloning Kit for sequencing (Thermo Fisher Scientiﬁc). Sanger sequencing was carried out for 10 clones per sample using the BigDye R⃝Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientiﬁc) and the ABI 3730 DNA Analyzer (Applied Biosystems). Sequencing ﬁles were then analyzed for methylation quantiﬁcation using the online tool BISMA – Bisulﬁte Sequencing DNA Methylation Analysis (Rohde et al., 2010) with lower threshold conversion rate at 95%, lower threshold sequence identity at 90%, upper threshold of N-sites at cytosine positions at 20% and per threshold gaps allowed at 20% as ﬁltering parameters. Methylation values were computed and diﬀerences between groups tested using Fisher’s Exact Test. Signiﬁcance was set at p < 0.05. Sequences of interest of unaﬀected and RCPS individuals were ampliﬁed by PCR (primer sequences in Supplementary Table S1), puriﬁed using the Illustra GFX PCR DNA and Gel Band Puriﬁcation kit (GE Healthcare) and cloned into the pGL4.24[luc2P/minP] vector (Promega), upstream of a minimal promoter and the luc2P gene. Sanger sequencing conﬁrmed all constructs: pGL4.24 vectors carrying control alleles with 4 repeats (3 CACA-20-nt and 1 CA-18-nt), 7 repeats (4 CACA-20-nt, 1 CA-18-nt, 1 CACA-20-nt, and 1 CA-18- nt), 10 repeats (7 CACA-20-nt, 1 CA-18-nt, 1 CACA-20-nt, and 1 CA-18-nt) and 12 repeats (1 CACA-20-nt, 10 CGCA- 20nt, and 1 CA-18-nt); pGL4.24 vectors carrying pathogenic alleles with 14 repeats (2 CACA-20nt, 10 CGCA-20nt, 1 CACA-20nt, and 1 CA-18nt) and 16 repeats (1 CACA-20nt, 13 CGCA-20nt, 1 CACA-20nt, and 1 CA-18nt). In order to investigate the individual eﬀect of each motif, diﬀering in the composition of central nucleotides, we also constructed vectors carrying sequences with only 1 CA-18-nt, 1 CACA-20-nt, and 1 CGCA-20nt, synthesized by Integrated DNA Technologies (IDT). 2www.urogene.org/methprimer/ Frontiers in Genetics \| www.frontiersin.org Methylation Assay Indeed, methylated CpGs are involved in gene repression especially when occurring at promoter/5′UTR (Feil and Fraga, 2012; Schübeler, 2015). Since EIF4A3 5′UTR motifs are CG- rich and the disease CGCA-20nt allele shows increased number of CpGs compared to control alleles, we evaluated DNA hypermethylation as a plausible mechanism behind EIF4A3 downregulation in RCPS patients. One microgram of genomic DNA from each sample were submitted to bisulﬁte conversion using EpiTect Bisulﬁte Conversion Kit (QIAgen). Bisulﬁte converted DNA was subsequently used for PCR, in which primers were designed April 2018 \| Volume 9 \| Article 149 Frontiers in Genetics \| www.frontiersin.org 3 Hsia et al. Hsia et al. EIF4A3 5′UTR Characterization FIGURE 1 \| Schematic representation of the 43 allele structures identiﬁed in the 380 unaffected individuals. The Brazilian RCPS patients showed at least two diﬀerent haplotypes. The haplotype associated with the pathogenic 14-repeat allele is diﬀerent from the one associated with the 16- repeat allele (Figure 2B), which in turn were not observed in any of the tested unaﬀected individuals (Figure 2C). The fact that the two aﬀected alleles with 14 and 16 repeats are embedded within diﬀerent haplotypes, undetected in unaﬀected individuals, suggests a distinct origin for these two diﬀerent-sized pathogenic alleles in our population. Next, we analyzed the homozygous (16 repeats) UK RCPS sample, which showed a diﬀerent haplotype from those observed in the Brazilian RCPS (Figure 2B). However, one UK RCPS haplotype is similar to a haplotype observed in three of the unaﬀected individuals carrying the disease-associated CGCA-20nt motif (Figures 2B,D). Besides, the alleles in these control individuals also show similarities in motif organization in relation to the allele in the UK RCPS patient, suggesting that these alleles possibly share a common origin (Figure 2E and Supplementary Table S2). Transcriptional Activity Is Inversely Correlated to Motif Number at EIF4A3 5′UTR To clarify the functional role of the allelic structure at EIF4A3 5′UTR, we generated constructs varying in size and composition (Figure 3), and carried out luciferase reporter assays. Inverse correlation between number of motifs and luciferase activity was observed (n = 4 independent experiments; p < 0.05; Figure 3A). Further, by investigating each motif individually, there was a discrete reduction in expression for the disease-associated CGCA-20nt, albeit not statistically signiﬁcant (n = 3 independent experiments; Figure 3B). In order to address whether the motif sequence plays a role in EIF4A3 gene expression, based on data shown in Figure 3A, we calculated the eﬀect per motif type on luciferase activity. Comparing the luciferase activity of alleles with similar composition of motifs, carrying 4 and 10 repeats (22.45% and 19.69%, respectively), we observed a decrease of 2.76% in expression, which represents a reduction of about 0.46% per CACA-20nt motif added in the allele structure. On the other hand, between the alleles carrying 12 and 16 repeats (17.8% and 11.96%, respectively), with comparable allelic structure, the diﬀerence was 5.84%, which means a reduction in luciferase activity of 1.46% per CGCA-20nt motif added (three times higher). Based on these results, we suggest that both size and allele sequence play a role in gene regulation. FIGURE 1 \| Schematic representation of the 43 allele structures identiﬁed in the 380 unaffected individuals. individuals, within alleles with 11 or more repeats (Figure 1). The largest allele (17 repeats) was found in heterozygosis in one control individual and contained 14 CGCA-20nt motifs. This allele must be pathogenic when in homozygosis. 5 UTR Hypermethylation Is Not Responsible for EIF4A3 Downregulation Responsible for EIF4A3 Downregulation Responsible for EIF4A3 Downregulation We used ﬁve SNPs for haplotype characterization and numbered them as following: SNP1 (rs11150824), SNP2 (rs2289534), SNP3 (rs3829612), SNP4 (rs10782008), and SNP5 (rs12943620), according to their relative genomic position on the annotated plus strand (GRCh38/hg38). (B) Haplotype analysis of ﬁve RCPS patients, four Brazilians, and one from the United Kingdom. Gray arrows indicate the haplotype associated with the 14-repeat alleles and black arrows indicate the haplotype associated with the 16-repeat alleles in Brazilian RCPS. UK RCPS presented a similar haplotype observed in the unaffected individuals with the disease CGCA-20nt motif (dotted arrow). (C) Haplotype analysis of four unaffected individuals without the disease CGCA-20nt motif. These individuals are homozygous for seven or eight repeats. (D) Haplotype analysis of four unaffected individuals with the disease CGCA-20nt motif. Dotted arrows indicate the haplotype present in three of these individuals. (E) Comparison between haplotypes of the UK RCPS patient, the three unaffected individuals with the CGCA-20nt motif and the three Brazilian RCPS patients. S is the sample ID, N is the total number of repeats for each allele and A is the allele structure (Figure 1 and Supplementary Table S2). Each bar represents an allele. FIGURE 2 \| (A) Schematic representation of the SNPs and microsatellite markers ﬂanking EIF4A3 (11,970 bp). We used ﬁve SNPs for haplotype characterization and numbered them as following: SNP1 (rs11150824), SNP2 (rs2289534), SNP3 (rs3829612), SNP4 (rs10782008), and SNP5 (rs12943620), according to their relative genomic position on the annotated plus strand (GRCh38/hg38). (B) Haplotype analysis of ﬁve RCPS patients, four Brazilians, and one from the United Kingdom. Gray arrows indicate the haplotype associated with the 14-repeat alleles and black arrows indicate the haplotype associated with the 16-repeat alleles in Brazilian RCPS. UK RCPS presented a similar haplotype observed in the unaffected individuals with the disease CGCA-20nt motif (dotted arrow). (C) Haplotype analysis of four unaffected individuals without the disease CGCA-20nt motif. These individuals are homozygous for seven or eight repeats. (D) Haplotype analysis of four unaffected individuals with the disease CGCA-20nt motif. Dotted arrows indicate the haplotype present in three of these individuals. (E) Comparison between haplotypes of the UK RCPS patient, the three unaffected individuals with the CGCA-20nt motif and the three Brazilian RCPS patients. S is the sample ID, N is the total number of repeats for each allele and A is the allele structure (Figure 1 and Supplementary Table S2). Each bar represents an allele. Responsible for EIF4A3 Downregulation We inspected methylation levels at the 5′UTR of EIF4A3, as the increased number of repeats in RCPS patients leads to gain of 37 CpG sites. We observed that both RCPS and controls did not show abundant methylation of this region (1.7% and 2.8% of methylated CpGs, respectively), with a discrete reduction of methylation in RCPS (p < 0.05) (Supplementary Table S3). There was no evident methylation variation at any speciﬁc CpG To understand the origin of the alleles containing the disease- associated CGCA-20nt motif, we initially characterized the haplotypes of 13 samples: ﬁve aﬀected individuals (four Brazilian and one from the United Kingdom) and eight control individuals (Supplementary Table S2), using ﬁve SNPs ﬂanking EIF4A3 and spanning 519 kb (Figure 2A). We observed a weak linkage disequilibrium in this block (D’ < 0.48). April 2018 \| Volume 9 \| Article 149 Frontiers in Genetics \| www.frontiersin.org 4 Hsia et al. EIF4A3 5′UTR Characterization FIGURE 2 \| (A) Schematic representation of the SNPs and microsatellite markers ﬂanking EIF4A3 (11,970 bp). We used ﬁve SNPs for haplotype characterization and numbered them as following: SNP1 (rs11150824), SNP2 (rs2289534), SNP3 (rs3829612), SNP4 (rs10782008), and SNP5 (rs12943620), according to their relative genomic position on the annotated plus strand (GRCh38/hg38). (B) Haplotype analysis of ﬁve RCPS patients, four Brazilians, and one from the United Kingdom. Gray arrows indicate the haplotype associated with the 14-repeat alleles and black arrows indicate the haplotype associated with the 16-repeat alleles in Brazilian RCPS. UK RCPS presented a similar haplotype observed in the unaffected individuals with the disease CGCA-20nt motif (dotted arrow). (C) Haplotype analysis of four unaffected individuals without the disease CGCA-20nt motif. These individuals are homozygous for seven or eight repeats. (D) Haplotype analysis of four unaffected individuals with the disease CGCA-20nt motif. Dotted arrows indicate the haplotype present in three of these individuals. (E) Comparison between haplotypes of the UK RCPS patient, the three unaffected individuals with the CGCA-20nt motif and the three Brazilian RCPS patients. S is the sample ID, N is the total number of repeats for each allele and A is the allele structure (Figure 1 and Supplementary Table S2). Each bar represents an allele. EIF4A3 5′UTR Characterization Hsia et al. Hsia et al. FIGURE 2 \| (A) Schematic representation of the SNPs and microsatellite markers ﬂanking EIF4A3 (11,970 bp). Frontiers in Genetics \| www.frontiersin.org DISCUSSION Expansions at non-coding regions have been extensively described in neurological disorders, and characterization of these regions have greatly contributed to the understanding of novel regulatory mechanisms (Gatchel and Zoghbi, 2005; Mirkin, 2007; La Spada and Taylor, 2010; McMurray, 2010; Russo et al., 2015; Usdin et al., 2015; Haeusler et al., 2016). Despite the great advances in genome sequence analysis, DNA of repetitive regions is still diﬃcult to be sequenced. In fact, the EIF4A3 5′UTR is not covered in GnomAD database, which reinforces the importance of characterizing this region through Sanger sequencing. ( ) The haplotype analyses were performed in order to get insights on the origin of the pathogenic alleles with increased number of repeats. Results revealed that the pathogenic alleles with 14 and 16 repeats of Brazilian patients have distinct origins, which in turn are diﬀerent from the haplotypes of the UK RCPS patient. These results suggest that the pathogenic alleles have arisen more than once. It is of note that the UK RCPS patient shares a common haplotype and also a similar motif structure with three unaﬀected individuals carrying the disease-associated CGCA- 20nt motif, suggesting a common ancestral among them. It is possible that, similarly to the alleles in the control population, the aﬀected alleles may have arisen also through unequal crossing. This hypothesis is also supported by the observation that the number of repeats at EIF4A3 5′UTR seems to be stable across generations (Favaro et al., 2014). This phenomenon is more comparable to the one observed in synpolydactyly, in which the poly-A at the 3′end of HOXD13 might have originated by unequal crossing over as it is quite stable when transmitted across generations (Warren, 1997), as opposed to dynamic mutations observed in neurological conditions, which have arisen only once (Virtaneva et al., 1997; Gatchel and Zoghbi, 2005; Mirkin, 2007; La Spada and Taylor, 2010; McMurray, 2010; DeJesus-Hernandez et al., 2011; Renton et al., 2011; Usdin et al., 2015; Haeusler et al., 2016). In this study, Sanger sequencing analysis of EIF4A3 5′UTR in 380 unaﬀected individuals revealed 43 diﬀerent alleles, with the most common alleles containing seven or eight repeats. Some of these alleles presented only one type of motif (CA- 18nt or CACA-20nt) with diﬀerent total number of repeats, while others presented a visible combination of these two common motifs, suggesting that these alleles may have originated through unequal crossing-over events. Responsible for EIF4A3 Downregulation FIGURE 2 \| (A) Schematic representation of the SNPs and microsatellite markers ﬂanking EIF4A3 (11,970 bp). We used ﬁve SNPs for haplotype characterization and numbered them as following: SNP1 (rs11150824), SNP2 (rs2289534), SNP3 (rs3829612), SNP4 (rs10782008), and SNP5 (rs12943620), according to their relative genomic position on the annotated plus strand (GRCh38/hg38). (B) Haplotype analysis of ﬁve RCPS patients, four Brazilians, and one from the United Kingdom. Gray arrows indicate the haplotype associated with the 14-repeat alleles and black arrows indicate the haplotype associated with the 16-repeat alleles in Brazilian RCPS. UK RCPS presented a similar haplotype observed in the unaffected individuals with the disease CGCA-20nt motif (dotted arrow). (C) Haplotype analysis of four unaffected individuals without the disease CGCA-20nt motif. These individuals are homozygous for seven or eight repeats. (D) Haplotype analysis of four unaffected individuals with the disease CGCA-20nt motif. Dotted arrows indicate the haplotype present in three of these individuals. (E) Comparison between haplotypes of the UK RCPS patient, the three unaffected individuals with the CGCA-20nt motif and the three Brazilian RCPS patients. S is the sample ID, N is the total number of repeats for each allele and A is the allele structure (Figure 1 and Supplementary Table S2). Each bar represents an allele. April 2018 \| Volume 9 \| Article 149 Frontiers in Genetics \| www.frontiersin.org 5 EIF4A3 5′UTR Characterization Hsia et al. FIGURE 3 \| Functional analysis of EIF4A3 5′UTR motifs. Graph depicting the relative luciferase activity, in percentage, of the constructs carrying different number of motifs (A) and of each motif independently (B). Data are represented as mean ± SEM of ﬁve experiments, which were performed in triplicate. Relative luciferase activity = [(experimental sample ratio) – (negative control ratio)/(positive control ratio) – (negative control ratio)]. ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05 and ns, non-signiﬁcant; one-way ANOVA with Tukey post hoc tests. FIGURE 3 \| Functional analysis of EIF4A3 5′UTR motifs. Graph depicting the relative luciferase activity, in percentage, of the constructs carrying different number of motifs (A) and of each motif independently (B). Data are represented as mean ± SEM of ﬁve experiments, which were performed in triplicate. Relative luciferase activity = [(experimental sample ratio) – (negative control ratio)/(positive control ratio) – (negative control ratio)]. ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05 and ns, non-signiﬁcant; one-way ANOVA with Tukey post hoc tests. Responsible for EIF4A3 Downregulation between RCPS and controls, as both presented low methylation levels. alleles (8/760) harbors the disease-associated CGCA-20nt motif. Interestingly, the largest alleles in this cohort (≥11 repeats), including one with 17 repeats, contained the disease CGCA- 20nt motif. These results suggest that RCPS could occur in any population containing alleles with the CGCA-20nt motif. Indeed, one of the patients here included is from United Kingdom (Bertola et al., 2017). Frontiers in Genetics \| www.frontiersin.org DISCUSSION These results show its polymorphic nature and conﬁrm the structural complexity, and uniqueness of this region, which was not comparable to any gene in which dynamic pathogenic expansions at non-coding regions had been reported (Brook et al., 1992; Campuzano et al., 1996; Moseley et al., 2006; Daughters et al., 2009; Galloway and Nelson, 2009; Sato et al., 2009; DeJesus-Hernandez et al., 2011; Kobayashi et al., 2011). In this enlarged cohort, 1% of the April 2018 \| Volume 9 \| Article 149 Frontiers in Genetics \| www.frontiersin.org 6 EIF4A3 5′UTR Characterization Hsia et al. ACKNOWLEDGMENTS We thank Dr. Meire Aguena and Dr. Antonia Cerqueira (IB- USP) for technical support. We also thank Dr. Marcelo Nobrega and Dr. Débora Sobreira (University of Chicago, United States) who donated the sequences for constructs used in the luciferase assay. FUNDING This work was funded by Centro de Pesquisa, Inovação e Difusão (CEPID, 2013/08028-1), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, 2015/21781-6), Conselho Nacional de Desenvolvimento Cientíﬁco e Tecnológico (CNPq, PQ10/2011), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, 1470032). This work was funded by Centro de Pesquisa, Inovação e Difusão (CEPID, 2013/08028-1), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, 2015/21781-6), Conselho Nacional de Desenvolvimento Cientíﬁco e Tecnológico (CNPq, PQ10/2011), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, 1470032). The inverse correlation between number of repeats and EIF4A3 expression levels suggests that the structure of the 5′UTR EIF4A3 may modulate the phenotype. Indeed, a broader phenotypic spectrum within RCPS has been observed, ranging from individuals with severe phenotype (homozygous for the 16-repeat allele or heterozygous for the 15- and 16-repeat alleles) to individuals with less severe skeletal involvements, harboring smaller number of motifs (homozygous for the 14- repeat allele or compound heterozygous for 14 repeats and a point mutation (c.809A>G) (Favaro et al., 2014; Bertola et al., 2017). Despite limitations in sample size, these results suggest that RCPS phenotypic variability depends upon the number and allele sequence of repeats at the 5′UTR of EIF4A3. TABLE S1 \| Primer sequences. TABLE S1 \| Primer sequences. TABLE S2 \| List of the 13 samples used in haplotype analysis and the description of motifs’ structure reported in each allele. TABLE S3 \| Methylation levels in the analyzed regions in RCPS patients and control individuals; p = 0.0461, Fisher’s Exact Test. AUTHOR CONTRIBUTIONS Next, we demonstrated that the number and allele sequence of motifs at 5′UTR is involved in EIF4A3 expression. These results thus suggest a potential cis-acting regulatory mechanism for these motifs on gene expression and conﬁrm our previous ﬁnding that the aﬀected alleles were associated with EIF4A3 downregulation in cells from diﬀerent tissues from RCPS patients, including peripheral blood (Favaro et al., 2014; Miller et al., 2017). DNA hypermethylation is not the mechanism for EIF4A3 downregulation in RCPS patients, as RCPS and control blood samples showed similar methylation levels. Hypomethylation, as observed at EIF4A3 5′UTR, is consistent with transcriptional activity (Baubec and Schübeler, 2014; Schübeler, 2015). There could be alternative mechanisms by which the increased number of motifs could repress EIF4A3 expression: the disease CGCA-20nt could create binding sites for repressor proteins and act as a silencer; or more complex mechanisms, including post-translational events, could be involved. Further functional studies are needed to pinpoint the exact molecular mechanism underlying EIF4A3 downregulation. DB, RP, and MZ provided the Brazilian DNA samples. AG and EW provided DNA sample and clinical information from the UK RCPS patient. GH, CM, and MP-B conceived and designed the study. GH performed the EIF4A3 5′UTR characterization and haplotype analyses. LB assisted with haplotype and linkage disequilibrium analyses. CM carried out the luciferase assay and LA performed the methylation assay. GH, CM, LA, LB, GK, and MP-B discussed and interpreted the results and wrote the manuscript. GH and CM have a major contribution in writing the manuscript. All authors contributed to the ﬁnal version and approved the manuscript. CONCLUSION The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fgene. 2018.00149/full#supplementary-material The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fgene. 2018.00149/full#supplementary-material In summary, we provide evidence supporting that the EIF4A3 5′UTR is highly polymorphic, comprising at least 43 diﬀerent alleles, which may have originated through extensive recombination in this region. Haplotype analysis in control and in aﬀected individuals suggests that there are more than one haplotype associated with the disease and RCPS alleles might have originated from unequal crossing-over events. We also provided further evidence that EIF4A3 5′UTR is a regulatory region, and that EIF4A3 downregulation in RCPS is not mediated by CpG methylation. Moreover, our ﬁndings provide insights to explain clinical variability in RCPS. FIGURE S1 \| Distribution of number of alleles per total number of repeats identiﬁed in the 380 samples of unaffected individuals. This analysis did not take in consideration the motifs’ structure. FIGURE S1 \| Distribution of number of alleles per total number of repeats identiﬁed in the 380 samples of unaffected individuals. This analysis did not take in consideration the motifs’ structure. REFERENCES (2012). Mixed sequence reader: a program for analyzing DNA sequences with heterozygous base calling. ScientiﬁcWorldJournal 2012:365104. doi: 10.1100/ 2012/365104 Genet. 38, 758–769. doi: 10.1038/ng1827 Muragaki, Y., Mundlos, S., Upton, J., and Olsen, B. R. (1996). Altered growth and branching patterns in synpolydactyly caused by mutations in HOXD13. Science 272, 548–551. doi: 10.1126/science.272.5261.548 Cummings, C. J., and Zoghbi, H. Y. (2000). 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Genet. 11, 247–258. doi: 10.1038/ nrg2748 Conﬂict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Li, L.-C., and Dahiya, R. (2002). MethPrimer: designing primers for methylation PCRs. Bioinformatics 18, 1427–1431. REFERENCES doi: 10.1093/bioinformatics/18.11.1427 Copyright © 2018 Hsia, Musso, Alvizi, Brito, Kobayashi, Pavanello, Zatz, Gardham, Wakeling, Zechi-Ceide, Bertola and Passos-Bueno. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 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https://openalex.org/W4362626796	https://figshare.com/articles/journal_contribution/Supplementary_Figure_3_from_Tolerance_of_Whole-Genome_Doubling_Propagates_Chromosomal_Instability_and_Accelerates_Cancer_Genome_Evolution/22529981/1/files/39993098.pdf	English	null	Supplementary Figure 8 from Tolerance of Whole-Genome Doubling Propagates Chromosomal Instability and Accelerates Cancer Genome Evolution	null	2,023	cc-by	1,102	Supplementary Figure 3: Dewhurst & McGranahan et al., Supplementary Figure 3 A) Types of segregation errors observed in diploid and tetraploid cells. Only cells with errors were included in t sis, and the percentage frequency of each type of error was calculated. Median number of errors scored for p =39, passage 50=37. Images were analysed with Softworx Explorer (Applied Precision, LLP) software. Se errors ere classified as described (23) Clone 8 Clone 9 Clone 10 Clone 11 Clone 16 Diploid Tetraploids 0% 20% 40% 60% 80% 100% 2 3 4 5 % frequency of colonies Chromosome 2 colony modes 0% 20% 40% 60% 80% 100% 2 3 4 5 6 % frequency of colonies Chromosome 8 colony modes C) A) passage 5 passage 50 % of all errors Bridges Acentrics Centrics Other 0 20 40 60 80 % of all errors proportion of all errors Bridges Acentrics Centrics Other 0 20 40 60 80 B) Segregation error types 0.0 0.2 0.4 0.6 0.8 1.0 Other Bridge Acentric Centric Tetraploids Diploids 504 270 p.val= 0.178 Diploids Tetraploids HCT-116 DC 8 DC-14 DC-25 TC-13 TC-16 TC-35 TC-17 TC3 TC4 Supplementary Figure 3: Dewhurst & McGranahan et al., Supplementary Figure 3 A) Types of segregation errors observed in diploid and tetraploid cells. Only cells with errors were included in t sis, and the percentage frequency of each type of error was calculated. Median number of errors scored for p =39, passage 50=37. Images were analysed with Softworx Explorer (Applied Precision, LLP) software. Se errors ere classified as described (23) Clone 8 Clone 9 Clone 10 Clone 11 Clone 16 Diploid Tetraploids 0% 20% 40% 60% 80% 100% 2 3 4 5 % frequency of colonies Chromosome 2 colony modes 0% 20% 40% 60% 80% 100% 2 3 4 5 6 % frequency of colonies Chromosome 8 colony modes C) A) passage 5 passage 50 % of all errors Bridges Acentrics Centrics Other 0 20 40 60 80 % of all errors proportion of all errors Bridges Acentrics Centrics Other 0 20 40 60 80 B) Segregation error types 0.0 0.2 0.4 0.6 0.8 1.0 Other Bridge Acentric Centric Tetraploids Diploids 504 270 p.val= 0.178 Diploids Tetraploids HCT-116 DC 8 DC-14 DC-25 TC-13 TC-16 TC-35 TC-17 TC3 TC4 Supplementary Figure 3: Dewhurst & McGranahan et al., Supplementary Figure 3: Dewhurst & McGranahan et al., Supplementary Figure 3: Dewhurst & McGranahan et al., A) passage 5 passage 50 % of all errors Bridges Acentrics Centrics Other 0 20 40 60 80 % of all errors Bridges Acentrics Centrics Other 0 20 40 60 80 Segregation error types Diploids Tetraploids HCT-116 DC 8 DC-14 DC-25 TC-13 TC-16 TC-35 TC-17 TC3 TC4 A) passage 50 % of all errors Bridges Acentrics Centrics Other 0 20 40 60 80 Diploids Tetraploids HCT-116 DC 8 DC-14 DC-25 TC-13 TC-16 TC-35 TC-17 TC3 TC4 passage 5 % of all errors Bridges Acentrics Centrics Other 0 20 40 60 80 % of all errors proportion of all errors B) 0.0 0.2 0.4 0.6 0.8 1.0 Other Bridge Acentric Centric Tetraploids Diploids 504 270 p.val= 0.178 Supplementary Figure 3 A) Types of segregation errors observed in diploid and tetraploid cells. Only cells with errors were included in this analy- sis, and the percentage frequency of each type of error was calculated. Median number of errors scored for passage 5 =39, passage 50=37. Images were analysed with Softworx Explorer (Applied Precision, LLP) software. Segregation errors were classified as described (23). Supplementary Figure 3: Dewhurst & McGranahan et al., Supplementary Figure 3 A) Types of segregation errors observed in diploid and tetraploid cells. Only cells with errors were included in t sis, and the percentage frequency of each type of error was calculated. Median number of errors scored for p =39, passage 50=37. Images were analysed with Softworx Explorer (Applied Precision, LLP) software. Se errors ere classified as described (23) Clone 8 Clone 9 Clone 10 Clone 11 Clone 16 Diploid Tetraploids 0% 20% 40% 60% 80% 100% 2 3 4 5 % frequency of colonies Chromosome 2 colony modes 0% 20% 40% 60% 80% 100% 2 3 4 5 6 % frequency of colonies Chromosome 8 colony modes C) A) passage 5 passage 50 % of all errors Bridges Acentrics Centrics Other 0 20 40 60 80 % of all errors proportion of all errors Bridges Acentrics Centrics Other 0 20 40 60 80 B) Segregation error types 0.0 0.2 0.4 0.6 0.8 1.0 Other Bridge Acentric Centric Tetraploids Diploids 504 270 p.val= 0.178 Diploids Tetraploids HCT-116 DC 8 DC-14 DC-25 TC-13 TC-16 TC-35 TC-17 TC3 TC4 B) Analysis of types of errors between diploids and tetraploids reveals no significant difference in prevalence of different types of segregation errors (P=0.178, Chi-squared test). Number of errors is shown on top of bars. C) Clonal FISH shows colony-to-colony variation in modal chromosome number. Frequency of colonies with the indicated modal chromosome number for chromosome 2 and chromosome 8 is shown for the indicated diploid and tetraploid clones of HCT-116_MLH1. Clone 8 Clone 9 Clone 10 Clone 11 Clone 16 Diploid Tetraploids 0% 20% 40% 60% 80% 100% 2 3 4 5 % frequency of colonies Chromosome 2 colony modes 0% 20% 40% 60% 80% 100% 2 3 4 5 6 % frequency of colonies Chromosome 8 colony modes C) proportion of all errors B) 0.0 0.2 0.4 0.6 0.8 1.0 Other Bridge Acentric Centric Tetraploids Diploids 504 270 p.val= 0.178 B) proportion of all errors Clone 8 Clone 9 Clone 10 Clone 11 Clone 16 Diploid Tetraploids 0% 20% 40% 60% 80% 100% 2 3 4 5 % frequency of colonies Chromosome 2 colony modes 0% 20% 40% 60% 80% 100% 2 3 4 5 6 % frequency of colonies Chromosome 8 colony modes C) C) Supplementary Figure 3 Supplementary Figure 3 Supplementary Figure 3 A) Types of segregation errors observed in diploid and tetraploid cells. Only cells with errors were included in this analy- sis, and the percentage frequency of each type of error was calculated. Median number of errors scored for passage 5 =39, passage 50=37. Images were analysed with Softworx Explorer (Applied Precision, LLP) software. Segregation errors were classified as described (23). ( ) B) Analysis of types of errors between diploids and tetraploids reveals no significant difference in prevalence of different types of segregation errors (P=0.178, Chi-squared test). Number of errors is shown on top of bars. C) Clonal FISH shows colony-to-colony variation in modal chromosome number. Frequency of colonies with the ndicated modal chromosome number for chromosome 2 and chromosome 8 is shown for the indicated diploid and tetraploid clones of HCT-116_MLH1.
https://openalex.org/W2736204032	http://www.nomos-elibrary.de/10.5771/0506-7286-1974-3-257.pdf	English	null	The Constitution of Bangladesh and a Short Constitutional History	Verfassung und Recht in Übersee	1,974	cc-by	10,375	The Birth of Bangladesh and Preconstitution Documents East Bengal (now Bangladesh) was always an uneasy partner in a hasty marriage. In the euphoria created during a somewhat panicky withdrawal of the British from Indian empire, it suddenly found itself as a part of a new State called Pakistan. Separated by over 1200 miles of Indian territory from its other wing it became a junior and neglected partner of a troubled new nation. Linguistically, culturally and anthropologically different from the members of the other part of Pakistan its people constituted majority in number but minority in influence. Speaking a language called Bengali - an extremely rich language in which the great Poet Rabindranath Tagore had already received a Nobel award - it found that even its linguistic rights were being denied. The majority of people in the then Pakistan spoke Bengali as Bengali was the mother tongue of aIl the people in East Bengal, but the pride of place was given to Urdu, a language not even understood in East Bengal. It was a standing complaint that economicaIly East Bengal was discriminated against by the Government of Pakistan; that although it produced a11 the jute and the tea which were the main foreign currency earner, very little of the foreign currency was utilised in East Bengal; that recruitment to the armed forces and civil services was heavily against East Bengal and that the economy was going from bad to worse. Even Yahya Khan the then President of Pakistan on November 8, 1971 stated "no one ever treated the Bengalees fairly . . . East Pakistan was down and out and we did not pay sufficient attention to its development". A common religion was the only link between East Bengal and West Pakistan. The people of East Bengal were too proud not to resist it or to take it lying down. But democratic methods of expression of protest were not available to them for very long. In 1958 martial law was clamped down on the whole of Pakistan including East Bengal and the country was since then under the management and domination of one military ruler after another. The spearhead of the movement of independence of East Bengal was a taIl and handsome Bengalee called Sheikh Mujibur Rehman, affectionately called Banga bandhu (Friend of Bengal) by his people. A live-wire and pleasant personality even as a student he was deeply involved in po li ti es and absolutely fearless. T H E C O N Sl'Il'Ul'I O N O F BAN GLA D E S H A N D A S H ORT C O N STITUTI O N AL H I S T ORY By S. C. SEN By S. C. SEN The Birth of Bangladesh and Preconstitution Documents In March 21, 1948 Mohamed Ali Jinnah, the founder of Pakistan, declared at a meeting in Dacca "that Urdu is going to be the lingua franca of this country. Anyone who says anything else is an enemy of Pakistan". No sooner had he said this than a sma11 group of students led by Mujibur Rehman protested. They wanted Bengali language to be given its rightful place and were not going to be buIldozed into accepting Urdu. They were promptly sent to jail. J ail visit had become a regular part later of the life of Mujibur Rehman. While in jail Mujibur Rehman was elected a Joint Secretary of the Awami League, the party which ultimately won in the national elections in 1954. Mujib had a short speIl 257 g/10.5771/0506-7286-1974-3-257, am 24.10.2024, 06:41:29 ess – - https://www.nomos-elibrary.de/agb 6-1974-3-257, am 24.10.2024, 06:41:29 ttps://www.nomos-elibrary.de/agb as a member of a Ministry. In 1956 when the new Constitution of Pakistan was proclaimed Mujibur Rehman along with other Awami League leaders rejected it as "a halfway provincial autonomy" and continued their campaign for total autonomy as weIl as the abolition of economic and financial discrimination between the two wings of Pakistan. On October 8, 1958 a military coup d' etat under Ayub Khan took place in Pakistan as a result of wh ich the 1956 Constitution was abrogated and the central and provincial governments dissolved. Mujibur Rehman was arrested and detained without trial for over a year. The authorities launched half a dozen criminal cases against hirn but the eh arges failed and he was released in December 1959. In 1962 Mujib was again imprisoned for leading the agitation against the imposition of the 1962 Constitution introduced by President Ayub Khan. He was again released after some time. In 1966 when the Government of Pakistan lifted some restrietions on political activity Mujibur Rehman announ ced a six-point programme in achieving full regional auto no my for East Bengal and his party started mobilising public opinion for attainment of this objective. Promptly he was arrested again by the military rulers and was later implicated in wh at was known as the notorious Agartala Conspiracy case and charged with treason. The prosecution failed and he was released in January 1969. The agitation led by Mujib was primarily responsible for toppling General Ayub Khan in March 1969. General Yahya Khan replaced Ayub Khan. The Birth of Bangladesh and Preconstitution Documents He pledged that he would hand over power to elected representatives of the people after holding general elections. Mujib and his party contested the elections on the basis of the six-point formula of the Awami party. The Six-point Formula 258 (6) A militia or paramilitary force, an ordnance factory, a military academy and the Naval headquarters would be set up in East Pakistan. (6) A militia or paramilitary force, an ordnance factory, a military academy and the Naval headquarters would be set up in East Pakistan. In December 1970 the general elections were held on the basis of the one-man one-vote franchise, the first ever such election in Pakistan. Mujibur Rehman and his party scored a sweeping victory. Out of 300 seats in the provincial assembly of East Bengal it captured 288 seats. Out of 313 seats in the national assembly it captured 167 seats. Victory of Mujibur Rehman in East Bengal was complete and he was entitled to become the Prime Minister of the whole of Pakistan as a result of his majority in the national assembly, a fact wh ich was not acceptable to some powerful persons and politicians in West Pakistan. p p p Events now began to happen at dramatic speed. The national assembly session was delayed from time to time by President Yahya Khan. People of East Bengal were not prepared to wait ad infinitum. Tempers of the people began rising high. The postponed meeting of the national assembly was fixed on the 3rd of March 1971. On the 1st of March 1971 the news reached Dacca that President Yahya Khan had again postponed the meeting of national assembly. There were violent demonstrations in Dacca against this postponement. Mujibur Rehman described the postponement of the national assembly as a direct conspiracy and launched a peaceful non-cooperation movement throughout East Bengal. He said "if the conspirators still think they can perpetuate their colonial rule they are living in a foo1's paradise . . . They will see his tory made if the conspirators fail to come to their senses." The students of Dacca University spearheaded a movement against continuance of martial law and massive demonstrations were held in the University and other educational institutions in Dacca. The cry of Free Bengal and Freedom for Bangladesh was now the cry all over East Bengal. By mid-night of the 1st of March Dacca was cut off from the outside world when posta!, telegraph, telephone and airport employees walked out. On the 2nd of March Mujibur Rehman announced a province-wide peaceful hartal from March 3rd to 6th to press people's demand for transfer of power. The Six-point Formula The six-point programme put forward by the Awami League was also the Party's election manifesto. (1) A federal form of Government would be established. (2) The Federal Government would control only defence and foreign policy, all other subjects being vested in the federating States. (3) Two separate, freely convertible currencies would be introduced for East and West Pakistan, or if a single currency was maintained, constitutional provision would be made to stop the flight of the capital from East to West Pakistan. There would be a separate banking reserve and separate fiscal and monetary policy for East Pakistan. (4) The States would have exclusive authority to levy taxes, federal expenses being met from a uniform percentage of all State taxes. (5) Separate external trade accounts would be maintained for each of the States, and foreign exchange earned from external trade would be at their disposal. Federal foreign exchange requirements would be met by the States on the basis of an equal percentage rate. Indigenous commodities would move between the States, free of taxation or tariffs. The States would be allowed to maintain trade representatives abroad and to negotiate trade agreements with other countries. (1) A federal form of Government would be established. (2) The Federal Government would control only defence and foreign policy, all other subjects being vested in the federating States. T f l ibl i ld b i d d f (3) Two separate, freely convertible currencies would be introduced for East and West Pakistan, or if a single currency was maintained, constitutional provision would be made to stop the flight of the capital from East to West Pakistan. There would be a separate banking reserve and separate fiscal and monetary policy for East Pakistan. y p y (4) The States would have exclusive authority to levy taxes, federal expenses being met from a uniform percentage of all State taxes. (5) Separate external trade accounts would be maintained for each of the States, and foreign exchange earned from external trade would be at their disposal. Federal foreign exchange requirements would be met by the States on the basis of an equal percentage rate. Indigenous commodities would move between the States, free of taxation or tariffs. The States would be allowed to maintain trade representatives abroad and to negotiate trade agreements with other countries. The Race Course Speech of Mujibur Rehman The Race Course Speech of Mujibur Rehman "I am before you today with a very heavy heart. You know and understand everything. We have done our best, and yet the streets of Dacca, Chittagong, Khulna, Rangpur and Jessore are drenched with the blood of my brothers. Today the people of Bengal want freedom; they want to live; they want I am before you today with a very heavy heart. You know and understand everything. We have done our best, and yet the streets of Dacca, Chittagong, Khulna, Rangpur and Jessore are drenched with the blood of my brothers. Today the people of Bengal want freedom; they want to live; they want their rights restored. gp J y Today the people of Bengal want freedom; they want to live; they want their rights restored. You e1ected Awami League to frame the constitution. We hoped that the National Assembly would sit and we would frame the constitution. Through this constitution, the people would achieve their economic, political and cultural freedom. The history of the last 23 years is the history of the agonised cry of the people of Bengal, it is a painful his tory of giving blood, it is the his tory of the tears of an oppressed people. We gave blood in 1952. In 1954, even though we won the e1ection, we were denied the opportunity to rule. By promulgating Martial Law in 1958, Ayub Khan kept us in bondage for ten years. In 1966, the six-point formula was submitted before the nation and for this many of my brothers were murdered. In 1969, in face of the mass movement, Ayub fell and Yahya came. Yahya said he would hand over power to the people and there would be a constitution. We accepted his promise. You all know what happened after that. But wh at did we get? Arms have been used against the unarmed people of Bengal. They have no weapOl1S. The arms we bought at our own cost to defend the country from external aggression, are being used to kill my innocent people. The suffering people are the targets of bullets. Whenever, we Bengalees, the majority of the nation, sought power to exercise our right to rule, a conspiracy was immediate1y hatched to stop it. They unleashed repression on uso Our struggle from now on is a struggle for emancipation for freedom. The Six-point Formula Military Governors replied by more shooting. Addressing a public meeting on the 3rd March Mujibur Rehman instructed his people not to pay taxes unless and until the power was transferred. He called for total immobilisation of the Government and of the system and called for closure of Government offices, industries and commercial establishments and markets. He also demanded return of the troops to their barracks and complete stoppage of all activities by troops. On the 6th of March President Yahya Khan promised in a broadcast that the inaugural session of the National Assembly would take place on the 25th of March. Simultaneously, some leaders in West Pakistan made it known that they will not join the National Assembly. Mujibur Rehman also stated that he had requested Yahya Khan to convene the National Assembly by the 1 5th of February, but Yahya Khan had refused to do that and had acted according to the dictates of minority leaders from West Pakistan. Mujibur Rehman was not consulted at all and he was not willing to act any more on the basis of the statements of President Yahya Khan. On the 7th of March Mujibur Rehman gave his historic speech at the Ramna Race Course in Dacca. Extracts from this speech are set out below: 259 / – The Declaration of Independence Whole of East Bengal was now virtually under Mujibur Rehman. Civil li fe had come to a dead halt. There was however great expectancy as to what was going to happen on the 25th of March 1971 when the National Assembly was to meet according to the President's declaration. Mujibur Rehman had however put four conditions for participating in the National Assembly deliberations. He asked for immediate end of Martial Law regime, withdrawal of troops from East Bengal, enquiry into killings of strikers and immediate handing over of the power to the people's representatives. When 25th of March arrived the situation had totally changed. It appeared that President Yahya Khan had already flown back to Rawalpindi and also had flown back the leaders of West Pakistan parties who had come to Dacca. Instead, large numbers of West Pakistani troops had arrived and the army let loose a reign of terror and repression. On the night of the 25th of March 1971 Mujibur Rehman declared that a sovereign independent People's Republic of Bangladesh had come into existence. This was a declaration of total independence. On the 26th of March 1971 Mujibur Rehman was arrested and flown to an unknown destination in West Pakistan. The war of independence of the people of Bangladesh started in right earnest and in full fury. It is not within the scope of this article to give any description of the war of independence of Bangladesh but certain important features of constitutional importance are noted below. The Race Course Speech of Mujibur Rehman Blood-stains of those killed have not yet dried arrd I cannot step on the blood of the martyrs to attend the National Assembly on March 25. Yahya Khan has convened the Assembly. But my demands are: Lift Martial Law; take the soldiers back to the barracks; investigate the mass killings; and transfer power to the elected representatives. After these, we will consider whether we could sit in the National Assembly or not. Before these demands are fulfilled there is no question of our sitting in the Assembly . . . There is a plot to finish off the people of Bangladesh. So please be careful . . . If they fire one single bullet, turn every horne in Bengal into a fortress. You will have to face the enemy with whatever you have. We must make hirn die of thirst and hunger, if necessary. The roads and ferries will have to be closed down. If I am not there to give you directions or if none of my colleagues is there for this purpose, you would continue yourse1ves. To the soldiers of the Pakistan Army I say this : You are my brothers, if you stay in the barracks, you will be left alone, but if you come to murder us, the consequences will be bad. Y ou will not be able to suppress seventyfive million people any more. Bengalees have learnt how to face death . . . nobody can suppress them . . . Be on your guard. Y ou should remember that the enemy has infiltrated 260 among uso They would try to create dissension among us und er cover. Bengalees, non-Bengalees, Hindus and Muslims are all our brothers. It is our responsibility to protect them all . . . There is still a possibility of our living like brothers with the people of West Pakistan, if there is a peaceful settlement. Otherwise there is none. If the Pakistan Army commits any more excesses we may never look at one another's face again . . . Be ready with whatever weapons you have in your hands. Since we have given blood once we will give more, but Bengal must be liberated . . . This is a struggle for survival, for emancipation and for freedom . . . Proclarnation of the Republic of Bangladesh On the 17th of April 1971 the Democratic Republic of Bangladesh was proclaimed as a formally constituted State to be run by a presidential form of Government. The Republic was proclaimed by its Vice-President Nazrul Islam as President Mujibur Rehman was in prison. Those present at the ceremony included Prime Minister Tazuddin Ahmed, members of his Cabinet and a large number of reporters and photographers from many countries. Proclarnation of the Republic of Bangladesh The declaration of independ ence and establishment of provisional Government of Bangladesh Republic was in the following terms : On April 10, 1971, the Provisional Government of Bangladesh in a meeting at Mujibnagar proclaimed independence by issuing the following declaration: Whereas free elections were held in Bangladesh from December 7, 1970 to "Whereas free elections were held in Bangladesh from December 7, 1970 to 261 5 /050 – January 17, 1971, to elect representatives for the purpose of framing a constitution; January 17, 1971, to elect representatives for the purpose of framing a constitution; And whereas at these elections the people of Bangladesh elected 1 67 out of 169 representatives belonging to the Awami League; And whereas at these elections the people of Bangladesh elected 1 67 out of 169 representatives belonging to the Awami League; And whereas the assembly so summoned was arbitrarily and illegally postponed for indefinite period; And whereas the assembly so summoned was arbitrarily and illegally postponed for indefinite period; And whereas General Yahya Khan summoned the elected representatives of the people to meet on March 3, 1971, for the purpose of framing a constitu tion; And whereas instead of fulfilling their promise and while still conferring with the representatives of the people of Bangladesh, Pakistan authorities declared an unjust and treacherous war; Whereas in the facts and circumstances of such treacherous conduct, Banga bandhu Sheikh Mujibur Rehman, the undisputed leader of 75 million of people of Bangladesh, in due fulfilment of the legitimate right of self-determi nation of the people of Bangladesh, duly made a declaration of independence at Dacca on March 26, 1971, and urged the people of Bangladesh to defend the honour and integrity of Bangladesh; And whereas in the conduct of a ruthless and savage war the Pakistani authorities committed and are still continuously committing numerous acts of genocide and unprecedented tortures, among others on the civilian and unarmed people of Bangladesh; And whereas the Pakistan Government by levying an unjust war and committing genocide and by other repressive measures made it impossible for the elected representatives of the people of Bangladesh to meet and frame a constitution, and give to themselves a government; And whereas the people of Bangladesh by their heroism, bravery and revolu tionary fervour have established effective control over the territories of Bangladesh; We, the elected representatives of the people of Bangladesh, as honour bound by the mandate given to us by the people of Bangladesh whose will is supreme, duly constituted ourselves into a constituent assembly and having held mutual consultations; And in order to ensure for the people of Bangladesh equality, human dignity and so ci al justice, declare and constitute Bangladesh to be sovereign people's republic and thereby confirm the declaration of independence already made by Bangabandhu Sheikh Mujibur Rehman; And do hereby affirm and resolve that till such time as a constitution is framed Bangabandhu Sheikh Mujibur Rehman shall be the President of the Republic and that Syed Nazrul Islam shall be the Vice-President of the Republic; And that the President shall be the Supreme Commander of all the armed forces of the Republic, shall exercise all the executive and legislative powers of the republic including the power to grant pardon, shall have the power to levy taxes and expend monies, shall have the power to summon and adjourn the constituent assembly; And do all other things that may be necessary to give to the people of Bangladesh an orderly and just government; we, the elected representatives 262 of the people of Bangladesh do further resolve that in the event of there being no President or the President being unable to enter upon his office, or being unable to exercise his powers and duties due to any reason whatsoever, the Vice-President shall have and exercise all the powers, duties and responsibilities, herein conferred on the President. Proclarnation of the Republic of Bangladesh We further resolve that we undertake to observe and give effect to all duties and obligations devolved upon us as a member of the family of nations and by the Charter of the Uni ted Nations. We further resolve that this proclarnation of independence shall be deemed to have come into effect from March 26, 1971. We further resolve that in order to give effect to this instrument we appoint Prof. M. Yusuf Ali our duly constituted potentiary and to give President and the Vice-President oaths of office." The war of independence of Bangladesh went on with mad fury. On the 3rd of December 1971 the dimensions were aggravated by Pakistan's declaration of war on India. The war however was of a short duration and on the 16th of December 1971 the Commander-in-Chief of the Pakistani Army in Bangladesh surrendered to the Commander-in-Chief of the Indian Army in Bangladesh. Mujibur Rehman however was still rotting in a jail in West Pakistan. On the 8th of January 1972 Mujibur Rehman was released from prison. He was flown to London wherefrom he arrived in Dacca via Delhi and was accorded a tumultous welcome by his people. Mujib promptly sat down to set his house in order and to formalise the constitutional documents of Bangladesh. On the 1 1 th of J anuary 1972 Mujibur Rehman issued the following provisional Constitution Order: The Provisional Constitution Order The Provision al Constitution of Bangladesh Order, 1972, promulgated by Sheikh Mujibur Rehman, envisages a Parliamentary democracy in the country. A Bangladesh Gazette Extraordinary notification dated J anuary 11, said, the nation's Bangladesh Constituent Assembly would consist of representa tives of the people of Bangladesh returned to the National and Provincial Assembly seats in the elections held in December 1970, January 1971, and March 1971, "not otherwise disqualified by or under any law". h The main points of the order were: (a) There shall be a Cabinet of Ministers with the Prime Minister as the Head, (b) The President shall, in exercise of all his functions, act in accordance with thc advice of the Prime Minister, (c) The President shall commission as Prime Minister a member of the Constituent Assembly who commands the confidence of the majority of the members of the Constituent Assembly. All other Ministers, Ministers of State and Deputy Ministers shall be appointed by the Prcsident on the advice of the Prime Minister, (d) In the event of a vacancy occurring in the office of the President at any time prior to the framing of the Constitution of the Constituent Assembly, the Cabinet shall appoint as President a citizen of Bangladesh, who will hold the office of President until another President enters upon the office in accordance with the Constitution as framed by 263 https://doi.org/10.5771/0506-7286-1974-3-257, am 24.10.2024, 06:41:29 Open Access – - https://www.nomos-elibrary.de/agb 6-1974-3-257, am 24.10.2024, 06:41:29 https://www.nomos-elibrary.de/agb the Constituent Assembly, (e) There sha11 be a High Court of Bangladesh consisting of a Chief Justice and so many other judges as may be appointed from time to time, (f) The Chief Justice of the High Court of Bangladesh sha11 administer an oath of office to the President and the President sha11 administer an oath of office to the Prime Minister, other Ministers, Ministers of State and Deputy Ministers. The form of the oath sha11 be as prescribed by the Cabinet. The Provision al Constitution of Bangladesh Order, 1972, was in pursuance of the proclarnation of Independence Order of April 10, 1971 made by the then Acting President, Syed Nazrul Islam. On the 14th of January 1972 Mujibur Rehman at a press conference gave the fo11owing policy statement: -7286-1974-3-257, am 24.10.2024, 06:41:29 - https://www.nomos-elibrary.de/agb Policy Statement of Mujibur Rebman Such a blueprint will involve institution al changes, in the fields of agriculture, industries and finance. These new institutions will seek to fully mobilise our material and human resources." "They will further seek to maximise production, consistent with the aim of narrowing down social and economic inequalities. While such a blueprint is being prepared with the utmost speed and certain tasks are being undertaken on an emergency basis - the first is relief and rehabilitation for which a comprehensive plan has been drawn up." "Mobilisation of resources from horne and abroad must be immediately undertaken. We urgently need assistance from abroad to supplement our own resources for this purpose. I appeal to all States, to the freedom-Ioving people of the world and to the international humanitarian organisations gene rally, to come to our assistance in all spheres, including assistance In maintaining supply of essential commodities which are urgently required." "I would like to express my gratitude and the gratitude of the people of Bangladesh to all freedom-Ioving States for their support, material and moral, in the national liberation struggle. 1 would like to record our special debt of gratitude to India, to Mrs. Indira Gandhi, Prime Minister of India, and her Government and her great people, for the total support extended by them to our struggle. I would similarly like to specially thank the Soviet Union and its great people for the consistent support extended by them to our cause, and for the invaluable role played by it in the United Nations. I would also like to express gratitude to Poland, Bulgaria and other East European countries, France and the U. K. I would also like to thank the freedom-Ioving people, journalists and leaders of thought and opinion throughout the world who supported our cause." "I thank those States that have accorded recognition to uso India, Bhutan, the German Democratic Republic, Bulgaria, Poland, Mongolia, Burma and other nations. The Bengali people have consistently supported freedom struggles throughout the world in Asia, Africa and Latin America. I, therefore, expect States all over the world, specially those who have tried to struggle for their independence, to extend recognition to the People's Republic of Bangladesh and to support its membership in the U. N. As regards our relations with Pakistan, I expect Mr. Policy Statement of Mujibur Rebman The fo11owing is the text of Sheikh Mujibur Rehman, Prime Minister of Bangladesh's statement at a news conference on 14th J anuary 1972 : "Bangladesh has now taken its place as an independent, sovereign State in the world community. In order to attain this cherished goal, millions have bad to lay down their lives and a11 sections of the people have undergone extreme suffering and made untold sacrifices. I reca11 with reverence the sacred memory of the millions of martyrs, who lost their lives and salute the heroes - young and old, peasants and workers, students and government servants, and in particular the brave sons of Bangladesh in the armed forces in the East Bengal Regiment, the former EPR, former Ansar, former Mujahids, police and a11 sections of the Mukti Bahini who uni ted together to resist the oppressor. " pp "The savages of the Pakistan fore es have left our country tota11y devastated. Systematic genocide has exterminated alm ost three million lives. Millions of hornes have been burnt down. Entire vi11ages have been wiped out. Among those that survived, millions were reduced to being refugees, having had to leave their hornes in order to save their lives and the honour of their women folk. Those who remained behind have suffered inhuman tortures and may have had their property looted." "As a result, the economy is in a shattered state. The most urgent task is that of reconstructing and restructuring the economy. The economy must be restored. Food, shelter and clothing must be provided. Educational institutions must once again function and education must be made accessible to a11 sections of the people. Relief and rehabilitation has to proceed on an emergency basis." "Out of the ashes that have been left by the barbarous hordes of Yabya Khan, a new society must be built up from its very foundations. The people of Bangladesh had always aspired to establish on their soil a just society, free of exploitation. The Bengali people have had to pay very dearly in order to attain this aim. Having regard to our population, our resources and the basic economic realities with which we are confronted, it will be possible to fulfil the aspirations of the people by establishing a 264 socialist economy. New planning machinery manned by the most competent professionals is undertaking the task of preparing a comprehensive blue print for a new economy. -7286-1974-3-257, am 24.10.2024, 06:41:29 - https://www.nomos-elibrary.de/agb Policy Statement of Mujibur Rebman Bhutto to accept the reality of independent, sovereign People's Republic of Bangladesh." "1 expect that the People's Republic of China which itself had attained liberation by armed struggle against warlords, feudal and colonial exploiters, should recognise the heroic success of our national liberation struggle. 1 hope that the People's Republic of China will now come forward to recognise the reality of the independent sovereign People's Republic of Bangladesh so that a basis may be established for mutual cooperation." "1 am deeply concious of the fact that it is necessary to maintain a framework of law and order in order to enable the people to undertake the task of nation-building. lt is but natural that those who have suffered from the cruelties of the Pakistan forces and their local collaborators are at the moment imbued with bitterness. Despite this, they have acted with 265 commendable restraint. 1 would like to ass ure our people that those who are responsible for committing atrocities and for collaboration will not go unpunished. At the same time it should not be forgotten that one of the fundamental aims of our national liberation struggle was to establish thc role of law and respect for fundamental human rights. The culprits will be duly punished in accordance with the due process of law. 1 would, therefore, appeal to all concerned to remain patient whilc thc due process of law takes its course." "I am taking immediate steps to ensurc that the High Court and the subordinate courts begin to function. I wou!d like to ensure that necessary steps are taken to correct some of the fundamental defects in the judicial system, with a view to elimination of corruption and delays. The principle of separation of the judiciary from the executive will be scrupulously observed." "While we set about to tackle the urgent task of relief and rehabilitation and economic reconstruction, we shall not delay a single moment more than is absolutely nccessary in convening the Constituent Assemb!y in order to place before it thc draft constitution of the People's Republic of Bangladesh. Thc preparation of the draft constitution is already under way. " "The strength of a uni ted and dctcrmined people has added a glorious chapter to the history of nationa! liberation struggles. Policy Statement of Mujibur Rebman Today the same strength and power of the people must be mobilised in order to liberate our people from poverty, hunger, illiteracy, disease and every form of exploitation. 1 am confident that with the total support which the people have given to my party and to mys elf we shall overcome the enormous problems with which we are confronted, and that we will be able to lay down the foundations of a just society, free from exploitation, for the attainment of which so many millions have laid down their lives and countless others have borne untold sufferings." A Constitution Committee was thereafter set up headed by the Law and Parliamentary Affairs Minister Dr. Kamal Hossain who was to sub mit the draft constitution to the Awami League Working Committee for its consideration before it was placed beforc the Constituent Assembly. The draft constitution was du!y prepared, approved and placed before the Constituent Assembly. The Constitution of Bangladesh On the 4th of November 1972 the Constitucnt Assembly of Bangladesh finalised its constitution and thus came into existence the constitutional charter of the youngest nation in Somh East Asia. After a violent and bloody struggle for independence was enshrined in a document the dreams and ambitions of a proud people. The salient features of the Constitution of Bangladesh are noted below. Resolution adopting the Constitution The resolution of the Constituent Assembly adopting the Constitution of Bangla desh may be summarised as follows: 266 "We the people of Bangladesh have declared our independence on the 26th of March 1971 and for the liberation of the nation we have raged a historic war and have established an independent and sovereign People's Republic of Bangladesh. We promise that the ideology wh ich inspired our freedom fighters and heroic martyrs to sacrifice their lives, viz., nationalism, socialism, democracy and secularism, these ideals shall be the main features of our Constitution. We further promise that the main objectives of our nation shall be to establish by democratic methods a socialist society free from exploitation of any kind which will ensure for every citizen the rule of law, basic human rights and political, economic and social equality, liberty and justice. We firmly declare that in order that we can prosper in independence and keep pace with the progress, hopes and aspirations of humanity and in order to enable us to advance the cause of international peace and cooperation and to play our full and rightful part therein it is our pious duty to keep the supremacy of this Constitution which is the expression of the will of the people of Bangladesh and the preservation, support and security and maintenance of the Constitution is our noble duty. We in our Constituent Assembly, on the 4th of November 1972, adopt, finalise and accept this Constitution of Bangladesh.» Text of the Constitution First part deals with the PresiJent (Rastrapati), his powers, his rights of exercising clemency, tenure of the office of the President, impeachment of the President and appointment of the Speaker to function in the office of the President during temporary absence of the President (articles 48 to 54). Second part is contained in articles 55 to 58 and provides for the Prime Minister and the Cabinet. It pro vi des also for the tenure of the office of the Prime Minister and other Ministers. Third part deals with the local-self Government and is contained in articles 59 and 60. The fourth part consists of provisions for the administration of defence and pro vi des for the Commander in-Chief, other officers and of war and is contained in articles 61 to 63. The fifth part consisting of only one article, article 64, provides for the office of the Attorney General. Chapter V deals with the legislature. This chapter is contained in articles 65 to 93. This chapter is divided into three parts, viz., legislature and parliament in part 1, legislation of laws and financial rules in part 2 and powers of passing ordinances in part 3. Part 1 contained in articles 65 to 79 pro vi des for establishment of the parliament (Sangsad), qualifications and disqualifications for becoming members of the parliament, vacation of offices of members of the parliament, the salaries and emoluments of members of the parliament (article 68), provisions for taking oath (article 69), vacation of offices of members of parliament on resigning from political parties or in case of voting against the mandate of the party (article 70), sessions of the parliament (article 72), the address of the President (article 73), Speaker and Deputy Speaker (article 74), rules of work, quorum and permanent committees of the parliament (articles 75 and 76), Nayapal (Ombudsman) (article 77), special rights and privileges of the parliament and members thereof (article 78), secretariat of the parliament (article 79). Part 2 of this chapter dealing with promulgation of laws and financial rules is contained in articles 80 to 92. Text of the Constitution The Constitution of Bangladesh consists of 1 1 Chapters and 4 Schedules. The chapters and schedules are as folIows. Chapter 1. This chapter consists of 7 articles and pro vi des for the republic, boundaries, the national language, the national song, the national flag and emblem, the capital, citizenship and supremacy of the Constitution. Bengali is the national language of Bangladesh and the Constitution is in Bengali language. The name of the State is People's Republic of Bangladesh (Gana Prajatantri Bangladesh). The capital of the State is Dacca. Chapter 11. This chapter consists of provisions which are the directive principles of the administration of the State. This is contained in articles 8 to 25. These articles deal with the directive principles, viz., nationalism socialism and freedom from the exploitation, democracy and human rights, secularism and freedom to practise religion, principles of ownership, liberation of farmers and labourers, development of villages and agricultural revolution, free and compulsory education, public health and morality, equality of opportunities, rights and obligations, duties of citizens and Government officials, separation of judiciary from the executive, national culture and heritage, national museums, international peace, security and cooperation and the development thereof. Chapter III. consists of fundamental rights. This chapter is composed of articles 26 to 47. This chapter deals with abrogation of laws not in conformity with fundamental rights (article 26), equality in the eye of law (article 27), elimination of discrimination on the ground of religion, etc. (article 28), equality of 267 opportunities in getting state employment (article 29), abolition of titles, honours and decorations (article 30), right to obtain protection of law and legal processes (article 31), security of life and property (article 32), safeguard against arrest and detention (article 33), elimination of compulsory labour (article 34), assuring justice and punishment in proper cases (article 35), freedom of movement and of meetings and freedom of thought, conscience and expressions (articles 36 to 40), freedom to practise trade, avocation or vocation (article 40), religious freedom (article 41), freedom to own properties (article 42), security of hornes and rights of privacy (article 43), enforcement of fundamental rights (article 44) and powers of State to enact laws by chan ging certain rights (articles 45 to 47). Chapter IV consists and provides for the Executive. This chapter is contamed in articles 48 to 74. This chapter has five parts. Text of the Constitution This deals and provides for procedure of enacting laws (article 80), money bills (article 8 1), prevention of im position of taxation without sanction of the parliament (article 83), consolidated funds and ac counts (article 84), control of currency (article 85), powers of the State to dis tribute funds (article 86), annual ac counts (article 87), charge on consolidated funds (article 88), provisions for allocation, etc. (article 91), accounts, loans and votes thereon (article 92). Part 3 consists only of one article, article 93 and provides for ordinance making powers. 268 Chapter VI provides for the judiciary. This chapter is contained in articles 94 to 1 17 and has three parts. Part 1 (articles 94 to 1 13) provides for the Supreme Court and establishment thereof, appointment of judges of the Supreme Court, tenure of such judges, appointment of additional judges, disabilities of jedges of the Supreme Court after retirement, jurisdiction of the High Court and powers of the High Court to issue writs, orders and directions for enforcement of fundamental rights, appellate powers of the High Court, advisory powers of the Supreme Court, powers of the Supreme Court as a court of record and its supervisory powers on other subordinate courts, etc. Part 2 contained in articles 1 14 to 116 makes provisions for the subordinate judiciary. Part 3 provides for establishment of administrative tribunals (article 1 1 7). A judge of the Supreme Court can only be removed from his office by an order of the President passed with support of a resolution of the Parliament having a two-thirds majority of members of the Parliament [article 96 (2)]. Chapter VII (articles 108 to 126) provides for election. This chapter provides for an Election Commission and Election Commissioner. The Election Commissioner is appointed by the President and holds his office for a tenure of 5 years. Election Commissioner is not removeable from his office excepting in the same manner as the removal of a judge of the Supreme Court. In article 122 the franchise is also fixed. A citizen of Bangladesh over the age of 1 8 years and of sound mind is entitled to exercise his franchise and vote at the elections. Chapter VIII (articles 127 to 1 32) provides for Auditor General and Comptroller. The Auditor General is not removeable excepting in the same manner as a Supreme Court judge. Text of the Constitution The Auditor General holds office up to the age of 60. The Auditor General has to make a report to the President and the President is to place it before the parliament. Chapter IX (articles 133 to 141) provides for the Executive of Bangladesh. This chapter is divided into two parts. Part 1 deals with the terms of appointment of civil servants, their tenure of service and provides safeguards against removal or demotion. Part 2 provides for a Public Service Commission. Members of the Commission are appointed by the President. Article 140 deals with the responsibil ities of the Commission. The Commission has to make an annual report to the President and the President arranges for its being placed before the parliament. Chapter X (article 142) provides for amendment of the Constitution. Under this article the Constitution can be amended only by a two-thirds majority of the parliament. An amendment passed by a requisite majority has to be placed before the President for his consent and if the President fails to give his consent within seven days then at the expiration of such time the President will be deemed to have given his consent to the same. Chapter XI (articles 143 to 153) makes provlSlons for D1 iscellaneous matters. Article 143 provides that all underground mineral rights as also rights within the territorial waters and the continental shelf are the property of the nation. Article 145 provides that all treaties and documents have to be expressed in the 269 name of the President of Bangladesh. Article 146 provides that suits and proceedings can be instituted against the Government of Bangladesh. Article 147 makes provisions for fixing the remuneration and emoluments of various holders of public offices including the President, the Prime Minister, Speaker and Deputy Speakers, Ministers, Judges of the Supreme Court, Accountant General and Comptroller, Election Commissioner and Members of the Public Service Commis sion. Other miscellaneous matters are dealt with in this chapter. There are four schedules to the Constitution. Schedule I provides for validation of certain laws. Various existing statutes and orders are validated specifically under this schedule. Schedule II provides for certain details as to the election of the President. President is elected by members of the parliament. The Chief Election Commissioner is entrusted with conduct of this election. Text of the Constitution Schedule III provides for and fixes the constitutional forms of the oaths to be administered to the President, the Ministers, Speaker and Deputy Speakers and members of parliament. It also provides for the oaths and declarations of various persons including the Chief Justice and other judges, the Chief Election Commissioner and Auditor General and Comptroller and members of the Public Service Commission. Schedule IV makes provisions for legislations and temporary rules and laws. It makes various provisions for and validates actions taken before the Constitution came into existence. Salient Features of the Constitution of Bangladesh Directive Principles Directive principles embodied in the Constitution provide the keynote to the nature of the Constitution. The four most important elements embodied in this chapter are that the directive principles of the Constitution are based on the principles of nationalism, socialism, democracy and secularism. The war of libera tion of Bangladesh was based on the aspiration of the Bengali people to get their independence. Bengalee nationalism has been made one of the directive principles of the Constitution. Bengali is the State language and the Constitution is written in Bengali language. The Constitution further pro vi des that the object of the Constitution is to establish a socialist society based on justice. The establishment of a democratic government and assurance of basic human rights and liberties is also made one of the directive principles of the Constitution. One of the most important features of the directive principles is secularism and freedom of all citizens to practise their religion. It also provides that all types of communalism have to be eradicated. The Constitution forbids making of any religion as a state religion, using religion for political purposes and pro vi des for abolition of discrimination against any person on the grounds of religion. As the Constitution of Pakistan has provided for making Islam the state religion this has been one of the most important departures from religion-based and non-secular constitutions. The above principles which have been made parts of the directive principles were enunciated by the Prime Minister Mujibur Rehman at the time of his struggle for independence. He thereby clearly substituted modernism for medievalism. The The above principles which have been made parts of the directive principles were enunciated by the Prime Minister Mujibur Rehman at the time of his struggle for independence. He thereby clearly substituted modernism for medievalism. The directive principles ensure that Bangladesh continues in a proper path for its 270 development as one of the modern secular democracies of the world. The chapter on directive principles makes various other interesting provisions. Article 1 3 provides for stateownership of all major fields. It also provides for cooperative ownership of property within the limits of law. It is clearly indicated that the rights of individual ownership can be limited or abridged by the State from time to time. Salient Features of the Constitution of Bangladesh Directive Principles Under article 14 it is one of the objects and responsibilities of the State that all labouring men, farmers and labourers are to be liberated from all forms of exploitation. Under article 15 one of the basic objects and responsibilities of the State is to have planned economic progress and material and cultural advancement of the citizen. It specifically pro vi des that the State is to take steps for providing food, clothing, shelter and economic assistance of citizens and recognises right to work and reasonable remuneration for the same, adequate rest, relaxation and holidays, and social security. The State is directed to take steps to eradicate unemployment, sickness and any economic difficulties arising out of sickness, widowhood, old age, etc. Principles of a modern welfare state are thereby entrenched in the Constitution. Article 16 also provides for elimination of differences between urban life and rural life. It specifically provides for development of villages, electrification, cottage industries, etc. In the background of the difficulties experienced by various countries by rapid urbanisation and growth of megalopolis this is an important provision. Subsequent actions of the Bangladesh Government also show that the Government is taking active steps to implement these directive principles. Article 17 pro vi des for free and compulsory education for all up to standards fixed by the State. Article 19 and 20 embody certain principles of socialism. Article 19 provides for equality of opportunities as also eradication of inequalities. Article 20 provides that for every ablebodied citizen work is a right, duty and an honour. It further incorporates the principle "from everyone according to his capacity and to everyone according to his work". It is interesting to note that the expression used here is that to everyone according to his "work" and not according to his "needs". It has been interpreted by some that the obligation of citizen to work conscientiously and the linking up of remuneration to work may have an effective bearing on movements for voluntary ceasing of work for political purposes. Article 25 puts also in the directive principles some of the international obligations of the State. Principles of sovereignty and honouring the concept of equality of States, non-interference in the international affairs of other States, peaceful settlement of international disputes, honouring international law and the charter of the United Nations are specifically provided for in article 25. Salient Features of the Constitution of Bangladesh Directive Principles This article also accepts as principles avoidance of use of force in international affairs, disarmament, co-existence and rights of every state to govern its affairs according to its own social, economic and political views, and elimination of imperialism, colonialism and discrimination on the ground of colour or complexion and pledges support to the oppressed p pl f th ld i th i t l i t h di i i ti Fundamental Rights The chapter on fundamental rights gives constitutional recognition and entrenches in the Constitution various human rights. It provides for total elimination of 271 any discrimination on grounds of religion, community, colour, sex or birth of any citizens. It however provides for power to the State to make laws for protection or advancement of women and children and also for advancement of any backward communities. This chapter also provides for assuring liberty of the people and provides that excepting by the due process of law no person can be deprived of his life or personal liberties. No one can be arrested without informing hirn of the cause of arrest and every arrested person must be given facilities for consulting his own lawyer and defending hirns elf. Every arrested person must be produced before a court of law within 24 hours and cannot be detained beyond 24 hours without an order of court. Under article 36 freedom of movement is ensured as a fundamental right. It is interesting to note that this freedom is not confined only to movement within the territory of Bangladesh but it also ensures to citizen the right to leave Bangladesh and re-enter the same. The chapter also provides for freedom of association, of meetings and of thought and expression. Freedom of press is also specifically provided for in article 39. It is interesting to note that in spite of accepting socialism as one of the basic principles article 42 clearly provides for right to own properties and to transfer the same. The State's power to nationalise private properties is preserved but compensation has to be given for the same. It is however specifically provided that no such action of the Government can be challenged in court on the basis that the compensation is not adequate. This clause is expected to avoid a lot of the controversy which arose in India on the ground that compensations offered by the State were not ade qua te or not a just equivalent of the properties taken. Article 44 pro vi des that action can be instituted directly in the Supreme Court for enforcing any of the fundamental rights. One of the controversies which may arise later is the right of the Parliament to amend the fundamental rights. This point had raised a nationwide controversy in India and was the cause of a constitutional storm. Fundamental Rights If the constitutional rights are considered to be human rights entrenched in the Constitution then they are not alienable rights nor has the Parliament the right to amend such provisions within the powers gran ted to it for amendment of the Constitution. It may be open to question whether in such a case a Constituent Assembly has to be re-convened for the purpose of considering such modifications. Parliament and Government Bangladesh under its Constitution is a parliamentary democracy with the Cabinet form of government. The President is the head of the State but the power of government lies with the Prime Minister. The Prime Minister selects his Council of Ministers. The Constitution provides for collective responsibility of the Cabinet and the Cabinet is responsible to the parliament. The Constitution provides for universal adult franchise, every person of sound mind and over the age of 18 being entitled to vote. The parliament is unicameral and there is parliamentary supremacy within the limits fixed by the written Constitution. The Constitution itself provides for parliament's power of amen ding the Constitution. 272 By HERBERT KRÜGER In 1973 a commission preparing a total revision of the Swiss Federal Constitution published a report ("Wahlen-Bericht"). The first part of this report, which deals with the conditions for a total revision of the Federal Constitution, could be called an instruction for constitution-making. The pattern of constitution-making has first of all to deal with the mode of procedure. But for lack of material the author is not able to draw any generalisation in this field. Therefore he attempts to show how the rightness of the contents of a constitution may be achieved. This depends on the situation in which constitution-making is attempted. Two main situations are distinguished, the ending of a revolution and constitutional change in an undisturbed state. After a revolution, the constitution has to establish a political order and to build a nation, whereas in the latter case continuity must be regarded as a high priority. The constitution is shaped by the ideas of the constitution-maker and his percep ti on of the realities of his country. It seems that a good constitution has to rely more on the social reality than on ideologies. Therefore it is necessary, that the constitution-maker analyzes the concrete situation of his state. The constitution however should not be a transformation of the reality into norms only, but should also achieve a better society. It is clear, that a constitution-maker will generally fail, if he is guided by mistrust and pessimism. The two main features of a constitution are: a programme of national integration and a programme of national representation. Integration means the building of a nation by integration of the individuals as citizens into the state. A constitution has to provide the possibilities for this process, namely the symbols of integration and the freedoms, which are necessary to perform the role of a citizen. Representation means that the citizens must form a collective will, for only collective eHorts can satisfy the great needs in our times. The constitution has to provide also for the formulation of a common will and a procedure, which guarantees that not any will but the best or at least the better will may prevail. Finally the constitution has to be a national authority in order to prohibit its destruction. This is the most difficult task of constitution making. By HERBERT KRÜGER A constitution can gain the necessary authority if it relies not on private ideals but on cosmopolitan ideals of the highest standard. Supreme Court and High Court The Supreme Court is under the Constitution the guardian of constitutional rights. In case of any violation of fundamental or constitutional rights action can be taken in the High Court or Supreme Court. The Supreme Court also has advisory jurisdiction and the President may refer any question to the Supreme Court for advice. There is total separation of the executive from the judiciary. The Chief Justice and Judges of the Supreme Court and High Court are appointed by the President and are not elective offices. The Judges hold office till the age of 62 and are not removeable except by an order of the President passed on the basis of a resolution of the parliament with two-thirds majority. 273 -7286-1974-3-257, am 24.10.2024, 06:41:29 - https://www.nomos-elibrary.de/agb https://doi.org/10.5 Open Access – – By W. WESSEL-$CHULZE Since independence there have been two dominant parties in Colombia: the Conservatives and the Liberals. For centuries each of them has governed the country until 1948. The following party strife, called the "violencia", has been one of the most violent and brutalized struggles of the twentieth century. At the brink of civil war and absolute chaos the military under the leaders hip of General Rojas Pinilla took over power. Being ousted by his colleagues in 1957 a military junta governed until August 1958. The party coalition of Conservatives and Liberals, called the "National Front", brought political stability with a system of parity and alternation. That meant equal share of all governmental offices and a change of the presidency between the two parties for sixteen years. Since independence there have been two dominant parties in Colombia: the Conservatives and the Liberals. For centuries each of them has governed the country until 1948. The following party strife, called the "violencia", has been one of the most violent and brutalized struggles of the twentieth century. At the brink of civil war and absolute chaos the military under the leaders hip of General Rojas Pinilla took over power. Being ousted by his colleagues in 1957 a military junta governed until August 1958. The party coalition of Conservatives and Liberals, called the "National Front", brought political stability with a system of parity and alternation. That meant equal share of all governmental offices and a change of the presidency between the two parties for sixteen years. Having returned from exile Rojas Pinilla looked for supporters of a populist policy in the parties. Between 1960-1970 Rojas and his daughter, Maria Eugenia, were successful in building a mass party. In the 1970 presidential and governmental elections the so called ANAPO lacked only 65 000 votes. In 1971 the ANAPO was officailly founded in the historie province city of Villa de Leyva. Th h f b ll d li di T Having returned from exile Rojas Pinilla looked for supporters of a populist policy in the parties. Between 1960-1970 Rojas and his daughter, Maria Eugenia, were successful in building a mass party. In the 1970 presidential and governmental elections the so called ANAPO lacked only 65 000 votes. In 1971 the ANAPO was officailly founded in the historie province city of Villa de Leyva. The ANAPO has often been called a "populist party". By S. C. SEN After the withdrawal of the British from the Indian Empire Bengal found itself as part of Pakistan, but the only link with West-Pakistan (1200 miles away) was religion. In 23 years of joint history the East was denied almost all rights. With the six-point formula of Bengal autol1omy Sheikh Mujibur Rehman gained an enormous victory in the 1970 election to the proposed National Assembly. Fearing majorisation by the East the party leaders of the West secured the postponement of the meeting of the National Assembly by President Yaya Khan. Over a campaign of non-cooperation answered by a reign of terror, this led to the Declaration of Independence and the civil war. 229 By the Provisional Constitution Order a Parliamentary System was set up. The Parliament consisted of the members elected to the National Assembly and acted also as Constituent Assembly. It approved the Constitution on the 4th of November 1972. The main features of the Constituion are a chapter on principles of State policy which are based on four elements, namely nationalism, socialism, democracy and secularism. Fundamental rights are guaranteed including the right of property. Bangladesh is a parliamentary democracy with a Cabinet form of government. The Parliament is unicameral. The Constitution provides also an independent judiciary as guardian of the constitutional rights. By W. WESSEL-$CHULZE according to Torcuato Di Tella the main elements of a populist movement or party are : a) a broad mass basis, b) an elite often dominated by a charismatic leader, c) a vague ideology. - The mass basis of the ANAPO is recruited from the marginal population of the big cities. In the elections of 1970 the ANAPO received 60 to 65 per cent of all votes of the lower classes. In the leadership are men from the middle and upper classes. At its top stood Rojas Pinilla as the charismatic leader until 1972. Now his daughter holds this position. In the ideology, written down in the "plataforma", the dispossessed people are promised everything from land to free education. On the other hand capital or property of the upper class will not be nationalized, c1ass-struggle is not forced. In the elections of the province parliaments in 1972 the ANAPO has lost half of all votes compared with those won in the elections of 1970. In the presidential elections in April 1974 the ANAPO candidate, Maria Eugenia Rojas de Moreno, was the third with not more than 450 000 votes. After this enormous loss of votes, especially in the lower classes, the fate of the ANAPO is unsecure. Surely it is not a competing third mass party any Ion ger. 230 5771/050 –
https://openalex.org/W4283806045	https://archimer.ifremer.fr/doc/00755/86741/95273.pdf	English	null	Third revision of the global surface seawater dimethyl sulfide climatology (DMS-Rev3)	Earth system science data	2,022	cc-by	17,485	Third revision of the global surface seawater dimethyl sulﬁde climatology (DMS-Rev3) Shrivardhan Hulswar1, Rafel Simó2, Martí Galí2,3, Thomas G. Bell4, Arancha Lana5, Swaleha Inamdar1,6, Paul R. Halloran7, George Manville7, and Anoop Sharad Mahajan1 1Indian Institute of Tropical Meteorology (IITM), Ministry of Earth Sciences, Pune, India 2Institut de Ciències del Mar (CSIC), Barcelona, Catalonia, Spain 3Barcelona Supercomputing Center (BSC), Barcelona, Catalonia, Spain 4Plymouth Marine Laboratory (PML), Plymouth, UK 5Institut Mediterrani d’Estudis Avançats (IMEDEA, UIB-CSIC), Esporles, Balearic Islands, Spain 6Institute of Environment and Sustainable Development, Banaras Hindu University, Varanasi, India 7College of Life and Environmental Sciences, University of Exeter, Exeter, UK Correspondence: Anoop Sharad Mahajan (anoop@tropmet.res.in) Received: 12 July 2021 – Discussion started: 28 September 2021 Revised: 10 May 2022 – Accepted: 24 May 2022 – Published: 5 July 2022 Abstract. This paper presents an updated estimation of the bottom-up global surface seawater dimethyl sul- ﬁde (DMS) climatology. This update, called DMS-Rev3, is the third of its kind and includes ﬁve signiﬁcant changes from the last climatology, L11 (Lana et al., 2011), that was released about a decade ago. The ﬁrst change is the inclusion of new observations that have become available over the last decade, creating a database of 873 539 observations leading to an ∼18-fold increase in raw data as compared to the last estimation. The second is signiﬁcant improvements in data handling, processing, and ﬁltering, to avoid biases due to different observation frequencies which result from different measurement techniques. Thirdly, we incorporate the dy- namic seasonal changes observed in the geographic boundaries of the ocean biogeochemical provinces. The fourth change involves the reﬁnement of the interpolation algorithm used to ﬁll in the missing data. Lastly, an upgraded smoothing algorithm based on observed DMS variability length scales (VLS) helps to reproduce a more realistic distribution of the DMS concentration data. The results show that DMS-Rev3 estimates the global annual mean DMS concentration to be ∼2.26 nM (2.39 nM without a sea-ice mask), i.e., about 4 % lower than the previous bottom-up L11 climatology. However, signiﬁcant regional differences of more than 100 % as com- pared to L11 are observed. The global sea-to-air ﬂux of DMS is estimated at ∼27.1 TgS yr−1, which is about 4 % lower than L11, although, like the DMS distribution, large regional differences were observed. The largest changes are observed in high concentration regions such as the polar oceans, although oceanic regions that were under-sampled in the past also show large differences between revisions of the climatology. Earth Syst. Sci. Data, 14, 2963–2987, 2022 https://doi.org/10.5194/essd-14-2963-2022 © Author(s) 2022. This work is distributed under the Creative Commons Attribution 4.0 License. Highlights. – The sea surface DMS concentration climatology was updated using an upgraded processing algorithm and the inclusion of new data. – Usage of monthly dynamic biogeochemical province bound- aries and DMS variability length scales (VLS) reduces the patchiness observed in surface mean concentrations seen in the older climatologies. – DMS-Rev3 estimates the global annual mean at 2.26 nM (2.39 nM without a sea-ice mask), approximately ∼7 % lower than the last DMS climatology (∼2 % lower without consider- ing sea ice), with much larger regional differences. Third revision of the global surface seawater dimethyl sulﬁde climatology (DMS-Rev3) Finally, DMS-Rev3 reduces the previously observed patchiness in high productivity regions. The new climatology, along with the algorithm, can be found in the online repository: https://doi.org/10.17632/hyn62spny2.1 (Mahajan, 2021). Published by Copernicus Publications. S. Hulswar et al.: DMS-Rev3 2964 concentrations, and more importantly, the ocean–atmosphere ﬂux on a global scale. To compute the ocean–atmosphere ﬂux, two basic methods have been adopted. The ﬁrst method, described here as the top-down approach method, relies on the dependency of DMS on various physical, chemi- cal, and biological parameters that correlate with the vari- ability of DMS, e.g., chlorophyll-a, photosynthetically ac- tive radiation (PAR), and nutrients. This method creates a parameterization-based seawater DMS inventory, which when fed with input ﬁelds and used in combination with an air–sea exchange parameterization, results in the ocean– atmosphere DMS ﬂux (Belviso et al., 2004; Bopp et al., 2004; Galí et al., 2018; Simó and Dachs, 2002; Vallina and Simó, 2007). These approaches provided statistical relation- ships needed to understand the mechanisms of the biogeo- chemical cycle of DMS, its formation and removal from the surface ocean. These seawater DMS parameterizations pro- duced in the past help to reproduce preliminary or local- ized DMS concentration ﬁelds but have shortcomings when applied on a global scale. For example, the SD02 algo- rithm (Simó and Dachs, 2002) was able to estimate val- ues accurately only in the tropical and temperate latitudes but underestimated DMS in low chlorophyll areas and along the Antarctic coast. The VS07algorithm (Vallina and Simó, 2007) was unable to reproduce the DMS–irradiance rela- tionship that depended on phytoplankton biomass, leading to overestimations or underestimations outside the subtrop- ical region. Another method applies a two-step approach, ﬁrst computing the DMSP concentrations (Galí et al., 2015) and using them to calculate the DMS concentrations utiliz- ing satellite-measured proxies (Galí et al., 2018). However, this satellite-based seawater DMS computation (DMSSAT) suffers from a negative bias in the Antarctic coastal re- gion during the productive season. The DMSSAT tends to underestimate observations by around 50 % in some re- gions of the Southern Ocean (Galí et al., 2018). A recent parameterization-based approach used an artiﬁcial neural network (ANN) to extrapolate seawater DMS observations into a global climatology (Wang et al., 2020). Third revision of the global surface seawater dimethyl sulﬁde climatology (DMS-Rev3) This approach using linear regressions showed that on a global scale, mixed layer depth (MLD – explaining ∼9 % of variance) and so- lar radiation (explaining ∼7 % of variance) are the strongest predictors of DMS. The ANN climatology captured 66 % of the raw data variance for the test DMS database (Wang et al., 2020). This approach however does not give much scientiﬁc insight into the relationships between biological and physi- cal parameters and processes controlling the DMS concen- trations. The concentrations are also underestimated in the higher latitudes and the episodic occurrence of higher DMS Highlights. Highlights. S. Hulswar et al.: DMS-Rev3 We updated the DMS database using the latest additions in the GSSDD, along with other published data that are not currently in the database, to reconstruct the monthly, sea- sonal, and annual DMS climatologies. Comparison with the L11 climatology demonstrates that the DMS-Rev3 addresses some of the concerns with previous climatologies. Further- more, shortcomings of the latest revision are discussed, along with the identiﬁcation of gaps that need to be addressed in subsequent versions. We updated the DMS database using the latest additions in the GSSDD, along with other published data that are not currently in the database, to reconstruct the monthly, sea- sonal, and annual DMS climatologies. Comparison with the L11 climatology demonstrates that the DMS-Rev3 addresses some of the concerns with previous climatologies. Further- more, shortcomings of the latest revision are discussed, along with the identiﬁcation of gaps that need to be addressed in subsequent versions. The second, a more widely used method is the bottom- up approach, which relies on the Global Surface Seawater DMS Database (GSSDD) (NOAA-PMEL, 2020), which con- sists of data contributed by research groups from all over the world. This database was established to consolidate all the surface seawater DMS measurements into a single compre- hensive dataset. These data are then modeled using a com- bination of smoothing and interpolation techniques to create a gridded seawater DMS concentration climatology, which is then converted into a ﬂux as mentioned above (Kettle et al., 1999; Lana et al., 2011). The ﬁrst attempt in creating the bottom-up climatology was made by Kettle et al. (1999). They used the then available dataset (15 617 observations across the global ocean) to create a global DMS climatol- ogy (hereafter called the K99). A decade later, this clima- tology was updated by Lana et al. (2011), using an updated DMS database (47 313 observations) and included some mi- nor changes in the computation algorithm (hereafter called the L11 climatology). Signiﬁcant differences were observed between the two climatologies: globally, the L11 estimated emission of DMS was 28 (17.6–34.4) TgS yr−1, about 17 % higher than the estimate calculated using K99. Regionally, large differences were observed, for example in the Indian Ocean, where L11 predicted higher values, and in the South- ern Ocean, where large longitudinal differences were re- ported. 2.1 Data consolidation and cleanup Since the last bottom-up DMS climatology (L11) was pub- lished about a decade ago by Lana et al. (Lana et al., 2011), the GSSDD database (NOAA-PMEL, 2020) has been con- tinuously updated with new observations and now consists of a total of 87 801 data points. This is a signiﬁcant increase in the number of data points (an increase of ∼85.6 %) com- pared to the number of data points used in the L11 climatol- ogy (47 313 – blue circles in Fig. 1). All the raw data used for the new climatology are also shown in Fig. S1. Most of these observations were made using the gas chromatog- raphy (GC) technique and have a temporal resolution rang- ing from 10 min to a data point every week for individual campaigns. Over the last decade, newer techniques that use high-resolution mass spectrometry have become more com- mon and led to a drastic increase in the number of avail- able data points. We added the raw high temporal resolution data (frequency as high as ∼1 s) from published campaigns, which make use of these new techniques (Behrenfeld et al., 2019; Jarníková et al., 2016; Royer et al., 2015; Wohl et al., 2020, 2022; Zavarsky et al., 2018b). On the addition of these high-resolution data, the consolidated raw dataset included 873 539 data points (∼1746 % increase over the number of data points in the L11 climatology). ) y ( ) However, over time, signiﬁcant shortcomings in L11 have been identiﬁed. First, it uses a single threshold for data se- lection using the 99.9 percentile of the data to remove any extreme values. This causes the relatively higher values in the open ocean to remain if they are under the threshold. These might represent a highly productive bloom in the re- gion and show anomalously high concentrations or be due to instrument-related issues. Such abnormal values cause re- gional patches of higher concentrations which are seen in the L11 climatology. The L11 climatology also uses static biogeochemical province boundaries for global data segrega- tion, which do not capture variability in the biogeochemical properties affecting DMS production, especially on seasonal scales. Since the number of available observations of sea sur- face DMS concentrations has increased, the data distribution over the oceans, especially in under-sampled regions like the remote oligotrophic oceans and the Southern Ocean has im- proved (Fig. 1). S. Hulswar et al.: DMS-Rev3 At present, the L11 (Lana et al., 2011) bottom-up climatology is considered as the primary reference product for global DMS seawater concentrations and is used as an in- put in numerous atmospheric chemistry models (Mahajan et al., 2015) and climate and Earth system models (ESMs). 1 Introduction Dimethyl sulﬁde (DMS) is a volatile compound found in the global oceans, and its biogeochemical cycle plays an impor- tant role in the Earth’s climate system (Andreae and Crutzen, 1997; Charlson et al., 1987). It is primarily a by-product of phytoplankton growth and marine microbial food web inter- actions (Simó, 2001). DMS is produced by the breakdown of the phytoplankton intracellular metabolite dimethylsulfo- niopropionate (DMSP), either in the algal cell or through microbial catabolism of the DMSP released due to physio- logical stress or mortality (Kiene et al., 2000; Stefels et al., 2007). This produced DMS is either oxidized by photochem- ical reactions or metabolized by bacteria (Toole et al., 2003), leaving a small portion that is released into the atmosphere as gaseous DMS (Galí and Simó, 2015; Simó, 2001). The DMS emitted from the surface ocean is responsible for up to 70 % of the natural sulfur emissions into the global at- mosphere (Andreae and Raemdonck, 1983; Carpenter et al., 2012). Oxidation of DMS takes place in the atmosphere and yields sulfuric and methanesulfonic acids, which eventually lead to the formation of sulfate aerosols that can grow to act as cloud condensation nuclei (CCN) (Andreae and Barnard, 1984; Pazmiño et al., 2005). New CCN can make clouds brighter, thus establishing a feedback loop between phyto- plankton and cloud albedo popularly known as the CLAW hypothesis (Charlson et al., 1987). While some studies based on large-scale observations of ocean surface DMS provided partial support for the CLAW hypothesis (Vallina and Simó, 2007), other studies based on model sensitivity analysis of the hypothesis have challenged it (Quinn and Bates, 2011; Woodhouse et al., 2010, 2013). However, even if the feed- back loop is not as strong as previously envisaged, DMS emissions contribute towards a large fraction of aerosols in the remote oceanic environment (Quinn et al., 2017) and its emissions need to be quantiﬁed accurately to improve our understanding of climate sensitivity, the current climate (Carslaw et al., 2013) and to improve the accuracy of future projections (Wang et al., 2021). Acknowledging the signiﬁcance of oceanic DMS, the sci- entiﬁc community has striven to reproduce an accurate rep- resentation of the global atmospheric and seawater DMS https://doi.org/10.5194/essd-14-2963-2022 Earth Syst. Sci. Data, 14, 2963–2987, 2022 2965 S. Hulswar et al.: DMS-Rev3 https://doi.org/10.5194/essd-14-2963-2022 2.2 Data uniﬁcation (spatial and temporal) This was done by ﬁrst sorting the data ac- cording to the updated dynamic Longhurst provinces (de- tailed in Sect. 2.3). Then the 99.9 percentile threshold was calculated for every province. This helped to identify and remove extreme values in each province, thereby reducing the number of data points to 872 523 (∼0.1 % data points were rejected). While this method might result in a nega- tive bias during blooms, the extreme values could add bias in the monthly ﬁrst-guess means of the related provinces due to the low number of data in each pixel. Details of the DMS concentration percentile thresholds for each province, along with the amount of data before and after applying the ﬁlters are given in Table 1. The range of thresholds for provinces that had sufﬁcient data (Sect. 2.4) was between ∼2 in the Red Sea to 344.1 nM close to Antarctica (Table 1). By com- parison, L11 used a single threshold of 148 nM for the global oceans, which ﬁltered out the higher values only in coastal and highly productive environments. The consolidated data contain observations made using vastly different measurement techniques. Until 2000, the GC-based instruments were widely used for the measure- ment of DMS, but later, high-frequency instruments based on high-resolution mass spectrometry (such as the Chemi- cal Ionization Mass Spectrometer (CIMS) and the Membrane Inlet Mass Spectrometer (MIMS)) started to become more common. This resulted in data with different spatial and tem- poral resolutions. We analyzed the sampling frequency of the observations (Fig. 2a) to identify the most common sampling frequency. It was found to be greater than or equal to 24 h when all the campaigns were considered. However, this is mostly because older campaigns reported data once a day or less frequently. All more recent campaigns have a much higher sampling frequency, at times as frequent as a data point every second. The high-resolution data collected using the mass spectrometry techniques contributed up to ∼59 % of the raw global database. However, the high-resolution data represent only 15 % of the campaigns (Fig. 2b and c). The climatology, which works on averages, can thus become biased towards the data procured using high-resolution in- struments. Hence, to avoid this bias and standardize the in- terpolation ﬁeld to facilitate further analysis, we binned all the data to frequencies of 1 min, 1 h, and 1 d. 2.1 Data consolidation and cleanup These regions had little data in the L11 cli- matology and were hence heavily reliant on interpolations (Tesdal et al., 2016). The newer data from these remote re- gions will help to make realistic estimates of concentrations, hence reducing dependence on the interpolated estimates. The ﬁrst step after consolidating all the available data was to ensure data quality. To do this, the values with incorrect location data were removed (in all about 83 data points had location on land and hence were removed). Unfortunately, as explained by Lana et al. (2011), there is no robust criteria or accepted method for the selection or elimination of historical data. However, intercomparison studies show that the histori- cal methods and new high-resolution methods reproduce data within a range of ±25 % (Bell et al., 2012; Swan et al., 2014). Hence, to avoid the undesirable effects that potentially erro- neous and extreme values might produce during the objective analysis, a two-step ﬁltering was conducted. The lower limit for the observations was set to 0.001 nM (typical detection limits are higher than the set lower limit). While we delete the data below this value to avoid erroneous data, inclusion of the deleted data with a ﬁxed lower limit affected the ﬁnal DMS climatology by <0.001 %. The K99 and L11 clima- tologies also applied a 99.9 percentile upper threshold ﬁlter Here we present the third revision of the bottom-up DMS climatology (DMS-Rev3), wherein we have amended the al- gorithm keeping in mind the shortcomings of K99 and L11. Earth Syst. Sci. Data, 14, 2963–2987, 2022 2966 S. Hulswar et al.: DMS-Rev3 Figure 1. Data from different sources were put together for creating the raw input dataset, which consisted of 873 539 points. The data in blue circles represent data that were used in the L11 DMS climatology, and the green represent the new data. Post quality control, and data uniﬁcation for addressing temporal and spatial sampling biases, 48 898 data points were used as an input for the DMS-Rev3 climatology calculation algorithm (red dots). Figure 1. Data from different sources were put together for creating the raw input dataset, which consisted of 873 539 points. The data in blue circles represent data that were used in the L11 DMS climatology, and the green represent the new data. 2.1 Data consolidation and cleanup Post quality control, and data uniﬁcation for addressing temporal and spatial sampling biases, 48 898 data points were used as an input for the DMS-Rev3 climatology calculation algorithm (red dots). 2.2 Data uniﬁcation (spatial and temporal) to remove the extremely high values which could be due to erroneous observations or a result of preferential sampling in phytoplankton blooms that could bias the climatology, as suggested by Galí et al. (2018). However, the range for DMS values is expected to be different in different biogeochemical regions. For example, peak values in the open ocean olig- otrophic regions can be up to 2 orders of magnitude lower than in highly productive coastal environments. Hence, a sin- gle threshold is not applicable for the global dataset. Instead, several thresholds need to be calculated for data segregated according to the biogeochemical properties that can affect DMS production. This was done by ﬁrst sorting the data ac- cording to the updated dynamic Longhurst provinces (de- tailed in Sect. 2.3). Then the 99.9 percentile threshold was calculated for every province. This helped to identify and remove extreme values in each province, thereby reducing the number of data points to 872 523 (∼0.1 % data points were rejected). While this method might result in a nega- tive bias during blooms, the extreme values could add bias in the monthly ﬁrst-guess means of the related provinces due to the low number of data in each pixel. Details of the DMS concentration percentile thresholds for each province, along with the amount of data before and after applying the ﬁlters are given in Table 1. The range of thresholds for provinces that had sufﬁcient data (Sect. 2.4) was between ∼2 in the Red Sea to 344.1 nM close to Antarctica (Table 1). By com- parison, L11 used a single threshold of 148 nM for the global oceans, which ﬁltered out the higher values only in coastal and highly productive environments. to remove the extremely high values which could be due to erroneous observations or a result of preferential sampling in phytoplankton blooms that could bias the climatology, as suggested by Galí et al. (2018). However, the range for DMS values is expected to be different in different biogeochemical regions. For example, peak values in the open ocean olig- otrophic regions can be up to 2 orders of magnitude lower than in highly productive coastal environments. Hence, a sin- gle threshold is not applicable for the global dataset. Instead, several thresholds need to be calculated for data segregated according to the biogeochemical properties that can affect DMS production. 2.2 Data uniﬁcation (spatial and temporal) After binning the data per minute, around 516 506 data points remain of which about 326 664 points belong only to higher-resolution datasets. This would result in a highly biased input dataset. Daily binning results in 7874 data points, signiﬁcantly re- ducing the number of points available for further analysis. Earth Syst. Sci. Data, 14, 2963–2987, 2022 https://doi.org/10.5194/essd-14-2963-2022 2967 S. Hulswar et al.: DMS-Rev3 Table 1. Details of the number of raw data points, the threshold used for ﬁltering extreme values, number of data points after binning, number of months with data in each biogeochemical province, before and after substitution are given. The provinces used as a donor where substitution was made are also listed. Provinces that were substituted (with data in less than ﬁve months) are highlighted in bold. Province Code Province Biome Ocean Threshold according No. of raw data points No. of data Binned data before substitution Substituted by Binned data after substitution number name to 99.9 percentile lost due to ﬁltering which province (nM) Before After No. No. No. No. 2.2 Data uniﬁcation (spatial and temporal) ﬁltering ﬁltering of data of months of data of months 1 ETRA Eastern tropical Trades Atlantic 8.8 126 125 1 234 6 234 6 Atlantic 2 APLR Austral polar Polar Antarctic 344.1 327 720 327 462 328 9790 8 9790 8 3 ANTA Antarctic Polar Antarctic 80.3 326 020 325 693 327 7816 9 7816 9 4 SANT Subantarctic Westerly Antarctic 8.0 45 225 45 180 45 3762 9 3762 9 water ring 5 SSTC South Westerly Antarctic 19.9 17 543 17 525 18 2420 11 2420 11 subtropical convergence 6 WTRA Western tropical Trades Atlantic 7.0 382 381 1 654 8 654 8 Atlantic 7 NEWZ New Zealand Coastal Paciﬁc 28.5 1465 1464 1 930 5 930 5 coast 8 AUSE East Coastal Paciﬁc 8.6 36 35 1 44 3 AUSE + NEWZ 758 7 Australian coast 9 SUND Sunda-Arafura Coastal Paciﬁc 6.9 777 775 2 416 4 SUND + AUSE 426 5 shelves 10 NATR North Atlantic Trades Atlantic 14.6 1749 1747 2 2526 12 2526 12 tropical gyre 11 CHIN China Sea Coastal Paciﬁc 16.4 830 829 1 700 8 700 8 12 CHIL Humboldt Coastal Paciﬁc 80.3 796 795 1 958 6 958 6 current Chile coast 13 CAMR Central Coastal Paciﬁc 5.4 130 129 1 112 1 CAMR + CCAL 4966 8 American coast 14 CCAL Coastal Coastal Paciﬁc 42.1 10 981 10 970 11 4854 7 4854 7 Californian current 15 ALSK Alaska Coastal Paciﬁc 154.9 4659 4654 5 4824 9 4824 9 coastal downwelling 16 ARCH Archipelagic Trades Paciﬁc 7.3 166 165 1 50 3 ARCH + TASM 326 6 deep basins 17 WARM Western Paciﬁc Trades Paciﬁc 8.5 10 429 10 419 10 2174 6 2174 6 warm pool 18 PEQD Paciﬁc Trades Paciﬁc 44.9 1395 1394 1 1682 10 1682 10 equatorial divergence https://doi.org/10.5194/essd-14-2963-2022 Earth Syst. Sci. Data, 14, 2963–2987, 2022 Earth Syst. Sci. Data, 14, 2963–2987, 2022 https://doi.org/10.5194/essd-14-2963-2022 S. Hulswar et al.: DMS-Rev3 2968 2968 S. Hulswar et al.: DMS-Re Province Code Province Biome Ocean Threshold according No. of raw data points No. of data Binned data before substitution Substituted by Binned data after substitution number name to 99.9 percentile lost due to ﬁltering which province (nM) Before After No. No. No. No. 2.2 Data uniﬁcation (spatial and temporal) ﬁltering ﬁltering of data of months of data of months 19 PNEC North Paciﬁc Trades Paciﬁc 6.9 11 325 11 314 11 1766 10 1766 10 equatorial counter current 20 NPTG North Paciﬁc Trades Paciﬁc 6.6 8129 8121 8 1270 11 1270 11 Tropical gyre 21 NAST W Northwest Atlantic Westerly Atlantic 8.6 1281 1280 1 1616 10 1616 10 subtropical gyre 22 SPSG South Paciﬁc Trades Paciﬁc 11.4 10 539 10 528 11 4768 8 4768 8 gyre 23 TASM Tasman Sea Westerly Paciﬁc 6.8 3996 3992 4 422 4 TASM + ARCH 448 6 24 C(O)CAL California Trades Paciﬁc 48.4 4811 4806 5 2850 8 2850 8 current 25 NPSW Northwest Westerly Paciﬁc 5.0 146 145 1 196 4 NPSW + NPSE 264 7 Paciﬁc subtropical 26 NPSE Northeast Westerly Paciﬁc 8.3 130 129 1 192 6 192 6 Paciﬁc subtropical 27 NPPF North Paciﬁc Westerly Paciﬁc 16.9 251 250 1 378 7 378 7 polar front 28 KURO Kuroshio Westerly Paciﬁc 10.8 697 696 1 728 4 KURO + NPSE 788 6 current 29 PSAW Western Paciﬁc Westerly Paciﬁc 11.0 252 251 1 290 5 290 5 subarctic gyres 30 PSAE Eastern Paciﬁc Westerly Paciﬁc 27.9 3043 3040 3 1288 5 1288 5 subarctic gyres 31 BERS North Paciﬁc Polar Paciﬁc 151.0 3856 3852 4 3928 9 3928 9 epicontinental sea 32 GFST Gulf Stream Westerly Atlantic 16.1 2073 2071 2 1460 11 1460 11 33 NADR North Atlantic Westerly Atlantic 27.1 4706 4701 5 3224 10 3224 10 Drift 34 AUSW Western Australian Coastal Indian 3.1 5997 5991 6 314 3 AUSW + AUSE + SUND 488 6 and Indonesian coast 35 INDE Western India Coastal Indian 220.5 98 97 1 176 8 176 8 coast 36 INDW Eastern India Coastal Indian 6.8 98 97 1 186 3 INDW + ARAB 322 8 coast 37 ARAB Northwest Arabian Westerly Indian 16.9 150 149 1 268 8 268 8 Sea upwelling 38 REDS Red Sea, Coastal Indian 2.0 14 13 1 26 1 REDS + ARAB 266 8 Arabian Gulf 39 EAFR Eastern India Coastal Indian 20.1 303 302 1 76 2 EAFR + ISSG 1112 7 coast https://doi.org/10.5194/essd-14-2963-2022 Earth Syst. Sci. Data, 14, 2963–2987, 2022 2969 S. Hulswar et al.: DMS-Rev3 Province Code Province Biome Ocean Threshold according No. of raw data points No. S. Hulswar et al.: DMS-Rev3 This resulted in a uniﬁed dataset of 48 898 data points for DMS-Rev3, re- ducing bias towards a particular measurement technique or campaign while ensuring that enough data points are avail- able in all the provinces for further analysis. Although keep- ing a higher temporal resolution would result in more data points, this calculation also helps reduce the disparity in the spatial resolution of the dataset, with the average distance between two consecutive data points for a single campaign coming to about 0.2◦(assuming a constant ship speed of 10 knots). In certain regions of the oceans, where the data availability is higher, a high-resolution climatology of up to 0.2◦spatial resolution could be attempted, but the majority of the world’s oceans are under-sampled or moderately sam- pled and hence a coarser resolution results in a more realis- tic climatology. Additionally, since most of the climate mod- els also work on 1◦spatial resolution or lower, this dataset was used to create a 1◦climatology like that produced earlier in K99 and L11. We tested the sensitivity of the climatol- ogy to the temporal resolution. While the global mean did not change signiﬁcantly depending on whether we used the minute, hour or daily binned data, the minute binned data re- sulted in regional means biased towards single campaigns in regions where high-resolution data are available. Since the data were not uniformly distributed across the oceans, the strategy adopted by the earlier K99 and L11 cli- matologies was to segregate the data in different provinces based on their biogeochemical properties (chlorophyll-a con- centrations, nitrate concentration, salinity, etc.) as deﬁned by Longhurst (2007) (Fig. S2a). Computing mean values repre- senting these provinces helped in creating a ﬁrst-guess global distribution, which is the ﬁrst step towards a climatology at a coarse resolution. However, satellite images for the biogeo- chemical parameters reveal that these features are highly dy- namic in terms of geographical extents (Devred et al., 2007; Oliver and Irwin, 2008; Reygondeau et al., 2013). Hence, us- ing a static province approach for the whole year (as in K99 and L11), though practical, has an inherent drawback of not accounting for the spatial/temporal changes in the biogeo- chemistry. This affects the estimations of DMS, especially along the borders of the provinces where static boundaries were used in K99 and L11. S. Hulswar et al.: DMS-Rev3 S. Hulswar et al.: DMS-Rev3 S. Hulswar et al.: DMS-Rev3 S. Hulswar et al.: DMS-Rev3 Table 2. Annual DMS ﬂux (TgS yr−1) per 10◦latitudinal band obtained from DMS-Rev3 is given in the table along with the annual DM ﬂux obtained from L11 for comparison. Latitude bands REV3 L11 90 to 80◦N 0.0 0.0 80 to 70◦N 0.1 0.1 70 to 60◦N 0.2 0.2 60 to 50◦N 0.8 0.9 50 to 40◦N 1.3 1.5 40 to 30◦N 1.5 1.5 30 to 20◦N 1.5 1.4 20 to 10◦N 2.4 2.6 10◦N to 0◦EQ 2.2 2.6 0◦EQ to −10◦S 2.0 2.2 −10 to −20◦S 3.4 3.5 −20 to −30◦S 3.0 3.0 −30 to −40◦S 2.5 2.7 −40 to −50◦S 2.6 2.8 −50 to −60◦S 2.4 2.1 −60 to −70◦S 1.0 0.9 −70 to −80◦S 0.1 0.1 −80 to −90◦S 0.0 0.0 Total 27.1 28.1 Hence, we binned the input data into hourly results, as it helped to degrade the high temporal resolution data to the same resolution as the rest of the historical data This resulted 2.3 A ﬁrst-guess monthly climatology according to dynamic Longhurst provinces S yr−1) per 10◦latitudinal band obtained from DMS-Rev3 is given in the table along with the annual DMS parison. Table 2. Annual DMS ﬂux (TgS yr−1) per 10◦latitudinal band obtained from DMS-Rev3 is given in the table along with the annual DMS ﬂux obtained from L11 for comparison. Latitude bands REV3 L11 90 to 80◦N 0.0 0.0 80 to 70◦N 0.1 0.1 70 to 60◦N 0.2 0.2 60 to 50◦N 0.8 0.9 50 to 40◦N 1.3 1.5 40 to 30◦N 1.5 1.5 30 to 20◦N 1.5 1.4 20 to 10◦N 2.4 2.6 10◦N to 0◦EQ 2.2 2.6 0◦EQ to −10◦S 2.0 2.2 −10 to −20◦S 3.4 3.5 −20 to −30◦S 3.0 3.0 −30 to −40◦S 2.5 2.7 −40 to −50◦S 2.6 2.8 −50 to −60◦S 2.4 2.1 −60 to −70◦S 1.0 0.9 −70 to −80◦S 0.1 0.1 −80 to −90◦S 0.0 0.0 Total 27.1 28.1 2.3 A ﬁrst-guess monthly climatology according to dynamic Longhurst provinces 2.3 A ﬁrst-guess monthly climatology according to dynamic Longhurst provinces Hence, we binned the input data into hourly results, as it helped to degrade the high temporal resolution data to the same resolution as the rest of the historical data. Earth Syst. Sci. Data, 14, 2963–2987, 2022 2.2 Data uniﬁcation (spatial and temporal) of data Binned data before substitution Substituted by Binned data after substitution number name to 99.9 percentile lost due to ﬁltering which province (nM) Before After No. No. No. No. ﬁltering ﬁltering of data of months of data of months 40 ISSG Indian South Trades Indian 12.5 13 574 13 560 14 1738 7 1738 7 subtropical gyre 41 MONS Indian monsoon Trades Indian 12.1 352 351 1 586 7 586 7 gyre 42 SARC Atlantic Polar Atlantic 66.7 1296 1295 1 1702 6 1702 6 sub-Arctic 43 BENG Benguela Coastal Atlantic 13.4 6399 6393 6 652 5 652 5 current coast 44 FKLD Southwest Coastal Atlantic 18.9 168 167 1 276 4 FKLD + BRAZ 364 7 Atlantic shelves 45 BRAZ Brazilian Coastal Atlantic 23.7 1060 1059 1 296 7 296 7 current coast 46 ARCT Atlantic Arctic Polar Atlantic 19.0 6311 6305 6 3572 9 3572 9 47 NAST E Northeast Atlantic Westerly Atlantic 19.7 5889 5883 6 4126 12 4126 12 subtropical gyre 48 CARB Caribbean Trades Atlantic 23.0 992 991 1 1318 9 1318 9 49 MEDI Mediterranean Sea Westerly Atlantic 60.5 625 624 1 864 11 864 11 50 NWCS Northwest Atlantic Coastal Atlantic 28.6 3329 3326 3 3102 11 3102 11 shelves 51 GUIA Guianas coast Coastal Atlantic 2.3 8 7 1 12 1 GUIA + WTRA 432 8 52 GUIN Guinea current Coastal Atlantic No data 0 0 0 0 ETRA 234 6 coast 53 CNRY Canary current Coastal Atlantic 32.0 73 72 1 138 6 138 6 coast 54 NECS Northeast Atlantic Coastal Atlantic 198.5 2661 2658 3 4376 11 4376 11 shelves 55 SATL South Atlantic Trades Atlantic 6.7 14 072 14 058 14 1866 9 1866 9 gyre 56 BPRL Boreal polar Polar Arctic 19.3 4006 4002 4 2970 8 2970 8 0.5194/essd-14-2963-2022 Earth Syst. Sci. Data, 14, 2963– Earth Syst. Sci. Data, 14, 2963–2987, 2022 Earth Syst. Sci. Data, 14, 2963–2987, 2022 2970 S. Hulswar et al.: DMS-Rev3 To address this, we make use of the dynamic Longhurst provinces based on the work of Rey- gondeau et al. (2013), who deﬁned the dynamic boundaries based on satellite data between 1997–2007 (Fig. S2b). The monthly data were segregated according to the changing geo- graphical extents for all the provinces and the means of these separated data were used for creating the ﬁrst-guess ﬁelds (Fig. S3). The advantage of using dynamic biogeochemical provinces is that they helped to resolve the environmental, biogeochemical dynamics better on a regional scale, espe- Earth Syst. Sci. Data, 14, 2963–2987, 2022 https://doi.org/10.5194/essd-14-2963-2022 2971 S. Hulswar et al.: DMS-Rev3 S. Hulswar et al.: DMS-Rev3 2971 Figure 2. (a) Frequency distribution of the sampling interval for the campaigns included in this study are shown. Note that the “24 h” bin represents temporal resolution lower than or equal to 24 h. (b) The type of measurement technique used to measure DMS during the individual campaigns. The unknown dataset/campaigns resemble the frequency of a GC instrument. (c) The number of raw data points according to the measurement technique is shown. The number of data is dominated by CIMS/MIMS based measurements, but the number of campaigns is dominated by the GC measurements. Figure 2. (a) Frequency distribution of the sampling interval for the campaigns included in this study are shown. Note that the “24 h” bin represents temporal resolution lower than or equal to 24 h. (b) The type of measurement technique used to measure DMS during the individual campaigns. The unknown dataset/campaigns resemble the frequency of a GC instrument. (c) The number of raw data points according to the measurement technique is shown. The number of data is dominated by CIMS/MIMS based measurements, but the number of campaigns is dominated by the GC measurements. moteness. This results in an uneven distribution of data in the different provinces with respect to space and time. Af- ter segregation, some provinces had data for all 12 months of the year, while others had as little as 1–2 months of data (Fig. 3; Table 1). Hence, there was a need to ﬁll the monthly gaps in some of the provinces with data from an appropri- ate “donor” province to provide sufﬁcient data for a valid in- terpolation for the annual variation in each province. S. Hulswar et al.: DMS-Rev3 Across all the provinces, it was seen that for provinces with only 1–2 months with data, substitution from (or merging with, in case the receiving province had no data at all) a sin- gle province would result in 5 months with data, provid- ing enough data for interpolation. Hence, if a province had data for less than 5 months, it was selected for substitu- tion/merging, thus all the “receiver” provinces had at least 5 months with data after substitution/merging. The data from the donor province were normalized by scaling up or down based on the ratio of concentrations of the donor and re- ceiver provinces for the common months. In case the receiv- ing province had no data at all, the data were substituted from the donor province. Details of the biogeochemical provinces that were substituted or merged are given in Table 1. cially in different seasons. The difference due to the inclu- sion of the seasonal variation in the province boundaries is discussed further in Sect. 3.2. The DMS-Rev3 also provides an option to create ﬁrst- guess ﬁelds using medians instead of means across the provinces. The median is not affected by the skewed dataset due to a few larger values. The median will also minimize the effect of the blooms that drive high DMS emissions. This approach thus helps to recreate background values bet- ter than using means, but can lead to an overall underesti- mation in regions where blooms are more common, but ob- servations have not been made frequently enough to capture them. Keeping this in mind, we used the province means for the calculation of the climatology, although the values using medians are also reported. Earth Syst. Sci. Data, 14, 2963–2987, 2022 https://doi.org/10.5194/essd-14-2963-2022 2.4 Data substitution, merging and interpolation The number of data points available per province differs greatly depending on the sampling carried out in that re- gion during a particular month. Since these are in situ ob- servations, some regions are adequately sampled, some are moderately sampled, while some are rarely or never sam- pled due to physical constraints like accessibility and re- In all, 14 provinces that needed substitution or merg- ing, i.e., provinces with less than 5 months that contained https://doi.org/10.5194/essd-14-2963-2022 Earth Syst. Sci. Data, 14, 2963–2987, 2022 2972 S. Hulswar et al.: DMS-Rev3 Figure 3. The data distribution in different biogeochemical provinces is different owing to the time, location, and frequency of observation. The black squares represent the monthly mean of data with standard deviations shown as error bars. The number of hourly binned observations in a month is shown below the x-axis. Figure 3. The data distribution in different biogeochemical provinces is different owing to the time, location, and frequency of observation The black squares represent the monthly mean of data with standard deviations shown as error bars. The number of hourly binned observation in a month is shown below the x-axis. Figure 3. The data distribution in different biogeochemical provinces is different owing to the time, location, and frequency of observation. The black squares represent the monthly mean of data with standard deviations shown as error bars. The number of hourly binned observations in a month is shown below the x-axis. data, were identiﬁed. The provinces off the eastern coast of Australia (TASM and the ARCH) were inter-substituted be- tween each other. The coastal province, AUSE was used to ﬁll the northern coastal province SUND. Although south of New Zealand, NEWZ was also updated with the data from AUSE due to biogeochemical similarities with AUSE. The data from AUSW, AUSE and SUND were merged to com- plete AUSW. In the northern Paciﬁc, NPSW and KURO were merged with NPSE. In equatorial regions, the coastal province on the east American coast, CAMR was substituted by CCAL following K99 and L11. The province FKLD re- ceived data from the adjacent coastal province BRAZ. The coastal Atlantic provinces GUIN and GUIA were substituted by the adjacent provinces ETRA and WTRA, respectively, following L11. Due to the usage of dynamic provinces, the province GUIN is present only in November, Decem- ber, January, and June. 2.4 Data substitution, merging and interpolation The Canary coastal current seems to highly inﬂuence the region occupied by GUIN province converting it to CNRY for the rest of the months. The In- dian Ocean coastal provinces INDE, REDS, and EAFR are under-sampled despite the importance for the highly popu- lated South Asian region’s respective countries. Data from the ARAB province are merged in the INDE and REDS provinces, while EAFR is merged with data from ISSG. The substitution or merging was done to ensure that the dependence on interpolation to estimate the values for the remaining months was as low as possible. However, even after this step, it was obvious that the data required some amount of interpolation as only two provinces had data for all 12 months (NATR and NASTE), while other provinces had data between 11 to 5 months (Fig. 3). Hence, to ob- tain monthly data for each province to estimate the month- to-month variability, it was interpolated to estimate the miss- ing monthly data following the recommendations of Lana et al. (2011). The above ensured that month-to-month variabil- ity was created for every province (Fig. 4). https://doi.org/10.5194/essd-14-2963-2022 Earth Syst. Sci. Data, 14, 2963–2987, 2022 2973 S. Hulswar et al.: DMS-Rev3 Figure 4. The substituted and interpolated monthly mean data (black squares) with respective standard deviations for each province are shown. The number of hourly binned observations in a month is shown below the x-axis, where 0 indicates that the data were interpolated. Figure 4. The substituted and interpolated monthly mean data (black squares) with respective standard deviations for each province shown. The number of hourly binned observations in a month is shown below the x-axis, where 0 indicates that the data were interpolate Figure 4. The substituted and interpolated monthly mean data (black squares) with respective standard deviations for each province are shown. The number of hourly binned observations in a month is shown below the x-axis, where 0 indicates that the data were interpolated. Earth Syst. Sci. Data, 14, 2963–2987, 2022 2.8 Uncertainties in the climatology Estimating the uncertainty in the climatology is difﬁcult due to the variety of methodologies and the lack of reported ob- servational uncertainties for most of the campaigns. The un- certainties in the DMS climatology were hence estimated from the point of data uniﬁcation (Sect. 2.2) and will there- fore be an underestimate of the total uncertainty in the ﬁnal climatology. First, the standard deviations were computed for the hourly binned observations. This standard deviation was further propagated while spatially averaging the data when creating the 1◦× 1◦bins. An additional standard deviation was computed for the ﬁrst-guess ﬁelds, which use the means across the biogeochemical provinces (Sect. 2.3). These stan- dard deviations from the hourly binned data, pixel binned data and province binned data were then propagated through the calculation of pooled standard deviations. The uncer- tainty introduced due to the smoothing using the Shuman and Barnes ﬁlters (Sect. 2.5) is difﬁcult to estimate, and hence the same smoothing ﬁlters were also applied to the standard de- viations to create a monthly and annual uncertainty database corresponding to the DMS concentrations. While these un- certainties are not a complete estimation of the errors, they provide an estimate of the range of DMS concentrations in the climatology. The calculated global monthly and annual standard deviations are shown in Fig. S7. Considering that large portions of the polar waters can be under sea ice at different times during the year, it is neces- sary to apply a sea-ice ﬁlter to modulate emissions in these regions. This can be done in two ways: (i) before creating the DMS climatology to ﬁlter out data that are under sea-ice regions; or (ii) after the climatology is created as a mask. A sea-ice monthly climatology (Fig. S6) was created using data obtained from the Defense Meteorological Satellite Program, F13 Special Sensor Microwave/Imager (SSM/I), and Scan- ning Multichannel Microwave Radiometer (SMMR). The DMS-Rev3 code replaces the data with zeros when consid- ered to be under sea ice, where sea ice is less than or equal to the set threshold. For example, the Antarctic coastal region is a DMS hotspot during the southern hemisphere summer and is a sea-ice covered region during the winter with no DMS emissions. An annual average ignoring these regions during winter would result in a hotspot with the annual average bi- ased towards the summer values. 2.5 Incorporation of observed VLS for smoothing the data points that it inﬂuences) is used as a “weight”. The ROI used by K99 and L11 was 555 km (∼5◦, close to the Rossby radius in the tropics). The main aim of using such a ﬁlter is to ensure that the DMS variability is captured in the climatology, but this also causes patchiness in the resultant climatology. We tested the sensitivity of the algorithm to the ROI value, different ﬁxed values of ROI: 555, 100, 75, 50, 25, 10, and 7.5 km. On a global scale, each ROI resulted in a dif- ferent global mean, but once the ROI dropped below 25 km, the mean value stabilized at ∼2.44 nM (Fig. S4). Although the global mean did not change by much, large regional dif- ferences were observed, with smaller ROI values showing less patchiness in the resultant climatology, indicating that choosing an appropriate ROI is crucial for an accurate esti- mation of the DMS distribution. The ﬁrst-guess database has a uniform distribution within a province as it is the mean value in the province. It also has a sharp and unrealistic transition at the boundaries. Hence, to create a realistic distribution of DMS values at the bound- aries, a Shuman ﬁlter, which is an unweighted 11-point smoothing ﬁlter (Shuman, 1957) was employed. On top of these smoothed ﬁrst-guess ﬁelds, a 1◦× 1◦spatially binned DMS concentration ﬁeld was superimposed by replacing the pixels where spatially binned data are available. This repli- cates the differences in the DMS concentrations within the individual biogeochemical provinces. The average DMS val- ues in a particular pixel (1◦× 1◦) contribute to the value of the pixel. However, there will be an inﬂuence of surrounding pixels on each of the pixels. This was accounted for using the Barnes ﬁlter (Barnes, 1964), which is a convergent weighted- average interpolation scheme where the radius of inﬂuence (ROI, which is the average distance between a grid point and The L11 climatology used the 555 km value since little information was available on DMS variability length scales (VLS) in the oceans, which is the distance over which one https://doi.org/10.5194/essd-14-2963-2022 Earth Syst. Sci. Data, 14, 2963–2987, 2022 2974 S. Hulswar et al.: DMS-Rev3 S. Hulswar et al.: DMS-Rev3 would expect the DMS concentration to signiﬁcantly change as one moves over the ocean surface. 2.6 Using sea-ice cover masks In addition to the oceanic sources of DMS, the presence of sea ice affects the emissions of DMS. The contribution of sea ice to the total DMS production during the melt pe- riod was simulated by Hayashida et al (2017) and showed episodic spikes of up to 8 µmol m−2 d−1. However, the exact extent of this contribution is not known due to the scarcity of ﬁeld measurements. Further model simulations highlighted the importance of addressing the sea-ice ecosystem sepa- rately for better DMS ﬂux estimates. The K99 and L11 DMS climatologies applied a sea-ice ﬁlter to mask out any emis- sions from areas covered with sea ice, which most likely un- derestimates the total DMS ﬂux. 2.8 Uncertainties in the climatology 2.8 Uncertainties in the climatology 2.7 Sea–air DMS ﬂux estimations The sea–air DMS ﬂux was estimated using the output of DMS-Rev3 to understand the impact of the new climatology. The estimations were carried out following the same pro- cedure as Lana et al. (2011) which used the Nightingale et al. (2000) parameterization. This parameterization is based on the DMS gas transfer velocity utilizing the wind speed at 10 m and the climatological sea surface temperature (SST). The inputs for calculating the ﬂux were for the same period as that used for L11 (1978–2008) and were obtained from the NCEP/NCAR reanalysis project (http://www.esrl.noaa.gov/, last access: 5 July 2021). This was necessary for a one-to-one comparison of DMS-Rev3 and L11 ﬂux estimations. 2.5 Incorporation of observed VLS for smoothing However, more recent studies based on high-frequency measurements (Asher et al., 2011; Royer et al., 2015) have been able to quantify the vari- ability in DMS and show that it occurs at a much smaller scale in the order of 15 to 50 km. These ﬁndings are con- sistent with initial ﬁndings from a new global VLS analy- sis of seawater DMS (using data from 2004–2019), which follows the method of Hales and Takahashi (2004) to give a global mean DMS VLS of ∼17 km (Fig. S5). The DMS- Rev3 climatology uses VLS results from 11 biogeochemical provinces to estimate the DMS VLS in other provinces and to create a simple DMS VLS distribution map. Monthly ge- ographical variability in VLS was estimated within the dy- namic province boundaries, after applying a Shuman ﬁlter as detailed above (Fig. 5). Monthly VLS distribution was then used for computing the convergent weighted-average inter- polation for DMS using the Barnes ﬁlter. The difference in the resultant DMS climatology due to the inclusion of the VLS information is discussed further in Sect. 3.3. data uniﬁcation, but before the creation of the climatology, the mask removed only 17 data points (88 data points for 30 % sea-ice cover threshold) from the 48 898 data points that are deemed to be under the sea ice. This does not lead to a signiﬁcant change in the overall calculations. However, if the mask is applied after the creation of the climatology, large portions from the polar provinces come under the mask and lead to changes in the monthly and the annual global mean values (detailed in Sect. 3.4). 2.7 Sea–air DMS ﬂux estimations 2.8 Uncertainties in the climatology A sea-ice threshold cutoff value of 50 % was used (which can be changed in the code). If the ﬁlter was applied after https://doi.org/10.5194/essd-14-2963-2022 Earth Syst. Sci. Data, 14, 2963–2987, 2022 2975 S. Hulswar et al.: DMS-Rev3 Figure 5. The DMS variability length scales (VLS) used for the weighted-average interpolation computation is shown. The VLS in 11 provinces was based on past observations, and the VLS in the other provinces was estimated by looking for biogeochemical similarities. Figure 5. The DMS variability length scales (VLS) used for the weighted-average interpolation computation is shown. The VLS in 11 provinces was based on past observations, and the VLS in the other provinces was estimated by looking for biogeochemical similarities. Figure 5. The DMS variability length scales (VLS) used for the weighted-average interpolation computa provinces was based on past observations, and the VLS in the other provinces was estimated by looking for b 3.1 Features in DMS-Rev3 3.1 Features in DMS-Rev3 3.1 Features in DMS-Rev3 3 Results and discussion a clear peak in December and January, followed by the ﬁrst minimum in April ∼2.19 nM (∼2.07 nM∗). The values then show a modest increase through the months of May– August (with values around ∼2.15 nM) due to the northern hemispheric summer. This can be attributed to the higher DMS values in the northern high latitudes. September shows the second minimum lowest mean DMS values of 1.72 nM (1.56 nM∗). The region south of 60◦S has the highest average concentrations, with large peaks seen in the high productivity regions such as the Weddell Sea and the Ross Sea. Outside this polar environment, higher values are seen on the south- west coast of Africa and the west coast of South America. El- evated concentrations are also observed in the Mediterranean Sea and the Arctic Ocean close to Norway and Greenland. The waters close to Alaska and California also show higher than the global mean concentrations. 3.1.1 Global Figure 6. Distribution of the monthly and annual DMS concentrations as estimated by DMS-Rev3 climatolo 3.1.1 Global Figure 6 shows the monthly and annual climatological distri- bution of surface seawater DMS concentrations as estimated by the DMS-Rev3 algorithm after the application of a 50 % sea-ice cover mask (the distribution without the application of the sea-ice mask is shown in Fig. S8). For the annual mean climatology, the DMS concentrations range between 0.1 to 7 nM, of which ∼85 % of the grid points contain DMS concentrations below 3 nM (Fig. S9). When globally aver- aged across all the oceans, DMS-Rev3 estimates the annual mean at 2.26 nM (2.39 nM with no sea-ice mask – henceforth nM∗indicates values without a sea-ice mask). If province medians are considered instead of the province means, the global annual average concentration reduces to 1.79 nM (1.73 nM∗), about ∼26 % lower than the L11 climatology (∼21 % lower than DMS-Rev3 calculated using means). The highest global averages are observed in November, Decem- ber, January, and February with average values of 2.51 nM (2.85 nM∗), 3.17 nM (3.46 nM∗), 3.32 nM (3.57 nM∗) and 2.48 nM (2.67 nM∗), respectively (Fig. S10). The higher val- ues in these months are mainly due to large concentrations in the southern hemisphere with coastal Antarctica dominat- ing the peak values. The global mean annual cycle shows A clear regional annual cycle is observed in most loca- tions, with the southern and northern hemispheres peaking during their respective summers. The range of values also differs according to the months, but higher values of as much as ∼8.8 to 14.7 nM (10–15.1 nM∗) are seen in the Antarctic coastal province (APLR), especially in November, Decem- ber, and January. However, even during these months, more than ∼60 % (83 % in November) of the grid points contain concentrations below 3 nM (Fig. S9). The ﬁnal output for in- dividual provinces is shown in Fig. S11. Earth Syst. Sci. Data, 14, 2963–2987, 2022 https://doi.org/10.5194/essd-14-2963-2022 2976 S. Hulswar et al.: DMS-Rev3 6. Distribution of the monthly and annual DMS concentrations as estimated by DMS-Rev3 climatology with a 50 % sea-ice mask. Figure 6. Distribution of the monthly and annual DMS concentrations as estimated by DMS-Rev3 climatology with a 50 % sea-ice mask. e monthly and annual DMS concentrations as estimated by DMS-Rev3 climatology with a 50 % sea-ice mask. Figure 6. Distribution of the monthly and annual DMS concentrations as estimated by DMS-Rev3 climatology with a 50 % sea-ice mask. 3.1.3 Extra-tropics The southern extratropical region gets split into three sectors: the Indian Ocean sector, the Atlantic Ocean sector, and the Paciﬁc Ocean sector. The northern extra-tropical region has two sectors: the Paciﬁc Ocean and the Atlantic Ocean. In the southern hemisphere, in the Indian Ocean extra- tropical region, the monthly variation observed in the po- lar province APLR is also noticed in the adjacent province ANTA, which shows peak monthly mean concentrations of 7.76 nM (8.32 nM∗) in December and 7.56 nM (7.69 nM∗) in January (Fig. S11). The adjacent provinces, SANT and SSTC, form a part of all the sectors mentioned above, al- though divided by the South American and the African con- tinents. These provinces follow a similar annual variability as the APLR and ANTA provinces, although the peak val- ues are lower (Fig. S11). These concentrations reﬂect the summertime productivity of the Southern Ocean during sum- mer. The province ISSG in the Indian Ocean sector, spread over the tropics and extra-tropics, follows a similar seasonal trend, although being further away from the polar region, it has much lower month-to-month variations. The highest concentrations are observed in the months of November– December–January. During the rest of the year, the values are lower and stay close to ∼2.2 nM, with a slight increase in June to 2.59 nM (Fig. S11). The province to the west of ISSG (EAFR) also has a similar annual trend with similar concentrations to those observed in the ISSG province. The slightly higher concentration in June in these two provinces is attributed to winter blooms occurring off the Madagascar coast (Dilmahamod et al., 2019). This trend is different in the AUSW province (east of ISSG), where the values are also higher during November (2.46 nM), December (3.19 nM), and January (3.24 nM), but a clear low is observed during August (1.05 nM) (Fig. S11). g ( ) ( g ) Provinces in the northern extra-tropical regions (BERS, PSAW, PSAE, NECS) typically show higher values in the northern hemispheric summer. The provinces in the North Atlantic extra-tropical gyre (NAST(W), NAST(E), GFST, NWCS, NECS and MEDI) typically show peaks during the northern summer. This pattern results from a combination of plankton seasonal species succession and short-term sun- light effects on DMS budgets, stimulating DMS production, inhibiting bacterial consumption, enhancing photolysis, etc. (Galí and Simó, 2015). 3.1.2 Polar of ice cover (Fig. 6). The concentrations increase through spring and summer until January, after which the values de- crease to April which has an average of 1.22 nM (1.23 nM∗). Over these months, since most of the APLR province is un- der sea ice, it does not make a large contribution to the global monthly mean DMS. Similarly, most of the polar province of BPLR also gets masked by sea ice during the northern win- ter. The peak DMS concentration was 4.31 nM (2.92 nM∗) during May (Fig. S11). In the North Atlantic region, phy- toplankton blooms dominate the Labrador and the Grand Banks coastal regions (ARCT province) in spring and sum- Regionally, the highest values were found in the polar biomes during their respective summers (60◦to 90◦both in the north- ern and southern hemispheres). The APLR showed the high- est levels of DMS with peak monthly averaged concentra- tions of 12.83 nM (13.34 nM∗) and 14.72 nM (15.16 nM∗) in December and January, respectively (Fig. S11). The DMS concentration was 0.58 nM in September. However, since the region is under the ice during this season, this value corre- sponds to the concentration limited to a smaller region to the north of the Weddell Sea, which stays exposed and out https://doi.org/10.5194/essd-14-2963-2022 Earth Syst. Sci. Data, 14, 2963–2987, 2022 2977 S. Hulswar et al.: DMS-Rev3 mer (Friedland et al., 2016), driving high DMS concentra- tions. A similar peak is observed in the SARC province with elevated DMS values during the same seasons (Fig. S11). radiation, wind stress and MLD similar to most provinces, but the variation is large for these variables (Chiswell et al., 2013). Variability in the DMS concentrations follows vari- ability in chlorophyll and sunlight with the highest concen- trations observed in summer, reducing in autumn with a de- creasing trend until the summer of the following year. The SSTC DMS concentrations are constant throughout the year, although from July to October DMS levels are consistently lower. This is most likely due to the autumn, winter and spring phytoplankton blooms observed in the TASM and NEWZ regions (Chiswell et al., 2013) (Fig. S11). 3.1.3 Extra-tropics These higher values in the North Atlantic provinces are mostly associated with the Phaeocystis pouchetii and coccolithophores blooms occurring in the months of May–July (Iida et al., 2002; Ma- trai et al., 2007; Matrai and Keller, 1993) (Figs. S11 and 6). In the Paciﬁc region, the BERS, PSAW, and PSAE provinces show higher values during May followed by lower concentrations in June and the concentrations rise again in July. A gradual decrease follows the summer peak, and the lowest values are observed during winter in December– January. In the Bering Sea and adjacent regions, blooms tend to form due to the dynamics of sea-ice retreat in spring (Jin et al., 2007; Sigler et al., 2014). This most likely contributes to the maxima observed in the respective provinces. Although signiﬁcant blooms were also reported in autumn (Ardyna et al., 2014), the magnitude of increase in the DMS concentra- tions is smaller after more data have been added. In the Paciﬁc region, the BERS, PSAW, and PSAE provinces show higher values during May followed by lower concentrations in June and the concentrations rise again in July. A gradual decrease follows the summer peak, and the lowest values are observed during winter in December– January. In the Bering Sea and adjacent regions, blooms tend to form due to the dynamics of sea-ice retreat in spring (Jin et al., 2007; Sigler et al., 2014). This most likely contributes to the maxima observed in the respective provinces. Although signiﬁcant blooms were also reported in autumn (Ardyna et al., 2014), the magnitude of increase in the DMS concentra- tions is smaller after more data have been added. In the southern hemispheric Atlantic Ocean sector, the provinces SATL, FKLD, BRAZ and BENG are a part of the South Atlantic Gyre. The mean values in these provinces decrease from approximately 2 nM in January to less than 1 nM in May (Fig. S11). The African coastal province, BENG shows higher values in January (4.09 nM) and March (3.59 nM) with lower concentrations in February (2.54 nM). This province does not show signiﬁcant variation throughout the year owing to the continual supply of nutrient-rich wa- ter due to an upwelling that supports primary production and hence DMS production (Jury and Brundrit, 1992) (Fig. S11). In the southern hemispheric Atlantic Ocean sector, the provinces SATL, FKLD, BRAZ and BENG are a part of the South Atlantic Gyre. https://doi.org/10.5194/essd-14-2963-2022 3.1.3 Extra-tropics The provinces NECS and MEDI show a similar trend to the polar province SARC. In addition to the summer peaks, an additional peak is also observed dur- ing the northern spring that has higher values than the peak in summer. The trends in the NECS province appear to be dom- inated by the spring blooms of the Baltic and the North Sea (April = 6.71 nM; May = 8.55 nM). A second bloom driven by dinoﬂagellates has been reported to occur in July (Hjerne et al., 2019) and coincides with a second peak in the DMS concentrations at 5.98 nM. The GFST and NWCS provinces show similar values and trends with twin maxima in spring and summer. Being a coastal province, NWCS shows consis- tently higher values than the GFST province. These higher values in the North Atlantic provinces are mostly associated with the Phaeocystis pouchetii and coccolithophores blooms occurring in the months of May–July (Iida et al., 2002; Ma- trai et al., 2007; Matrai and Keller, 1993) (Figs. S11 and 6). PSAW, PSAE, NECS) typically show higher values in the northern hemispheric summer. The provinces in the North Atlantic extra-tropical gyre (NAST(W), NAST(E), GFST, NWCS, NECS and MEDI) typically show peaks during the northern summer. This pattern results from a combination of plankton seasonal species succession and short-term sun- light effects on DMS budgets, stimulating DMS production, inhibiting bacterial consumption, enhancing photolysis, etc. (Galí and Simó, 2015). The provinces NECS and MEDI show a similar trend to the polar province SARC. In addition to the summer peaks, an additional peak is also observed dur- ing the northern spring that has higher values than the peak in summer. The trends in the NECS province appear to be dom- inated by the spring blooms of the Baltic and the North Sea (April = 6.71 nM; May = 8.55 nM). A second bloom driven by dinoﬂagellates has been reported to occur in July (Hjerne et al., 2019) and coincides with a second peak in the DMS concentrations at 5.98 nM. The GFST and NWCS provinces show similar values and trends with twin maxima in spring and summer. Being a coastal province, NWCS shows consis- tently higher values than the GFST province. 3.1.3 Extra-tropics The mean values in these provinces decrease from approximately 2 nM in January to less than 1 nM in May (Fig. S11). The African coastal province, BENG shows higher values in January (4.09 nM) and March (3.59 nM) with lower concentrations in February (2.54 nM). This province does not show signiﬁcant variation throughout the year owing to the continual supply of nutrient-rich wa- ter due to an upwelling that supports primary production and hence DMS production (Jury and Brundrit, 1992) (Fig. S11). In the southern hemispheric Paciﬁc Ocean sector, the provinces AUSE, TASM and NEWZ cover the eastern and southern coastal region of Australia and coastal New Zealand. The primary production in TASM and NEWZ shows an annual cycle controlled by the availability of solar In the southern hemispheric Paciﬁc Ocean sector, the provinces AUSE, TASM and NEWZ cover the eastern and southern coastal region of Australia and coastal New Zealand. The primary production in TASM and NEWZ shows an annual cycle controlled by the availability of solar 3.1.4 Tropics 3.1.4 Tropics In the Indian Ocean sector of the tropics, the province MONS is in the central Indian Ocean with coastal provinces of REDS, INDW and INDE to the north, ARAB to the west, https://doi.org/10.5194/essd-14-2963-2022 Earth Syst. Sci. Data, 14, 2963–2987, 2022 2978 S. Hulswar et al.: DMS-Rev3 AUSW to the east, and ISSG to the south. The annual vari- ation in the MONS province is like the ISSG province, al- though with the peak value observed in February instead of December (3.66 nM). The INDE and REDS provinces show a similar monthly variation as the ARAB province. This region experiences the southwest and northeast monsoons during the periods between June–September and October– December. The Somali current ﬂowing through the ARAB and INDE provinces is characterized by seasonal changes in- ﬂuenced by the southwest and northeast monsoons. During June–September, the warm southwest monsoon moves the coastal waters north-eastward, creating a coastal upwelling (McCreary et al., 1996). This could be a reason for the higher mean DMS concentrations observed in these provinces dur- ing June and July (∼2.8 nM). However, these variations in DMS are not observed in the INDW province which is on the eastern coast of the Indian peninsula and not directly affected by the upwelling. In November, the coastal Indian province INDW shows higher values than the ARAB province, most likely due to the northeast monsoon winds increasing con- tinental aerosol deposition over the ocean surface, thereby increasing productivity due to increased nutrient availability (Figs. S11 and 6). The Atlantic Ocean tropical sector is comprised of the provinces CARB, NATR, CNRY, ETRA, WTRA, GUIN, GUIA, and SATL. The major gyres in the northern and southern tropics are NATR and SATL. The gyres show elevated DMS concentrations during August (NATR) and March (SATL). However, in the SATL province, the peak concentration is observed during the transition from sum- mer to autumn (March = 2.64 nM). In the SATL province, the open ocean sees the conﬂuence of the Brazilian–Malvinas current which signiﬁcantly affects the productivity of the re- gion. During most months, DMS concentrations are in the range of 1–2.5 nM in this province, with the lowest observed in May (0.83 nM). The NATR province has higher values throughout the year compared to SATL, except in March. 3.1.4 Tropics In the ETRA and WTRA provinces, a clear annual peak is seen in August, which coincides with the maximum extent and magnitude of phytoplankton blooms (July–September) in the eastern equatorial Atlantic Ocean (Pérez et al., 2005). The in- puts from the Amazon and Congo rivers are a major source of nutrients in this area throughout the year, with peak in- put between May and September. Similarly, in the GUIA and the CARB provinces, the Amazon and the Orinoco river in- puts inﬂuence the seasonal variability of primary production (Forget et al., 2011). This is seen in the peak concentrations of DMS in CARB (September = 2.74 nM) and GUIA (Au- gust = 2.75 nM). In the eastern Atlantic, the GUIN province shows a seasonal presence, with the CNRY province oc- cupying its location as seen in the dynamic biogeochemi- cal province map (Figs. S1, S11). Three characteristic peaks are seen in the CNRY province in March (3.86 nM), August (3.34 nM) and November (2.45 nM) (Figs. S11 and 6). In the northern tropical Paciﬁc Ocean, the province NPTG shows higher values in August (2.34 nM), while the lowest levels are observed in January (∼1 nM). DMS concentra- tions increase from a low in January to 1.61 nM in April, de- creasing slightly to 1.29 nM in May and increasing thereafter until August. In PNEC, there is no signiﬁcant annual trend as the mean DMS concentrations stay in the 2–2.6 nM range with one lower value in May (1.59 nM). From May to Au- gust, PNEC and NTPG show an increase in DMS (Figs. S11 and 6). 3.2 Differences due to usage of dynamic biogeochemical province boundaries ) In the southern tropical Paciﬁc region, the provinces SPSG, CHIL, PEQD, and PNEC are seen to have little vari- ability unlike those seen in the Indian Ocean. The mean DMS values in the SPSG province are higher in January and March (∼3.3 nM), with a drop in February to 2.63 nM. The mean then drops to 1.39 nM in April followed by an increase to 2.74 nM in July. From June onwards, the mean DMS con- centrations are ∼2.6 nM (2.34 to 2.9 nM) up until Decem- ber (2.9 nM). During the southern summer months, CHIL is inﬂuenced by increased DMS production with higher mean concentrations in December (4.93 nM) and January (7.53 nM). This is also observed in the PEQD province as the mean DMS is slightly higher in December (3.38 nM) and January (3.14 nM) compared to the other months. The Chile coastal “Humboldt” current is one of the most pro- ductive currents (Penven et al., 2005), and this may be driving the higher DMS concentrations during May–June (May = 4.27 nM, June = 5.61 nM) in CHIL. Since the cur- rent ﬂows northward from CHIL to the province PEQD, the effect is seen as a slight increase during June (3.35 nM) in PEQD (Figs. S11 and 6). Dynamic biogeochemical province boundaries as deﬁned by Reygondeau et al. (2013) were used in the DMS-Rev3 clima- tology instead of static boundaries to incorporate the monthly variability of surface properties in the world’s oceans. In this section, we describe the differences caused by using the dy- namic province boundaries as compared to the static bound- aries (Figs. 7 and S12a). As expected, most of the differ- ences are seen at the province boundaries when compared to the output using static provinces. Major differences are seen in the polar regions and smaller differences in the extra- tropical and tropical oceans. When globally averaged, the an- nual difference caused in the climatology by using the dy- namic province boundaries is only about 0.02 nM (∼5.1 %) (Table S1), however, regional differences are up to ±5 nM (±100 %) (Figs. 7 and S12a). The sea-ice mask excludes data from the Arctic region when sea-ice cover exceeds 50 %. ern winter (∼40 % to 60 %), while it ranged from 0 % to ∼−40 % for the northern summer. The Southern Ocean region showed the greatest regional differences in November where concentrations differed by >4 nM (+100 %), while the largest reduction was observed in January (−4 nM; −50 %). Both large positive and negative differences are seen in this region during the summer months with a negligible difference during the rest of the year. 3.2 Differences due to usage of dynamic biogeochemical province boundaries If the sea-ice mask is ig- nored, the use of dynamic provinces generates signiﬁcant differences in the Arctic Ocean region, with increases of 1– 3 nM (+100 %) during February–March and decreases of 1 Earth Syst. Sci. Data, 14, 2963–2987, 2022 https://doi.org/10.5194/essd-14-2963-2022 2979 S. Hulswar et al.: DMS-Rev3 Figure 7. Differences between the monthly and annual mean DMS concentrations estimated using dynamic and static biogeoc province boundaries (dynamic −static). Figure 7. Differences between the monthly and annual mean DMS concentrations estimated using dynamic and static biogeochemical province boundaries (dynamic −static). ern winter (∼40 % to 60 %), while it ranged from 0 % to ∼−40 % for the northern summer. The Southern Ocean region showed the greatest regional differences in November where concentrations differed by >4 nM (+100 %), while the largest reduction was observed in January (−4 nM; −50 %). Both large positive and negative differences are seen in this region during the summer months with a negligible difference during the rest of the year. ern winter (∼40 % to 60 %), while it ranged from 0 % to ∼−40 % for the northern summer. to 3 nM (−40 % to −60 %) during April (Fig. S12b). The winter and spring months show a positive change in the DMS concentrations as compared to the summer months where a reduction in the DMS concentrations is observed (Fig. 7). The Arctic region, especially the Nordic Seas where ice-free regions exist even during the winter season, showed a pos- itive difference with a maximum of 100 % change during February. The difference stayed positive during the north- The Southern Ocean region showed the greatest regional differences in November where concentrations differed by >4 nM (+100 %), while the largest reduction was observed in January (−4 nM; −50 %). Both large positive and negative differences are seen in this region during the summer months with a negligible difference during the rest of the year. The Southern Ocean region showed the greatest regional differences in November where concentrations differed by >4 nM (+100 %), while the largest reduction was observed in January (−4 nM; −50 %). Both large positive and negative differences are seen in this region during the summer months with a negligible difference during the rest of the year. https://doi.org/10.5194/essd-14-2963-2022 Earth Syst. Sci. Data, 14, 2963–2987, 2022 S. Hulswar et al.: DMS-Rev3 S. 3.5 Differences between DMS-Rev3 and L11 3.5 Differences between DMS-Rev3 and L11 Figure 9 shows the differences between the DMS- Rev3 and L11 climatologies (DMSREV3−DMSL11) and Fig. S14 shows the percentage difference ((DMSREV3−DMSL11) × 100/DMSL11) between the two. The most noticeable difference is the reduced patch- iness in the data distribution. This is mainly due to the improvements made in the interpolation routine and the use of observed DMS VLS in the Barnes ﬁlter (Sect. 2.5). For the global annual climatology mean, 73 % of the grid points show a positive difference (Figs. 9, S15), ∼51 % data show differences up to ±1 nM (±10 %), and ∼81 % of data have differences within ±2 nM (±30 %) (Fig. S15). Only 0.24 % of data show differences equal to or above 100 % (>5 nM). Overall, the DMS-Rev3 climatology estimates a global annual average DMS concentration that is 0.05 nM less than the L11 climatology. The largest mean difference is seen in November (+0.31 nM), and the lowest difference is seen in September (−0.03 nM) (Table S1). When the monthly differences are considered, large re- gional differences show up between the VLS and 555 km ﬁl- ters. For instance, during the southern hemispheric summer (December, January, February), large differences of above ±5 nM (±100 %) are seen in the Southern Ocean. This sug- gests that the use of a ﬁxed value of 555 km led to severe over- and underestimations in this region. During February, the entire Indian Ocean region shows patches of both posi- tive and negative differences. The central Atlantic region also shows positive differences during the months from March to May, with the largest differences observed in March ∼2 nM (>100 %). The Paciﬁc Ocean shows the least differences amongst all the world’s oceans. The Arctic region also shows differences of above 100 %, especially during the summer season. Overall, the largest differences are observed in the high concentration polar regions, with smaller differences seen close to the tropics. Most of the large differences are found in the polar re- gions, especially in the Southern Ocean. The equatorial oceans show differences less than ±0.5 nM, except in parts of the Indian and Paciﬁc oceans, where differences of up to −1 nM are observed (Fig. 9). The two climatologies agree well in the oligotrophic regions of the oceans where lower DMS concentrations are observed. 3.5 Differences between DMS-Rev3 and L11 Monthly, the largest dif- ferences are seen in the Southern Ocean during the months of November–February, with the DMS-Rev3 climatology dis- playing higher values in November throughout the South- ern Ocean. For the rest of the southern hemisphere summer, positive and negative differences of up to 5 nM are seen in the Southern Ocean, where the negative differences coincide with the patches that were a feature in the L11 climatology. 3.3 Differences due to usage of variability length scales (VLS) The other major change in DMS-Rev3 was the use of DMS VLS, which led to signiﬁcant changes, reduc- ing the patchiness that was a feature of past clima- tologies. In this section, we present the effect of using VLS compared to the ﬁxed value of 555 km when ap- plying the Barnes ﬁlter (see Sect. 2.5). The differences (DMSVLS−DMS555) are shown in Fig. 8 and the percent- age differences ((DMSVLS−DMS555) × 100/DMS555) are shown in Fig. S13. When globally averaged, the annual DMS concentration increased by 0.025 nM (+7.4 %) when using the VLS while applying the Barnes ﬁlter (Table S1). Annu- ally, the largest differences of up to ±4 nM (±90 %) were ob- served in the coastal Antarctic region and smaller increases were observed in certain regions of the Atlantic and Paciﬁc oceans. However, over most of the world’s oceans, the differ- ence was within ±0.3 nM (±5 %). 3.2 Differences due to usage of dynamic biogeochemical province boundaries Hulswar et al.: DMS-Rev3 2980 ter the data uniﬁcation, before creating the DMS-Rev3 cli- matology. The most noticeable changes are when the ﬁlter is applied after the creation of climatology. The global annual DMS reduces from 2.39 to 2.37 nM (0.8 % reduction when using a 95 % sea-ice mask) and further down to 2.21 nM (∼8 % reduction when using a 20 % sea-ice mask). The re- lationship with the sea-ice mask and the averaged annual global DMS concentrations is now shown in Fig. S17. The relationship between the percent threshold sea ice and the DMS concentration is linear, showing that the polar regions contribute heavily to the annual global average. For the rest of the regions in the extra-tropics and tropics, regional differences of between ±2 nM (up to 100 %) can be seen, changing throughout the year. Most of the world’s oceans show differences of ±20 %. Clear patches with larger differences can be observed, such as in the Atlantic Ocean where the differences are around −2 nM (−60 % to −80 %). A few patches are also observed in the northern and equa- torial Paciﬁc Ocean, where large differences are seen in dif- ferent locations depending on the month (Fig. 7). Globally, the largest difference is seen during November, where the use of dynamical province boundaries leads to an increase of 0.36 nM in the globally averaged concentration, while the smallest difference is seen during May (−0.003 nM). For this study, a threshold of 50 % was considered for the sea-ice cover. The sea-ice cover removes a major part of the province of BPLR and parts of the BERS, SARC and ARCT provinces were also masked, reducing the monthly means of the respective provinces. Similarly, in the southern region, the province APLR was also masked by the sea ice. The southern hemisphere winter shows the presence of sea ice to a much larger extent, covering areas of APLR and ANTA provinces. 3.4 Differences due to sea-ice cover There is a marginal change in the global annual mean DMS (<1%) when applying the sea-ice cover at 50% threshold af- https://doi.org/10.5194/essd-14-2963-2022 Earth Syst. Sci. Data, 14, 2963–2987, 2022 2981 S. Hulswar et al.: DMS-Rev3 Figure 8. Differences in the DMS concentrations caused using variability length scales (VLS) instead of a ﬁxed value for th inﬂuence as used by L11 (555 km) for the weighted average interpolation computation. (VLS – 555 km). 4 DMS ﬂux Ocean region, which covers ∼7 % of oceans with in the range from 10–20 µmol S m−2 d−1. The So Figure 8. Differences in the DMS concentrations caused using variability length scales (VLS) instead of a ﬁxed value for the radius of inﬂuence as used by L11 (555 km) for the weighted average interpolation computation. (VLS – 555 km). https://doi.org/10.5194/essd-14-2963-2022 4 DMS ﬂux Ocean region, which covers ∼7 % of oceans with emissions in the range from 10–20 µmol S m−2 d−1. The Southern Pa- ciﬁc Ocean shows higher values as compared to the North- ern Paciﬁc, although the Eastern Paciﬁc shows more ﬂux as compared to the western Paciﬁc, mainly due to the coastal provinces of CHIL, CAMR, CCAL, and ALSK which show particularly large ﬂuxes. The North Atlantic region, mainly in the GFST and NADR, shows a higher ﬂux compared to the Ocean region, which covers ∼7 % of oceans with emissions in the range from 10–20 µmol S m−2 d−1. The Southern Pa- ciﬁc Ocean shows higher values as compared to the North- ern Paciﬁc, although the Eastern Paciﬁc shows more ﬂux as compared to the western Paciﬁc, mainly due to the coastal provinces of CHIL, CAMR, CCAL, and ALSK which show particularly large ﬂuxes. The North Atlantic region, mainly in the GFST and NADR, shows a higher ﬂux compared to the The global sea–air DMS ﬂux (Fig. 10) reveals that ∼93 % of the world oceans emit DMS in the range of 0– 10 µmol S m−2 d−1, with a few hotspots in the world’s coastal regions like the provinces of BENG, ARAB, along with oceanic provinces like the conﬂuence of PEQD and SPSG in the Paciﬁc, and the ISSG province in the central Indian https://doi.org/10.5194/essd-14-2963-2022 Earth Syst. Sci. Data, 14, 2963–2987, 2022 2982 S. Hulswar et al.: DMS-Rev3 Figure 9. Differences between the monthly and annual mean DMS concentrations of the Rev3 and L11 climatologies (Rev3 −L11). Figure 9. Differences between the monthly and annual mean DMS concentrations of the Rev3 and L11 climatologies (Rev3 −L11). etween the monthly and annual mean DMS concentrations of the Rev3 and L11 climatologies (Rev3 −L11). Figure 9. Differences between the monthly and annual mean DMS concentrations of the Rev3 and L11 clim South Atlantic. The equatorial Indian Ocean region shows lower values than the rest of the Indian Ocean. The Arabian Sea shows a higher ﬂux of DMS compared to the Bay of Bengal. The total global annual DMS ﬂux is estimated at 27.1 TgS yr−1, about 3.5 % lower than the L11 climatology. The DMS ﬂux variability follows the variability in DMS con- centration, which is signiﬁcant at the regional scale. summer. The northern hemisphere summer shows elevated DMS emissions in only a few regions. 4 DMS ﬂux Additional regions are visible as hotspots in the open ocean (e.g., in the North At- lantic and Central Indian Ocean) as well as in coastal regions like those observed off the Chile coast, South of Alaskan coast in the Paciﬁc Ocean, and the North-western Arabian Sea. The hotspot observed in the Central Indian Ocean re- gion is present almost through all seasons, although it seems to be weakening during the northern spring. With an annual South Atlantic. The equatorial Indian Ocean region shows lower values than the rest of the Indian Ocean. The Arabian Sea shows a higher ﬂux of DMS compared to the Bay of Bengal. The total global annual DMS ﬂux is estimated at 27.1 TgS yr−1, about 3.5 % lower than the L11 climatology. The DMS ﬂux variability follows the variability in DMS con- centration, which is signiﬁcant at the regional scale. The entire oceanic region to the south of ∼30◦S seems to be a major source of DMS during the southern hemisphere Earth Syst. Sci. Data, 14, 2963–2987, 2022 https://doi.org/10.5194/essd-14-2963-2022 2983 S. Hulswar et al.: DMS-Rev3 S. Hulswar et al.: DMS Rev3 Figure 10. Distribution of the monthly and annual DMS ﬂux as estimated by DMS-Rev3 climatology with 50 % sea-ice mask. Figure 10. Distribution of the monthly and annual DMS ﬂux as estimated by DMS-Rev3 climatology with 5 ibution of the monthly and annual DMS ﬂux as estimated by DMS-Rev3 climatology with 50 % sea-ice mask. average of ∼2.76 nM, this region is above the global aver- age, increasing the annual DMS ﬂux (∼14 µmol S m−2 d−1). speed of about ∼>10 m s−1 seems to be one of the main factors to suppress the DMS ﬂuxes. The optimum response was observed in the range of 5–8 m s−1 approximately com- paring the wind speed climatology and DMS ﬂuxes in the re- gion where the DMS ﬂuxes were found to be higher over the global oceans. This explains most of the regional ﬂux vari- ability as observed in the DMS-Rev3 output. The evidence of this is seen in the Antarctic coastal region, which seems to be a major source of DMS during the southern summer. But average of ∼2.76 nM, this region is above the global aver- age, increasing the annual DMS ﬂux (∼14 µmol S m−2 d−1). Since the Nightingale et al. 6 Conclusion Disclaimer. Publisher’s note: Copernicus Publications remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. An updated global sea surface DMS concentration climatol- ogy was created by upgrading the processing algorithm initi- ated by Kettle et al. (1999) and Lana et al. (2011), along with the inclusion of new data compiled from various sources. The global annual average concentration reduced to 2.26 nM, al- though large differences of up to 5 nM were observed on re- gional scales during certain months. This is an important dif- ference considering the effect of regional emissions on the total impact of DMS on the Earth’s radiative budget (Fid- des et al., 2018; Mahajan et al., 2015; Thornhill et al., 2021; Woodhouse et al., 2013). The global sea–air ﬂux of DMS is estimated at 27.1 TgS yr−1 which is similar to L11 (a 3.5 % decrease). The use of dynamic province boundaries allowed the estimation of more realistic annual trends in different re- gions. The patchiness in the climatology identiﬁed in pre- vious estimates was reduced by region-speciﬁc data exclu- sion (Sect. 2.2) and the usage of the observation-based VLS (Sect. 2.5). The use of dynamic province boundaries and VLS was not important at the annual and global scales but resulted in large differences at regional scales. Although this climatology shows signiﬁcant improvements in the estima- tion of seawater DMS concentrations, it still suffers from a lack of continuous observations, especially in certain parts of the world’s oceans. Focus can be given speciﬁcally to the provinces NEWS, AUSE, AUSW, SUND, and ARCH which are present around Australia, including the Great Bar- rier Reef and the Indonesian archipelago. Along with these regions, the northern and eastern regions of the Arabian sea (EAFR, INDE and REDS provinces) also need extensive sampling for reducing the dependence on substitution and interpolation. Another major uncertainty is the contribution in regions affected by sea ice. The DMS-Rev3 essentially re- moves data from sea-ice regions according to a ﬁxed percent- Acknowledgements. We thank all the groups that have con- tributed their measurements to the Global Surface Seawa- ter Dimethylsulﬁde (DMS) Database (https://saga.pmel.noaa.gov/ dms/, last access: 18 February 2020). Financial support. Indian Institute of Tropical Meteorology (IITM) is funded by the Ministry of Earth Sciences (MOES), Government of India. Thomas G. 6 Conclusion Bell contribution to this study was supported by the NASA North Atlantic Aerosols and Ma- rine Ecosystems Study (grant no. NNX15AF31G). George Manville (with input from Thomas G. Bell and Paul R. Halloran) contributed the VLS analysis, which is part of his PhD (NERC industrial CASE studentship NE/R007586/1). Review statement. This paper was edited by Kirsten El- ger and reviewed by Patricia Matrai, Murat Aydin, and Giuseppe M. R. Manzella. 4 DMS ﬂux (2000) parameterization uses the wind speed and the SST for estimation of the gas transfer from sea to air, the DMS ﬂux hence calculated shows a di- rect relationship between the wind speed and SST. However, wind speed is a major driving force of the gas transfer ve- locity, which is a major determinant of the ﬂux. As observed by Bell et al. (2013) and Zavarsky et al. (2018a), the wind Since the Nightingale et al. (2000) parameterization uses the wind speed and the SST for estimation of the gas transfer from sea to air, the DMS ﬂux hence calculated shows a di- rect relationship between the wind speed and SST. However, wind speed is a major driving force of the gas transfer ve- locity, which is a major determinant of the ﬂux. As observed by Bell et al. (2013) and Zavarsky et al. (2018a), the wind https://doi.org/10.5194/essd-14-2963-2022 Earth Syst. Sci. Data, 14, 2963–2987, 2022 2984 S. Hulswar et al.: DMS-Rev3 S. Hulswar et al.: DMS-Rev3 due to the higher wind speed over the region, the contribution to the DMS ﬂux is lower than one would expect. This region also has lower SST, which further reduces the gas transfer. age, neglecting the contribution of these regions. Thus, al- though this new climatology is a major upgrade from the past estimates, and matches the estimates using top-down meth- ods, further improvements are needed in the future, with the main limiting factor being data availability. The differences in the ﬂuxes with respect to L11 show a similar pattern to that seen in the distribution of oceanic DMS on monthly and annual scales (Fig. S16). This means that al- though the change in the global annual average of DMS ﬂux is not substantial, the DMS-Rev3 climatology has large re- gional changes (Table 2) due to a reduction in the patchiness of the oceanic DMS concentrations. Most of the high lati- tudes show a reduced ﬂux, while the tropical regions show an enhanced ﬂux. Supplement. The supplement related to this article is available online at: https://doi.org/10.5194/essd-14-2963-2022-supplement. Author contributions. ASM and RS devised the research. SH, SI and ASM wrote the new algorithm based on the original algorithm by AL. TGB, PRH and GM did the analysis for DMS variability length. MG provided scientiﬁc insights to improving the algorithm. SH and ASM wrote the paper with inputs from all the authors. 5 Code and data availability The data used for creating the climatology, along with the algorithm, can be found in the online repository: https://doi.org/10.17632/hyn62spny2.1 (Mahajan, 2021). Competing interests. The contact author has declared that nei- ther they nor their co-authors have any competing interests. https://doi.org/10.5194/essd-14-2963-2022 References Andreae, M. O. and Raemdonck, H.: Dimethyl Sulﬁde in the Sur- face Ocean and the Marine Atmosphere: A Global View, Science, 221, 744–747, http://www.jstor.org/stable/1691026 (last access: September 2020), 1983. Andreae, M. O. and Barnard, W. R.: The marine chem- istry of dimethylsulﬁde, Mar. Chem., 14, 267–279, https://doi.org/10.1016/0304-4203(84)90047-1, 1984. Earth Syst. Sci. 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https://openalex.org/W4291192244	https://amt.copernicus.org/articles/15/5383/2022/amt-15-5383-2022.pdf	English	null	Comment on amt-2022-128	null	2,022	cc-by	14,971	Correspondence: Helen M. Worden (hmw@ucar.edu) These comparison results give us conﬁdence in the use of TROPESS/CrIS CO proﬁles and er- ror characterization for continuing the multi-decadal record of satellite CO observations. Correspondence: Helen M. Worden (hmw@ucar.edu) Correspondence: Helen M. Worden (hmw@ucar.edu) Received: 16 April 2022 – Discussion started: 28 April 2022 Revised: 12 August 2022 – Accepted: 2 September 2022 – Published: 22 September 2022 Abstract. The new single-pixel TROPESS (TRopospheric Ozone and its Precursors from Earth System Sounding) pro- ﬁle retrievals of carbon monoxide (CO) from the Cross-track Infrared Sounder (CrIS) are evaluated using vertical proﬁles of in situ observations from the National Oceanic and Atmo- spheric Administration (NOAA) Global Monitoring Labora- tory (GML) aircraft program and from the Atmospheric To- mography Mission (ATom) campaigns. The TROPESS op- timal estimation retrievals are produced using the MUSES (MUlti-SpEctra, MUlti-SpEcies, MUlti-Sensors) algorithm, which has heritage from retrieval algorithms developed for the EOS/Aura Tropospheric Emission Spectrometer (TES). TROPESS products provide retrieval diagnostics and error covariance matrices that propagate instrument noise as well as the uncertainties from sequential retrievals of parameters such as temperature and water vapor that are required to estimate the carbon monoxide proﬁles. The validation ap- proach used here evaluates biases in column and proﬁle val- ues as well as the validity of the retrieval error estimates using the mean and variance of the compared satellite and aircraft observations. CrIS–NOAA GML comparisons had biases of 0.6 % for partial column average volume mixing ratios (VMRs) and (2.3, 0.9, −4.5) % for VMRs at (750, 511, 287) hPa vertical levels, respectively, with standard de- viations from 9 % to 14 %. CrIS–ATom comparisons had bi- ases of −0.04 % for partial column and (2.2, 0.5, −3.0) % for (750, 511, 287) hPa vertical levels, respectively, with stan- dard deviations from 6 % to 10 %. The reported observational errors for TROPESS/CrIS CO proﬁles have the expected be- havior with respect to the vertical pattern in standard devi- ation of the comparisons. These comparison results give us conﬁdence in the use of TROPESS/CrIS CO proﬁles and er- ror characterization for continuing the multi-decadal record of satellite CO observations. viations from 9 % to 14 %. CrIS–ATom comparisons had bi- ases of −0.04 % for partial column and (2.2, 0.5, −3.0) % for (750, 511, 287) hPa vertical levels, respectively, with stan- dard deviations from 6 % to 10 %. The reported observational errors for TROPESS/CrIS CO proﬁles have the expected be- havior with respect to the vertical pattern in standard devi- ation of the comparisons. 1 Introduction Carbon monoxide (CO) is a useful tracer of atmospheric pol- lution, with direct emissions from incomplete combustion such as biomass and fossil fuel burning as well as secondary production from the oxidation of methane (CH4) and volatile organic compounds (VOCs). Atmospheric CO distributions have a seasonal cycle that is mainly driven by photochem- ical destruction, which allows CO to build up over winter and early spring in higher latitudes. The lifetime of CO of weeks to months (e.g., Holloway et al., 2000) is long enough to allow observations of pollution plumes and their subse- Atmos. Meas. Tech., 15, 5383–5398, 2022 https://doi.org/10.5194/amt-15-5383-2022 © Author(s) 2022. This work is distributed under the Creative Commons Attribution 4.0 License. TROPESS/CrIS carbon monoxide proﬁle validation with NOAA GML and ATom in situ aircraft observations Helen M. Worden1, Gene L. Francis1, Susan S. Kulawik2, Kevin W. Bowman3, Karen Cady-Pereira4, Dejian Fu3, Jennifer D. Hegarty4, Valentin Kantchev3, Ming Luo3, Vivienne H. Payne3, John R. Worden3, Róisín Commane5, and Kathryn McKain6,7 1Atmospheric Chemistry Observations and Modeling (ACOM), National Center for Atmospheric Research (NCAR), Boulder, CO, USA 3Jet Propulsion Laboratory/California Institute for Technology, Pasadena, CA, USA 4 3Jet Propulsion Laboratory/California Institute for Technology, Pasadena, CA, USA 4 5Dept. of Earth and Environmental Sciences, Lamont-Doherty Earth Observatory, Columbia University, Palisades, NY, USA 6Cooperative Institute for Research in Environmental Sciences (CIRES), University of Colorado, Boulder, CO, USA 7Global Monitoring Division (GMD), National Oceanic and Atmospheric Administration, Boulder, CO, USA Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. H. M. Worden et al.: TROPESS/CrIS carbon monoxide proﬁle validation 5384 cursors from Earth System Sounding) Science Data Process- ing System (Bowman, 2021). TROPESS is a NASA project that provides a framework for consistent data processing of ozone and ozone precursors across different satellite in- struments. TROPESS retrievals use single-FOV radiances in sequential optimal estimation retrievals (Rodgers, 2000) of temperature, water vapor, effective cloud parameters, ozone, CO, and other trace gases, allowing for full characterization of the vertical retrieval sensitivity with an averaging kernel and error covariance (Bowman et al., 2006). TROPESS/CrIS CO products differ from other available CrIS CO data prod- ucts that combine nine FOVs to obtain a single cloud-cleared radiance and corresponding retrieval of atmospheric parame- ters such as the NOAA Unique Combined Atmospheric Pro- cessing System (NUCAPS) (Gambacorta et al., 2014, 2017; Nalli et al., 2020) and the Community Long-term Infrared Microwave Combined Atmospheric Product System (CLIM- CAPS) (Smith and Barnet, 2020). quent long-range transport, but short enough to distinguish the plumes against background seasonal distributions (e.g., Edwards et al., 2004, 2006; Hegarty et al., 2009, 2010). As a dominant sink for the hydroxyl radical (OH), CO plays a critical role in atmospheric reactivity (e.g., Lelieveld et al., 2016) and is considered a short-lived climate pollutant (SLCP) because of its impacts on methane lifetime as well as carbon dioxide and ozone formation (e.g., Myhre et al., 2014; Gaubert et al., 2017). Published by Copernicus Publications on behalf of the European Geosciences Union. Global observations of tropospheric CO from satellites started in 2000 with the NASA Earth Observing Sys- tem (EOS) Measurement of Pollution in the Troposphere (MOPITT) instrument on Terra (Drummond et al., 2010), followed by the EOS Atmospheric Infrared Spectrometer (AIRS, McMillan et al., 2005) on Aqua launched in 2002, the Scanning Imaging Absorption Spectrometer for Atmo- spheric Chartography (SCIAMACHY, de Laat et al., 2006) on Envisat launched in 2002, the EOS Tropospheric Emis- sion Spectrometer (TES, Beer et al., 2006) on Aura launched in 2004, the Infrared Atmospheric Sounding Interferometer (IASI, Clerbaux et al., 2009) on the MetOp series beginning in 2006, the Cross-track Infrared Sounder (CrIS, Gambacorta et al., 2014) on the Suomi National Polar-orbiting Partner- ship (SNPP) satellite launched in 2011, and most recently the Joint Polar Satellite System (JPSS) series, TROPOMI on the Sentinel-5 precursor in 2017 (Borsdorff et al., 2018), and the Fourier transform spectrometer (FTS-2) on the Greenhouse gases Observing SATellite-2 (GOSAT-2, Suto et al., 2021) launched in 2018. Satellite CO observations are assimilated for reanalyses and operational air quality forecasting (e.g., Gaubert et al., 2016; Inness et al., 2019; Miyazaki et al., 2020) and have been used in inverse modeling analyses to es- timate emissions and attribute sources for co-emitted species such as CO2 (e.g., Kopacz et al., 2010; Jiang et al., 2017; Liu et al., 2017; Zheng et al., 2019; Gaubert et al., 2020; Byrne et al., 2021; Qu et al., 2022). Trend analyses of satel- lite CO observations (e.g., Worden et al., 2013; Buchholz et al., 2021) show a general decline of atmospheric CO over the satellite record globally and in most regions, but with a slow- ing of this decrease in recent years that emphasizes the need for continued satellite CO observations that are validated and have reliable error characterization. TROPESS data products report a separate matrix for the observational error terms along with the total retrieval error covariance that includes the contribution of smoothing error. This is important for evaluation of retrieval errors using in situ proﬁles since the validation comparison removes the ef- fect of smoothing in the retrieval by applying the retrieval averaging kernel and a priori to the in situ proﬁle before dif- ferencing (Rodgers and Connor, 2003). Similar comparisons were performed in the recent validation study for the MUSES single-FOV CO retrievals from the Aura Atmospheric In- frared Sounder (AIRS) of Hegarty et al. (2022). Published by Copernicus Publications on behalf of the European Geosciences Union. Section 2 describes the TROPESS retrievals and CO data products in more detail, and Sect. 3 describes the validation in situ data from the National Oceanic and Atmospheric Ad- ministration (NOAA) Global Monitoring Laboratory (GML) aircraft network and the Atmospheric Tomography Mission (ATom) campaigns. The validation methods are presented in Sect. 4, and results are shown in Sect. 5 with a summary and conclusions in Sect. 6. 2.1 TROPESS retrieval approach TROPESS data processing (Bowman, 2021) produces re- trievals of temperature, water vapor, and trace gases such as ozone (O3), methane (CH4), carbon monoxide (CO), ammo- nia (NH3), and peroxyacetyl nitrate (PAN) from single and multiple instruments including AIRS and OMI as well as CrIS and TROPOMI. The MUSES retrieval algorithm used in TROPESS was developed with heritage from Aura/TES retrieval processing. Bowman et al. (2021) describe the se- quential MUSES retrievals of temperature, water vapor, and effective cloud properties for each FOV that are necessary for the retrieval of CO. Each step in the sequence includes an iterative retrieval with a forward model and updated estimate of the state vector of atmospheric parameters following the maximum a posteriori (MAP) method. The forward model for radiative transfer at CrIS TIR wavelengths uses optimal spectral sampling (OSS, Moncet et al., 2015), which includes effective cloud optical depth and height parameters (Eldering et al., 2008; Kulawik et al., 2006). Here we analyze TROPESS/CrIS TIR-only CO retrievals that use the 2181–2200 cm−1 spectral range. A priori proﬁles for TROPESS CO retrievals are taken from the model clima- tology used in Aura/TES processing (MOZART, Brasseur et al., 1998), with monthly variation over a 30◦latitude and 60◦ longitude grid. The a priori uncertainty covariance matrix used to constrain the retrieval is the same as used for MO- PITT proﬁles (Deeter et al., 2010) with 30 % uncertainty for vertical CO parameters at all levels and correlation lengths corresponding to 100 hPa between them in the troposphere. As stated in the Introduction, the TROPESS single-FOV products are different from the NUCAPS and CLIMCAPS products that combine nine FOVs in a retrieval from a sin- gle cloud-cleared radiance (Susskind et al., 2003). These multiple-FOV products have the advantage of increased global coverage in the presence of partially cloudy scenes but with coarser spatial resolution. Figure 2 shows an exam- ple of SNPP CLIMCAPS (Barnet, 2019) compared to SNPP TROPESS/CrIS CO products (daytime only) on 13 Septem- ber 2018 over the Pole Creek ﬁre in Utah. For CLIMCAPS, trace gas products with less than 1 DFS report mass mix- ing ratio (MMR) on a single level at the retrieval pressure with peak sensitivity, which is 500 hPa for CO. We converted MMR to VMR for Fig. 2. 2.2 TROPESS/CrIS CO data examples 2.2 (δ = 0.625 cm−1), these ﬁner-resolution spectral radiances have been available since 4 December 2014. Here we only utilize the full-spectral-resolution CrIS data. Figure 1 shows an example of TROPESS/CrIS CO data for 12 September 2020 when there were signiﬁcant ﬁres in the western US. These retrievals are from a special data col- lection that processed scenes selected from 0.25◦× 0.25◦ latitude–longitude subsampling to enable throughput with the available computing capacity (Bowman et al., 2021). The data in this collection are pre-ﬁltered for quality (see Sect. 2.1), and Fig. 1a shows all available day and night re- trievals. Figure 1b shows the data after higher cloudy scenes are removed (i.e., cloud tops with pressure < 700 hPa and cloud effective optical depth > 0.1). For reference, Fig. 1c shows the mid-tropospheric average CO volume mixing ra- tio (VMR) for the a priori proﬁles used in the retrievals, and Fig. 1d shows a NASA Worldview (https://worldview. earthdata.nasa.gov/, last access: 14 September 2022) im- age from SNPP/VIIRS (Visible Infrared Imaging Radiome- ter Suite) with clouds and smoke shown in true color and red areas indicating ﬁre and thermal anomalies. Since verti- cal proﬁle retrievals using TIR radiances have sensitivity to CO mainly in the free troposphere, Fig. 1 shows individual retrievals with average VMR from vertical layers between 700 and 350 hPa. When all scenes are included, the average number of degrees of freedom for signal (DFS) is 0.99 for the CrIS CO observations in Fig. 1a, and when cloudy scenes are removed (Fig. 1b) the average DFS is 1.14 for the remaining CrIS observations. H. M. Worden et al.: TROPESS/CrIS carbon monoxide proﬁle validation H. M. Worden et al.: TROPESS/CrIS carbon monoxide proﬁle validation 2 TROPESS/CrIS single-ﬁeld-of-view CO proﬁle retrievals The ﬁrst Cross-track Infrared Sounder (CrIS) was launched 28 October 2011 on the SNPP satellite into a sun- synchronous polar orbit with an altitude near 830 km and an Equator-crossing time (ascending node) near 13:30 LT. CrIS is a Fourier transform spectrometer (FTS) operating in three spectral bands between 648 and 2555 cm−1. This includes the R-branch of the thermal infrared (TIR) CO (0–1) fun- damental band above 2155 cm−1. After launch, spectral ra- diance data that included the CO band were collected us- ing a spectral resolution of 2.5 cm−1. This resolution was relatively coarse and signiﬁcantly limited the vertical sen- sitivity of CO retrievals (Gambacorta et al., 2014). Follow- ing the decision to collect data at full spectral resolution In this study, we evaluate the biases and reported un- certainties of single-ﬁeld-of-view (FOV) CO retrievals from the Cross-track Infrared Sounder (CrIS) on board the SNPP satellite launched in October 2011. CrIS is a Fourier trans- form spectrometer (FTS) that has continuation instruments on the current and planned JPSS series with JPSS1/NOAA- 20 launched in 2017 and planned launches in 2022, 2028, and 2032 (https://www.nesdis.noaa.gov/about/our-ofﬁces/ joint-polar-satellite-system-jpss-program-ofﬁce, last access: 14 September 2022). The CrIS CO retrievals evaluated here use the MUSES (MUlti-SpEctra, MUlti-SpEcies, MUlti- Sensors) algorithm (Fu et al., 2016, 2018, 2019) and are pro- cessed with the TROPESS (TRopospheric Ozone and its Pre- https://doi.org/10.5194/amt-15-5383-2022 Atmos. Meas. Tech., 15, 5383–5398, 2022 5385 https://doi.org/10.5194/amt-15-5383-2022 2.1 TROPESS retrieval approach Panel (d) shows the NASA Worldview SNPP/VIIRS image for 14 September 2020 with clouds and smoke (true color) as well as ﬁre thermal anomalies (red). Figure 1. SNPP TROPESS/CrIS and SNPP/VIIRS observations for 14 September 2020. Panel (a) shows the average CO VMR for 700 to 350 hPa for all processed TROPESS CO retrievals with good data quality (see text). Panel (b) shows the same free troposphere CO averages as (a) but with cloudy scenes removed (see text). Panel (c) shows the average TROPESS a priori CO VMR for 700 to 350 hPa. Panel (d) shows the NASA Worldview SNPP/VIIRS image for 14 September 2020 with clouds and smoke (true color) as well as ﬁre thermal anomalies (red). Figure 2. SNPP observations of the Pole Creek ﬁre in Utah, USA, on 13 September 2018. The Great Salt Lake is in the upper left of each panel, and state borders with Idaho, Wyoming, and Colorado are indicated by solid straight lines. Dotted lines indicate a 1◦latitude by 1◦ longitude grid, with the top left corner at 42◦N, −113◦E. Panel (a) shows CLIMCAPS/CrIS CO at 500 hPa (MMR converted to VMR). Panel (b) shows the TROPESS/CrIS tropospheric CO column average VMR, and panel (c) shows the corresponding NASA Worldview SNPP/VIIRS image with clouds and smoke (true color) as well as ﬁre thermal anomalies (red). Figure 2. SNPP observations of the Pole Creek ﬁre in Utah, USA, on 13 September 2018. The Great Salt Lake is in the upper left of each panel, and state borders with Idaho, Wyoming, and Colorado are indicated by solid straight lines. Dotted lines indicate a 1◦latitude by 1◦ longitude grid, with the top left corner at 42◦N, −113◦E. Panel (a) shows CLIMCAPS/CrIS CO at 500 hPa (MMR converted to VMR). Panel (b) shows the TROPESS/CrIS tropospheric CO column average VMR, and panel (c) shows the corresponding NASA Worldview SNPP/VIIRS image with clouds and smoke (true color) as well as ﬁre thermal anomalies (red). WE-CAN (Western Wildﬁre Experiment for Cloud Chem- istry, Aerosol Absorption, and Nitrogen) campaign. creases by 1/λ4, is completely negligible and Mie scattering would be signiﬁcant only for particles larger than ∼λ/π = 1.5 µm, (e.g., Seinfeld and Pandis, 1998), while the size dis- tribution for biomass burning smoke particles peaks around 0.3 µm (e.g., Reid et al., 2005). For the same Pole Creek ﬁre in Fig. 2, Juncosa Calahorrano et al. 2.1 TROPESS retrieval approach This is compared to the tropo- spheric column average VMR from TROPESS, so the back- ground VMR values are close but do not represent the same retrieved quantities. CrIS retrieval center locations are shown by the circles in Fig. 2a and b, which are not intended to represent the spatial extent of the observations. The CLIM- CAPS retrievals show elevated CO from the ﬁre, but these combined FOV retrievals would give an overestimate of the plume width and do not distinguish the larger plume from the smaller ﬁres to the east in Colorado. The TROPESS CO products have quality ﬂags for screen- ing cases that did not converge or that have unphysical results. This screening checks the magnitude and spectral structure of radiance residuals, cloud retrieval characteris- tics, and deviation of surface emissivity from a priori val- ues. Speciﬁcally, retrievals with good data quality of 1 have radiance residual standard deviation less than 12 times the ra- diance error, an absolute value of the radiance residual mean less than 0.7 times the radiance error, KdotDL (the normal- ized dot product of the Jacobians and the radiance residual) less than 0.8, LdotDL (the normalized dot product of the ra- diance and the residual) less than 0.6, cloud-top pressures below 90 hPa, mean cloud optical depths less than 50, cloud variability (variation with respect to wavenumber) less than 3, and mean surface emissivity that did not change by more than 0.06. These threshold values are based on comparisons with in situ data and other satellite data to determine when retrievals are valid. We note that retrievals of CO in the presence of smoke are not signiﬁcantly affected by scattering for infrared ob- servations at wavelengths λ ∼4.6 µm, such as in the CrIS CO band. This is because Rayleigh scattering, which de- https://doi.org/10.5194/amt-15-5383-2022 Atmos. Meas. Tech., 15, 5383–5398, 2022 H. M. Worden et al.: TROPESS/CrIS carbon monoxide proﬁle validation 5386 H. M. Worden et al.: TROPESS/CrIS carbon monoxide proﬁle validation Figure 1. SNPP TROPESS/CrIS and SNPP/VIIRS observations for 14 September 2020. Panel (a) shows the average CO VMR for 700 to 350 hPa for all processed TROPESS CO retrievals with good data quality (see text). Panel (b) shows the same free troposphere CO averages as (a) but with cloudy scenes removed (see text). Panel (c) shows the average TROPESS a priori CO VMR for 700 to 350 hPa. 2.1 TROPESS retrieval approach (2021) showed how SNPP/CrIS single-pixel MUSES retrievals of acyl peroxy nitrates, also known as PAN, along with CO, can be used to follow ﬁre plume chemical evolution. After subtracting background amounts, the normalized excess mixing ratios (NEMRs) of PAN with respect to CO, computed from the CrIS observations for this plume, were consistent with in situ aircraft observations of smoke plumes from the summer 2018 4 Validation methodology depending on aircraft limitations at each site. Flask samples are then sent for laboratory analysis of a multitude of trace gases including CO, which was measured with vacuum UV– ﬂuorescence spectroscopy during the time period of this anal- ysis. CO mixing ratios are reported relative to the WMO X2014A scale (https://gml.noaa.gov/ccl/co_scale.html, last access: 14 September 2022) and have reproducibility ∼1 ppb (Sweeney et al., 2015). NOAA GML aircraft proﬁles of CO have been used for the long-term validation of the MOPITT CO record, with updated validation for each new data version (Deeter et al., 2019, and references therein). For the current analysis, we use NOAA GML aircraft network observations of CO collected during 2016 and 2017 from seven locations (Table 1). 4.1 Data selection, coincidence criteria, and vertical extension of aircraft proﬁles TROPESS/CrIS CO proﬁles are selected for comparison if they have retrieval quality of 1 and effective cloud optical depth less than 0.1 to ensure non-cloudy CrIS observations. We then ﬁnd all eligible CrIS and aircraft proﬁle pairs within 9 h and 50 km distance. This has been a standard coincidence distance criterion for several validation studies (e.g., Deeter et al., 2019, 2022; Hegarty et al., 2022). Tang et al. (2020) found very little sensitivity in MOPITT CO validation results for 25, 50, 100, and 200 km coincidence except for the cases with a 25 km radius that resulted in an insufﬁcient number of matches for meaningful statistics. The Tang et al. (2020) study also tested the time coincidence criterion (12, 6, 2, and 1 h) with similar conclusions. Application of the 9 h–50 km coincidence criteria yielded 2092 CrIS–aircraft proﬁle pairs for NOAA GML ﬂights from 2016 and 2017 and 1052 pro- ﬁle pairs for the ATom 1–4 campaigns. Since the aircraft pro- ﬁles used for validation do not span the full vertical range of satellite-retrieved proﬁles, we must extend these with a rea- sonable approximation of atmospheric CO to facilitate the comparison as described below in Sect. 4.2. Here we use the TROPESS a priori proﬁles (from model climatology, de- scribed above) to extend the in situ proﬁles above the highest altitude sampled. The a priori proﬁle is scaled to match the CO abundance of the aircraft measurement at the highest al- titude. The choices of model and approach for extending the aircraft proﬁles are examined more in Tang et al. (2020) and Hegarty et al. (2022), with similar conclusions that the im- pacts apply mostly to bias estimates in the middle to upper troposphere. Martìnez-Alonso et al. (2022) compute the un- 3.1 NOAA GML aircraft network Spanning 3 decades, NOAA GML aircraft network vertical proﬁle observations are taken on semi-regular ﬂights (ap- proximately one per month) at ﬁxed sites mostly in North America except for one site in Rarotonga, Cook Islands (Sweeney et al., 2015). These ﬂights collect air samples using an automated ﬂask system to obtain vertical proﬁles for each trace gas measured from near the surface to around 400 hPa, Atmos. Meas. Tech., 15, 5383–5398, 2022 https://doi.org/10.5194/amt-15-5383-2022 H. M. Worden et al.: TROPESS/CrIS carbon monoxide proﬁle validation Table 1. Aircraft in situ validation observations used in this study. NOAA/GML network ﬂask/UV spectrometer (±1 ppb CO) Code/site name Latitude (◦N) Longitude (◦W) Dates available RTA/Raratonga −21.25 159.83 2000–2021 TGC/offshore Corpus Christi,TX 27.73 96.86 2003–2021 CMA/offshore Cape May, NJ 38.83 74.32 2005–2022 THD/Trinidad Head, CA 41.05 124.15 2003–2022 NHA/offshore Portsmouth, NH 42.95 70.63 2003–2022 ESP/Estevan Pt., BC 49.38 128.54 2002–2021 ACG/Alaska Coast Guard 57.74 152.50 2009–2021 NASA/ATom QCLS (±0.15 ppb CO) ATom 1–4 Paciﬁc 75 to −65 150 to 70 Jul 2016, Jan 2017, Sep 2017, Apr 2018 ATom 1–4 Atlantic −75 to 80 65 to 20 Aug 2016, Feb 2017, Oct 2017, May 2018 https://gml.noaa.gov/ccgg/aircraft/ (last access: 14 September 2022); https://espo.nasa.gov/atom/content/ATom (last access: 14 September 2022). H. M. Worden et al.: TROPESS/CrIS carbon monoxide proﬁle validation 5387 H. M. Worden et al.: TROPESS/CrIS carbon monoxide proﬁle validation Table 1. Aircraft in situ validation observations used in this study. Table 1. Aircraft in situ validation observations used in this study. 3.2 ATom aircraft campaigns The Atmospheric Tomography Mission (ATom) was de- signed to study air masses in the most remote regions of the Paciﬁc and Atlantic Ocean in each season (Thompson et al., 2022), which also makes the data valuable for validat- ing satellite CO observations over a range of latitudes, with mostly background CO concentrations, except for where transported pollution plumes were encountered (Deeter et al., 2019, 2022; Martínez-Alonso et al., 2020; Hegarty et al., 2022). We use CO proﬁles from the quantum cascade laser spectrometer (QCLS) on ATom campaigns 1–4 (see Table 1). These NASA DC-8 ﬂights obtained vertical proﬁles from 0.2 to 12 km altitude (∼290 hPa) by ascending or descend- ing approximately every 220 km. CO was measured at 1 Hz with QCLS reproducibility around 0.15 ppbv (McManus et al., 2010; Santoni et al., 2014). The QCLS data were cal- ibrated to the X2014A CO WMO scale maintained by the NOAA GML. https://doi.org/10.5194/amt-15-5383-2022 Atmos. Meas. Tech., 15, 5383–5398, 2022 H. M. Worden et al.: TROPESS/CrIS carbon monoxide proﬁle validation H. M. Worden et al.: TROPESS/CrIS carbon monoxide proﬁle validation 5388 Figure 3. Examples of TROPESS/CrIS CO averaging kernel (A) (a) and the validation process (b). The colors of the averaging kernel indicate the pressure level (66 levels from 1017.45 to 0.1 hPa) corresponding to each row, with the surface-level row also indicated by the squares. The number of degrees of freedom for signal (DFS), given by the sum of the diagonal (i.e., trace) of this averaging kernel, is 1.26. The right panel shows the CrIS CO proﬁle retrieval (solid red line) with total error (dashed red lines), observation error (dotted red lines), a priori proﬁle (solid cyan line with squares), and diagonal uncertainty (dashed cyan lines). The closest ATom aircraft proﬁle had 10.4 km–3.5 h coincidence. The original ATom proﬁle (dashed grey line) is interpolated to the CrIS vertical grid (solid grey with squares) and transformed by the instrument operator to give ATom ˆxval (Eq. 1) (solid black line with squares). Figure 3. Examples of TROPESS/CrIS CO averaging kernel (A) (a) and the validation process (b). The colors of the averaging kernel indicate the pressure level (66 levels from 1017.45 to 0.1 hPa) corresponding to each row, with the surface-level row also indicated by the squares. The number of degrees of freedom for signal (DFS), given by the sum of the diagonal (i.e., trace) of this averaging kernel, is 1.26. 4.3 Evaluating TROPESS CO reported observational errors Following Bowman et al. (2006, 2021), for retrieved parameter ˆx (e.g., CO abundance) with a priori co- variance Sa, radiance measurement covariance Se, Jaco- bian matrix K = ∂L ∂x , radiance L(x), gain matrix G = KT S−1 e K + S−1 a −1KT S−1 e , and averaging kernel A = GK, the a posteriori error covariance can be written as the sum of 3.2 ATom aircraft campaigns The right panel shows the CrIS CO proﬁle retrieval (solid red line) with total error (dashed red lines), observation error (dotted red lines), a priori proﬁle (solid cyan line with squares), and diagonal uncertainty (dashed cyan lines). The closest ATom aircraft proﬁle had 10.4 km–3.5 h coincidence. The original ATom proﬁle (dashed grey line) is interpolated to the CrIS vertical grid (solid grey with squares) and transformed by the instrument operator to give ATom ˆxval (Eq. 1) (solid black line with squares). This operation accounts for both the broad vertical resolution (or “smoothing”) of remotely sensed measurements and the inﬂuence of the a priori, which is especially important in the vertical ranges where satellite observations have low sensi- tivity to CO abundance. Figure 3 shows an example of the averaging kernel A and a validation comparison with Eq. (1) applied to an ATom in situ proﬁle. This operation accounts for both the broad vertical resolution (or “smoothing”) of remotely sensed measurements and the inﬂuence of the a priori, which is especially important in the vertical ranges where satellite observations have low sensi- tivity to CO abundance. Figure 3 shows an example of the averaging kernel A and a validation comparison with Eq. (1) applied to an ATom in situ proﬁle. certainty introduced by this extension explicitly using NOAA AirCore in situ balloon proﬁles that sample into the strato- sphere (Karion et al., 2010). This uncertainty is computed for validation using aircraft proﬁles (with top samples around 400 hPa for NOAA/GML) by comparing MOPITT proﬁles to truncated and extended AirCore proﬁles vs. the true full Air- Core proﬁles. The comparison error introduced by the ex- tension was at most 3 % around 300 hPa and much less than the standard deviation of MOPITT and full AirCore proﬁle differences (∼7%–10 %) in the upper troposphere. We also note that for ATom proﬁles, the highest-altitude samples are normally taken around 12 km (∼200 hPa), and the proﬁle ex- tension therefore has a minimal impact on tropospheric vali- dation results. https://doi.org/10.5194/amt-15-5383-2022 4.2 Comparison of TROPESS satellite and aircraft observations Relative differences (%) in single CrIS retrievals with coincident NOAA GML ˆxval proﬁles (grey) and the average percent difference with 1σ horizontal bars (red). Both day and night CrIS observations are included for coincidence search, with 1866 day and 266 night comparison pairs found. Figure 5. Latitude dependence of CO partial column average VMR (ppb) for TROPESS/CrIS retrievals and NOAA GML ˆxval (a) as well as bias difference statistics (b) shown by box–whisker symbols representing minimum and maximum values (whisker), lower quar- tile (box bottom), median (white stripe), and upper quartile (box top). A minimum of ﬁve comparisons per bin was required. Figure 5. Latitude dependence of CO partial column average VMR (ppb) for TROPESS/CrIS retrievals and NOAA GML ˆxval (a) as well as bias difference statistics (b) shown by box–whisker symbols representing minimum and maximum values (whisker), lower quar- tile (box bottom), median (white stripe), and upper quartile (box top). A minimum of ﬁve comparisons per bin was required. Figure 5. Latitude dependence of CO partial column average VMR (ppb) for TROPESS/CrIS retrievals and NOAA GML ˆxval (a) as well as bias difference statistics (b) shown by box–whisker symbols representing minimum and maximum values (whisker), lower quar- tile (box bottom), median (white stripe), and upper quartile (box top). A minimum of ﬁve comparisons per bin was required. Figure 4. Relative differences (%) in single CrIS retrievals with coincident NOAA GML ˆxval proﬁles (grey) and the average percent difference with 1σ horizontal bars (red). Both day and night CrIS observations are included for coincidence search, with 1866 day and 266 night comparison pairs found. pressure rather than spectral resolution. After accounting for retrieval differences in a priori proﬁles and covariances be- tween MOPITT and IASI (another FTS instrument), George et al. (2015) ﬁnd a similar positive bias for MOPITT in the upper troposphere. operator in Eq. (1) to the in situ aircraft proﬁle, we are ac- counting for the smoothing error term. Thus, we expect dif- ferences between ˆxval and our retrieved ˆx to be due to obser- vational error terms (Eq. 3) and to geophysical differences from the sampling of different air masses and surface loca- tions because of imperfect coincidence. Table 2 gives the mean bias and standard deviations for selected pressures and partial column average VMRs over different observing conditions (land, ocean, day, and night). 4.2 Comparison of TROPESS satellite and aircraft observations Sˆx = Ssmoothing + Sobservational , (2) with Ssmoothing = (I −Axx)Sa(I −Axx)T and Sobservational = Snoise + Scross−state + Ssystematic, (3) where Snoise = GSeGT , Scross−state = P b_retAxsSb_ret a AT xs, and (2) In order to account for the satellite observational and re- trieval approach, including prior information, when compar- ing satellite retrieval products to in situ measurements of CO, we apply the instrument operator to convert the in situ proﬁle into the values that would be retrieved for the same air mass assuming the satellite instrument and retrieval (Jones et al., 2003, Rodgers and Conner, 2003, Worden et al., 2007): (3) Ssystematic = X bGKbSb(GKb)T . (4) Ssystematic = X bGKbSb(GKb)T . ˆxval = xa + A(xval −xa), (1) (4) (1) In this notation, b variables are parameters that are held constant in the CO retrieval (such as temperature and wa- ter vapor) but affect the radiance observation and are prop- agated through Jacobian Kb, while b_ret variables are re- trieved along with CO (such as surface emissivity) and have corresponding off-diagonal terms in the full retrieval aver- aging kernel matrix. When we apply the satellite instrument where xval is the aircraft or sonde in situ proﬁle being used for validation (following extension, described above, and lin- ear interpolation to the satellite vertical grid), xa is the a pri- ori proﬁle used in the TROPESS retrieval, A is the averaging kernel matrix that describes the observation and retrieval ver- tical sensitivity to the true state, and ˆxval is the in situ valida- tion proﬁle transformed by the satellite instrument operator. https://doi.org/10.5194/amt-15-5383-2022 Atmos. Meas. Tech., 15, 5383–5398, 2022 H. M. Worden et al.: TROPESS/CrIS carbon monoxide proﬁle validation 5389 Figure 4. Relative differences (%) in single CrIS retrievals with coincident NOAA GML ˆxval proﬁles (grey) and the average percent difference with 1σ horizontal bars (red). Both day and night CrIS observations are included for coincidence search, with 1866 day and 266 night comparison pairs found. Figure 5. Latitude dependence of CO partial column average VMR (ppb) for TROPESS/CrIS retrievals and NOAA GML ˆxval (a) as well as bias difference statistics (b) shown by box–whisker symbols representing minimum and maximum values (whisker), lower quar- tile (box bottom), median (white stripe), and upper quartile (box top). A minimum of ﬁve comparisons per bin was required. Figure 4. 5.1 TROPESS/CrIS CO comparisons with NOAA GML aircraft data After extending the in situ proﬁles vertically (described in Sect. 4.1) and applying Eq. (1), we compute the differences between satellite retrievals and transformed aircraft proﬁles. Figure 4 shows the bias (% relative difference) of the CrIS CO retrieved proﬁles with respect to NOAA GML aircraft proﬁles (ˆxval). A similar pattern of positive bias in the lower to middle troposphere and negative bias in the upper tro- posphere is observed for MUSES/AIRS proﬁles compared to NOAA GML ﬂights (Hegarty et al., 2022). However, MOPITT (version 9, TIR-only data) comparisons to NOAA GML (Deeter et al., 2022) have almost the opposite vertical bias pattern with a negative bias (−1.6 %) in the lower to middle troposphere and a positive bias (0.6 %) in the upper troposphere. Since TROPESS and MOPITT retrievals both use optimal estimation algorithms and a similar prior CO error covariance, this different vertical bias pattern is most likely due to instrument differences. MOPITT uses gas ﬁl- ter correlation radiometry instead of spectroscopy to detect CO absorption in the atmosphere with corresponding differ- ences in vertical sensitivity that are determined from gas cell Figure 5 shows how the observed partial column average VMRs and CrIS retrieval bias with respect to NOAA GML ˆxval proﬁles vary with latitude, and Fig. 6 shows how these vary with time. No signiﬁcant bias dependence on latitude is observed for the NOAA GML ﬂight sites. Although a bias drift of −0.007 ± 0.001 % d−1 is detected, we recognize that our comparison time range is not sufﬁcient for a reliable es- timate of bias drift, and more years of comparisons would be required. 4.2 Comparison of TROPESS satellite and aircraft observations The partial column refers to the CO column between the min- imum and maximum ﬂight altitudes of each aircraft proﬁle. The average VMR over this range is computed by interpo- lating both the CrIS retrieval and the aircraft ˆxval proﬁle to these end points. Since aircraft ﬂights normally occur during daytime, there are fewer coincident pairs for CrIS night re- trievals. Tang et al. (2020) ﬁnd larger bias and variance for nighttime MOPITT data in comparisons with in situ aircraft data, especially for ﬂights over urban regions, suggesting that more night validation ﬂights are needed to properly evaluate night satellite retrievals. Atmos. Meas. Tech., 15, 5383–5398, 2022 5.2 TROPESS/CrIS CO validation with ATom Both day and night CrIS observations are included. Figure 6. Time dependence of CO partial column average VMR Figure 6. Time dependence of CO partial column average VMR (ppb) for TROPESS/CrIS retrievals and NOAA GML ˆxval (a) as well as bias difference statistics (b) shown by box–whisker symbols representing minimum and maximum values (whisker), lower quar- tile (box bottom), median (white stripe), and upper quartile (box top). A minimum of ﬁve comparisons per bin was required. The dashed line indicates a ﬁt for bias drift (see text). Figure 6. Time dependence of CO partial column average VMR (ppb) for TROPESS/CrIS retrievals and NOAA GML ˆxval (a) as well as bias difference statistics (b) shown by box–whisker symbols representing minimum and maximum values (whisker), lower quar- tile (box bottom), median (white stripe), and upper quartile (box top). A minimum of ﬁve comparisons per bin was required. The dashed line indicates a ﬁt for bias drift (see text). Figure 7. Relative differences (%) in single CrIS retrievals with coincident ATom ˆxval proﬁles (grey) and the average percent dif- ference with 1σ horizontal bars (red). Latitude ranges are indicated in each panel along with the number of comparison pairs. Both day and night CrIS observations are included. ﬁles for all latitudes and three latitude ranges: 30◦S to 30◦N, 90 to 30◦S, and 30 to 90◦N. The vertical behavior of the bias is similar to the above CrIS comparisons with NOAA GML ﬂights, with a positive bias in the lower troposphere and a negative bias in the upper troposphere, and is also sim- ilar to the MUSES/AIRS CO proﬁles compared to ATom ﬂights (Hegarty et al., 2022). However, for MOPITT V9T comparisons to ATom ﬂights (Deeter et al., 2022), the verti- cal bias pattern is again mostly opposite, with a negative bias (∼4 %) in the lower to middle troposphere and a positive bias (∼2 %) in the upper troposphere. This TROPESS/CrIS CO bias also differs from Nalli et al. (2020), who examined the bias of NUCAPS proﬁles (including CO) with respect to ATom in situ proﬁles. That study, using the multiple-FOV NUCAPS retrievals, found a small positive bias (∼2 %) for SNPP/CrIS CO with respect to ATom CO at all tropospheric vertical levels after applying their averaging kernels. lected pressures and partial column average VMRs over dif- ferent observing conditions (land, ocean, day, and night) and latitude ranges. 5.2 TROPESS/CrIS CO validation with ATom Figure 7 shows the bias (% relative difference) of the CrIS CO retrieved proﬁles with respect to ATom ˆxval in situ pro- https://doi.org/10.5194/amt-15-5383-2022 Atmos. Meas. Tech., 15, 5383–5398, 2022 H. M. Worden et al.: TROPESS/CrIS carbon monoxide proﬁle validation 5390 H. M. Worden et al.: TROPESS/CrIS carbon monoxide proﬁle validation Table 2. Bias and standard deviation (SD) for comparisons of SNPP TROPESS/CrIS CO retrievals and in situ CO proﬁles from NOAA GML ﬁghts 5390 Table 2. Bias and standard deviation (SD) for comparisons of SNPP TROPESS/CrIS CO retrievals and in situ CO proﬁles from NOAA GML ﬁghts. % bias % SD % bias % SD % bias % SD % bias % SD No. Obs. type 750 hPa 750 hPa 511 hPa 511 hPa 287 hPa 287 hPa column column pairs All 2.29 9.84 0.92 11.20 −4.48 13.76 0.57 8.56 2092 Land 3.04 10.85 −0.044 11.95 −6.15 13.97 1.24 9.46 853 Ocn 1.78 9.04 1.58 10.59 −3.33 13.49 0.11 7.84 1239 Day 1.97 9.79 0.13 10.93 −5.37 13.32 0.23 8.77 1866 Ngt 4.94 9.86 7.36 11.27 2.81 15.05 3.41 5.82 266 Figure 6. Time dependence of CO partial column average VMR (ppb) for TROPESS/CrIS retrievals and NOAA GML ˆxval (a) as well as bias difference statistics (b) shown by box–whisker symbols representing minimum and maximum values (whisker), lower quar- tile (box bottom), median (white stripe), and upper quartile (box top). A minimum of ﬁve comparisons per bin was required. The dashed line indicates a ﬁt for bias drift (see text). Figure 7. Relative differences (%) in single CrIS retrievals with coincident ATom ˆxval proﬁles (grey) and the average percent dif- ference with 1σ horizontal bars (red). Latitude ranges are indicated in each panel along with the number of comparison pairs. Both day and night CrIS observations are included. Table 2. Bias and standard deviation (SD) for comparisons of SNPP TROPESS/CrIS CO retrievals and in situ CO proﬁles from NOAA GML ﬁghts. % bias % SD % bias % SD % bias % SD % bias % SD No. Obs. 5.2 TROPESS/CrIS CO validation with ATom type 750 hPa 750 hPa 511 hPa 511 hPa 287 hPa 287 hPa column column pairs All 2.29 9.84 0.92 11.20 −4.48 13.76 0.57 8.56 2092 Land 3.04 10.85 −0.044 11.95 −6.15 13.97 1.24 9.46 853 Ocn 1.78 9.04 1.58 10.59 −3.33 13.49 0.11 7.84 1239 Day 1.97 9.79 0.13 10.93 −5.37 13.32 0.23 8.77 1866 Ngt 4.94 9.86 7.36 11.27 2.81 15.05 3.41 5.82 266 Figure 6. Time dependence of CO partial column average VMR (ppb) for TROPESS/CrIS retrievals and NOAA GML ˆxval (a) as well as bias difference statistics (b) shown by box–whisker symbols representing minimum and maximum values (whisker), lower quar- tile (box bottom), median (white stripe), and upper quartile (box top). A minimum of ﬁve comparisons per bin was required. The dashed line indicates a ﬁt for bias drift (see text). Figure 7. Relative differences (%) in single CrIS retrievals with coincident ATom ˆxval proﬁles (grey) and the average percent dif- ference with 1σ horizontal bars (red). Latitude ranges are indicated in each panel along with the number of comparison pairs. Both day and night CrIS observations are included. n (SD) for comparisons of SNPP TROPESS/CrIS CO retrievals and in situ CO proﬁles from NOAA GML ation (SD) for comparisons of SNPP TROPESS/CrIS CO retrievals and in situ CO proﬁles from NOAA GML Table 2. Bias and standard deviation (SD) for comparisons of SNPP TROPESS/CrIS CO retrievals and in situ CO proﬁles from NOAA GML ﬁghts. Table 2. Bias and standard deviation (SD) for comparisons of SNPP TROPESS/CrIS CO retrievals and in situ ﬁghts. Table 2. Bias and standard deviation (SD) for comparisons of SNPP TROPESS/CrIS CO retrievals and in situ CO proﬁles from NOAA GML ﬁghts. Day 1.97 9.79 0.13 10 Ngt 4.94 9.86 7.36 11 Figure 6. Time dependence of CO partial column average VMR (ppb) for TROPESS/CrIS retrievals and NOAA GML ˆxval (a) as well as bias difference statistics (b) shown by box–whisker symbols representing minimum and maximum values (whisker), lower quar- tile (box bottom), median (white stripe), and upper quartile (box top). A minimum of ﬁve comparisons per bin was required. The dashed line indicates a ﬁt for bias drift (see text). Figure 7. Relative differences (%) in single CrIS retrievals with coincident ATom ˆxval proﬁles (grey) and the average percent dif- ference with 1σ horizontal bars (red). Latitude ranges are indicated in each panel along with the number of comparison pairs. 5.2 TROPESS/CrIS CO validation with ATom Blue triangles in- dicate CrIS CO partial column average values over land and At- lantic Ocean scenes; red diamonds indicate CrIS values over Paciﬁc Ocean scenes. Figure 8. Latitude dependence of CO partial column average VMR (ppb) for TROPESS/CrIS retrievals and ATom ˆxval (a) and bias dif- ference statistics (b) shown by box–whisker symbols representing minimum and maximum values (whisker), lower quartile (box bot- tom), median (white stripe), and upper quartile (box top). A mini- mum of ﬁve comparisons per bin was required. Figure 9. Latitude dependence of partial column average CO for each ATom campaign. Black squares show ATom ˆxval partial col- umn average values over Atlantic Ocean scenes; black circles in- dicate ATom values over Paciﬁc Ocean scenes. Blue triangles in- dicate CrIS CO partial column average values over land and At- lantic Ocean scenes; red diamonds indicate CrIS values over Paciﬁc Ocean scenes. Figure 9. Latitude dependence of partial column average CO for each ATom campaign. Black squares show ATom ˆxval partial col- umn average values over Atlantic Ocean scenes; black circles in- dicate ATom values over Paciﬁc Ocean scenes. Blue triangles in- dicate CrIS CO partial column average values over land and At- lantic Ocean scenes; red diamonds indicate CrIS values over Paciﬁc Ocean scenes. Figure 8. Latitude dependence of CO partial column average VMR (ppb) for TROPESS/CrIS retrievals and ATom ˆxval (a) and bias dif- ference statistics (b) shown by box–whisker symbols representing minimum and maximum values (whisker), lower quartile (box bot- tom), median (white stripe), and upper quartile (box top). A mini- mum of ﬁve comparisons per bin was required. Figure 10. Bias of CrIS partial column average CO vs. CO amount for NOAA GML ﬂights in the top panel and ATom ﬂights in the bottom panel with box–whisker symbols in 5 ppb bins. Linear re- gression results are shown in the legend boxes. al., 2021) when assessing the contributions to this latitude dependence in TROPESS/CrIS CO bias. In Fig. 9, we examine the seasonal behavior of CO sam- pled by ATom and CrIS in mostly remote ocean regions. In the high-latitude Southern Hemisphere (SH), we see the low- est values in summer and fall (Jan–Feb and Apr–May) as expected due to the chemical destruction of CO in a region with few local combustion sources. 5.2 TROPESS/CrIS CO validation with ATom In the tropics, we ﬁnd high values corresponding to African and South American biomass burning plumes over the Atlantic in all seasons ex- cept Northern Hemisphere (NH) spring. Lower values of CO in the tropics for NH summer and winter correspond to pro- ﬁles over the Paciﬁc Ocean (e.g., Strode et al., 2018; Bour- geois et al., 2020). The close alignment of the CrIS and ATom ˆxval partial column average values in Fig. 9 indicates that CrIS is able to capture the seasonal, latitudinal, and hemi- spherical variations observed by ATom. Figure 10. Bias of CrIS partial column average CO vs. CO amount for NOAA GML ﬂights in the top panel and ATom ﬂights in the bottom panel with box–whisker symbols in 5 ppb bins. Linear re- gression results are shown in the legend boxes. 5.4 Evaluation of TROPESS/CrIS CO retrieval observational errors Here we compare the observed variance of differences be- tween retrieved CrIS CO proﬁles and in situ aircraft pro- ﬁles, after applying Eq. (1), with the TROPESS reported ob- servational errors deﬁned in Eqs. (3) and (4). As described in Sect. 4.3, we expect the differences between retrieved CrIS and aircraft CO proﬁles (ˆxval) to have a variance due to the combination of observational errors and geophysical variation from imperfect coincidence. Figure 11 shows com- parisons of individual and average computed observational fractional errors to the standard deviation (SD) of CrIS– ˆxval proﬁle differences as well as the diagonal for the a pri- ori covariance and the SD of prior–ˆxval proﬁle differences. As expected, the average observational errors are less than 5.2 TROPESS/CrIS CO validation with ATom As described above, the partial column aver- age VMR is computed over the altitude ranges of each air- craft proﬁle. Due to the nature of the ATom campaign, there are fewer observations over land. Figure 8 shows how the observed partial column average VMRs and CrIS retrieval bias with respect to ATom ˆxval pro- ﬁles vary with latitude. It appears that tropical and North- ern Hemisphere subtropical latitude ranges have a slightly higher positive bias than what is observed for higher lati- tudes, potentially indicating a TROPESS/CrIS retrieval issue with water vapor or some other interferent that is not fully characterized and requires further investigation. For exam- ple, Deeter et al. (2018) found that an empirical correction to MOPITT radiances resulting from a linear dependence on water vapor removed most of the latitude-dependent bias in MOPITT CO proﬁles. Another gas interferent in the TIR CO band is N2O, and we will also need to consider the latitude- dependent N2O anomalies observed by ATom (Gonzalez et CrIS CO comparisons with ATom have less variance than comparisons with NOAA GML, especially for 90 to 30◦S. Table 3 gives the mean bias and standard deviations for se- Atmos. Meas. Tech., 15, 5383–5398, 2022 https://doi.org/10.5194/amt-15-5383-2022 H. M. Worden et al.: TROPESS/CrIS carbon monoxide proﬁle validation H. M. Worden et al.: TROPESS/CrIS carbon monoxide proﬁle validation 5391 H. M. Worden et al.: TROPESS/CrIS carbon monoxide pr Figure 8. Latitude dependence of CO partial column average VMR (ppb) for TROPESS/CrIS retrievals and ATom ˆxval (a) and bias dif- ference statistics (b) shown by box–whisker symbols representing minimum and maximum values (whisker), lower quartile (box bot- tom), median (white stripe), and upper quartile (box top). A mini- mum of ﬁve comparisons per bin was required. p Figure 8. Latitude dependence of CO partial column average VMR (ppb) for TROPESS/CrIS retrievals and ATom ˆxval (a) and bias dif- ference statistics (b) shown by box–whisker symbols representing minimum and maximum values (whisker), lower quartile (box bot- tom), median (white stripe), and upper quartile (box top). A mini- mum of ﬁve comparisons per bin was required. Figure 9. Latitude dependence of partial column average CO for each ATom campaign. Black squares show ATom ˆxval partial col- umn average values over Atlantic Ocean scenes; black circles in- dicate ATom values over Paciﬁc Ocean scenes. 5.3 Dependence on CO amount For both the NOAA GML and ATom ﬂights we ﬁnd a small negative dependence of TROPESS/CrIS retrieval bias with respect to CO amount, with magnitude less than 0.1 % ppb−1. Figure 10 shows how the partial column average VMR bias varies with CO VMR for the two validation data sources, and we can also see how ATom ﬂights sampled air with lower CO concentrations. Figure 10 indicates that TROPESS/CrIS CO average column VMRs have very little dependence on CO amount, and we ﬁnd similar results for CrIS-retrieved CO at vertical levels 511 and 750 hPa (shown in the Supplement). https://doi.org/10.5194/amt-15-5383-2022 Atmos. Meas. Tech., 15, 5383–5398, 2022 5392 H. M. Worden et al.: TROPESS/CrIS carbon monoxide proﬁle validation Table 3. Bias and standard deviation (SD) for comparisons of SNPP TROPESS/CrIS CO retrievals and in situ CO proﬁles from ATom ﬂight campaigns 1–4. H. M. Worden et al.: TROPESS/CrIS carbon monoxide proﬁle validation 5392 Table 3. Bias and standard deviation (SD) for comparisons of SNPP TROPESS/CrIS CO retrievals and in situ CO proﬁles from ATom ﬂight campaigns 1–4. Table 3. Bias and standard deviation (SD) for comparisons of SNPP TROPESS/CrIS CO retrievals and in situ CO proﬁles from ATom ﬂ ampaigns 1–4. Latitude % bias % SD % bias % SD % bias % SD % bias % SD No. Obs. type range (◦) 750 hPa 750 hPa 511 hPa 511 hPa 287 hPa 287 hPa col. col. 5.3 Dependence on CO amount pairs All all 2.21 8.46 0.54 8.12 −2.95 10.24 −0.035 5.91 1052 Land all 1.20 4.15 −0.49 7.59 −2.95 10.46 −0.79 7.09 102 Land 30◦S–30◦N – – – – – – – – 1 Land 30–90◦N 1.22 4.27 −0.69 7.76 −3.25 10.70 −0.91 7.32 95 Land 90–30◦S 0.12 0.29 0.89 2.35 1.84 4.65 0.67 1.86 6 Ocn all 2.32 8.79 0.65 8.17 −2.95 10.21 0.046 5.76 950 Ocn 30◦S–30◦N 4.32 10.80 3.96 6.75 −0.86 11.67 2.33 5.44 418 Ocn 30–90◦N 0.75 6.01 −2.28 8.70 −5.03 8.51 −2.22 6.34 310 Ocn 90–30◦S 0.74 6.85 −1.46 7.5 −3.98 8.57 −1.09 3.49 222 Day all 2.62 8.76 0.53 7.91 −3.21 9.81 0.010 5.85 782 Day 30◦S–30◦N 4.94 11.42 3.55 6.57 −2.01 10.99 2.23 5.16 300 Day 30–90◦N 0.91 5.76 −1.63 8.62 −4.33 9.22 −1.68 6.74 331 Day 90–30◦S 1.79 6.90 −0.72 6.71 −3.11 8.12 −0.70 2.91 151 Ngt all 1.03 7.39 0.57 8.71 −2.21 11.36 −0.17 6.08 270 Ngt 30◦S–30◦N 2.79 8.82 5.02 7.07 2.03 12.73 2.59 6.09 119 Ngt 30–9◦N 0.68 5.15 −3.16 7.93 −5.88 8.45 −2.98 5.84 74 Ngt 90–30◦S −1.35 5.94 −2.73 8.58 −5.25 9.15 −1.73 4.30 77 Table 3. Bias and standard deviation (SD) for comparisons of SNPP TROPESS/CrIS CO retrievals and in situ CO proﬁles from ATom ﬂight campaigns 1–4. Table 3. Bias and standard deviation (SD) for comparisons of SNPP TROPESS/CrIS CO retrievals and in situ CO proﬁles from ATom ﬂight campaigns 1–4. 6 Summary and conclusions SD(CrIS–ˆxval), but in some vertical ranges, they are much less and could be underestimated via instrument and system- atic error assumptions in the TROPESS retrieval as Hegarty et al. (2022) suggest. Additional studies to test the sensitivity of the comparison variance to a range of coincidence crite- ria are needed to conﬁrm a retrieval underestimate, but these would require several repeated validation measurements for the same observing conditions. This study used in situ observations from routine NOAA GML ﬂights and the four ATom campaigns to evaluate TROPESS single-pixel CO retrievals from the SNPP/CrIS FTS instrument. We ﬁnd the following. 1. The single-FOV CrIS product provides improved repre- sentation of CO in smoke plumes compared to retrievals that combine multiple FOVs. Despite the potential for underestimated observational er- rors, the general behavior of the error comparison is what we expect from Eq. (1), and we can see the retrieval inﬂuence on the shape of SD(CrIS–ˆxval). Near the surface, where there is less retrieval sensitivity as indicated by the averaging kernel, we see that SD(prior–ˆxval) becomes smaller than SD(CrIS– ˆxval). This is expected for vertical ranges with less retrieval sensitivity since the priori contribution becomes more domi- nant in ˆxval. In contrast, for the middle troposphere where we have the most sensitivity for TIR remote sensing, it is clear that SD (CrIS – ˆxval) represents an improvement over SD (prior–ˆxval). In Fig. 12, the error comparison is shown sep- arately for three ATom latitude ranges, and we can see that the agreement between observational errors and SD (CrIS– ˆxval) is closest for ATom ﬂights in the mostly clean middle- to high-latitude Southern Hemisphere, where it is most likely that the aircraft and satellite are observing similar air masses with background CO concentrations. These results give con- ﬁdence that TROPESS single-retrieval error characterization can be used to weight data for averaging and inverse analysis applications. 2. Comparisons with aircraft in situ proﬁles (after exten- sion, interpolation, and application of Eq. 1) show that biases have a vertical dependence in the troposphere that is consistent for both sets of in situ data with av- erage biases that are positive (∼2.3 %) in the lower tro- posphere and negative (∼−4.5 %) in the upper tropo- sphere. 3. Atmos. Meas. Tech., 15, 5383–5398, 2022 6 Summary and conclusions For reference, and the standard deviation of CrIS prior with aircraft ˆxval is in cyan, and the a priori fractional uncertainty (0.3) is shown in cyan with triangles. Figure 12. Same as Fig. 11 but for three ATom latitude ranges. Figure 12. Same as Fig. 11 but for three ATom latitude ranges. 6. There is a small negative dependence (magnitude < 0.1 % ppb−1) of CrIS bias on CO amount. 6. There is a small negative dependence (magnitude < 0.1 % ppb−1) of CrIS bias on CO amount. 6. There is a small negative dependence (magnitude < 0.1 % ppb−1) of CrIS bias on CO amount. long-term records of satellite CO observations are harmo- nized and used together for computing trends, data assimila- tion, or other analyses. For example, with the 22-year record of MOPITT CO proﬁles, this is especially important when combining datasets since the vertical bias pattern for MO- PITT data with respect to in situ observations has a positive bias in the upper troposphere and negative bias in the lower to middle troposphere with the opposite behavior compared to the TROPESS/CrIS vertical bias pattern. 7. Comparisons of computed observational errors and standard deviations of retrieval–aircraft comparison dif- ferences show expected vertical behavior and demon- strate signiﬁcant improvement over the standard devia- tion of prior–aircraft differences in vertical ranges with higher retrieval sensitivity. Future validation of the TROPESS/CrIS CO products will include a longer time record of comparisons and quantiﬁca- tion of bias drift for CrIS on SNPP and on the JPSS satellite series. The validation results presented here demonstrate that these products are suitable for tropospheric CO data analy- ses. The bias at all vertical levels is < 10 %, and error char- acterization for single retrievals can be used to weight data for averaging and applications such as data assimilation and inverse modeling. TROPESS/CrIS CO biases detected in this study are in gen- eral much smaller than comparison standard deviations. We therefore make no recommendations for automated bias cor- rections in data processing, similar to other validation stud- ies for satellite CO retrievals (e.g., Deeter et al., 2019, 2022). 6 Summary and conclusions Small biases (0.6 % and −0.04 % for NOAA GML and ATom, respectively) are observed for the CrIS CO par- tial column average VMR corresponding to the aircraft proﬁle vertical ranges. 3. Small biases (0.6 % and −0.04 % for NOAA GML and ATom, respectively) are observed for the CrIS CO par- tial column average VMR corresponding to the aircraft proﬁle vertical ranges. 4. No signiﬁcant latitude dependence of CrIS CO column bias is found for the NOAA GML comparisons, but comparisons with ATom, which better covered a range of latitudes, have a slightly more positive bias for trop- ical scenes that could indicate a small, uncharacterized retrieval dependence on water vapor or another interfer- ent species. 4. No signiﬁcant latitude dependence of CrIS CO column bias is found for the NOAA GML comparisons, but comparisons with ATom, which better covered a range of latitudes, have a slightly more positive bias for trop- ical scenes that could indicate a small, uncharacterized retrieval dependence on water vapor or another interfer- ent species. 5. CrIS CO retrievals capture the seasonal and spatial vari- ations observed by ATom. 5. CrIS CO retrievals capture the seasonal and spatial vari- ations observed by ATom. Atmos. Meas. Tech., 15, 5383–5398, 2022 https://doi.org/10.5194/amt-15-5383-2022 H. M. Worden et al.: TROPESS/CrIS carbon monoxide proﬁle validation H. M. Worden et al.: TROPESS/CrIS carbon monoxide proﬁle validation 5393 Figure 11. Error comparison of CrIS observational error estimates and the standard deviation (SD) of CrIS ˆxval (in black) for NOAA GML ﬂights (a) and ATom ﬂights (b). Single-proﬁle CrIS observational error estimates are plotted in red, with the average in dark blue with triangles. For reference, and the standard deviation of CrIS prior with aircraft ˆxval is in cyan, and the a priori fractional uncertainty (0.3) is shown in cyan with triangles. Figure 11. Error comparison of CrIS observational error estimates and the standard deviation (SD) of CrIS ˆxval (in black) for NOAA GML ﬂi h ( ) d AT ﬂi h (b) Si l ﬁl C IS b i l i l d i d i h h i d k bl i h Figure 11. Error comparison of CrIS observational error estimates and the standard deviation (SD) of CrIS ˆxval (in black) for NOAA GML ﬂights (a) and ATom ﬂights (b). Single-proﬁle CrIS observational error estimates are plotted in red, with the average in dark blue with triangles. https://doi.org/10.5194/amt-15-5383-2022 References The peer-review process was guided by an independent editor, and the authors also have no other competing interests to declare. T., Clark, H., Commane, R., Daube, B., Diskin, G. W., Elkins, J. W., Gao, R.-S., Gaudel, A., Hintsa, E. J., Johnson, B. J., Kivi, J. W., Gao, R.-S., Gaudel, A., Hintsa, E. J., Johnson, B. J., Kivi, R., McKain, K., Moore, F. L., Parrish, D. D., Querel, R., Ray, E., Sánchez, R., Sweeney, C., Tarasick, D. W., Thompson, A. M., Thouret, V., Witte, J. C., Wofsy, S. C., and Ryerson, T. B.: Global-scale distribution of ozone in the remote troposphere from the ATom and HIPPO airborne ﬁeld missions, Atmos. Chem. Phys., 20, 10611–10635, https://doi.org/10.5194/acp-20- 10611-2020, 2020. Disclaimer. Publisher’s note: Copernicus Publications remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Bowman, K. W.: TROPESS CrIS-SNPP L2 Carbon Monoxide for West Coast Fires HiRes, Standard Product V1, Green- belt, MD, USA, Goddard Earth Sciences Data and In- formation Services Center (GES DISC), NASA [data set], https://doi.org/10.5067/Y3MAIEUNDTBX, 2021. Acknowledgements. This research was conducted at the National Center for Atmospheric Research (NCAR), which is sponsored by the National Science Foundation. Part of this research was carried out at the Jet Propulsion Laboratory (JPL), California Institute of Technology, under a contract with the National Aeronautics and Space Administration. The NOAA GML aircraft observations are supported by NOAA and CIRES. The ATom aircraft data were sup- ported by the NASA Airborne Science Program and Earth Science Project Ofﬁce. We acknowledge the use of imagery from the NASA Worldview application (https://worldview.earthdata.nasa.gov/, last access: 17 September 2022), part of the NASA Earth Observing System Data and Information System (EOSDIS). We thank Ben- jamin Gaubert for his NCAR internal review of the paper. Bowman, K. W., Rodgers, C. D., Kulawik, S. S., Worden, J., Sarkissian, E., Osterman, G., Steck, T., Lou, M., Eldering, A., and Shephard, M.: Tropospheric emission spectrometer: retrieval method and error analysis, IEEE Trans. Geosci. Remote Sens., 44, 1297–1307, https://doi.org/10.1109/TGRS.2006.871234, 2006. Bowman, K. W., Worden, J. R., Herman, R., Cady-Pereira, K., Natraj, V., Payne, V. H., Worden, H. M., and Kulawik, S. S.: TROPESS Level 2 Algorithm Theoretical Basis Document (ATBD) V1, 2021 at: https://docserver.gesdisc.eosdis.nasa. gov/public/project/TROPESS/TROPESS_ATBDv1.1.pdf, last access: 12 April 2022. Brasseur, G. P., Hauglustaine, D. A., Walters, S., Rasch, P. J., Muller, J. 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TROPESS/CrIS CO products are available via the GES DISC from the NASA TRopospheric Ozone and its Pre- cursors from Earth System Sounding (TROPESS) project at https://doi.org/10.5067/I1NONOEPXLHS (Bowman, 2021). Review statement. This paper was edited by Gabriele Stiller and re- viewed by two anonymous referees. 6 Summary and conclusions This is unlike other TROPESS products such as CH4 (Ku- lawik et al., 2021) for which a bias correction is more ap- propriate given the size of bias detected as well as the at- mospheric lifetime (∼10 years for methane) and reduced at- mospheric variability compared to CO. Each analysis using TROPESS/CrIS CO data must consider the variability of CO over the domain of interest and ascertain whether the biases observed here could affect numerical conclusions. The bi- ases reported from this study will need to be included when Data availability. The NOAA GML data were obtained from https://doi.org/10.7289/V5N58JMF (Sweeney et al., 2021). 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https://openalex.org/W4288343417	https://zenodo.org/records/3236738/files/Toc%20%20-%20%20-%20%20ijdkp%20May.pdf	English	null	Current Issue: May 2019, Volume 9, Number 2/3	Zenodo (CERN European Organization for Nuclear Research)	2,019	cc-by	18	Applying Neural Networks for Supervised Learning of Medical Data Ourida Ben Boubaker, Jouf University, Kingdom of Saudi Arabia
https://openalex.org/W2791254386	https://halshs.archives-ouvertes.fr/halshs-01785750/document	English	null	Prenatal care and socioeconomic status: effect on cesarean delivery	Health economics review	2,018	cc-by	9,675	To cite this version: Carine Milcent. Healthcare for Migrants in Urban China: A New Frontier. China perspectives, 2010, 2010 (4). halshs-00754683v7 1 I would like to thank Wei Zhong and all of the participants at the CASS seminars (Beijing), Beijing Normal University, and the Sciences Politics seminar at BNU (Beijing). I am also grateful to Thomas Serrurier, Pierre Miege, Andrew Clark, and Richard Balme for helpful comments. HAL Id: halshs-00754683 https://pjse.hal.science/halshs-00754683v7 Submitted on 26 Mar 2014 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Keywords: Healthcare access, Health insurance, Social Capital, China, Migrants. Healthcare for Migrants in Urban China: A New Frontier Carine Milcent1 JEL Classification: D82, H52, I18. Keywords: Healthcare access, Health insurance, Social Capital, China, Migrants. Abstract How can healthcare access for Chinese migrants be improved? Migrant workers face two types of healthcare-access exclusion in the workplace: one is financial (because of their income) and the other is social (because of the lack of social networks in the city). We use 2006 data from a survey of rural migrant workers conducted in five of the most economically-advanced cities. The empirical findings support the hypothesis of a return to the hometown for migrant workers with poor health. Apart from residency permits and income, the social integration of migrant workers is also found to be a decisive factor in healthcare access. JEL Classification: D82, H52, I18. 1 1 2 S. I. Hossain, “Tackling Health Transition in China,” The World Bank: World Bank, Policy Research Paper No. 1813, 1997; National Bureau of Statistics, China Statistical Yearbook 2002, Beijing, China Statistical Press, 2002. X. Liu and Y. Yi, The Health Sector in China: Policy and Institutional Review, background paper for the orld Bank China Health Study, 2004. Available at the website: http://siteresources.worldbank.org/ NTEAPREGTOPHEANUT/Resources/502734.1129734318233/policyandinstitutionalreview-final.pdf. 3 Central Intelligence Agency, World Factbook 2010, 2010, https://www.cia.gov/library/publications/the-world- factbook/geos/ch.html. 2002. 3 Central Intelligence Agency, World Factbook 2010, 2010, https://www.cia.gov/library/publications/the-world- factbook/geos/ch.html. 4 X. Liu and Y. Yi, The Health Sector in China: Policy and Institutional Review, background paper for the World Bank China Health Study, 2004. Available at the website: http://siteresources.worldbank.org/ Central Intelligence Agency, World Factbook 2010, 2010, https://www.cia.gov/library/publications/the-wo ctbook/geos/ch.html. X. Liu and Y. Yi, The Health Sector in China: Policy and Institutional Review, background paper for the No. 1813, 1997; National Bureau of Statistics, China Statistical Yearbook 2002, Beijing, China Statistical Press, 2002. 3 Central Intelligence Agency, World Factbook 2010, 2010, https://www.cia.gov/library/publications/the-world- factbook/geos/ch.html. 4 X. Liu and Y. Yi, The Health Sector in China: Policy and Institutional Review, background paper for the World Bank China Health Study, 2004. Available at the website: http://siteresources.worldbank.org/ INTEAPREGTOPHEANUT/Resources/502734.1129734318233/policyandinstitutionalreview-final.pdf. Introduction The growth in Gross Domestic Product (GDP) has been accompanied by an expansion in healthcare expenditure. From under 3 percent of GDP in 1980, this grew to 5.3 percent in 2000,2 increasing even more rapidly in urban than in rural areas. As the percentage of migrants in cities increases, their access to education and healthcare has become an increasingly central issue in China. Who has healthcare coverage? This paper proposes some answers by focusing on rural-urban migrant workers with formal jobs and in regular situations. On top of their existing 103 million urban migrants, Chinese cities continue to experience a steady migration inflow. In terms of health, one consequence is a falling proportion of the rural population living in extreme poverty, and improvements in the level of nutrition. Life expectancy at birth in 2010 was 74.51 years,3 which is eight years higher than the world average. As the disastrous famine in the late 1950s resulted in high mortality, the current life expectancy figure represents an enormous improvement within China. Other health indicators such as the infant mortality rate, the under-five mortality rate, and the maternal mortality rate have also registered improvements.4 However, this expansion of healthcare is for the most part unequally distributed. Economic reforms have had positive effects on GDP and the level of nutrition, but have led to the collapse of the public healthcare network and universal access to basic healthcare. The result is an increase in healthcare inequality. The World Health Organization (WHO) periodically ranks countries according to the inequality of their healthcare systems, in particular their healthcare insurance system. According to the last WHO country classification, the Chinese health sector ranked close to the bottom in terms of the fairness of financial contributions. To help understand this deterioration, we here present a brief description of the healthcare-insurance system before the economic reforms and its subsequent evolution. In China, before the economic reforms, the healthcare-insurance system was not homogeneous. In urban areas, it depended on large state-owned enterprises (SOE) that took care of workers' 2 medical needs; healthcare in rural areas was determined locally. However, out-of-pocket expenses were close to zero, whatever the healthcare setup. In urban areas, the dismantling of the large state-owned enterprises (SOE) led to healthcare supplied by smaller-size units. These units quickly ran into difficulties financing their own social insurance. 6 Adam Wagstaff, Eddy van Doorslaer, Hattem van der Burg, Samuel Calonge, Terkel Christiansen, Guido Citoni, Ulf-G Gerdtham, Mike Gerfin, Lorna Gross, and Unto Hakinnen, “Equity in the Finance of Health Care: Some Further International Comparisons,” Journal of Health Economics, 18 (3), 1999, pp. 263-290. 7 Gerald Bloom and Xingyuan Gu, “Health Sector Reform: Lessons from China,” Social Science and Medicine, 45(3), 1997, pp. 351-360. 8 Xingzhu Liu and Anne Mills, “Financing Reforms of Public Health Services in China: Lessons for Other Nations,” Social Science and Medicine, 54(11), 2002, pp. 1691-1698. 9 5 Angus Deaton, “Health, Inequality and Economic Development,” Journal of Economic Literature, 41(1), 2003, pp. 113-158. 6 ( ) pp 9 Sukhan Jackson, Adrian C. Sleigh, Li Peng, and Liu Xi-Li, “Health Finance in Rural Henan: Low Premium Insurance Compared to the Out-of-Pocket System,” China Quarterly, 181, 2005, pp. 137-157. Introduction There are two main reasons for this: first, they were smaller than before, so the insurance pool was based on a smaller number of individuals; second, they were responsible for making a profit (with no intervention from the state), but part of that profit was used for social insurance. Facing the risk of collapse in the economic framework, the government was forced to react. It introduced a kind of social insurance with far lower coverage than before. In rural areas, farmers were themselves made responsible for social insurance through a pooling system at the village level. The rapid privatisation of farming was therefore disconnected from the financial burden of social insurance for rural inhabitants. Farms were not obliged to use part of their profits for social insurance, and there was therefore no risk of collapse for the agricultural sector. As such, the government did not have to step in to provide an insurance system for the rural population as it did for the urban population. The result was a degradation and sometimes complete disappearance of the healthcare-insurance system in rural areas. There is by now a large body of literature addressing the question of health inequality around the world.5 Various methods of analysis have been developed, some making use of advances in the literature on income distribution and taxation. Works have focused on individual countries as well as on comparing many different countries.6 In the case of China, however, there is very little literature. Bloom and Gu (1997)7 analyse the causes of increasing medical care costs and the deterioration of preventive programmes in some poor areas. Liu and Mills (2002)8 also focus on the reduction in demand for preventive services. A growing number of papers have considered the effect of the introduction of the New rural Cooperative Medical Scheme (NCMS). Jackson et al. (2005)9 compare the introduction of NCMS with the out-of-pocket system in Henan Province. Wagstaff et al. (2009) also analyse the introduction 3 of the NCMS, but for different provinces in China, and provide an extensive review of this new literature. However, empirical studies on healthcare and migrants have attracted almost no attention in existing research. In this paper, we highlight migrants' healthcare exclusion due to both income and their lack of integration in the city. The household registration system (hukou) is interwoven with distribution of services and job opportunities. 10 Nansheng Bai and Yupeng He, “Huiliu haishi waichu - Anhui Sichuan ersheng nongcun waichu laodongli huiliu yanjiu” (Return or emigrate? A study on rural return migration in Anhui and Sichuan provinces), Shehuixue yanjiu, vol. 3, 2002, pp. 64-78; Z. Q. Zhang, Y. Zhou, S. X. Lu and Y. H. Chen, “Return migration of rural labourer from Western China: Causes and strategies,” Stat Res, 24, 2007, pp. 9-15 (in Chinese). Introduction Most peasants who enter cities are not granted urban citizenship and are treated as "outsiders" to the urban society. The hukou system in fact also has consequences on the income effect. Hukou increases costs significantly, and represents a cost barrier to access equivalent to income loss. We shed light on the importance of both material and social capital, or life conditions, for migrant workers in providing greater access to healthcare. Above all, we provide new evidence on the decisive role of social networks on migrants’ healthcare access. Two pieces of work in sociology used qualitative methods to underline the effect of health on the decision to return home.10 Our empirical findings support the hypothesis of the return home for migrants with health problems. We are the first to highlight this pattern of exclusion using an empirical strategy and to emphasise the importance of social networks, the residence registration system, and the support of family and relatives as a factor in the outcome of treatment in compelling migrants to leave the city. In Section 2, we present the Chinese context. The description of the data and some preliminary statistics on migrants and healthcare are given Section 3. The econometric model is presented in Section 4. In sections 5 and 6, we present the first econometric results and explore migrants’ healthcare access depending on their health status. Section 7 concludes the paper. The Chinese context China's economic reforms have produced remarkable growth in GDP. This growth has been particularly concentrated in certain areas, mainly the major cities in the eastern and south- eastern regions, leading to massive internal migration. Since the late 1970s, the economic take-off in some urban centres has created a huge demand for labour in these areas and triggered a wave of migration. The general GDP growth across the country led to rising labour demand at the local level in most areas, but much less 4 4 so than in these industrial centres.11 The forecast is that by 2020 more than 50 percent of China's total population will live in towns and cities. This implies that in coming years about three hundred million farmers will leave the rural areas. This will be the largest migration mankind has ever experienced. This internal migration raises a number of issues. One core issue in China is the conflict between the greater welfare associated with economic reforms and the deterioration of healthcare insurance. While economic reforms have had positive effects on GDP and the level of nutrition, they have also led to the collapse of both the public healthcare network and universal access to basic healthcare. The result has been an increase in healthcare inequality. There is another element that may also play a role in determining migrants' healthcare access and healthcare expenditure. In Chinese hospitals, family and relatives play a crucial role in the treatment and recovery process. Healthcare providers furnish only minimal accommodation and catering. When someone is in hospital, relatives must be present to support the patient through meals and help with their treatment. The presence of relatives also affects the way in which hospital staff treat the patient. The presence of family and relatives in the city can therefore also be a key component of healthcare access in the city. In theory, migrants are supposed to claim medical care benefits in the locations where they are registered. Even if most migrants do not have access to urban health insurance, some cities such as Shanghai have developed specific health insurance schemes for migrants. These local policies are increasingly common in China, but still cover only a small percentage of migrants, and the level of coverage is very low. Hence, the hukou system and the need for family support in hospitals push migrants to return to their place of registration. , ( ), , pp 12 Nansheng Bai and Yupeng He, “Huiliu haishi waichu-Anhui Sichuan ersheng nongcun waichu laodongli huiliu yanjiu,” art. cit., 2002; Z. Q. Zhang, Y. Zhou, S. X. Lu and Y. H. Chen, “Return migration of rural labourer from Western China: Causes and strategies,” art. cit., 2007; Xiaojiang Hu, Sarah Cook, and Miguel A Salazar, “Internal migration and health in China,” The Lancet, 372(9651), 2008, pp. 1717-1719. 11 Zai Liang, Yiu Por Chen, and Yanmin Gu, “Rural industrialisation and internal migration in China,” Urban Studies, 39(12), 2002, pp. 2175-2187. 12 The Chinese context Sociological literature based on hometown surveys indicates this pattern. Health conditions that reduce the capacity to work (including old age, pregnancy, and recent childbirth) push migrants to return home to seek family support and to avoid the high medical and living costs in cities.12 To soften the distinction between the insured and the uninsured, we should underline that the level of coverage is often fairly low even for the insured. Leaving rural areas therefore does not produce urban dwellers. A survey conducted by Fudan University on 30,000 migrant workers in major Chinese cities revealed that only 7.6 5 percent of them are satisfied with their life in the city, and that 68 percent are convinced that the city will not accept their integration.13 The healthcare system is organised such that hospitals capable of treating serious health conditions are found only in larger cities. For very severe health conditions, migrants can obtain some help from the local government in their place of work or from their employing firms. Migrants may thus prefer to stay in the city in order to obtain adequate healthcare. In this paper, we will therefore distinguish acute health problems from other types of disease or injury. 13 “Most migrant workers in cities unhappy: Survey,” China Daily, 14 January 2008, http://www.chinadaily. com.cn/bizchina/2008-01/14/content_6391702.htm. The survey on five cities y f We use data from the Survey of Rural Migrant Workers Health and Social-economic Status in 2006. This survey was conducted by the National Population and Family Planning Commission of China and the Institute of Economics of the Chinese Academy of Social Sciences (CASS). The sample is composed of 2,398 interviewees with health and social- economic status information. The interviewees are rural-urban migrant workers. Rural-urban migrant workers are defined as workers who migrate from rural areas to cities. They come from rural areas and have rural hukou: they do not have permanent urban hukou from the place where they work. As noted above, the type of hukou has serious implications in terms of public service access, including health insurance. The rural-urban migrant workers were selected by their work units. This sample was constructed to be representative of rural-urban migrant workers with a formal job and in a regular situation. Therefore, this sample is composed of migrants in socio-economic situations that are called non-critical. The sample excludes migrants with informal jobs and self-employed migrant workers. The survey covers fives cities: Chongqing, Dalian, Shanghai, Shenzhen, and Wuhan. These five cities represent five of China's geographical regions, each with their own economic characteristics. All five of these cities have economic indices amongst the highest in the country and contain substantial numbers of rural migrant workers. Dalian represents Northeast China; Shanghai represents East China and is the economic hub of the country; Wuhan represents Central China; Shenzhen represents South China and is archetypal of the Pearl River Delta region, the biggest manufacturing base for exports; Chongqing represents 6 6 Southwest China and is one of the biggest cities in the country; Northwest China is less developed and contains fewer rural migrant workers. Tables 1 and 2 show the sample characteristics. The average age is about 27 and there are no old people in the sample, which is typical of Chinese migrant characteristics. Migrants are younger in Dalian and Shenzhen than in the other three cities. On average, they are older in Shanghai and Chongqing. One explanation may come from the types of work in which migrants are employed. In Dalian, Wuhan, Shenzhen, and Chongqing, the sample mainly covers workers in industrial production lines, where jobs are better suited for younger workers. In Shanghai, the sample mainly comes from small companies and service industries where physical requirements may disqualify older migrants. < Insert Table 2 > In China, compulsory education lasts for nine years. In most provinces, these nine years of education include six years of primary school (or elementary school) and three years of junior middle school. Most children start school at the age of seven. After junior middle school, they can choose to leave the education system. Beyond junior middle school there are three types of education: senior middle school (and then college after passing a national exam), vocational middle school, and technical middle school. Most migrants come into the labour market after graduating from junior middle school, and some join the labour market after graduating from vocational middle school or technical middle school; very few come into the labour market after graduating from high school. Dalian has the greatest variance in terms of the level of education, with the smallest percentage of elementary school migrants and the highest percentage of vocational middle school migrants. The survey on five cities The share of male and female workers is similar across cities. 14 Fan Ming, Health economics: the effects of health on labour market presentation, Beijing, Social Science Literature Press [Chinese version], 2002. g, ff Literature Press [Chinese version], 2002. Healthcare use In this paper, we have two sources of information on healthcare expenditure: healthcare expenditures in the last month and average healthcare expenditures per year. These come from information declared by the migrant herself. Healthcare expenditures over the last month are commonly used as an index of healthcare access. Here, however, the percentage of individuals with a positive value is very low, and the results using this variable are open to discussion.15 The second source of information has a number of advantages: the share of individuals declaring such healthcare expenditures is significant, and expenditure over a year is a more general measure of healthcare access. The health indices listed in Table 3 measure health conditions that limit the capacity to work. Therefore, a priori, all those who answered "Yes" to at least one of the questions should go to visit a doctor.16 Table 5 shows that only few of them actually do so. In the data, 61 percent of individuals declare a positive average healthcare expenditure figure during the year in question. Dalian is the city where migrants have the least healthcare use. < Insert Table 5 > q 16 In urban China, people visit doctors in hospitals. Therefore, in this paper "to go to the hospital" and "to visit a doctor" mean the same thing. Health status Health in this survey is measured by questions on whether health reduces the capacity to work. This seems apt, as Chinese migrants often ignore health problems unless they affect their capacity to work. These questions were designed by Fan Ming (2002)14 to rank incapacity or injuries that limit migrant labour supply. Following Fan Ming (2002), a health indicator (Health1) is constructed from six objective health questions, of which five are Yes/No and the other is on a scale from one to five. The index is equal to the weighted sum of 7 these variables. Health1 gives information on health conditions limiting work, and takes values from 0 (very good health) to 30 (very poor health conditions limiting work). The questions and weights are shown in Table 3. 15 These results are available on request. 16 < Insert Table 3 > Table 4 displays migrants’ health conditions. Globally, the population should be in good health, as young urban migrants are there to supply labour. In this survey, Wuhan is the city where migrants have the worst health, and Shanghai the city where migrants are in the best health, independent of the index that is analysed. Health insurance Migrants have to claim for insurance reimbursement in the location where they are enrolled. Here, health insurance refers to subscribing to health insurance in the city of employment. This insurance is mainly subscribed to by the firm. The term “health insurance” excludes NCMS insurance, to which migrants subscribe in their hometown. 8 Table 5 shows that the level of insurance varies according to the city. While 80 percent of migrants are insured in Dalian, in Chongqing only 18 percent of migrants are. In addition, healthcare use can depend on having health insurance. In Shanghai and Wuhan, insured individuals have more healthcare use than others do, while in Dalian, Wuhan, and Shenzhen insured individuals have less healthcare use than others. Therefore, the effect of insurance does not seem to be unambiguous. As noted above, there is no national-level health insurance system for migrant workers in China. Provincial governments and some city governments have their own policies regarding migrant health insurance. The five cities in our survey have their own health insurance systems according to local institutions. Specifically, Dalian and Shanghai provide health insurance for migrant workers, with a scheme different from the regular insurance systems for workers with urban hukou. In Shanghai, the government has a comprehensive social insurance system for migrant workers, including three types of insurance: health insurance, pensions, and work-related injury insurance. At the time of this survey, Shenzhen was in the process of setting up a health insurance system for migrant workers. Wuhan and Chongqing are different from the other three cities in having no health insurance system for migrant workers. However, migrant workers in these two cities, as well as in the three others, have the option of joining the health insurance system for city workers. There are two main benefits for migrant workers in subscribing to a migrant health insurance system rather than to a regular health insurance system. First, the fee for the city worker insurance system is higher than that for migrant health insurance; migrant workers, who earn less than city workers (on average) cannot necessarily afford the higher fee. Second, if a migrant worker participates in the city worker insurance scheme, she cannot then transfer her insurance benefits to other cities (or areas). However, we know that one of the key characteristics of Chinese migrant workers is that they frequently move. Health insurance Migrants often retain links with their hometown through children, land rights, and so on. If they participate in one city's insurance scheme, they cannot subsequently transfer their insurance benefits to another location. 19 We also have information on firm ownership (state-owned firms, joint-venture firms, private Chinese firms, and foreign firms). However, these variables are not good candidates. Indeed, in China, firm ownership is correlated with healthcare access. State-owned firms often have agreements with certain hospitals (for instance, with military hospitals that are on average of better quality), independent of any participation in an insurance program. Estimation issues Who uses healthcare? We would like to measure healthcare access itself, i.e. the possibility of a migrant obtaining access to any form of healthcare. As we are not able to obtain a direct measure of healthcare access, we set up a proxy using the presence of healthcare expenditures 9 during the year. When any healthcare expenditure is observed, we consider the migrant to have access to healthcare. HCA is a binary variable indicating whether the migrant has access to health care, i.e. had positive healthcare expenditure. We then identify the determinants of migrants’ health consumption by using a probit model. Since we can see from the preliminary statistics that the city has an effect on healthcare use, we also control the model for the city characteristic effects.17 Table 6 displays descriptive statistics for all the independent variables used in the estimations. p y 18 Hugh R. Waters, “Measuring equity in access to health care,” Social Science and Medecine, 51(4), 20 599-612. 17 The Hausman test performed for each model (each column) supports the city fixed-effect assumption. The term “fixed effect probit model” is mainly used when individual fixed effects are considered. Our model can also be viewed as a probit model with city dummies. 18 Hugh R. Waters, “Measuring equity in access to health care,” Social Science and Medecine, 51(4), 2000, pp. 599 612 17 The Hausman test performed for each model (each column) supports the city fixed-effect assumption. term “fixed effect probit model” is mainly used when individual fixed effects are considered. Our model be viewed as a probit model with city dummies. 20 As noted above, some works on rural areas suggest that migrants return to their hometowns when they need healthcare. Nansheng Bai and Yupeng He, “Huiliu haishi waichu-Anhui Sichuan ersheng nongcun waichu laodongli huiliu yanjiu,” art. cit., 2002; Z. Q. Zhang, Y. Zhou, S. X. Lu, and Y. H. Chen, “Return migration of rural labourer from Western China: Causes and strategies,” art. cit., 2007. First empirical results First empirical results In the following, we do not take hukou into account in the model, as all of the migrants in this survey are in the same situation: they do not have permanent hukou to work in the urban area. We show the results using the health index as defined by Fan (Health1). The health-status index is recoded into four groups: Very good health; Good/Fair health; Bad health and Very bad health. However, the results are qualitatively similar when we consider instead a vector of dummy variables for questions about health (as listed in Table 3). Table 7 displays the results from the econometric model. We find that migrant remittances increase healthcare use. This causality is surprising. Poor health induces higher medical expenditure and so less remittance. Indeed, migrants' remittances reduce their net income. We might then expect a negative effect on healthcare use due to lower income. < Insert Table 6 > In a second step, we focus on migrants who have health trouble. In the case of migrants who have a health trouble according to the Fan’s health index, we should observe use of healthcare. This is the definition of healthcare access given by Waters (2000).18 On a population where we should observe use of healthcare, the absence of healthcare consumption is interpreted as a healthcare access issue. When any healthcare expenditure is observed, we consider the migrant to have access to healthcare. The dummy variable for the presence of healthcare expenditure is then interpreted as healthcare access. In Waters’s paper, need for healthcare is defined by the self-reported health index. Need for healthcare is here measured by Fan's health index. If unmeasured variables that affect the outcome are also correlated with the migrant’s participation in health insurance (e.g. unobserved migrant characteristics), the results of the probit estimation are likely to be inconsistent. To ensure consistent estimates, we employ instrumental variable (IV) estimation by using instruments that are uncorrelated with unobserved individual characteristics but correlated with health insurance. The firm-size index is a good candidate as an instrument. Indeed, the migrant's participation in a health insurance program will depend on the firm's subscription to health insurance and other such characteristics of the firm. Controlling for industry (manufacturing, construction, or services), the size of the firm is plausibly uncorrelated with unobserved individual characteristics.19 10 < Insert Table 7 > Healthcare insurance has no positive and significant effect on healthcare use. Controlling only for demographic variables and the level of health, we find no effect of insurance on healthcare use, and when we control for income and migrants' remittances (Table 7, Column 5), we even obtain a negative significant effect. At first sight, this is worrying. The aim of healthcare insurance is to improve healthcare use. In Section 2, we explained the possibility of selection bias in the data due to inter alia legislative aspects (the hukou and its consequences). To test for selection bias is to test for migrant healthcare exclusion. However, having insurance does not mean being well covered. The level of coverage may differ according to the type of healthcare and the gravity of the illness. In China, the level of coverage is often low. Hence, as long as migrants consider their health condition as not being an issue for doing their job, they may ignore it. As the degree of gravity worsens, they react, which does not necessarily mean visiting a doctor in the location where they work; they can instead return to their hometown. They do so partly because in their hometown they benefit from family support, and partly also because they may benefit from health insurance there. Recall that public-insurance benefits can only be claimed in the locations where migrants are registered. Migrants are excluded from the health system program set up for urban citizens with the "right" hukou in their city of employment.20 Because our survey is conducted in the workplace, these migrants will therefore not have been interviewed. 11 While migrants may prefer to return to their hometown for more severe health conditions, there is another factor that may prevent migrants with poor health from leaving the cities. As described above, the Chinese health system is organised in such a way that hospital quality varies with the degree of urbanisation, with cities offering better quality healthcare. Given a certain degree of severity in a health condition,21 staying in the city may be a better option for migrants, as it gives them access to an adequate quality of healthcare. Moreover, the level of co-payment is not so low for more severe health conditions, as municipalities or firms where the migrants work may provide financial subsidies in this situation, regardless of hukou. < Insert Table 7 > We therefore propose two models dependant on the gravity of the health condition. As health indices do not capture lower degrees of severity, one model (Model A) considers migrants with health conditions that do not require high-quality healthcare, i.e. health conditions that do not require visiting a city hospital; the second model (Model B) considers migrants with more acute health conditions.22 We have no variable in hand to identify migrants who may eventually leave the city to deal with health problems. However, we know that there is potentially some subset of the population that is not present in our data due to incidental attrition. Comparing the results for migrants with different degrees of health problems allows us to examine the presence of selection bias. e Health1 variable; these produce very similar results. The results presented here pertain We have also considered the unweighted index, which does not change the results. 21 When the degree of gravity is severe. 22 g g y 22 We examined different thresholds to define Models A and B. We used the third quartile and the ninth decile of the Health1 variable; these produce very similar results The results presented here pertain to the third quartile examined different thresholds to define Models A and B. We used the third quartile and the ninth decile ealth1 variable; these produce very similar results. The results presented here pertain to the third quartile. p y p p q 23 We have also considered the unweighted index, which does not change the results. en the degree of gravity is severe. examined different thresholds to define Models A and B. We used the third quartile and the ninth decile g g y 22 We examined different thresholds to define Models A and B. We used the third quartile and the ninth decile of the Health1 variable; these produce very similar results. The results presented here pertain to the third quartile. 23 We have also considered the unweighted index which does not change the results When the degree of gravity is severe. 22 We examined different thresholds to define Models A and B. We used the third quartile and the ni q the Health1 variable; these produce very similar results. The results presented here pertain to the third quartile. 23 We have also considered the unweighted index, which does not change the results. 24 The results without the NCMS index are unchanged. These are available on request. As noted above, the term health insurance is for subscription to health insurance in the city of employment. Migrants' exclusion from healthcare access g f Columns 1 to 3 of Table 8 display the results of Model B. Regarding demographic variables, age is positively associated with healthcare access, and female migrants have better healthcare access than do men. To control for hardship of work, we include variables for industry and type of firm. Only working for a state-owned firm has an effect on healthcare use. Before the economic reforms, all those working in state-owned firms had complete healthcare coverage. The situation now is less clear, as this access has gradually deteriorated and migrants usually do not have access to this insurance. However, with the challenge of migrants' healthcare use, these firms are encouraged to facilitate healthcare access for migrants with more severe health conditions. The level of income acts as a push factor on healthcare use. The lower the income, the less likely the migrant is to use healthcare. The financial domain is then one determinant of migrants' exclusion from healthcare use. We here focus on more severe health conditions. The optimal behaviour is therefore to stay in the city in order to benefit from better-quality hospital care. First of all, we find that education, migrant remittances, and health insurance all play their expected role: education increases healthcare access; remitting to the rural family has a negative effect on healthcare use; and insurance positively and significantly influences healthcare use. Hence, on the sample where we suspect selection to be less of a problem, we obtain the expected correlations. Healthcare use: A behaviour that depends on health status We present the results according to Fan's index.23 However, we may still have variations in health within a given level of this health index. We thus control for internal variations in health via the vector of the six variables for each of the six health questions in both Models A and B. However, the results are not sensitive to the inclusion of the vector in the regressions. Moreover, in this survey, health insurance subscription is decided by the firm. The results could therefore be affected by the non-exogeneity of the effect of health insurance on healthcare use. As explained above, we resolve this issue with instrumental-variable estimation. The results of such estimation are displayed in Column 6 of each Table. What about the potential non-exogeneous effect of NCMS insurance on healthcare use in the city? In China, the system of insurance is associated with one location. Migrants cannot 12 transfer insurance benefits to another location. Because the NCMS is associated with the hometown, NCMS subscription may have an effect on the decision to return to the hometown, but is not a substitute for the city insurance system. Because we focus on the city of employment (the place where the survey was conducted), the subscription to NCMS insurance should not be an issue here.24 < Insert Table 9 > Second, remitting has a positive and significant effect on healthcare that was not found with Model B. We explain this result as reflecting selection. Migrants who remit are those who have contact with their hometown. Therefore, these are the ones who leave the place of work when their health is bad enough. This selection effect therefore leads to a sample with only a small percentage that has healthcare use and remit. As income plays a strong positive role in healthcare use, those who do not have healthcare use are the poorest, whatever the level of remittance. Health insurance has a significant negative effect on healthcare use. This effect can also be viewed as reflecting selection. With Model B, we found a positive effect of health insurance. This result suggests that the level of coverage for migrants in the city of employment is not enough to allow them to have healthcare use there. Migrants who can afford to return to the hometown and who still have contacts in the hometown therefore prefer to return to the hometown. In addition, Table 5 indicated that the percentage of insured migrants in Dalian is about three times that in the other cities, and that mean health expenditures in Dalian are about 80 percent higher than those in the other cities. Even so, migrants have less healthcare use than in the other cities. To control for any specific Dalian effect, we thus add an interaction variable between the index for being a migrant in Dalian and being insured. Adding a specific Dalian effect does not change the results. Likewise, all of the models have been re-estimated without the observations from Dalian, which did not change the results. The selection bias thus appears clearly. Health insurance and education have a negative impact on healthcare use, whereas remitting induces greater healthcare use. These results are counterintuitive and are opposite those in Model B. In terms of selection bias, however, these results make sense. Due to this bias, the sub-sample of migrants with healthcare use is composed of those who do not have enough contact with the hometown to return there. 25 We present the determinants in the next section. < Insert Table 8 > Table 9 displays the results from Model A. First, education has a negative and significant effect on healthcare access. With Model B, we found that education has a positive and significant effect on healthcare use. In the light of potential selection, when people have health problems, but not very severe ones, the higher the level of education, the more likely it is they will return to their hometown. Those who do not leave the city of employment are those who are not excluded from the healthcare system for some reason that we have not yet 13 identified,25 or those who cannot leave, possibly for financial reasons or due to a lack of family support in the hometown. The term "floating" for migrants is sometimes exaggerated. Some no longer have any contact with their hometown. Integration into the city Integration into the city In Model B, the sample is composed of individuals who have health problems that limit their ability to work. Therefore, healthcare use can be interpreted as healthcare access.26 We now add indices of integration into the city to the model (in Columns 4 to 6 of Tables 8 and 9). The survey includes a number of different questions on life conditions. We introduce expenditures as life-condition indicators. Specifically, we use clothing, transportation, housing, and education expenditures as proxies for life conditions. With respect to education, the presence of such expenses means that a child lives with the migrant. In China, the majority of migrant children live in the rural area with their grandparents. Apart from financial reasons, another consideration is that migrant children have no access to public schools due to hukou. Individuals only have access to public services in the town of their residency permit. As children only have access to public schools in the place where they are registered, migrant children who live with their parents are obliged to attend private schools. In addition to the subscription fee, parents have to pay additional fees for not having the right hukou. Therefore, education expenses are a good proxy for better life conditions. Likewise, spending money on transport says a lot about life conditions. In China, migrants in extreme situations do not have houses. They live in tents or prefab housing close to their job. Therefore, transportation expenses suggest that migrants live in better conditions. In conclusion, education and transportation expenses are proxies for integration into the city. In Model B (Table 8), all of the expenses have a positive effect on healthcare access except for transportation expenditures. As noted above, migrants with transportation expenditures are more likely to have real accommodation rather than a tent or prefab housing close to their place of work. However, transportation expenditure necessarily reduces migrants' disposable income (going to work leads to transportation expenditures). Controlling for income, the type of job, and other types of expenditures, this may explain the negative effect. We have also introduced control variables for the type of work contract and ownership of durable goods: motorbike, bicycle, VCD/DVD, TV, washing machine, mobile phone, microwave or oven, refrigerator, gas stove, and computer. Controlling for social capital and expenditures, these new variables have no impact on healthcare access. g g q y 27 We do not present the results controlling for "the way in which the migrant obtained their job," family structure, and the ownership of durable goods, as none of the estimated coefficients were significant, and their inclusion does not change the other results. < Insert Table 9 > Therefore, our empirical findings highlight two forms of exclusion: i) exclusion for financial reasons -- migrants cannot afford healthcare use or the cost of returning to their hometown; ii) the results confirm the hypothesis of exclusion from healthcare use in the city of employment for migrants with bad health. 14 Integration into the city Hugh R. Waters, “Measuring equity in access to health care,” art. cit., 2000. Integration into the city Moreover, we have information on family structure, but this has no effect on migrants' healthcare access either.27 15 With respect to the social networks, we apply information on the presence of networks, the number of acquaintances or friends, and the way in which the migrant obtained her job. Having friends or acquaintances and being in a better socio-economic situation are two different phenomena. Friends or acquaintances can provide help in case of worsening health. Social networks thus present one reason to remain in the city despite the hukou, a legislative incentive to leave, and regardless of the individual's health. Social integration has a very significant effect on healthcare access, with a marginal effect of about 60 percent. We also find that having friends or acquaintances acts as a significant positive factor in healthcare access, with a marginal effect of 67.3 percent. On the selected sample, the social network also explains healthcare use. In Model A, we find that having friends or acquaintances acts as a significant positive factor in healthcare use. This positive and significant effect of social networks is echoed in Model B, where the marginal effect is 45 percent. One of the main results of this paper is then the importance of the network and social capital on the healthcare access of migrant workers. Networks act as a push factor in determining healthcare access and allow migrants to obtain access to healthcare regardless of hukou. Conclusion This paper uses data from the Survey of Rural Migrant Workers' Health and Social-economic Status in 2006, covering five cities in China: Chongqing, Dalian, Shanghai, Shenzhen, and Wuhan. These five cities are among China's most industrialised and with the most rural migrant workers. They represent China's five geographical regions with their respective economic characteristics. Using this dataset, the results highlight two exclusion factors: a financial factor (income) and a social factor (the social networks in the city). We present new evidence on the determinants of healthcare use for migrant workers. While financial support is one of the key factors, as expected, another is the social network and the feeling of being socially integrated. This sheds light on the ability of social networks to tackle exclusion from healthcare access. Age and being a woman increases the need for healthcare use. The propensity to use healthcare is higher for migrants working in state-owned firms. In addition, remitting to the rural family has a negative effect on the propensity for healthcare use. Finally, education and health insurance contribute to a higher propensity for healthcare use. There is no national-level health insurance system in China. The right to healthcare 16 insurance and other benefits depends on a permanent residence registration system, the hukou. Individuals are only supposed to claim medical care benefits in the locality where they are registered. Migrants are therefore excluded from urban public health insurance and have to return to their place of registration in order to take advantage of rural public health insurance. Due to these legislative factors, migrants are excluded from healthcare use in their city of employment. In addition, in Chinese hospitals, family and relatives play a crucial role during treatment, as hospitals provide only a minimum level of accommodation and catering. When someone is in hospital, relatives need to be present to support the patient with meals and in helping with their treatment, not to mention the psychological impact of family support on patient recovery. The presence of relatives is therefore an important factor in how individuals recover. Hence, the hukou emphasises both income and the need for family support in hospitals as factors pushing migrants to return to their place of registration. However, these "push factors" are softened by the organisation of the Chinese health system, in which hospital quality varies according to the level of urbanisation, with cities offering the best quality of healthcare. Conclusion Individuals with severe health conditions will have access to adequate healthcare quality in a city hospital, so staying in the city may be the best option for these migrant workers. In this paper, we thus distinguish two models of healthcare use according to the gravity of the health condition. As long as migrants do not consider their health problems to be an issue in terms of doing their job, they may ignore them. When the gravity of their condition becomes an issue for their work, they react. Reacting does not necessarily mean a visit to a doctor in their city of employment. As noted above, the hukou acts as a “push” factor on income and the lack of social network to exclude them from healthcare use in the city, and they may then return to their hometown. As our survey is conducted in the city of employment, migrant workers who return to their hometowns do not appear in this survey. This selection bias is apparent in our empirical results; migrants who can afford to return to their hometown and who still have contacts in their hometown do indeed return there. Apart from residency permits and income, we last show that social networks (having friends or acquaintances) stand out as a significant determinant of healthcare access. This highlights the importance of migrant networks in healthcare access. Feeling socially integrated also has a strong positive effect on healthcare access for migrant workers with poor health. As such, social integration seems to be a key factor influencing healthcare access. But what about those who cannot afford to return to the hometown and those who no longer have contact with relatives in their hometown? Should they remain outside the healthcare system in 17 their city of employment without any other options? Targeting this population will allow us to define the specificity of this population (the younger tier, the older tier, females?) and to establish an adequate healthcare-access policy. In this paper, we use data from migrants selected by their work units. These migrants have formal jobs and are in a regular and non-critical situation. The study does not cover the bottom tier of the migrant population. Our results thus give a much less critical picture than the reality. However, even within this rosier picture we have identified some difficult situations that indicate implementation of an adequate and efficient health policy as a priority. Conclusion One efficient policy to improve migrants' healthcare access in China would be to focus on improving their social integration in the city in which they work. One way is to build up migrants' social capital by improving their networks. Resident committees may be the perfect vector for such a policy. Resident committees are defined at the neighbourhood level in urban areas in China, and are the place where nominated residents manage neighbourhood affairs and state-citizen interactions. To data, migrants have not always been well integrated into these committees, even if they have a "real" house. Encouraging committees to include migrants could therefore be a good way of improving migrants' healthcare access. We have shown the importance of integration for migrants' healthcare access. However, we have not explained the mechanism behind this result. It would be of interest to determine the channel through which integration affects healthcare access: via better information, for example? Via interactions with local workers influencing the behaviour of migrant workers? It is also important to know whether this integration process affects only healthcare access or other domains of life as well, such as children's education. 18 Table 1: Gender and age by city City Male Age Total % Mean Std. dev. No. Obs. Dalian 26.03 24.90 5.29 366 Shanghai 53.78 31.20 9.29 543 Wuhan 57.73 28.56 9.05 466 Shenzhen 45.38 26.36 7.23 498 Chongqing 51.24 30.85 8.35 525 Five Cities 48.00 28.65 8.46 2 398 Source: CASS 2006 Migrants’ survey, 5 cities, 2,398 observations. Table 1: Gender and age by city Table 2: Education structure Table 2: Education structure % Dalian Shanghai Wuhan Shenzhen Chongqing Five Cities Illiterate 0 2.03 0.43 0.40 1.71 1.00 Elementary School 1.37 14.55 8.80 9.04 13.14 9.97 Junior Middle School 54.37 53.59 57.94 63.45 52.57 56.38 Senior Middle School 12.84 18.05 19.96 16.27 18.48 17.35 Vocational Middle School 19.95 1.47 2.36 2.81 2.67 5.00 Technical Middle School 7.38 4.60 8.80 6.63 7.62 6.92 Junior College 3.83 4.24 1.07 1.00 3.81 2.79 College 0.27 1.47 0.64 0.20 0 0.54 Total 366 543 466 498 525 2 398 Source: see Table 1. Note: HCE: health care expenditure. Conclusion Table 3: Measuring Health Condition of Migrant Workers Question Variable format Weight (w) Difficulty in raising hand and bending in the last month Yes/No If yes, w=6; if No, w=0 Difficulty in walking 1 km in the last month Yes/No If yes, w=6; if No, w=0 Difficulty in climbing stairs in the last month Yes/No If yes, w=6; if No, w=0 Feeling headache or dizziness often in past three months Yes/No If yes, w=3; if No, w=0 Feeling extremely tired in the last month Yes/No If yes, w=4; if No, w=0 Feeling stressed in the last month 1st to 6th levels w=0, …, w=5 Table 3: Measuring Health Condition of Migrant Workers Table 4: Health Distribution by City Table 4: Health Distribution by City Province p25 p50 p75 p90 Mean Std. Dev. Dalian 0 0 4 8 2.2 3.3 Shanghai 0 0 1 5 1.5 3.6 Wuhan 0 1 4 9 3.1 4.9 Shenzhen 0 1 4 8 2.7 4.2 Chongqing 0 0 3 8 2.4 4.4 Total 0 0 3 8 2.3 4.2 Source: See Table 1. Note: The index presented here is Health1, Fan's index, from good health (low values) to bad health (higher values). Source: See Table 1. Note: The index presented here is Health1, Fan's index, from good health (low values) to bad health (higher values). Table 5: Healthcare Consumption and Insurance by City City Healthcare Consumption Having Health Insurance (%) All sample Insured Not insured (%) of HCE > 0 Mean HCE Std. dev. HCE (%) of HCE > 0 (%) of HCE > 0 Dalian 41.6 963.9 4 116.2 32.8 44.5 80.0 Shanghai 63.0 563.6 1 114.8 64.7 57.9 28.2 Wuhan 60.9 470.5 846.0 63.4 50.8 27.2 Shenzhen 55.0 493.9 1 088.6 53.5 57.7 14.4 Chongqing 78.1 515.1 1 405.5 80.4 77.9 18.3 Total 61.0 560.5 1724.6 63.9 53.9 31.0 Source: See Table 1 Table 5: Healthcare Consumption and Insurance by City Table 6: Descriptive Statistics of Independent Variables Variable All sample Mean Std Dev. Conclusion Female 52.0 0.50 Age (30-40) 30.0 0.46 Age over 40 12.6 0.33 Junior School 56.4 0.50 High School 29.2 0.45 College 3.3 0.18 Income 1 434€ 910.80 Remittance 212€ 263.53 Health Insurance 33.3 0.47 NCMS Insurance 21.2 0.41 Fixed-term contract 54.8 0.76 Firm size (from 1-small to 3-large) 2.28* 0.26 Sector Construction 7.2 0,49 Manufacturing 60.2 0,30 Ownership State-owned 10.1 0,25 Collective 6.9 0,43 Foreign or JV 25.3 0,50 Expenditure > Q25 Clothing 22.3 0,42 Transportation 8.6 0,28 Education 79.6 0,40 House 37.3 0,48 More than 2 acquaintances 79.8 0.40 Feels socially integrated 55.9 0.50 Source: See Table 1. Firm size 1 is small, 2 is medium and 3 is large. Table 6: Descriptive Statistics of Independent Variables Table 7: Healthcare Consumption for the Whole Sample (1) Probit (2) Probit (3) Probit (4) Probit (5) Probit Good/Fair health 0.216* 0.214 0.232 0.234 0.240 (0.0990) (0.0992) (0.0996) (0.0998) (0.105) Bad health 0.577* 0.575* 0.596* 0.590* 0.706* (0.181) (0.182) (0.182) (0.182) (0.197) Very bad health 0.632* 0.634* 0.677* 0.675* 0.615 (0.242) (0.242) (0.245) (0.245) (0.251) Gender 0.200 0.197 0.279* 0.267* 0.250 (0.0942) (0.0950) (0.0971) (0.0974) (0.101) Age (30-40) 0.419* 0.420* 0.371* 0.356* 0.345* (0.106) (0.108) (0.107) (0.110) (0.115) Age over 40 0.463* 0.462* 0.504* 0.475* 0.452* (0.153) (0.157) (0.154) (0.158) (0.160) Junior School -0.119 -0.186 -0.174 (0.161) (0.163) (0.165) High School -0.0627 -0.181 -0.149 (0.175) (0.179) (0.183) College 0.500 0.272 0.327 (0.311) (0.327) (0.337) Log (Income) 0.472* 0.458* 0.397* (0.129) (0.133) (0.140) Log (Remittances) 0.0311 (0.0128) Health Insurance -0.113 -0.139 -0.175 -0.181 -0.238 (0.108) (0.109) (0.110) (0.111) (0.114) Number of provinces 5 5 5 5 5 Notes: The reference for health status is “Very good health.” Standard errors in parentheses. * p<0.01, p<0.05, * p<0.1. Notes: The reference is migrants who are illiterate or with an elementary level of education, working in the Service Industry, in a large private Chinese ﬁrm. Within-variation in health status is controlled for with the vector of dummy variables for each health question. Standard errors in parentheses. * p<0.01, p<0.05, * p<0.1. The instrumented variable in column (6) is health insurance. Conclusion Table 7: Healthcare Consumption for the Whole Sample Table 8: Healthcare Consumption for Severe Health Conditions (Model B) (1) Probit (2) Probit (3) Probit (4) Probit (5) Probit (6) IV model Gender 0.543* 0.555* 0.705 0.848 0.852 0.852 (0.318) (0.321) (0.334) (0.352) (0.352) (0.339) Age (30-40) 1.271* 1.292* 1.220* 0.905 0.905 1.008 (0.364) (0.369) (0.378) (0.428) (0.429) (0.414) Age over 40 0.950 0.983 0.722 0.874 0.877 1.021** (0.462) (0.464) (0.480) (0.545) (0.544) (0.514) Junior school 0.730* 0.805* 0.713 0.536 0.544 0.702 (0.436) (0.443) (0.458) (0.488) (0.488) (0.479) High school 1.069** 1.032* 1.133** 0.903 0.922 0.939* (0.532) (0.533) (0.551) (0.576) (0.573) (0.561) College 1.648 1.324 1.888 1.678 1.671 1.714 (1.254) (1.224) (1.271) (1.272) (1.273) (1.277) Log (Income) 0.771* 0.755* 0.906 0.986 0.996** 0.740 (0.403) (0.403) (0.436) (0.467) (0.467) (0.457) Log (Remittance) -0.0272 -0.0236 -0.0336 -0.0371 -0.0363 -0.0469 (0.0394) (0.0398) (0.0406) (0.0432) (0.0432) (0.0414) Health Insurance 0.357 0.0620 0.851* 0.913* 0.892* 0.919* (0.393) (0.421) (0.453) (0.478) (0.474) (0.530) NCMS -0.124 -0.130 -0.0702 (0.383) (0.410) (0.384) Construction Ind. 0.471 0.546 0.550 0.380 (0.695) (0.719) (0.720) (0.687) Manufacturing Ind. -0.186 -0.224 -0.226 0.00424 (0.449) (0.479) (0.479) (0.446) State-owned ﬁrm 1.311 1.274 1.257 0.797 (0.582) (0.588) (0.585) (0.549) Collective ﬁrm 0.267 0.292 0.287 0.294 (0.599) (0.618) (0.618) (0.573) Foreign and JV ﬁrm -0.622 -0.431 -0.434 -0.657 (0.469) (0.493) (0.493) (0.495) Fixed-term contract -0.200 -0.180 -0.315 (0.344) (0.338) (0.352) Clothing expenditures 0.545 0.551 0.607 (0.405) (0.404) (0.390) Transport expenditures -1.628 -1.619 -1.464 (0.783) (0.781) (0.669) Education expenditures 0.543 0.542 0.305 (0.448) (0.448) (0.432) Housing expenditures 0.227 0.224 0.0628 (0.329) (0.329) (0.322) Acquaintances 0.274 0.262 0.322* (0.410) (0.409) (0.166) Feeling socially integrated 0.529* 0.531* 0.473* (0.319) (0.318) (0.256) InsuranceDalian 2.071* (1.041) Number of provinces 5 5 5 5 5 5 tes: The reference is migrants who are illiterate or with an elementary level of education, working in the rvice Industry, in a large private Chinese ﬁrm. Within-variation in health status is controlled for with the ctor of dummy variables for each health question. Standard errors in parentheses. Conclusion * p<0.01, p<0.05, * Table 8: Healthcare Consumption for Severe Health Conditions (Model B) Table 9: Healthcare Consumption for Bad Health Conditions (Model A) (1) Probit (2) Probit (3) Probit (4) Probit (5) Probit (6) IV model Gender 0.229 0.227 0.241** 0.146 0.145 0.100 (0.108) (0.108) (0.109) (0.113) (0.113) (0.107) Age (30-40) 0.233* 0.232* 0.210* 0.00881 0.00905 0.00515 (0.124) (0.124) (0.125) (0.137) (0.137) (0.131) Age over 40 0.370 0.370 0.305* 0.228 0.233 0.327* (0.172) (0.172) (0.175) (0.183) (0.183) (0.174) Junior school -0.350* -0.351* -0.355 -0.481* -0.482* -0.456 (0.180) (0.180) (0.181) (0.187) (0.187) (0.179) High school -0.344* -0.345* -0.337* -0.461 -0.459 -0.474 (0.199) (0.199) (0.200) (0.206) (0.206) (0.199) College 0.133 0.132 0.103 0.0162 0.0160 0.0478 (0.356) (0.356) (0.358) (0.364) (0.364) (0.353) Log (Income) 0.372 0.371 0.329 0.121 0.118 0.103 (0.150) (0.150) (0.154) (0.164) (0.164) (0.158) Log (Remittance) 0.0385* 0.0385* 0.0396* 0.0494* 0.0494* 0.0482* (0.0136) (0.0136) (0.0137) (0.0141) (0.0141) (0.0133) Health Insurance -0.308 -0.314 -0.305 -0.338* -0.336*** -0.133* (0.120) (0.126) (0.125) (0.130) (0.130) (0.076) NCMS 0.0958 0.0596 0.00852 (0.128) (0.130) (0.123) Construction Ind. 0.472 0.561 0.566 0.622* (0.218) (0.223) (0.223) (0.220) Manufacturing Ind. 0.171 0.161 0.166 0.210 (0.151) (0.154) (0.154) (0.146) State-owned ﬁrm 0.0239 0.0135 0.0156 -0.0173 (0.181) (0.185) (0.185) (0.181) Collective ﬁrm 0.192 0.119 0.118 0.115 (0.218) (0.222) (0.222) (0.210) Foreign and JV ﬁrm 0.107 0.0903 0.0830 -0.102 (0.182) (0.186) (0.185) (0.188) Fixed-term contract -0.0655 -0.0668 -0.151 (0.117) (0.117) (0.112) Clothing expenditures -0.191 -0.192 -0.214* (0.136) (0.136) (0.128) Transport expenditures -0.503* -0.506* -0.460* (0.187) (0.187) (0.176) Education expenditures -0.461* -0.458* -0.417* (0.152) (0.152) (0.146) Housing expenditures -0.376* -0.377* -0.386* (0.119) (0.119) (0.114) Acquaintances 0.380* 0.384* 0.372* (0.133) (0.133) (0.125) Feeling socially integrated 0.0633 0.0614 0.0234 (0.111) (0.110) (0.105) Insurance*Dalian 0.0654 (0.401) Number of provinces 5 5 5 5 5 5 Notes: See Table 8 Table 9: Healthcare Consumption for Bad Health Conditions (Model A)
https://openalex.org/W4320020335	https://jurnal.unimed.ac.id/2012/index.php/jgkp/article/download/41892/20017	English	null	AN ANALYSIS OF ILLOCUTIONARY ACT ON SONG LYRICS OF BRUNO MARS’S DOO-WOOPS & HOOLIGANS ALBUM	JGK (Jurnal Guru Kita)	2,022	cc-by	3,162	AN ANALYSIS OF ILLOCUTIONARY ACT ON SONG LYRICS OF BRUNO MARS’S DOO-WOOPS & HOOLIGANS ALBUM Ambar Wulan Sari, Tengku Winona Emelia Universitas Muhammadiyah Sumatera Utara Surel: ambarwulan@umsu.ac.id, t.winona@umsu.ac.id, Abstract: An Analysis Of Illocutionary Act On Song Lyrics Of Bruno Mars’s Doo-Woops & Hooligans Album. Illocutionary act is an act performed in saying something. Illocutionary acts is an utterance with some kind of function in mind. As a part of written language, literary works such as play, novel, film, short story, and song can also be analyzed by using illocutionary act theoryThe study discussed about the use of Illocutionary Act to determine the lyric of the song’s meaning. The objectives of this study were to : (1) To describe the types of illocutionary speech acts are contained on song lyrics Doo-Woops & Holligans Album. And (2) To find out the functions of the illocutionary act found in the song lyrics Doo-Woops & Holligans Album. This study applied a descriptive qualitative method to analyze the data. The source of the data was obtained from Bruno Mars’s Doo-Woops & Holligans Album. The data was taken the lyrics of 10 songs from Bruno Mars’s Doo-Woops & Holligans Album to find the types and functions of Illocutionary Act . The technique in analyzing the data was using the theory of Miles & Huberman, that is data condensation, data display, and conclusion drawing and verfication. There were then found out the song lyrics of Doo-Wops & Hooligans Album by Bruno Mars use most of representative illocutionary acts and the song lyrics of Doo- Wops & Hooligans Album by Bruno Mars use most of collaborative illocutionary acts’s function. Keywords: Illocutionary cct, Bruno Mars, Doo-Woops and, Holligans album Keywords: Illocutionary cct, Bruno Mars, Doo-Woops and, Holligans album Abstrak: Tindak ilokusi adalah tindakan yang dilakukan dalam mengatakan sesuatu. Tindak ilokusi adalah tuturan yang memiliki fungsi tertentu. Sebagai bagian dari bahasa tulis, karya sastra seperti drama, novel, film, cerpen, dan lagu juga dapat dianalisis dengan menggunakan teori tindak ilokusi Kajian ini membahas tentang penggunaan Illocutionary Act untuk menentukan makna lirik lagu. Tujuan dari penelitian ini adalah untuk : (1) Mendeskripsikan jenis-jenis tindak tutur ilokusi yang terdapat pada lirik lagu Album Doo-Woops & Holligans. Dan (2) Untuk mengetahui fungsi tindak ilokusi yang terdapat pada lirik lagu Album Doo-Woops & Holligans. Penelitian ini menggunakan metode deskriptif kualitatif untuk menganalisis data. Sumber data diperoleh dari Album Doo-Woops & Holligans milik Bruno Mars. Data diambil lirik 10 lagu dari Album Doo- Woops & Holligans Bruno Mars untuk mengetahui jenis dan fungsi Illocutionary Act. Teknik analisis data menggunakan teori Miles & Huberman yaitu kondensasi data, penyajian data, penarikan kesimpulan dan verifikasi. Kemudian ditemukan lirik lagu Album Doo-Wops & Hooligans karya Bruno Mars sebagian besar menggunakan tindak ilokusi representatif dan lirik lagu Album Doo-Wops & Hooligans karya Bruno Mars sebagian besar menggunakan fungsi tindak ilokusi kolaboratif. An Analysis Of Illocutionary Act On Song Lyrics Of Bruno Mars’s Doo-Woops & Hooligans Album. (Hlm. 166-172) 166 Kata kunci: Illocutionary act, Bruno Mars, album Doo-Woops and Holligans 1. INTRODUCTION Speech act theory developed during the middle of the twentieth century out of sense of dissatisfaction on the part of writers such as J. L. Austin. He defined speech acts as the actions performed in saying something. Speech act theory says that the action performed when an utterance is produced can be analysed on the three different levels. These are, Locutionary act, Illocutionary act, and Perlocutionary act. Firstly, locutionary act is roughly equivalent to uttering certain sentence with a certain sense and reference, which again is roughly Kata kunci: Illocutionary act, Bruno Mars, album Doo-Woops and Holligans Kata kunci: Illocutionary act, Bruno Mars, album Doo-Woops and Holligans Mars’s Doo-Woops & Hooligans Album. (Hlm. that the action performed when an utterance is produced can be analysed on the three different levels. These are, Locutionary act, Illocutionary act, and Perlocutionary act. Firstly, locutionary act is roughly equivalent to uttering certain sentence with a certain sense and reference, which again is roughly Kata kunci: Illocutionary act, Bruno Mars, album Doo-Woops and Holligans 1. INTRODUCTION Speech act theory developed during the middle of the twentieth century out of sense of dissatisfaction on the part of writers such as J. L. Austin. He defined speech acts as the actions performed in saying something. Speech act theory says An Analysis Of Illocutionary Act On Song Lyrics Of Bruno Mars’s Doo-Woops & Hooligans Album. (Hlm. 166-172) 166-172) 166 equivalent to “meaning” in traditional sense. Secondly, Illocutionary act such as informing, ordering, warning, undertaking, Thirdly, Perlocutionary acts achieveby saying something, such as convincing, persuading, deterring, and even, say, surprising or misleading (Smith, 2003). equivalent to “meaning” in traditional sense. Secondly, Illocutionary act such as informing, ordering, warning, undertaking, Thirdly, Perlocutionary acts achieveby saying something, such as convincing, persuading, deterring, and even, say, surprising or misleading (Smith, 2003). listener to feel the emotions with the song, song is like communicating with listeners through lyrics that are in the form of words or sentences. Songs as communication media are often used as a medium for delivering messages. Song lyrics can be seen as one of the written works of art whose shape is similar to poetry. The language in the song lyrics is a language that is condensed, shortened, and given a rhythm with a coherent sound and the choice of figurative and imaginative words. misleading (Smith, 2003). Illocutionary act is an act performed in saying something. Illocutionary acts is an utterance with some kind of function in mind. When analyzing an utterance, it does not only deal with what do the sentence means, but also what kind of act does a speaker performs in uttering a sentence. Illocutionary act is a very important part of speech act because illocutionary act becomes the main central of communication. Illocutionary acts becomes the basic of analysis in pragmatic comprehension (Gunarwan, 2007). g g Thus, illocutionary act can be applied in the song lyrics in order to get the intended message of the song and also what kind of message which is delivered in the song lyrics because everyone may have different interpretation about the meaning of sentence in the song lyrics. It depends on the speaker’s meaning and also the context. In order to successfully deliver this intended message, the hearer should understand the context and the speaker’s idea, and by analyzing illocutionary act found in the song lyrics of this album, we can get to that goal. An Analysis Of Illocutionary Act On Song Lyrics Of Bruno Mars’s Doo-Woops & Hooligans Album. (Hlm. 166-172) An Analysis Of Illocutionary Act On Song Lyrics Of Bruno Mars’s Doo-Woops & Hooligans Album. (Hlm. 166-172) 1. INTRODUCTION It means that, the hearer will know the purpose of the speaker if s/he understands the illocutionary act of the utterance. The interpretation of the illocutionary acts are concerned with force. for example: “I will go to campus at 8.am”. In this utterance, the speaker has made an action of “promise” via language to go to the campus at 8 a.m. But Illocutionary acts are also act performed to utter the intention behind the speaker’s words that indicates the speaker’s purpose in saying something whether to state, recommends, offers, promises, etc. It means that illocutionary act can also be found on written language as written language also contains writer’s intention or purpose to say to the reader. As a part of written language, literary works such as play, novel, film, short story, and song can also be analyzed by using illocutionary act theory. the researcher has several song lyrics from Bruno Mars as the object of his study because besides the song lyrics have never been studied, Bruno Mars’s song lyrics have their own peculiarities, namely stories that describe daily life, unity, friendship, romance, and love. Album Doo- Wops & Hooligans album is chosen cause his debut studio peaked at number three on the US Billboard 200 and topped the charts in Canada, Germany, Ireland, the Netherlands and in the United Kingdom. This album produced number one successful singles titled "Just the Way You Are", "Grenade" and "The Lazy Song". "Just the Way You Are", won a Grammy Award for Best Male Pop Vocal Performance. In 2011, Mars recorded a single entitled Song is a type of literary work that writer compose the language into lyrics and add melodies to create melodious. The song can make the An Analysis Of Illocutionary Act On Song Lyrics Of Bruno Mars’s Doo-Woops & Hooligans Album. (Hlm. 166-172) 166-172) 167 Woops & Holligans Album that are being analyzed (Chih-Pei HU, Yan- Yi Chang, 2017). "It Will Rain" for The Twilight Saga: Breaking Dawn - Part 1. "It Will Rain" for The Twilight Saga: Breaking Dawn - Part 1. From the description above, it can be seen that the lyrics of the Bruno Mars’s song is an interesting material to study. In addition, the songwriter also uses several language styles that affect the beauty of the song lyrics. Based on background of this study, the writer are eager to conduct a research with the title, “An Analysis Of Illocutionary Act On Song Lyrics Of Bruno Mars’s Doo- Woops & Holligans Album” . The sources of the data in this research are the song lyrics of Bruno Mars’s Doo-Woops & Holligans Album. This album contains 10 songs. Those songs are (1) Grenade, (2) Just The Way You Are, (3) Our First Time, (4) Runaway Baby, (1) Grenade, (2) Just The Way You Are, (3) Our First Time, (4) Runaway Baby, (5) The Lazy Song, (6) Marry You, (7) Talking to the Moon, (8) Liquor Store Blues, (9) Count on me, and (10) The Other Side. The data are in the form of sentence and clause from single lyric or combine lyrics which contain of illocutionary act. The problem of this research are identified as follow, The types of illocutionary speech acts are contained on song lyrics Doo-Woops & Holligans Album and The functions of the illocutionary act found in the song lyrics Doo- Woops & Holligans Album. The technique of collecting data is used by researcher to collect the data that is needed and related to the discussion in the research. In this study, the writer used documentation method. Documentation explaining the technique is to look for data about things or variables in the form of notes, transcripts, books, The aims of this research is : To describe the types of illocutionary speech acts are contained on song lyrics Doo-Woops & Holligans Album and To find out the functions of the illocutionary act found in the song lyrics Doo-Woops & Holligans Album. newspapers, magazines, inscription, and agenda for information embodied data relating to the discussion (Sudaryono, 2016). 2. METHOD OF RESEARCH 2. METHOD OF RESEARCH In this study, the writer used design of qualitative descriptive method which is a method of research that attempt to describe and interpret the objects in accordance with reality. The qualitative research is a means for exploring and understanding the meaning individuals or groups ascribe to a social or human problem. The descriptive method is implemented because the data analysis is presented descriptively, the writer used song lyrics from Bruno Mars’s Doo- After collecting data, the researcher would implement to analyze data, there are three concurrent flows of activity: (1) data condensation, refers to the process of selecting, focusing, simplifying, abstracting, and/or transforming the data that appear in the full corpus (body) of written-up field notes, interview, transcriptions, documents, and other empirical materials (2) data display an organized, compressed assembly of information that allows conclusion drawing and action and An Analysis Of Illocutionary Act On Song Lyrics Of Bruno Mars’s Doo-Woops & Hooligans Album. (Hlm. 166-172) 166-172) 168 (3) conclusion drawing/verification, . Conclusions are also verified as the analyst proceeds. Verification may be as brief as a fleeting second thought crossing the analyst’s mind during writing, with a short excursion back to the field notes Miles & Saldana, 2014). types of illocutionary acts and their functions, the researcher tries to analyze the lyrics one by one and one by one stanza to be categorized as illocutionary acts. As an answer to the first problem formulation, the researcher discusses the types of illocutionary speech acts are contained on song lyrics Doo-Woops & Holligans Album with details using the table below: An Analysis Of Illocutionary Act On Song Lyrics Of Bruno Mars’s Doo-Woops & Hooligans Album. (Hlm. 166-172) 169 3. DATA AND ANALYSIS From the analysis of the data findings above which determine the An Analysis Of Illocutionary Act On Song Lyrics Of Bruno Mars’s Doo-Woops & Hooligans Album. (Hlm. 166-172) 170 Table 4.1 Data Finding of Illocutionary Acts’s Types No Title of Song Illocutionary Act’s Type Frequency Number of Data 1 Grenade Representative 9 15 Directive 2 Comissive 1 Expressive 2 Declarative 1 2 Just The Way You Are Representative 6 16 Directive 1 Comissive 3 Expressive 6 3 Our First Time Representative 10 18 Directive 3 Comissive 1 Expressive 4 4 Runaway Baby Representative 14 17 Directive 4 Comissive 1 Expressive 1 5 The Lazy Song Representative 10 16 Directive 3 Comissive 1 Expressive 2 6 Marry You Representative 9 17 Directive 6 Comissive 1 7 Talking to the Moon Representative 12 15 Directive 2 Expressive 1 8 Liqour Stores Blue Representative 14 20 Directive 2 Comissive 2 Expressive 2 9 Count on Me Representative 9 16 Directive 1 Comissive 6 10 The Other Side Representative 15 24 Directive 5 Comissive 4 TOTAL 174 Table 4.1 shows that the total numbers of data are 174 data from 10 songs containing Illocutionary Acts’s Type. In the we can see that the most dominant s of Illocutionary Acts found in the Hooligans Album by Bruno Mars use most of representative illocutionary acts, which are 108 data. Table 4.1 shows that the total numbers of data are 174 data from 10 songs containing Illocutionary Acts’s Type. In the e we can see that the most dominant es of Illocutionary Acts found in the Hooligans Album by Bruno Mars use most of representative illocutionary acts, which are 108 data. Illocutionary Acts’s Type. In the table we can see that the most dominant types of Illocutionary Acts found in the album is representative with 108 data (62%), followed by directive with 29 data (38,2%), comissive with 20 Then for the illocutionary act function of the song lyrics of Doo-Wops & Hooligans Album by Bruno Mars which was analyzed by researchers to answer the second problem formulation, the detailed description is in the table below: data (11,5%), expressive with 16 data (9,8%) and declarative with 1 data (0,6%). From the data finding that shows in the table, the researcher can concluded that the song lyrics of Doo-Wops & Table 4.2 Table 4.2 Data Finding of Illocutionary Acts’s Function No Title of Song Illocutionary Act’s Function Frequency Number of Data 1 Grenade Competitive 1 6 Collaborative 2 Conflictive 3 2 Just The Way You Are Convivial 3 3 3 Our First Time Convivial 3 3 170 4 Runaway Baby Competitive 2 4 Convivial 1 Collaborative 1 5 The Lazy Song Competitive 2 4 Convivial 1 Collaborative 1 6 Marry You Convivial 1 1 7 Talking to the Moon Competitive 1 3 Convivial 1 Collaborative 1 8 Liqour Stores Blue Competitive 1 4 Collaborative 3 9 Count on Me Convivial 3 3 10 The Other Side Competitive 1 7 Collaborative 6 TOTAL 38 declarative with 1 data (0,6%), it’s can concluded that the song lyrics of Doo- Wops & Hooligans Album by Bruno Mars use most of representative illocutionary acts. declarative with 1 data (0,6%), it’s can concluded that the song lyrics of Doo- Wops & Hooligans Album by Bruno Mars use most of representative illocutionary acts. Table 4.2 shows that the total numbers of data are 38 data from 10 songs containing Illocutionary Acts’s Type. In the table we can see that the most dominant types of Illocutionary Acts found in the album is collaborative with 15 data (39,5%), followed by convivial with 14 data (36,8%), competitive with 6 data (15,8%), and conflictive with 3 data (7,9%). From the data finding that shows in the table, the researcher can concluded that the song lyrics of Doo-Wops & Hooligans Album by Bruno Mars use most of collaborative illocutionary acts’s function, which are 15 data. 2. The functions of the illocutionary act found in the song lyrics Doo-Woops & Holligans Album are collaborative with 15 data (39,5%), followed by convivial with 14 data (36,8%), competitive with 6 data (15,8%), and conflictive with 3 data (7,9%), it’s can concluded that the song lyrics of Doo-Wops & Hooligans Album by Bruno Mars use most of collaborative illocutionary acts’s function, b. Suggestion b. Suggestion Based on the results, the researcher proposes some suggestions as follows: b. Suggestion Based on the results, the researcher proposes some suggestions as follows: Smith, Barry. (2003) “John Searle: From Speech Acts To Social Reality”, In Barry Smith (Ed.), John Searle. Cambridge : Cambridge University Press, 1. To the future researchers who want to conduct the similar study can undertake the research with deeper discussions about the social goal in the functions of illocutionary act. The researcher suggests this research needed to be done next in understanding the functions of illocutionary acts in order to generate a more perfect study. Y. C. PH. and Chang. (2017). John W, Creswell, Research Design: Qualitative, Quantitative, and Mixed Methods Approaches. Journal of Social and Administrative Sciences, vol. 4, no. June. pp. 3–5 2. It is also expected from the future researchers to provide more explanation related with the study of illocutionary functions about the linguistic behavior: positive and negative politeness which is needed to be explained clearly of how it is consider as positive or negative politeness in the utterances of the speakers. 3. There are more objects of study that can be conducted with illocutionary acts as the frame work. The other researchers can inquire in different area such as advertisement, speech, movie and any other objects to analyze not only the category but also the function of illocutionary act to present different result. a. Conclusion Based on the findings and data analysis, there were some conclusion that could be described as follows: An Analysis Of Illocutionary Act On Song Lyrics Of Bruno Mars’s Doo-Woops & Hooligans Album. (Hlm. 166-172) 171 1. The types of illocutionary speech acts are contained on song lyrics Doo- Woops & Holligans Album are representative with 108 data (62%), directive with 29 data (38,2%), comissive with 20 data (11,5%), expressive with 16 data (9,8%) and 171 Pendidikan. Yogyakarta: Andi. An Analysis Of Illocutionary Act On Song Lyrics Of Bruno Mars’s Doo-Woops & Hooligans Album. (Hlm. 166-172) 172 REFERENCES Gunarwan, Asim. (2007). Pragmatik Teori & Kajian Nusantara. Jakarta : Universitas Atma Jaya. Miles, M.B, Huberman, A.M, & Saldana, J. (2014). Qualitative Data Analysis, A Methods Sourcebook, Edition 3. USA: Sage Publications. Sudaryono. (2016). Metode Penelitian Sudaryono. (2016). Metode Penelitian An Analysis Of Illocutionary Act On Song Lyrics Of Bruno Mars’s Doo-Woops & Hooligans Album. (Hlm. 166-172) 172
https://openalex.org/W4283687645	https://www.ojsstikesbanyuwangi.com/index.php/PHJ/article/download/223/170	Indonesian	null	Gambaran Status Gizi Balita Pada Masa Pandemi Covid-19 Di Wilayah Kerja Puskesmas Kaliwates	Professional Health Journal	2,022	cc-by-sa	2,961	Abstrak Pendahuluan: Indonesia termasuk negara dengan tingkat kematian anak tertinggi akibat Covid-19 yaitu 2,5 persen. Salah satu penyebab nya antara lain rendahnya tingkat pemeriksaan kesehatan anak, status anak dengan kelainan bawaan dan anak dengan status gizi buruk. Peningkatan imunitas perlu dilakukan untuk mencegah angka kesakitan pada anak. Salah satunya dengan mengonsumsi makanan dengan gizi seimbang, menjaga protokol kesehatan, memakai masker dan menghindari kerumunan. Tujuan dari penelitian ini adalah melihat dan mendeskripsikan status gizi pada anak pada masa pandemi Covid-19. Metode: Metode penelitian yang digunakan yaitu deskriptif (cross sectional). Populasi pada penelitian ini yaitu seluruh balita di Posyandu Aster 11 dan 15 sebanyak 91 anak. Metode pengumpulan data dilakukan dengan cara melakukan pengukuran TB dan BB. Hasil pengukuran kemudian dianalisa dengan menggunakan tabel kategori status gizi balita menurut WHO. Analisis data menggunakan rumus distribusi frekuensi. Hasil: analisis berdasarkan TB/U (45% dengan kategori sangat pendek, 9% pendek, 46% normal), analisa berdasarkan BB/U (4% dengan kategori gizi buruk, 18% gizi kurang, 77% gizi baik dan 1% gizi lebih), analisa berdasarkan BB/TB (2% dengan kategori sangat kurus, 3% kurus, 65% normal, 30% gemuk). Kesimpulan: Dari hasil analisis tersebut diatas menyatakan bahwa sebagian besar balita memiliki status gizi yang baik meskipun ada balita dengan kategori status gizi buruk dan ada balita dengan kategori sangat pendek (stunting). Kata kunci : Gizi, Bayi dan Balita, Covid-19 PROFESIONAL HEALTH JOURNAL Volume 3, No. 2, Bulan Juni Tahun 2022 (Hal. 124-130) https://www.ojsstikesbanyuwangi.com/index.php/PHJ Ririn Handayania, Yuningsiha, Laila Karimatu Zalikaa a) Fakultas Ilmu Kesehatan Prodi Kebidanan Program Sarjana Universitas dr.Soebandi Email korespondensi: ririnhandayani89@uds.ac.id Abstract Introduction: Indonesia is one of the countries with the highest child mortality rate due to Covid-19 which is 2.5 percent. One of the causes of the high child mortality rate is the low level of child health examinations, the status of children with congenital disorders and children with poor nutritional status. Increasing immunity needs to be done to prevent morbidity in children. One of them is by eating balanced nutrition, maintaining health protocols, wearing masks and avoiding crowds. The purpose of this study was to identify the nutritional status of toddlers during the Covid-19 pandemic. Methode: Research of methods is descriptive, cross sectional approach. Subjects in this study were all toddlers in Posyandu Aster 11 and 15 as much 91 children. Methods of collecting data by measuring TB and BB. Measurement results analyzed using a table of categories of nutritional status of toddlers to WHO. Result: Data analysis used the frequency distribution. Analysis result were based on TB/U (45% in the very short category, 9% short, 46%). ), analysis based on BB/TB (2% with very thin category, 3% thin, 65% normal, 30% fat). Concluision: Research conclution that most of toddlers have good nutritional status, although there are toddlers of poor nutritional status and there are toddlers with category of very short (stunting). PENDAHULUAN dinyatakan sebagai pandemi oleh WHO (WHO, 2020). Peta data sebaran covid per tanggal 26 November 2020 secara globlal telah menyebar di 220 Negara, jumlah Ririn Handayani dkk., Gambaran Status Gizi Balita Virus Covid-19 merupakan jenis penyakit infeksi yang disebabkan oleh virus novel Coronavirus. Covid-19 telah 124 PROFESIONAL HEALTH JOURNAL Volume 3, No. 2, Bulan Juni Tahun 2022 (Hal. 124-130) https://www.ojsstikesbanyuwangi.com/index.php/PHJ terkonfirmasi 60.074.174 dan jumlah meninggal 1.416.292. Di indonesia jumlah kasus positif 516.753, kasus sembuh 433.649, kasus meninggal 16.352 (covid19.go.id). Di jawa timur jumlah kasus positif 60.190, kasus sembuh 53.131, kasus meninggal 4.275 (infocovid19.jatimprov.go.id/). Sedangkan di jember, jumlah kasus positif 2.216, kasus sembuh 1.642, kasus meninggal 93 (jemberkab.go.id). terkonfirmasi 60.074.174 dan jumlah meninggal 1.416.292. Di indonesia jumlah kasus positif 516.753, kasus sembuh 433.649, kasus meninggal 16.352 (covid19.go.id). Di jawa timur jumlah kasus positif 60.190, kasus sembuh 53.131, kasus meninggal 4.275 (infocovid19.jatimprov.go.id/). Sedangkan di jember, jumlah kasus positif 2.216, kasus sembuh 1.642, kasus meninggal 93 (jemberkab.go.id). kekurangan gizi (stunting/wasting) yang telah tertuang dalam RPJMN 2020-2024. Penurunan angka kekurangan gizi tersebut dilakukan melalui konseling dan suplementasi gizi ibu hamil, promosi dan konseling PMBA, pemantauan tumbuh kembang balita, pemberian vitamin A dan PMT bagi balita dengan gizi kurang (Kemenkes RI, 2020). Mengingat pentingnya pemantauan gizi bayi balita pada masa pandemi covid- 19, makan penulis ingin melihat status gizi balita pada masa Pandemi Covid-19 di Wilayah Kerja Puskesmas Kaliwates Jember dalam rangka deteksi dini kegawatan status gizi pada balita dengan harapan mampu menurunkan angka kesakitan akibat adanya virus Covid-19. Indonesia merupakan pemegang rekor tertingi angka kematian anak akibat Covid-19 (2,5%) (Komarudin, 2020). IDAI menyatakan terdapat 11 ribu anak Indonesia yang terpapar Covid-19. Dari data tersebut terdapat 584 kasus anak yang terpapar Covid-19. p p Gejala awal yang mungkin dialami anak yang terpapar Covid-19 adalah diare, kejang, shock, dan bahkan ada yang tanpa memiliki gejala. Tingginya angka kematian anak dapat dipicu oleh beberapa faktor antara lain rendahnya tingkat pemeriksaan kesehatan, anak dengan kelainan bawaan dan anak dengan status gizi buruk, serta penanganan yang terlambat. Salah satu langkah yang perlu dilakukan untuk menjaga imunitas pada anak antara lain dengan mengonsumsi makanan dengan gizi seimbang yang meliputi protein, lemak, karbohidrat, vitamin C (Komarudin, 2020). METODE Metode penelitian yang digunakan yaitu deskriptif (cross sectional). Populasi dalam penelitian ini adalah seluruh balita di Posyandu Aster 11 dan 15 sebanyak 91 anak. Teknik pengumpulan sampel adalah total sampling. Sampel yang digunakan adalah seluruh balita di Posyandu Aster 11 dan 15 yang bersedia enjadi responden yaitu sebanyak 91 anak. Metode pengumpulan data dilakukan dengan cara melakukan pengukuran TB dan BB. Hasil pengukuran kemudian dianalisa dengan menggunakan tabel kategori status gizi balita menurut WHO. Analisis data menggunakan rumus distribusi frekuensi. Presiden RI telah menetapkan Kepres No.11 Tahun 2020 PP No. 21 Tahun 2020, yang isinya meliputi pemenuhan kebutuhan dasar penduduk melalui pemenuhan kebutuhan pangan, pemenuhan pelayanan kesehatan dan pemenuhan kebutuhan sehari-hari, termasuk pemenuhan kebutuhan pelayanan kesehatan masyarakat yang telah dilaksanakan di fasilitas tingkat pertama (PUSKESMAS) (Kemenkes RI, 2020). . PROFESIONAL HEALTH JOURNAL Volume 3, No. 2, Bulan Juni Tahun 2022 (Hal. 124-130) https://www.ojsstikesbanyuwangi.com/index.php/PHJ pada penelitian ini berdasarkan berat badan menurut umur didapatkan 4% balita dengan kategori status gizi buruk, 18% balita dengan kategori status gizi kurang, 77% balita dengan kategori gizi baik, 1% balita dengan kategori status gizi lebih. Dari hasil analisis status gizi berdasarkan BB/U didapatkan 4 balita (4%) dengan kategori status gizi buruk, 16 balita (18%) dengan kategori status gizi kurang, 70 balita (77%) dengan kategori gizi baik, 1 balita (1%) dengan kategori status gizi lebih. Tabel 2 Status Gizi Balita Berdasarkan TB/U Kategori L (TB/U) P (TB/U) Total % SGT PENDEK (<-3SD) 18 23 41 45 PENDEK (-3SD s.d < -2SD) 5 4 9 10 NORMAL (>-2SD) 22 19 41 45 TOTAL 45 46 91 100 Dari hasil analisis status gizi berdasarkan TB/U didapatkan 41 balita (45%) dengan kategori sangat pendek, 8 balita (9%) dengan kategori pendek, 42 balita (46%) dengan kategori normal. Tabel 2 Status Gizi Balita Berdasarkan Tabel 2 Status Gizi Balita Berdasarkan TB/U Pertumbuhan bayi dan balita di Posyandu Aster 11 dan 15 diwilayah kerja Puskesmas Kaliwates tumbuh dengan baik hal ini ditunjukkan dengan adanya balita (sebagian besar) 77% dengan status gizi baik meskipun ada 4% balita dengan kategori gizi buruk dan 18% balita dengan kategori gizi kurang. Menurut Setyorini (2021), Faktor-faktor yang dapat mempengarui status gizi pada bayi dan balita antara lain tingkat kemiskinan/sosial ekonomi, PMT pada anak, tingkat kekooperatifan orang tua dalam pemberian asuhan (Setyorini, 2021). Dari hasil analisis status gizi berdasarkan TB/U didapatkan 41 balita (45%) dengan kategori sangat pendek, 8 balita (9%) dengan kategori pendek, 42 balita (46%) dengan kategori normal. Tabel 3 Status Gizi Balita Berdasarkan TB/BB Tabel 3 Status Gizi Balita Berdasarkan TB/BB Kategori L (BB/TB) P (BB/TB) Total % SGT KURUS (<-3SD) 1 1 2 2 KURUS (-3SD s.d < -2SD) 1 2 3 3 NORMAL (-2SD s.d 2SD) 31 28 59 65 GEMUK (>2SD) 12 15 27 30 TOTAL 45 46 91 100 Dari hasil analisis status gizi berdasarkan BB/TB didapatkan 2 balita (2%) dengan kategori sangat kurus, 3 balita (3%) dengan kategori kurus, 59 balita (65%) dengan kategori normal, 27 balita (1%) dengan kategori gemuk Tabel 3 Status Gizi Balita Berdasarkan Jika kita lihat kembali dari hasil pengkajian RISKESDAS pada Tahun 2018 tentang status gizi pada balita usia 0-59 bulan, telah ditemukan balita dengan status gizi buruk sebesar 3,9% dan balita dengan status gizi kurang sebesar 13,8% (Profil Kesehatan Indonesia, 2019). . PROFESIONAL HEALTH JOURNAL Volume 3, No. 2, Bulan Juni Tahun 2022 (Hal. 124-130) https://www.ojsstikesbanyuwangi.com/index.php/PHJ Data tersebut semakin mendukung hasil penelitian ini bahwa sampai saat ini masih ada balita dengan status gizi buruk dan dapat kita simpulkan bahwa masalah tentang status HASIL Tabel 1 Status Gizi Balita Berdasarkan BB/U Tabel 1 Status Gizi Balita Berdasarkan BB/U Kategori L (BB/U) P (BB/U) Total % GIZI BURUK (<-3SD) 1 3 4 4 GIZI KURANG (-3SD s.d < -2SD) 10 6 16 18 GIZI BAIK (-2SD s.d 2SD) 34 36 70 77 GIZI LEBIH (>2SD) 0 1 1 1 TOTAL 45 46 91 100 Ririn Handayani dkk., Gambaran Status Gizi Balita Salah satu upaya pemenuhan kebutuhan pelayanan kesehatan pada anak dimulai dengan penurunkan angka 125 . PROFESIONAL HEALTH JOURNAL Volume 3, No. 2, Bulan Juni Tahun 2022 (Hal. 124-130) https://www.ojsstikesbanyuwangi.com/index.php/PHJ PEMBAHASAN Pengukuran status gizi bayi dan balita pada penelitian ini dilakukan sesuai dengan Standar WHO. Dimana status gizi balita dapat diukur melalui tiga kategori yaitu (1) status gizi yang dilihat berdasarkan BB/U; (2) status gizi dilihat berdasarkan TB/U, dan (3) status gizi dilihat berdasarkan BB/TB. Hasil dari pengukuran status gizi 126 Ririn Handayani dkk., Gambaran Status Gizi Balita . PROFESIONAL HEALTH JOURNAL Volume 3, No. 2, Bulan Juni Tahun 2022 (Hal. 124-130) https://www.ojsstikesbanyuwangi.com/index.php/PHJ . PROFESIONAL HEALTH JOURNAL Volume 3, No. 2, Bulan Juni Tahun 2022 (Hal. 124-130) https://www.ojsstikesbanyuwangi.com/index.php/PHJ lebih rentan terjangkit penyakit infeksi, seperti cacingan, infeksi saluran pernafasan atas (ISPA), diare (Kartikawati, 2011). gizi pada bayi dan balita masih belum tertangani dengan tuntas. Kegiatan pelayanan gizi yang dapat dilakukan dimulai dari pelayanan konseling dan pemberian suplementasi gizi pada ibu hamil, pemantauan tumbuh kembang balita, pemberian suplemen vitamin A dan PMT Balita dengan status gizi kurang, serta penanganan balita gizi buruk (Kemenkes RI, 2020). gizi pada bayi dan balita masih belum tertangani dengan tuntas. Kegiatan pelayanan gizi yang dapat dilakukan dimulai dari pelayanan konseling dan pemberian suplementasi gizi pada ibu hamil, pemantauan tumbuh kembang balita, pemberian suplemen vitamin A dan PMT Balita dengan status gizi kurang, serta penanganan balita gizi buruk (Kemenkes RI, 2020). Indikator status gizi lain yang didapat dari hasil pengukuran TB/U didapatkan 41 balita (45%) dengan kategori sangat pendek, 8 balita (9%) dengan kategori pendek, 42 balita (46%) dengan kategori normal. Dari hasil pengukuran tersebut dapat kita lihat terdapat 41 balita (45%) dengan kategori sangat pendek dan 8 balita (9%) dengan kategori pendek yang artinya diwilayah Posyandu Aster 11 dan 15 tedapat 49 balita stunting yang membutuhkan penanganan lebih lanjut untuk mencegah kodisi yang semakin buruk pada balita tersebut. Efek jangka panjang terkait dengan masalah status gizi dapat mempengaruhi derajat kesehatan anak serta mampu mempengaruhi pertumbuhan fisik dan mental pada anak. Saat ini kita tahu bahwa Indonesia memegang rekor tertinggi tingkat kematian anak akibat Covid-19 (2,5 %). Tanda gejala awal yang dialami pada anak yang terpapar Covid-19 yaitu diare, kejang, shock atau dengan tanpa gejala. Tingginya angka kematian anak di Indonesia dapat dipicu oleh beberapa faktor yaitu rendahnya tingkat pemeriksaan pada bayi dan balita, adanya penyakit bawaan yang diderita oleh bayi dan balita dan adanya bayi dan balita yang menderita gizi buruk, serta penanganan yang terlambat. Status bayi dan balita dengan gizi buruk inilah yang menjadi kekhawatiran kita sebagai tenaga kesehatan akan resiko dari penularan Covid-19 karena balita akan memiliki status imun yang rendah jika status gizi yang dimiliki juga rendah (buruk). Menurut Kartika (2011) bayi dan balita dengan status gizi rendah (kurang) Faktor - faktor yang dapat mempengaruhi Stunting antara lain faktor keturunan, dimana pertumbuhan fisik anak juga dapat dipengaruhi oleh tinggi badan orang tua (Zottarelli, 2014). Penelitian serupa telah dilakukan oleh Fitriahadi (2018) ibu dengan kondisi fisik yang pendek kemungkinan dapat melakirkan anak dengan stunting. Hal ini dapat Ririn Handayani dkk., Gambaran Status Gizi Balita 127 . PROFESIONAL HEALTH JOURNAL Volume 3, No. . PROFESIONAL HEALTH JOURNAL Volume 3, No. 2, Bulan Juni Tahun 2022 (Hal. 124-130) https://www.ojsstikesbanyuwangi.com/index.php/PHJ Dimana masih adanya masalah kekurangan gizi bahkan gizi buruk (deficiency, undernutrition), akan tetapi juga terdapat masalah terkait dengan kelebihan gizi, kelebihan berat badan bahkan kegemukan. Dari hasil pengkajian yang dilakukan oleh peneliti ditemukan 27 balita (1%) dengan kategori gemuk. Hal ini juga menjadi beban kita sebagai tenaga kesehatan untuk mengatasi masalah tersebut karena kegemukan bahkan obesitas pada usia anak-anak dapat memiliki kecenderungan menderita hal yang sama pada masa dewasa dan akan mengakibatkan berbagai macam penyakit kronis akibat kegemukan seperti kencing manis dan penyakit jantung. Beberapa ahli menyarankan usaha pencegahan obesitas telah dimulai pada awal masa anak-anak (Yusac, 2007). Dimana masih adanya masalah kekurangan gizi bahkan gizi buruk (deficiency, undernutrition), akan tetapi juga terdapat masalah terkait dengan kelebihan gizi, kelebihan berat badan bahkan kegemukan. Dari hasil pengkajian yang dilakukan oleh peneliti ditemukan 27 balita (1%) dengan kategori gemuk. Hal ini juga menjadi beban kita sebagai tenaga kesehatan untuk mengatasi masalah tersebut karena kegemukan bahkan obesitas pada usia anak-anak dapat memiliki kecenderungan menderita hal yang sama pada masa dewasa dan akan mengakibatkan berbagai macam penyakit kronis akibat kegemukan seperti kencing manis dan penyakit jantung. Beberapa ahli menyarankan usaha pencegahan obesitas telah dimulai pada awal masa anak-anak (Yusac, 2007). berhubungan dengan kondisi patologis orang tua antara lain defisiensi hormon pertumbuhan. Berbeda dengan kejadian stunting bukan karena kondisi patologis, misalnya karena masalah gizi atau infeksi, maka kemungkinan besar anak dapat tumbuh normal tanpa stunting. Kategori terahir dari hasil pengkajian yang dilakukan oleh peneliti tentang status gizi berdasarkan BB/TB didapatkan 2 balita (2%) dengan kategori sangat kurus, 3 balita (3%) dengan kategori kurus, 59 balita (65%) dengan kategori normal, 27 balita (1%) dengan kategori gemuk. Status gizi dengan kategori sangat kurus maupun dengan kategori kurus berdasarkan BB/TB menggambarkan suatu proses patologis yang terjadi sekarang sehingga menyebabkan kehilangan berat badan yang signifikan sebagai konsekuensi dari puasa akut atau adanya penyakit yang berat. Proporsi berat badan terhadap tinggi badan yang buruk lebih rentan terhadap infeksi dan mortalitas (WFP, 2005). Berdasarkan indeks BB/TB pada kelompok umur bayi dan balita vegetarian memiliki proporsi tubuh yang normal antara berat badan dan tinggi yaitu sebesar 63,2% (Pramita, 2014). . PROFESIONAL HEALTH JOURNAL Volume 3, No. 2, Bulan Juni Tahun 2022 (Hal. 124-130) https://www.ojsstikesbanyuwangi.com/index.php/PHJ 2, Bulan Juni Tahun 2022 (Hal. 124-130) https://www.ojsstikesbanyuwangi.com/index.php/PHJ PROFESIONAL HEALTH JOURNAL Volume 3, No. 2, Bulan Juni Tahun 2022 (Hal. 124-130) https://www.ojsstikesbanyuwangi.com/index.php/PHJ Stunting pada Balita usia 24-59 bulan di Provinsi Nusa Tenggara Timur (Analisis data Riskesdas 2010). Skripsi: Depok: FKM UI. KESIMPULAN DAN SARAN Pemenuhan gizi pada bayi dan balita sangat penting dan membutuhkan penanganan yang serius karena dapat menyebabkan terjadinya masalah pertumbuhan dan perkembangan pada anak. Untuk itu perlu dilakukan pemantauan secara berkala terkait dengan pertumbuhan dan perkembangan pada anak sebagai upaya deteksi dini kelainan pada anak. DAFTAR PUSTAKA Direktorat Gizi Masyarakat (2018). Buku Saku Pemantauan Status Gizi. Indonesia kini menghadapi beban ganda terkait dengan masalah gizi masyarakat. Ririn Handayani dkk., Gambaran Status Gizi Balita 128 PROFESIONAL HEALTH JOURNAL Volume 3, No. 2, Bulan Juni Tahun 2022 (Hal. 124-130) https://www.ojsstikesbanyuwangi.com/index.php/PHJ Ririn Handayani dkk., Gambaran Status Gizi Balita KEMENKES RI Dwihestie, Rosida and Kesehatan, (2020). Gambaran Prevalensi Balita Stunting. Jurnal Ilmiah Permas: Jurnal Ilmiah STIKES Kendal Volume 10 No 4, Hal 615–624, Oktober 2020 Dwihestie, Rosida and Kesehatan, (2020). Gambaran Prevalensi Balita Stunting. Jurnal Ilmiah Permas: Jurnal Ilmiah STIKES Kendal Volume 10 No 4, Hal 615–624, Oktober 2020 Hidayah, N. R. (2011). Faktor-faktor yang Berhubungan dengan Kejadian Stunting pada Balita usia 24-59 bulan di Provinsi Nusa Tenggara Timur (Analisis data Riskesdas 2010). Skripsi: Depok: FKM UI. Kemenkes RI (2019). Profil Kesehatan Indonesia. Jakarta: Kemenkes RI Kemenkes RI (2020). Panduan Gizi Seimbang Pada Masa Pandemi Covid-19. Gerakan Masyarakat Hidup Sehat, KEMENKES RI Kemenkes RI (2020). Panduan Gizi Seimbang Pada Masa Pandemi Covid-19. Gerakan Masyarakat Hidup Sehat, KEMENKES RI Kartikawati, P. (2011). Faktor Yang Mempengaruhi Kejadian Stunted Growth Pada Anak Balita Di Wilayah Kerja Puskesmas Arjasa Kabupaten Jember. Skripsi: Jember. Kemenkes RI (2020). Pedoman Pelayanan Gizi Pada Masa Tanggap Darurat Covid-19. Jakarta: Kemenkes RI. Komarudin. (2020). IDAI: Tingkat Kematian Anak Akibat Covid-19 di Indonesia Tertinggi di Asia Pasifik. https://www.liputan6.com/lifestyle/re ad/4330555/idai-tingkat-kematian- anak-akibat-covid-19-di-indonesia- tertinggi-di-asia-pasifik Komarudin. (2020). IDAI: Tingkat Kematian Anak Akibat Covid-19 di Indonesia Tertinggi di Asia Pasifik. https://www.liputan6.com/lifestyle/re ad/4330555/idai-tingkat-kematian- anak-akibat-covid-19-di-indonesia- tertinggi-di-asia-pasifik Komarudin. (2020). IDAI: Tingkat Kematian Anak Akibat Covid-19 di Indonesia Tertinggi di Asia Pasifik. https://www.liputan6.com/lifestyle/re ad/4330555/idai-tingkat-kematian- anak-akibat-covid-19-di-indonesia- tertinggi-di-asia-pasifik Satriawan (2018). Penanganan Masalah Stunting di Indonesia. Tim Nasional Percepatan Penanggulanagna Kemiskinan. Ririn Handayani dkk., Gambaran Status Gizi Balita Fitriahadi, E. (2018). Hubungan Tinggi Badan Ibu Dengan Kejadian Stunting Pada Balita Usia 24-59 Bulan. Jurnal Keperawatan dan Kebidanan Aisyiyah. ISSN 2477-8184. 14(1). 15- 24.https://ejournal.unisayogya.ac.id/e journal/index.php/jkk/article/view/54 5 Hidayah, N. R. (2011). Faktor-faktor yang Berhubungan dengan Kejadian Ririn Handayani dkk., Gambaran Status Gizi Bali Fitriahadi, E. (2018). Hubungan Tinggi Badan Ibu Dengan Kejadian Stunting Pada Balita Usia 24-59 Bulan. Jurnal Keperawatan dan Kebidanan Aisyiyah. ISSN 2477-8184. 14(1). 15- 24.https://ejournal.unisayogya.ac.id/e journal/index.php/jkk/article/view/54 5 Hidayah, N. R. (2011). Faktor-faktor yang Berhubungan dengan Kejadian Fitriahadi, E. (2018). Hubungan Tinggi Badan Ibu Dengan Kejadian Stunting Pada Balita Usia 24-59 Bulan. Jurnal Keperawatan dan Kebidanan Aisyiyah. ISSN 2477-8184. 14(1). 15- 24.https://ejournal.unisayogya.ac.id/e journal/index.php/jkk/article/view/54 Setyorini, Catur; Lieskusumastuti, Anita D .(2021). Description Of Nutritional Status Of Infant And Childhood In The Time Of Covid-19 In Kalurahan Jetis Sukoharjo. Avicenna : Journal of Health Research, Vol 4 No 1. Maret 2021 (118 - 127) World Food Programme (2005). A Manual: Measuring and Interpreting 129 . PROFESIONAL HEALTH JOURNAL Volume 3, No. 2, Bulan Juni Tahun 2022 (Hal. 124-130) https://www.ojsstikesbanyuwangi.com/index.php/PHJ Malnutrition and Mortality. Rome: WFP. Malnutrition and Mortality. Rome: WFP. Yussac, Aristato (2007). Prevalensi obesitas pada anak usia 4-6 tahun dan hubungannya dengan asupan dan pola makan. Majalah kedokteran Indonesia 2007: 57(2). Zottarelli, L.K, Sunil, T.S, Rajaram, S. (2014). Influence of Parental and Socioeconomics Factors on Stunting in Children Under 5 Years in Egypt. Easten Mediterranean Health Journal. dalam http://www.emro.who.int/emhi/10. 130 Ririn Handayani dkk., Gambaran Status Gizi Balita
https://openalex.org/W2999155565	https://europepmc.org/articles/pmc6966092?pdf=render	English	null	RNA CoSSMos 2.0: an improved searchable database of secondary structure motifs in RNA three-dimensional structures	Database	2,020	cc-by	3,930	Abstract The RNA Characterization of Secondary Structure Motifs, RNA CoSSMos, database is a freely accessible online database that allows users to identify secondary structure motifs among RNA 3D structures and explore their structural features. RNA CoSSMos 2.0 now requires two closing base pairs for all RNA loop motifs to create a less redundant database of secondary structures. Furthermore, RNA CoSSMos 2.0 represents an upgraded database with new features that summarize search ﬁndings and aid in the search for 3D structural patterns among RNA secondary structure motifs. Previously, users were limited to viewing search results individually, with no built-in tools to compare search results. RNA CoSSMos 2.0 provides two new features, allowing users to summarize, analyze and compare their search result ﬁndings. A function has been added to the website that calculates the average and representative structures of the search results. Additionally, users can now view a summary page of their search results that reports percentages of each structural feature found, including sugar pucker, glycosidic linkage, hydrogen bonding patterns and stacking interactions. Other upgrades include a newly embedded NGL structural viewer, the option to download the clipped structure coordinates in ∗.pdb format and improved NMR structure results. RNA CoSSMos 2.0 is no longer simply a search engine for a structure database; it now has the capability of analyzing, comparing and summarizing search results. Database URL: http://rnacossmos.com Database URL: http://rnacossmos.com Database URL: http://rnacossmos.com RNA CoSSMos 2.0: an improved searchable database of secondary structure motifs in RNA three-dimensional structures Katherine E. Richardson, Charles C. Kirkpatrick and Brent M. Znosko* Saint Louis University, Department of Chemistry, 3501 Laclede Avenue, St. Louis, MO 63103 USA *Corresponding author: Tel: (314) 977-8567; Fax: (314) 977-2521; Email: brent.znosko@slu.edu Citation details: Richardson,K.E., Kirkpatrick,C.C. and Znosko,B.M. RNA CoSSMos 2.0: an improved searchable database of secondary structure motifs in RNA three-dimensional structures. Database (2020) Vol. 2020: article ID baz153; doi:10.1093/database/baz153 tion details: Richardson,K.E., Kirkpatrick,C.C. and Znosko,B.M. RNA CoSSMos 2.0: an improved searchable database econdary structure motifs in RNA three-dimensional structures. Database (2020) Vol. 2020: article ID baz153; 10.1093/database/baz153 Citation details: Richardson,K.E., Kirkpatrick,C.C. and Znosko,B.M. RNA CoSSMos 2.0: an improved searchable database of secondary structure motifs in RNA three-dimensional structures. Database (2020) Vol. 2020: article ID baz153; doi:10.1093/database/baz153 Received 24 June 2019; Revised 12 November 2019; Accepted 13 December 2019 Database, 2020, 1–6 doi: 10.1093/database/baz153 Database Update © The Author(s) 2020. Published by Oxford University Press. Page 1 of 6 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. ( b t f it ti ) © The Author(s) 2020. Published by Oxford University Press. Introduction The two main improvements to the RNA CoSSMos database are new functionalities to (i) calculate an average and representative structure of the results and (ii) provide a summary page with percentages of structural features among the results. Additional improvements include (iii) the addition of a sec- ond closing base pair requirement for loop motifs, (iv) the replacement of Jmol by NGL Viewer for 3D structure view- ing (14–17), (v) the option to download the coordinates of the clipped structures in ∗.pdb format and (vi) the addition of the model numbers of NMR results to the database. The RNA CoSSMos website has been moved to a faster webhost and is now available at http://rnacossmos.com. Currently, there are several databases that contain 3D structures for secondary structure motifs. The Structural Classification of RNA (SCOR) database was created through a manual classification and inspection of RNA structures (2, 3). The SCOR database provides 3D struc- tures for hairpin and internal loops deposited in the Protein Data Bank (PDB) before 15 May 2003. RNA Fragments Search Engine and Database (RNA FRABASE) 2.0 (4, 5), which is updated monthly, converts PDB files of RNA struc- tures to a dot-bracket notation using the RNAView pro- gram (6) to identify base pairs. RNA FRABASE 2.0 allows users to search for RNA 3D structure fragments using the user input sequence and dot-bracket secondary structure notation (4, 5). The RNA 3D Motif Atlas (7) uses Find RNA 3D (FR3D) (8) to search for hairpin and internal loops and classify the loops into 3D structure motifs. FR3D uses a base-centered approach in which each base is represented by a single center point to compare RNA 3D structures (8). The RNA Characterization of Secondary Structure Motifs (RNA CoSSMos) database was first released in 2011 with weekly automatic updates (9). To create the RNA CoSSMos database, the RNA-containing 3D structures in the PDB (10) were searched for internal, bulge and hairpin loops using MC-Search (11, 12). MC-Search uses user-defined base pairing interactions and subgraph isomorphism algorithms to search PDB files for RNA secondary structure motifs (11, 12). Subsequently, MC-Annotate (11, 12) was used to determine structural information including sugar puckers, glycosidic linkages, hydrogen bonding patterns and stacking interactions within the loop and closing base pair. MC-Annotate uses homogenous transformation matrices to calculate base–base interactions (11, 12). Introduction RNA folds, gain knowledge of its biological functions and design potential therapeutics. A variety of approaches have been taken in predicting RNA 3D structure from sequence, with template-based approaches resulting in a particularly A deep understanding of the primary, secondary and ter- tiary structures of RNA and the relationship between these levels of structure is essential to understand and predict how (page number not for citation purposes) (page number not for citation purposes) Database, Vol. 2020, Article ID baz153 Page 2 of 6 provided by MC-Annotate that includes not only base- pairing interactions but additional structural information including sugar puckers, glycoside linkages and stacking interactions. The option to search the database using any combination of PDB information, experimental parameters, motif and/or sequence allows users to highly customize their database viewing. high level of accuracy (1). A template-based approach uses solved RNA 3D structures or fragments as templates to build an initial structure of the structure to be predicted. A critical area for improvement of RNA 3D structure pre- diction is the prediction of non-Watson–Crick interactions, such as those found in loops (1). Therefore, the utility of a database that contains 3D structure templates for secondary structure motifs remains high. Herein, we use the term ‘motif’ to describe a secondary structure element, such as an internal loop, bulge loop or hairpin loop. This definition of a secondary structure motif does not necessarily imply sequence similarity, 3D structural similarity nor a shared biological function. Since the RNA CoSSMos release in 2011, the RNA- containing structures in the PDB have increased from 2156 to over 3909, leading to subsequent growth of the RNA CoSSMos database. An RNA CoSSMos search returns results which can be viewed individually on the website. For searches that return a large number of results, individual viewing can become a daunting task. Additionally, studies that search for tertiary structural patterns among secondary structure motifs, such as those performed on single mismatches (13), have shown the need for users to be able to further analyze and summarize their search results. RNA CoSSMos 2.0 is no longer simply a search engine for a struc- ture database; it now has the capability of analyzing, com- paring and summarizing the search results. Introduction The results of MC-Search and MC-Annotate were pre-compiled into an online database for fast retrieval with an intuitive user-interface. RNA CoSSMos was designed to complement similar databases previously created. What continues to make the RNA CoSSMos database unique is the intuitive graphical user interface that does not require knowledge of a specialized syntax and the detailed structural annotation New results analysis features In addition to viewing search results individually, users can now further explore their search results in the ‘Summarize Results’ tab. Two features have been added to the website to allow users to obtain a summary of their search results: (i) an average structure feature and (ii) a results summary feature. These features (described below) can be chosen together or independently, depending on the needs of the user. Users can further customize their summary by selecting which structures in their search results should be included in the summary calculation. If the user’s search contains ≤350 results, they can select the desired results using check boxes. Alternatively, the user can download the search results as a delimited spreadsheet, edit the file to select for results to include in the summary and then upload the file to the RNA Page 3 of 6 Database, Vol. 2020, Article ID baz153 Figure 1. The RNA CoSSMos average structure page showing the average and representative structure for all GNRA tetraloops. A total of 4724 GNRA tetraloops were included in the calculations using the backbone and three atoms from each base. Figure 1. The RNA CoSSMos average structure page showing the average and representative structure for all GNRA tetraloops. A total of 4724 GNRA tetraloops were included in the calculations using the backbone and three atoms from each base. CoSSMos website. An option is also available to include all of the search results in the summary calculations. bonding patterns and stacking interactions within the loop and closing base pairs across all of the selected RNA CoSSMos search results. These percentages are reported for individual features (e.g. the sugar pucker of the first base in the loop) and combinations of structural features (e.g. the residue conformations of the first and last bases in the loop and hydrogen-bonding interactions between these bases). The tables utilize the residue numbering sys- tem described in this paper and on the RNA CoSSMos FAQ wiki page (http://rnacossmos.com/faq2.php). For user convenience, the tables can be expanded and collapsed so that users can focus on the structural annotations of greatest interest (Figure 2). Additionally, all of the tables can be downloaded as a zipped file that contains individual delimited spreadsheets for each table. Average structure. The average structure feature calculates an average structure of all of the selected search results using a Python script. New results analysis features Users can customize this calculation by selecting which atom coordinates they would like included in the calculation. Atom coordinate options are (i) phos- phate atoms only, (ii) phosphate-sugar backbone atoms or (iii) phosphate-sugar backbone and bases. If users select the ‘phosphate-sugar backbone and bases’ option, all of the atom coordinates from the backbone and three atom coordinates from each base (N9, C8 and C4 for purines and N1, C2 and C6 for pyrimidines) are used in the calculation to allow users to calculate average structures for results with differing sequences. To calculate the average structure, the coordinates are superimposed using the Kabsch algorithm (18) to calculate the optimal rotation matrix. The average structure (which consists of the selected coordinates) is available for viewing on the website, and the coordinates can be downloaded as a ∗.pdb file. In addition to the average structure, the root mean square deviation (RMSD) of each result structure compared to the average structure is calculated, and the result that has the smallest RMSD to the average structure is reported as the representative structure. The representative structure can also be viewed on the website and downloaded as a ∗.pdb file (Figure 1). New requirement for two closing base pairs Extensive use of the previous RNA CoSSMos database returned results containing substantial overlap between loop motifs as well as loop instances with one or no closing base pair. For example, residues A1386–A1396 in the structure of the large ribosomal subunit in complex with virginiamycin M (PDB ID 1N8R) contained a hairpin of seven nucleotides in residues A1387–A1395, a hairpin of three nucleotides in residues A1388–A1392 and a bulge of two nucleotides in residues A1387–A1388 and A1392– A1395 (Figure 3). To reduce the overlap and improve the integrity of the database, a new set of MC-Search input descriptors were written that require two A-U, C-G, G-C, G-U, U-A or U-G closing base pairs. The requirement of Results summary. The results summary compiles all the indi- vidual MC-Annotate results into tables that report per- centages of sugar puckers, glycosidic linkages, hydrogen Page 4 of 6 Database, Vol. 2020, Article ID baz153 re 2. The RNA CoSSMos results summary page (A) collapsed and (B) expanded for ‘Sugar Pucker’ and ‘Glycosidic Conformation’. closing base pairs for secondary structures has been second 5′ closing base of the ‘B’ strand or ‘bottom’ strand Figure 2. The RNA CoSSMos results summary page (A) collapsed and (B) expanded for ‘Sugar Pucker’ and ‘Glycosidic Conformation’. Figure 2. The RNA CoSSMos results summary page (A) collapsed and (B) expanded for ‘Sugar Pucker’ and ‘Glycosidic Conformation’. second 5′ closing base of the ‘B’ strand or ‘bottom’ strand is assigned the next position where the ‘A’ strand terminated, then the remaining bases in the ‘B’ strand are sequentially assigned from the 5′ to 3′ end. two closing base pairs for secondary structures has been shown to improve the accuracy of RNA folding (19). With these new input descriptors, residues A1386–A1396 in PDB ID 1N8R will only contain a hairpin of seven nucleotides in the RNA CoSSMos 2.0 database. Improved structure viewing Residue numbering system. In order to compare the structures and structural features of the results, a common residue numbering system was necessary. The residue ID numbers within each result were renumbered as follows (Figure 4). For hairpin loops, the second 5′ closing base is assigned as the first base,with the remaining bases assigned sequentially from the 5′ to the 3′ end. For internal and bulge loops, the second 5′ closing base of the ‘A’ strand or ‘top’ strand is assigned as the first base, with the remaining bases in this strand assigned sequentially from the 5′ to the 3′ end. The RNA CoSSMos previously utilized a Jmol applet (20) for 3D structure viewing. For RNA CoSSMos 2.0, the Jmol applet has been replaced by NGL Viewer (http://nglviewer. org) which does not require the use of Java or plugins. The NGL molecular viewer has replaced the Jmol viewer on the individual detailed results pages and is also utilized to view the average and representative structures. Previously, users could download a snapshot of the Jmol viewer. Now, users can download a clipped ∗.pdb file of the motif structure Database, Vol. 2020, Article ID baz153 Page 5 of 6 Figure 3. The secondary structure representation of residues A1386– A1396 in PDB ID 1N8R drawn with forna (24). The previous RNA CoSSMos database contained a hairpin of seven nucleotides in residues A1387–A1395, a hairpin of three nucleotides in residues A1388–A1392, and a bulge of two nucleotides in residues A1387–A1388 and A1392– A1395. RNA CoSSMos 2.0 only contains a hairpin of seven nucleotides in residues 1386–1396. models are denoted in the database with an ‘∗’ after the model number. Users can view the detailed results of the individual models or can utilize the new summary features to learn about structural information within the ensemble or across multiple selected ensembles.When a user wishes to utilize the ‘Summarize Results’ tab with results that contain NMR structures, the user is given the option to include all of the models within the ensemble or only the representative model in the summary calculations. Example utilization The previous RNA CoSSMos database contained a hairpin of seven nucleotides in residues A1387–A1395, a hairpin of three nucleotides in residues A1388–A1392, and a bulge of two nucleotides in residues A1387–A1388 and A1392– A1395. RNA CoSSMos 2.0 only contains a hairpin of seven nucleotides in residues 1386–1396. Figure 4. An illustration of the numbering system used for (A) a tetraloop and (B) a 1 × 1 nucleotide internal loop. Figure 4. An illustration of the numbering system used for (A) a tetraloop and (B) a 1 × 1 nucleotide internal loop. in which the residue ID numbers have been renumbered according to the residue numbering system described in this paper. This will allow users to view and manipulate the clipped structures on their own computers using their preferred software in addition to 3D structure viewing on the RNA CoSSMos website. Additional viewing options allow users to see individual motifs and representative structures embedded within the global structure found in the ∗.pdb file. Example utilization Hairpins that contain four bases in the loop are commonly referred to as tetraloops. Tetraloops with the sequence GNRA (N = any nucleotide, R = A or G) often form an unusually stable 3D fold that is well-documented (21, 22). To demonstrate the usefulness of the new analysis features of the RNA CoSSMos database, we conducted a search for tetraloops with the GNRA loop sequence. The RNA CoSSMos database currently contains 4724 tetraloops with the GNRA loop sequence. Utilizing the original version of the RNA CoSSMos database, users could then explore the detailed results pages of the 4724 results individu- ally on the website or by downloading a tab-delimited file that could be explored using spreadsheet software on their computer. There were no tools available on CoSSMos to compare structures or summarize results. Now, using the updated RNA CoSSMos database, users can compare and/or summarize the results using the ‘Average Structure’ and ‘Results Summary’ features. The average and repre- sentative structures of the 4724 tetraloops were calcu- lated using the backbone and three atoms from each base (Figure 1). These structures can be viewed on the website or can be downloaded as templates for RNA structure prediction. Furthermore, the structural features of all 4724 tetraloops were tabulated using the ‘Results Summary’ fea- ture, showing that approximately 90, 85 and 95% of the GNRA tetraloops contain hydrogen bonding between the G nucleobase and A nucleobase, base stacking of N on R, and base stacking of R on A, respectively, three features characteristic of the GNRA fold (23). A similar analysis utilizing the new features has identified three new tetraloop sequence families (25). We anticipate that the new features can be utilized for a variety of similar and novel purposes. Figure 3. The secondary structure representation of residues A1386– A1396 in PDB ID 1N8R drawn with forna (24). The previous RNA CoSSMos database contained a hairpin of seven nucleotides in residues A1387–A1395, a hairpin of three nucleotides in residues A1388–A1392, and a bulge of two nucleotides in residues A1387–A1388 and A1392– A1395. RNA CoSSMos 2.0 only contains a hairpin of seven nucleotides in residues 1386–1396. Figure 3. The secondary structure representation of residues A1386– A1396 in PDB ID 1N8R drawn with forna (24). Availability 12. Lemieux,S. and Major,F. (2002) RNA canonical and non- canonical base pairing types: a recognition method and complete repertoire. Nucleic Acids Res., 30, 4250–4263. The RNA CoSSMos 2.0 database is freely available online and is now hosted at a new URL http://rnacossmos.com. We will maintain the redirection from the previously used URL (http://cossmos.slu.edu) for the foreseeable future, but users are encouraged to update their links accordingly. 13. Davis,A.R., Kirkpatrick,C.C. and Znosko,B.M. (2011) Struc- tural characterization of naturally occurring RNA single mis- matches. Nucleic Acids Res., 39, 1081–1094. 14. Rose,A.S., Bradley,A.R., Valasatava,Y. et al. (2018) NGL viewer: web-based molecular graphics for large complexes. Bioinfor- matics, 34, 3755–3758. Funding National Institutes of Health (2R15GM085699-03). Funding for open access charge: National Institutes of Health. 17. Tiemann,J.K.S., Guixà-González,R., Hildebrand,P.W. et al. (2017) MDsrv: viewing and sharing molecular dynamics sim- ulations on the web. Nat. Methods, 14, 1123. 18. Kabsch,W. (1976) A solution for the best rotation to relate two sets of vectors. Acta Crystallogr., 32, 922–923. Improved NMR structure handling The new RNA CoSSMos database continues to provide users with a unique online tool to search for 3D structure characteristics of RNA secondary structure motifs using a simple and intuitive graphical user interface but now allows its users to explore and compare structural features and patterns among their customized set of search results Model numbers corresponding to those in the original PDB structure have been added to the RNA CoSSMos 2.0 database for ensemble structures solved by NMR. For each NMR ensemble within each motif, the model that is closest to the average of the ensemble results is selected as the representative model for the ensemble. The representative Database, Vol. 2020, Article ID baz153 Page 6 of 6 in addition to viewing the results individually. Additional updates not described here to improve ease of use, user customization and database completeness and accuracy have been made to ensure that the database remains a reliable and valuable tool for the scientific community. In 2014, the PDBx/mmCIF file format became the standard PDB format to allow for large structures that cannot be rep- resented in the ∗.pdb file format. Currently, ∼6% of RNA containing structures are only offered in the PDBx/mmCIF file format. Future versions of the RNA CoSSMos database will incorporate the PBDX/mmCIF file format, and future directions of the database will be driven by feedback from the users. As the PDB continues to grow, so will the RNA CoSSMos database and its capabilities. 6. Yang,H.W., Jossinet,F., Leontis,N. et al. (2003) Tools for the automatic identification and classification of RNA base pairs. Nucleic Acids Res., 31, 3450–3460. 7. Parlea,L.G., Sweeney,B.A., Hosseini-Asanjan,M. et al. (2016) The RNA 3D motif atlas: computational methods for extraction, organization and evaluation of RNA motifs. Methods, 103, 99–119. 8. Sarver,M., Zirbel,C.L., Stombaugh,J. et al. (2008) FR3D: finding local and composite recurrent structural motifs in RNA 3D structures. J Math Biol., 56, 215–252. structures. J Math Biol., 56, 215–252. 9. Vanegas,P.L., Hudson,G.A., Davis,A.R. et al. (2012) RNA CoSS- Mos: characterization of secondary structure motifs-a search- able database of secondary structure motifs in RNA three- dimensional structures. Nucleic Acids Res., 40, D439–D444. 10. Berman,H.M., Westbrook,J., Feng,Z. et al. (2000) The protein data bank. Nucleic Acids Res., 28, 235–242. 11. Gendron,P., Lemieux,S. and Major,F. (2001) Quantitative analy- sis of nucleic acid three-dimensional structures. J Mol Biol., 308, 919–936. Acknowledgements 15. Rose,A.S. and Hildebrand,P.W. (2015) NGL viewer: a web application for molecular visualization. Nucleic Acids Res., 43, W576–W579. The authors would like to thank Miranda Adams and Sharear Saon for their assistance testing earlier prototypes of this database. 16. Nguyen,H., Case,D.A. and Rose,A.S. (2017) NGLview– interactive molecular graphics for Jupyter notebooks. Bioinfor matics, 34, 1241–1242. References 19. Mathews,D.H., Andre,T.C., Kim,J. et al. (1997) In Leontis, N.B. and SantaLucia, J. (eds.), Molecular Modeling of Nucleic Acids. ACS Symposium Series, Vol. 682, pp. 246–257. 1. Miao,Z.C., Adamiak,R.W., Blanchet,M.F. et al. (2015) RNA- Puzzles Round II: Assessment of RNA structure prediction programs applied to three large RNA structures. RNA, 21, 1066–1084. 20. Hanson,R. (2010) Jmol - a paradigm shift in crystallographic visualization. J Appl Cryst., 43, 1250–1260. 2. Klosterman,P.S., Tamura,M., Holbrook,S.R. et al. (2002) SCOR: a structural classification of RNA database. Nucleic Acids Res., 30, 392–394. 21. Correll,C.C. and Swinger,K. (2003) Common and distinctive features of GNRA tetraloops based on a GUAA tetraloop struc- ture at 1.4 angstrom resolution. RNA, 9, 355–363. 3. Tamura,M., Hendrix,D.K., Klosterman,P.S. et al. (2004) SCOR: structural classification of RNA, version 2.0. Nucleic Acids Res., 32, D182–D184. 22. Heus,H.A. and Pardi,A. (1991) Structural features that give rise to the unusual stability of RNA hairpins containing GNRA loops. Science, 253, 191–194. 23. Bottaro,S. and Lindorff-Larsen,K. (2017) Mapping the universe of RNA tetraloop folds. Biophys J., 113, 257–267. 4. Popenda,M., Szachniuk,M., Blazewicz,M. et al. (2010) RNA FRABASE 2.0: an advanced web-accessible database with the capacity to search the three-dimensional fragments within RNA structures. BMC Bioinf., 11, 231. 24. Kerpedjiev,P., Hammer,S. and Hofacker,I.L. (2015) Forna (force- directed RNA): simple and effective online RNA secondary structure diagrams. Bioinformatics, 31, 3377–3379. 5. Popenda,M., Blazewicz,M., Szachniuk,M. et al. (2008) RNA FRABASE version 1.0: an engine with a database to search for the three-dimensional fragments within RNA structures. Nucleic Acids Res., 36, D386–D391. 25. Richardson,K.E., Adams,M.S., Kirkpatrick,C.C. et al. (2019) Identification and characterization of new RNA tetraloop sequence families, Biochemistry, 58, 4809–4820.
https://openalex.org/W2031247685	https://europepmc.org/articles/pmc4117422?pdf=render	English	null	Different Inward and Outward Conduction Mechanisms in NaVMs Suggested by Molecular Dynamics Simulations	PLOS computational biology/PLoS computational biology	2,014	cc-by	9,891	Abstract Competing Interests: The authors have declared that no competing interests exist. * Email: anna.stary@univie.ac.at As reviewed recently [24], sodium ions were illustrated to spontaneously traverse the SF into the cavity with energy barriers between ,2–5 kcal/mol. Compared to potassium coordination in KV channels, sodium ions partially or fully preserve their first hydration shells [13,15–17,20,22]. A loosely coupled knock-on mechanism with an average ion occupancy around two in the SF was predicted during ion conduction [14,15,21,22]. The incoming ion repulses the present ion out of the SF. The wide radius ($9 A˚ ) of the SF enables double occupancy of ions at the same level [20,21]. Further, Na+ vs. K+ [15,16,20,20–22] and Na+ vs. Ca2+ [17,20] discrimination studies were carried out. These studies revealed higher energy barriers in the SF for K+ and Ca2+ compared to Na+. Subsequently, non-equilibrium simulations were performed to investigate conduction under applied mem- brane potentials and to study kinetics [21,22]. The estimated inward conductance rate successfully reproduced electrophysiolo- gy data [22]. Song Ke, E. N. Timin, Anna Stary-Weinzinger* Department of Pharmacology and Toxicology, University of Vienna, Vienna, Austria July 2014 \| Volume 10 \| Issue 7 \| e1003746 Citation: Ke S, Timin EN, Stary-Weinzinger A (2014) Different Inward and Outward Conduction Mechanisms in NaVMs Suggested by Molecular Dynamics Simulations. PLoS Comput Biol 10(7): e1003746. doi:10.1371/journal.pcbi.1003746 PLOS Computational Biology \| www.ploscompbiol.org Abstract Rapid and selective ion transport is essential for the generation and regulation of electrical signaling pathways in living organisms. Here, we use molecular dynamics (MD) simulations with an applied membrane potential to investigate the ion flux of bacterial sodium channel NaVMs. 5.9 ms simulations with 500 mM NaCl suggest different mechanisms for inward and outward flux. The predicted inward conductance rate of ,2763 pS, agrees with experiment. The estimated outward conductance rate is 1563 pS, which is considerably lower. Comparing inward and outward flux, the mean ion dwell time in the selectivity filter (SF) is prolonged from 13.560.6 ns to 20.161.1 ns. Analysis of the Na+ distribution revealed distinct patterns for influx and efflux events. In 32.065.9% of the simulation time, the E53 side chains adopted a flipped conformation during outward conduction, whereas this conformational change was rarely observed (2.760.5%) during influx. Further, simulations with dihedral restraints revealed that influx is less affected by the E53 conformational flexibility. In contrast, during outward conduction, our simulations indicate that the flipped E53 conformation provides direct coordination for Na+. The free energy profile (potential of mean force calculations) indicates that this conformational change lowers the putative barriers between sites SCEN and SHFS during outward conduction. We hypothesize that during an action potential, the increased Na+ outward transition propensities at depolarizing potentials might increase the probability of E53 conformational changes in the SF. Subsequently, this might be a first step towards initiating slow inactivation. Editor: Emad Tajkhorshid, University of Illinois, United States of America Received December 17, 2013; Accepted June 13, 2014; Published July 31, 2014 Copyright: 2014 Ke et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by The Austrian Science Fund (FWF) Project W1232. The funders had no role in study design, d decision to publish, or preparation of the manuscript. Funding: This work was supported by The Austrian Science Fund (FWF) Project W1232. The funde decision to publish, or preparation of the manuscript. supported by The Austrian Science Fund (FWF) Project W1232. The funders had no role in study design, data collection and analysis eparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Different Inward and Outward Conduction Mechanisms in NaVMs Suggested by Molecular Dynamics Simulations Song Ke, E. N. Timin, Anna Stary-Weinzinger* Author Summary To explore the underlying differences between inward and outward ion permeation, we plotted the ion probability density map across the pore region from all four simulations. Several favorable ion-interacting sites (SEX, SHFS, SCEN and SIN) from periplasm to cytoplasm were assigned as proposed previously by Payandeh et al. [4]. The ion-interacting sites across the SF were determined by measuring the axial distance (Z axis) along certain atoms from 25.00 to 10.25 A˚ as shown in Figure 3. Side chain oxygens of S54 from all four chains were taken as the origin (Z = 0.0 A˚ ) of the SF. Additionally, in two previous studies, a site with an energy barrier (,2 kcal/mol) distinguishing between site SHFS and SCEN was identified [17,22]. In this study, we refer to this site as ‘‘SBAR’’, indicating this barrier (2.75#Z,4.75 A˚ ). Voltage gated sodium channels are essential components of living cell membranes. They regulate the cell potential by facilitating permeation of ions across the membrane. In the past decades, studies revealed that the bacterial selectivity filter (SF) exhibits a constricted architecture lined with electronegative carboxyl oxygens of four glutamic acid side chains (EEEE motif), which repulse anions but attract Na+ ions. Crystal structures enable the investigation of structural dynamics with computational methods. Ion selectivity and conduction mechanisms between Na+, K+ and Ca2+ are progressively elucidated by molecular dynamics simulations and free energy calculations. The structural dynamics of the protein, especially the flexibility of SF and its fundamental role in kinetics underpinning ion selectivity, conduction and channel gating are less well understood. To shed light on this question, we use computational simulations to simulate ion conduction with membrane potentials. Our results suggest different dynamical behaviors of the EEEE locus and distinct ion distribution patterns in the SF with respect to permeating directionalities. These findings indicate a novel mechanism in differentiating reciprocal transitions of ion flow, preventing large sodium efflux during action potential initiation and may further suggest that increased flipping propensities at depolarizing poten- tials, might initially trigger channel slow inactivation. During influx (Figure 4A and B), short-lived Na+ binding at site SEX was observed (2.660.5 ns) in an asymmetrical manner. SHFS is the dominant site with the highest ion density. At this site, ions tended to be directly coordinated with side chain oxygens of E53 and S54 in an off-axis manner. Single channel conductance The four-fold symmetrical structure of NaVMs (pdb identifier: 4F4L) was generated using chain A (splayed outward by 25u rotation about its Y-bond at position T84), which creates an open pore with a diameter of ,14 A˚ [7]. As described by Ulmschneider et al. [22], a harmonic restraint was exerted on the S5 and S6 TM helices to keep the gate in the open conformation throughout simulations. This structure was then embedded into a POPC lipid patch and duplicated in the Z direction (pore axis). A constant charge imbalance of four elementary charges (4 e) across each lipid bilayer between the central electrolyte bath and the two outer ones was maintained during simulation (supplementary movie S1) [27]. To investigate the influence of the presence of Na+ ions on E53 side chain dynamics, three repeated simulations without ions in the SF (‘‘no salt’’) were performed. Irrespective of the directionality of the applied potentials, the flip probability is less than 0.6% in all simulations (Figure 5C). This indicates, that a depolarizing potential per se does not significantly influence the number of E53 flipping events. This suggests that the combination of local positive charge carried by outward Na+ flux in the SF especially at sites SHFS and SBAR and the outward attracting membrane potential might collectively induce the rotation of the x2 angle from ,290u to ,60u. Four times 500 ns double-patch MD simulations with 500 mM NaCl were performed, with the first 100 ns treated as equilibration. Figure 1 shows the cumulative ion conducting events from MD simulations with depolarized and hyperpolar- ized membrane potentials of DV: 5656126 mV (Figure 2A). The estimated sodium current in the inward direction is c = 2763 pS. This value agrees with previously observed single channel conductance measurements (c,33 pS) [22]. Our double bilayer simulations enabled us to further estimate outward conduction, which amounts to c = 1563 pS. Interestingly, this process is distinguishably slower than inward ion flux (P,0.01, N = 4). Structural dynamics of glutamic acid Conformational isomerization of the E53 side chains has been reported previously [17,23]. Generally, the glutamic acid side chain might adopt two main conformations (Figure 5A and B): inward-facing (x2 angle ,60u, flipped) and outward-facing (x2 angle ,290u, non-flipped). In our simulations, during influx, flipping events were observed only in 2.760.5% of the simulation time, thus the E53 side chain mainly adopted a non-flipped conformation. In contrast, during efflux 32.065.9% flipping events were observed (P value = 0.015, see Figure 5C). A more detailed investigation of this flipping events revealed that 80% of these changes occurred in only one of the four glutamic acid side chains (‘‘one-flip’’) (Figure 5D). Author Summary Additionally, a less densely populated configuration was observed in the center of this site consistent with previous studies [21,24]. Moving inward from site SHFS, ions further translocated transiently via site SBAR (1.560.3 ns) to site SCEN (3.460.3 ns). Subsequently, ions reciprocally traversed between sites SCEN and SIN. These results are in good agreement with previous simulation studies [14,15,17,21,22]. Our simulations revealed a distinct ion distribution pattern for efflux compared to influx as shown in Figure 4C and D. After entering into site SIN from the cytosol, ions mainly populated sites SCEN and SBAR with extended dwell times compared to inward conduction, (SCEN: 11.961.1 ns vs. 3.460.3 ns; SBAR: 8.260.9 ns vs. 1.560.3 ns) suggesting a putative barrier for efflux between sites SBAR and SHFS. Additionally, during efflux, Na+ ions tended to traverse in an on-axis manner through the filter. It is not clear to which extend structural changes at the EEEE locus in Nav channels are crucial for conductance and inactivation in Nav channels. To investigate these issues, we conducted MD simulations using the open conformation of the bacterial sodium channel homologue NaVMs (Magnetococcus sp. (strain MC-1)) [7] pore domain focusing on the structural changes of the SF during inward and outward conduction. Ion distribution patterns and kinetics Author Summary E53 Dynamics Facilitate NaVMs Efflux E53 Dynamics Facilitate NaVMs Efflux Introduction Na+ flux through voltage gated sodium channels (NaV) is crucial for initiating action potentials in the membranes of electrically excitable cells. They mediate a variety of biological functions such as muscle contraction, propagation of nerve impulses, release of hormones and many more [1]. As a consequence, mutations in NaV channels lead to a variety of channelopathies, such as congenital epilepsy, cardiac arrhythmias or chronic pain [2,3]. Recently, homotetrameric crystal structures of several bacte- rial NaV channels were successfully resolved [4–10], providing a tremendous opportunity to investigate the structure and function of these channels on the atomistic level. They are composed of four membrane spanning subunits and contain six transmembrane (TM) helices per subunit. Helices S1 to S4 form the voltage sensing module. Helices S5, P1 segments, the selectivity filter (SF) region, P2 segments and S6 helices, lining the inner pore cavity, form the pore module. The SF of most bacterial channels contains the amino acid sequence TLESW. The four glutamic acid side chains [11] form a high field strength binding site (HFS) [12] which is essential for ion selectivity. In eukaryotic sodium channels, this site consists of the amino acids motif DEKA. A recent study by Chakrabarti et al. [23], suggested that conformational changes at the EEEE motif (corresponding to E177 in NaVAb) might play an important role in ion conduction. However, this observation was not reported in other simulations, except for simulations using Ca2+ as a charge carrier [17]. In K+ channels, subtle structural changes in the SF, involving rotations around a highly conserved glycine residue result in different non- conductive conformations [25,26]. This regulation of ion flow by conformational changes of the selectivity filter is termed C-type inactivation. The molecular mechanisms underlying ion conduction and selectivity in NaV are beginning to emerge. Computational methods, particularly molecular dynamics (MD) simulations are extensively adopted to address these questions [13–23]. July 2014 \| Volume 10 \| Issue 7 \| e1003746 PLOS Computational Biology \| www.ploscompbiol.org 1 PLOS Computational Biology \| www.ploscompbiol.org Ionic binding modes and conduction mechanism during inward conduction A detailed investigation of the ionic binding modes and their relations to free energy profiles enabled us to describe mechanisms July 2014 \| Volume 10 \| Issue 7 \| e1003746 PLOS Computational Biology \| www.ploscompbiol.org 2 E53 Dynamics Facilitate NaVMs Efflux Figure 1. Ion flux in double bilayer simulations without dihedral restraints on E53 (n = 4). A) Average ion flux count of inward conduction through the SF over time (color: blue). B) Average ion flux count of outward conduction through the SF over time (color: red). Error estimations shown in the figure are S.E.M. doi:10.1371/journal.pcbi.1003746.g001 Figure 1. Ion flux in double bilayer simulations without dihedral restraints on E53 (n = 4). A) Average ion flux count of inward conduction through the SF over time (color: blue). B) Average ion flux count of outward conduction through the SF over time (color: red). Error estimations shown in the figure are S.E.M. doi:10.1371/journal.pcbi.1003746.g001 through the SF over time (color: blue). B) Average ion flux count of outward conduction through the SF over time (color: red). Error estimations shown in the figure are S.E.M. doi:10.1371/journal.pcbi.1003746.g001 Figure 2. Transmembrane voltages comparison. A) Electrostatic potential across the simulation box calculated from the simulations without dihedral restraints on E53. B) Electrostatic potential across the simulation box calculated from the snapshots (t.5 ns) extracted from the simulation in Figure 2A without ion conduction events in neither inward nor outward directions C) Electrostatic potential across the simulation box calculated ure 2. Transmembrane voltages comparison. A) Electrostatic potential across the simulation box calculated from the simulations without dral restraints on E53. B) Electrostatic potential across the simulation box calculated from the snapshots (t.5 ns) extracted from the simulation in re 2A without ion conduction events in neither inward nor outward directions. C) Electrostatic potential across the simulation box calculated m the ‘‘no salt’’ simulations. (Error estimations shown in the figure are S.D.). 0.1371/journal.pcbi.1003746.g002 Figure 2. Transmembrane voltages comparison. A) Electrostatic potential across the simulation box calculated from the simulations wit dihedral restraints on E53. B) Electrostatic potential across the simulation box calculated from the snapshots (t.5 ns) extracted from the simulati Figure 2A without ion conduction events in neither inward nor outward directions. C) Electrostatic potential across the simulation box calcu from the ‘‘no salt’’ simulations. (Error estimations shown in the figure are S.D.). doi:10 1371/journal pcbi 1003746 g002 Figure 2. Transmembrane voltages comparison. Ionic binding modes and conduction mechanism during inward conduction At site SHFS, the probe ions (yellow) mainly distributed in an off-axis manner, the first coupling Na+ ions (blue) may occupy site SCEN (IN), and there existed a second binding site for coupling ions at site SEX (Figure 6A, II). Subsequently, the probe ions distributed in the middle of channel axis when traversing the short lived site SBAR, with the other two coupling ions populating sites SEX and SIN (CAV) respectively (Figure 6A, III). The probe ions then occupied site SCEN in both on-axis and off-axis manners, other coupling ions in the SF were distributed mainly at sites SIN and SEX. Only a few coupled ions occupied sites SHFS and SBAR (Figure 6A, IV). When probe ions faced the energy barrier at site SBAR, a delicate tightly-coupled ‘‘knock-off’’ conducting mechanism oc- curred. Initially, E53 started to flip and one of the carboxyl oxygens started to coordinate the probe ions (Figure 7A, III9 and S5, B). Compared to ions located in the close energy wells (Figure 7A, III), the probe ions were meanwhile expulsed by the outward movements of approaching coupling ions at site SIN (Figure 7A, III9). If this knock-off mechanism was successful, the probe ions would then migrate to site SHFS, as a result, the coupling ions would move outward to site SCEN simultaneous (Figure 7A, II9 and IV9). At this time, two carboxyl oxygens of the flipped E53 side chain tended to coordinate with the probe ions and coupling ions respectively (Figure 7A, II9 and IV9 and S5, B). Afterwards, ions left site SHFS promptly (t = 2.6 ns) into the periplasm via site SEX. In these ionic binding modes, under hyperpolarized potential, the coupling ions in the SF generally demonstrated a loosely coupled knock-on mechanism with only a few of them present in the adjacent binding sites to the probe ions (Figure 6A, II–IV). This is in agreement with a study by [21,22], where it was shown that during inward conduction the ions displayed a combination of mono-ionic and multi-ionic mechanism with an overall occupancy of 1.8 ions in the pore region. The flipping probability analysis indicates that the conforma- tional changes of the E53 side chains play a minor role for ion inward conduction as shown in Figure 6A, II9–IV9. Ionic binding modes and conduction mechanism during inward conduction A) Electrostatic potential across the simulation box calculated from the simulations without dihedral restraints on E53. B) Electrostatic potential across the simulation box calculated from the snapshots (t.5 ns) extracted from the simulation in Figure 2A without ion conduction events in neither inward nor outward directions. C) Electrostatic potential across the simulation box calculated from the ‘‘no salt’’ simulations. (Error estimations shown in the figure are S.D.). doi:10.1371/journal.pcbi.1003746.g002 July 2014 \| Volume 10 \| Issue 7 \| e1003746 July 2014 \| Volume 10 \| Issue 7 \| e1003746 PLOS Computational Biology \| www.ploscompbiol.org 3 E53 Dynamics Facilitate NaVMs Efflux Figure 3. Annotations of the SF ion interacting site. SF backbone atoms are shown as blue sticks, E53 and S54 side chain atoms are also shown to determine the site SHFS (only two opposite subunits are shown for clarity). In addition, sodium ions are depicted with yellow spheres. Sites SEX (2 5.00#Z,0.00 A˚), SHFS (0.00#Z,2.75 A˚), SBAR (2.75#Z,4.75 A˚), site SCEN (4.75#Z,7.75 A˚) and site SIN (7.75#Z,10.25 A˚) and the length of each site were annotated at the lateral sides of the figure. doi:10.1371/journal.pcbi.1003746.g003 Figure 3. Annotations of the SF ion interacting site. SF backbone atoms are shown as blue sticks, E53 and S54 side chain atoms are also shown to determine the site SHFS (only two opposite subunits are shown for clarity). In addition, sodium ions are depicted with yellow spheres. Sites SEX (2 5.00#Z,0.00 A˚), SHFS (0.00#Z,2.75 A˚), SBAR (2.75#Z,4.75 A˚), site SCEN (4.75#Z,7.75 A˚) and site SIN (7.75#Z,10.25 A˚) and the length of each site were annotated at the lateral sides of the figure. doi:10.1371/journal.pcbi.1003746.g003 were tightly coupled with ions at site SBAR (Figure 7A, III and V) corresponding to two energy wells in Figure 7B, III and V). When probe ions located at site SCEN, the coupling ions distributed in the upper part of the cavity (Figure 7A, IV) which corresponds to the energy well at site SCEN (Figure 7B, IV). Generally, the translocation of probe ions from the cytoplasm to site SBAR is readily stepwise by a tight knock-off mechanism without significant energy barriers. At all three energy wells (Figure 7B III–V) the E53 side chains maintained non-flipped conformations. regarding different conducting directionalities (Figure 6 and 7). During inward conduction, the largest barrier in the SF occurs between sites SHFS and SBAR which amounts to 2.1 kcal/mol (Figure 6B). Ionic binding modes and conduction mechanism during inward conduction If the attempt to overcome the barrier failed, the aforemen- tioned mechanism was easily reversed, the probe ions and coupling ions occupied the two stable energy wells at sites SBAR with the coupling ions at site SIN (Figure 7A, III and V) and site SCEN with the coupling ions in the cavity (Figure 7A, IV) again. That is the reason why ions stayed longer in sites SBAR and SCEN. One the one hand, larger Pi values (flip inducing probability of number of probe ions, see methods for details) values of sites SBAR, SCEN and SHFS indicated the flipped conformations of E53 were crucial (Pi.90%) in overcoming the dual energy barriers between SCEN, SBAR and SHFS. On the other hand, smaller Pt values July 2014 \| Volume 10 \| Issue 7 \| e1003746 Ionic binding modes and conduction mechanism during outward conduction A) Ion distribution of inward simulations labeled with respective interacting sites [4], lower left: 2-D (axial and radial distribution) density; lower right: 1-D axial distribution along the pore axis; upper left: 1-D radial distribution from the center of the pore axis. B) Inward dwell time in respective interacting sites (158 ion conduction events from four simulations were taken for analysis). C) Ion distribution of outward simulations labeled with respective interacting sites [4], lower left: 2-D (axial and radial distribution) density; lower right: 1-D axial distribution along the pore axis; upper left: 1-D radial distribution from the center of pore axis. D) Outward dwell time in respective interacting sites (79 ion conduction events from four simulations were taken for analysis). doi:10.1371/journal.pcbi.1003746.g004 (flipping time probability for all probe ions, see methods for details) values indicated that the flipping events were easily reversible. Because of these flipping events, the major ion distribution for outward conduction is limited to the center of the channel axis during translocation within the SF. channel. The outward conduction with ‘‘one-flip’’ simulation displayed an increased efflux rate compared to simulations without dihedral restraints on E53, where the flipping events would be reversible when conducting ions (Figures S1, S2, S3, S4). Interestingly, if E53 was restrained to a ‘‘non-flip’’ configuration, sodium ions translocation slowed down (Figure 8B). These results suggest a clear influence of filter dynamics on the efflux rate. Ionic binding modes and conduction mechanism during outward conduction Compared to inward permeation, the maximum energy barrier during outward conduction amounted to 2.3 kcal/mol between sites SBAR and SHFS. It is interesting that the free energy difference between sites SCEN and SHFS is 2.2 kcal/mol, which is close to the largest energy barrier (Figure 7B). In addition, the ionic binding modes demonstrate a distinct conduction mechanism compared to Na+ influx. Traversing outward from the cavity, ions at site SIN July 2014 \| Volume 10 \| Issue 7 \| e1003746 July 2014 \| Volume 10 \| Issue 7 \| e1003746 PLOS Computational Biology \| www.ploscompbiol.org 4 E53 Dynamics Facilitate NaVMs Efflux Figure 4. Ion distribution probability density map. A) Ion distribution of inward simulations labeled with respective interacting sites [4], lower left: 2-D (axial and radial distribution) density; lower right: 1-D axial distribution along the pore axis; upper left: 1-D radial distribution from the center of the pore axis. B) Inward dwell time in respective interacting sites (158 ion conduction events from four simulations were taken for analysis). C) Ion distribution of outward simulations labeled with respective interacting sites [4], lower left: 2-D (axial and radial distribution) density; lower right: 1-D axial distribution along the pore axis; upper left: 1-D radial distribution from the center of pore axis. D) Outward dwell time in respective interacting sites (79 ion conduction events from four simulations were taken for analysis). doi:10.1371/journal.pcbi.1003746.g004 Figure 4. Ion distribution probability density map. A) Ion distribution of inward simulations labeled with respective interacting sites [4], lower left: 2-D (axial and radial distribution) density; lower right: 1-D axial distribution along the pore axis; upper left: 1-D radial distribution from the center of the pore axis. B) Inward dwell time in respective interacting sites (158 ion conduction events from four simulations were taken for analysis). C) Ion distribution of outward simulations labeled with respective interacting sites [4], lower left: 2-D (axial and radial distribution) density; lower right: 1-D axial distribution along the pore axis; upper left: 1-D radial distribution from the center of pore axis. D) Outward dwell time in respective interacting sites (79 ion conduction events from four simulations were taken for analysis). doi:10.1371/journal.pcbi.1003746.g004 Figure 4. Ion distribution probability density map. E53 conformation determines efflux rate Comparison of the free energy profiles from outward simula- tions of these three types of configurations revealed that the largest energy barrier of the ‘‘non-flip’’ simulations is increased from 2.3 kcal/mol (non-restraint) to 3.4 kcal/mol (Figure 9A and C). The lowest energy well at site SBAR was also replaced by site SCEN. The energy profile of ‘‘one-flip’’ simulations indicated that the energy barrier between sites SCEN and SHFS was diminished, To further explore the correlation between flux directionality and E53 conformation, we performed two sets of inward and outward conduction simulations (four times 300 ns) with dihedral restraints to maintain ‘‘non-flip’’ and ‘‘one-flip’’ configurations during sampling. The influx rate was independent of the E53 conformations as shown in Figure 8A. This observation disagrees with recent data from Chakrabarti et al. [23] on the NaVAb July 2014 \| Volume 10 \| Issue 7 \| e1003746 PLOS Computational Biology \| www.ploscompbiol.org PLOS Computational Biology \| www.ploscompbiol.org 5 E53 Dynamics Facilitate NaVMs Efflux Figure 5. E53 conformational changes. A) x1-x2 angle distribution of the inward conductance simulations. B) x1-x2 angle distribution of the outward conductance simulations. C) The probability of flipping events of E53 side chains, the inward conduction is depicted as blue dotted histogram. The outward conduction is depicted as red dotted histogram (a flip is defined when at least one out of four subunits was shifted to flipping state in an analyzing window over time from four simulations); Control simulations with ions not in the SF (‘‘no salt’’) at hyperpolarized and depolarized membrane potentials are depicted as blue and red solid histograms, respectively. Error estimations shown in the figure are S.E.M. D) The distribution of E53 flipping numbers from all flipping events during outward conduction. doi:10.1371/journal.pcbi.1003746.g005 Figure 5. E53 conformational changes. A) x1-x2 angle distribution of the inward conductance simulations. B) x1-x2 angle distribution of the outward conductance simulations. C) The probability of flipping events of E53 side chains, the inward conduction is depicted as blue dotted histogram. The outward conduction is depicted as red dotted histogram (a flip is defined when at least one out of four subunits was shifted to flipping state in an analyzing window over time from four simulations); Control simulations with ions not in the SF (‘‘no salt’’) at hyperpolarized and depolarized membrane potentials are depicted as blue and red solid histograms, respectively. Error estimations shown in the figure are S.E.M. E53 conformation determines efflux rate D) The distribution of E53 flipping numbers from all flipping events during outward conduction. doi:10.1371/journal.pcbi.1003746.g005 although the original energy barrier increased slightly by 0.3 kcal/ mol (Figure 9A and B). Therefore, the outward conduction would be more straightforward without reversible backward translocation compared to the simulations without the dihedral restraints from a kinetic point of view. vestibule. Ions will then translocate via sites SHFS, SBAR, SCEN and SIN spontaneously to complete a conduction event [21–23]. Details of conduction are only partially understood. Large conformational changes of the glutamic acid side chains were described recently [23]. However, its role for conduction is still under discussion [24]. To gain further insights into these questions, we performed MD simulations to compare the different binding patterns and characterize the structural dynamics of glutamic acids during ion permeation. Double bilayer simulations with the open NaVMs structure enabled us to investigate influx and efflux separately. The calculated inward conductance rate is in good agreement with a previously reported experiment and computational data [22]. The July 2014 \| Volume 10 \| Issue 7 \| e1003746 PLOS Computational Biology \| www.ploscompbiol.org Discussion The mechanism of ion conduction and selectivity of bacterial voltage gated sodium channels is gradually emerging. The inverted tepee shape architecture of the SF lined with TLESW sequence enables sodium influx at the diffusion rate. The glutamic acid side chains are responsible for recruiting Na+ ions from the outer July 2014 \| Volume 10 \| Issue 7 \| e1003746 PLOS Computational Biology \| www.ploscompbiol.org 6 E53 Dynamics Facilitate NaVMs Efflux gure 6. Inward ionic binding modes and PMF. A) Overlay of the conformational space of the probe ions (yellow), coupling sodium ions ( d E53 carboxyl oxygen distributions (transparent spheres with different intensity) for different ionic binding modes at different interaction sit apshot of a typical E53 sidechain conformation is shown as stick representation (red, carboxyl oxygens; yellow, sidechain carbons), I–V are no ding modes and II9–IV9 are flipping binding modes for sites SHFS, SBAR and SCEN. Arrows indicate the direction of ion conduction; B) 1-D PM ward conduction in SF region, the largest free energy barrier is labeled by a terminal peak and well, corresponding positions of typical bin odes on the energy profile are labeled. Error estimations shown in the figure are S.E.M. :10.1371/journal.pcbi.1003746.g006 Figure 6. Inward ionic binding modes and PMF. A) Overlay of the conformational space of the probe ions (yellow), coupling sodium ions (blue) and E53 carboxyl oxygen distributions (transparent spheres with different intensity) for different ionic binding modes at different interaction sites. A snapshot of a typical E53 sidechain conformation is shown as stick representation (red, carboxyl oxygens; yellow, sidechain carbons), I–V are non-flip binding modes and II9–IV9 are flipping binding modes for sites SHFS, SBAR and SCEN. Arrows indicate the direction of ion conduction; B) 1-D PMF of inward conduction in SF region, the largest free energy barrier is labeled by a terminal peak and well, corresponding positions of typical binding modes on the energy profile are labeled. Error estimations shown in the figure are S.E.M. doi:10.1371/journal.pcbi.1003746.g006 July 2014 \| Volume 10 \| Issue 7 \| e1003746 PLOS Computational Biology \| www.ploscompbiol.org PLOS Computational Biology \| www.ploscompbiol.org 7 E53 Dynamics Facilitate NaVMs Efflux igure 7. Outward ionic binding modes and PMF. A) Overlay of the conformational space of the probe ions (yellow), coupling sodium io blue) and E53 carboxyl oxygen distributions (transparent spheres with different intensity) for different ionic binding modes at different interacti tes. July 2014 \| Volume 10 \| Issue 7 \| e1003746 Discussion The simulations without restraints of E53 are depicted as solid line, the ones with the ‘‘one-flip’’ restraints are shown as dotted line and the ones with the ‘‘non-flip’’ restraints are shown as dashed line. B) Average ion flux count through the SF over time of outward conduction (color: red). Error estimations shown in the figure are S.E.M. doi:10.1371/journal.pcbi.1003746.g008 binding modes [23]. Remarkably, detailed investigations of the structural dynamics of E53 in our study revealed distinct isomerization patterns between forward and backward transloca- tions respectively. When the ion moved into the SF from the outer vestibule under hyperpolarized membrane potential, the E53 remained mostly in the non-flipped conformation. In contrast, during outward conduction, the flipping occurrence increased significantly with a typical ‘‘one-flip’’ configuration (Figure 5C and D) when coordinating ions occupied the SF (Figure 7). In our simulations, the depolarized and hyperpolarized membrane potentials of approximately DV: 565 mV enabled the detailed investigation of ion permeation directionalities. This was not possible in previous simulations at ,0 mV [23]. The conductive, open gate structure used in this study may also reduce the repulsive effect which could have been induced by ions present in the cavity in previous simulations with a closed gate [23]. In addition, different forcefields used in these two studies may also play a substantial role for these discrepancies. As reported by Cordomi et al [29]), compared to the combination of OPLS-AA protein with Berger lipids parameters, combining Amber99sb protein and Berger lipids gives more accurate free energies of solvation in water and water to cyclohexane transfer with respect to experimental data for glutamic acid side chains. This may explain the reduced flexibility of the glutamic acid side chain dynamics observed in our study. Further, the force field discrepancies might explain the contrasting results for the ‘‘no salt’’ simulations in these two studies. In the study by Chacrabarti et al [23], the E side chains are more favorable to form flipped conformations even in the ‘‘no salt’’ conformation. This is in contrast to our simulations, where flipping events occurred rarely (Figure 5C) in the ‘‘no salt’’ simulations. estimated outward conductance rate obtained from MD simula- tions is predicted to be markedly lower than inward permeation (1563 pS vs 2763 pS, Figure 1). Ion translocation between sites SBAR and SHFS is substantially prolonged (8.260.9 ns vs 1.560.3 ns, Figure 4) during Na+ efflux. Discussion A snapshot of a typical E53 sidechain conformation is shown as stick representation (red, carboxyl oxygens; yellow, sidechain carbons), I–V a on-flip binding modes and II9–IV9 are flipping binding modes for sites SHFS, SBAR and SCEN. Arrows indicate the direction of ion conduction; B) 1 MF of outward conduction in SF region, the largest free energy barriers are labeled by terminal peaks and wells, corresponding positions of typi nding modes on the energy profile are labeled. Error estimations shown in the figure are S.E.M. oi:10.1371/journal.pcbi.1003746.g007 LOS Computational Biology \| www ploscompbiol org 8 July 2014 \| Volume 10 \| Issue 7 \| e10037 Figure 7. Outward ionic binding modes and PMF. A) Overlay of the conformational space of the probe ions (yellow), coupling sodium ions (blue) and E53 carboxyl oxygen distributions (transparent spheres with different intensity) for different ionic binding modes at different interaction sites. A snapshot of a typical E53 sidechain conformation is shown as stick representation (red, carboxyl oxygens; yellow, sidechain carbons), I–V are non-flip binding modes and II9–IV9 are flipping binding modes for sites SHFS, SBAR and SCEN. Arrows indicate the direction of ion conduction; B) 1-D PMF of outward conduction in SF region, the largest free energy barriers are labeled by terminal peaks and wells, corresponding positions of typical binding modes on the energy profile are labeled. Error estimations shown in the figure are S.E.M. doi:10.1371/journal.pcbi.1003746.g007 July 2014 \| Volume 10 \| Issue 7 \| e1003746 July 2014 \| Volume 10 \| Issue 7 \| e1003746 PLOS Computational Biology \| www.ploscompbiol.org 8 E53 Dynamics Facilitate NaVMs Efflux Figure 8. Influences of different flipping states at Dq = 4e (n = 4). A) Average ion flux counts through the SF over time of inward conduction (color: blue). The simulations without restraints of E53 are depicted as solid line, the ones with the ‘‘one-flip’’ restraints are shown as dotted line and the ones with the ‘‘non-flip’’ restraints are shown as dashed line. B) Average ion flux count through the SF over time of outward conduction (color: red). Error estimations shown in the figure are S.E.M. doi:10.1371/journal.pcbi.1003746.g008 Figure 8. Influences of different flipping states at Dq = 4e (n = 4). A) Average ion flux counts through the SF over time of inward conduction (color: blue). PLOS Computational Biology \| www.ploscompbiol.org July 2014 \| Volume 10 \| Issue 7 \| e1003746 Discussion From the energetic point of view, this would imply a potential barrier. This agrees with previous two-ion free energy calculation studies, revealing a higher energy barrier in this region for outward current compared to inward conduction (DG: 4.6 kcal/mol vs 0.4 kcal/mol, Stock et al. [21]; DG: 3.560.5 kcal/mol and 2.460.3 kcal/mol, Furini and Domene [15]). In our studies, this barrier is also higher for outward conduction (DG: 2.3 kcal/mol vs. 2.1 kcal/mol). In agreement with previous studies [21,22] during inward conduction, our simulations revealed that ion translocations in the SF generally involve a loosely coupled knock on mechanism with an average ion occupancy of 1.8 (Figure 6). A possible outward conduction mechanism was described by Stock et al., [21] using a ‘‘fully activated-open’’ NaVAb channel structure [28]. They have found a third ion denoted k, directly coupling with the probe ions triggering outward conduction by a ‘‘nudging’’ collision effect. Similar results were obtained in our study, which shows that the coupling ions directly couple with the probe ions by a tight ‘‘knock-off’’ mechanism. Moreover, our simulations further elucidated that this ‘‘knock-off’’ mechanism is highly dependent on the conformational isomerization of the glutamic acid side chains in the SF. In other words, to overcome the energy barriers of outward conduction, at least one of the glutamic acid side chains has to be flipped to an inward facing conformation (Figure 7). A recent simulation study under ,0 mV membrane potential with a closed gate NaVAb structure suggested that Na+ in- and outward movement involves variable configurations of multiple glutamic acid side chains giving rise to non-simple degenerated ion While the inward flow exhibited indistinguishable flux rates irrespective of the E53 conformation (Figure 8A), efflux displayed July 2014 \| Volume 10 \| Issue 7 \| e1003746 PLOS Computational Biology \| www.ploscompbiol.org PLOS Computational Biology \| www.ploscompbiol.org 9 E53 Dynamics Facilitate NaVMs Efflux Figure 9. PMF comparison for outward conduction. A) PMF of the simulations without restraints of E53 is depicted as solid line. (B) PMF of the simulations with ‘‘one-flip’’ restraints. C) PMF of the simulations with ‘‘non-flip’’ restraints. The largest free energy barriers in each figure are labeled by terminal peaks and wells. Error estimations shown in the figure are S.E.M. doi:10.1371/journal.pcbi.1003746.g009 Figure 9. PMF comparison for outward conduction. A) PMF of the simulations without restraints of E53 is depicted as solid line. (B) PMF of the simulations with ‘‘one-flip’’ restraints. Discussion C) PMF of the simulations with ‘‘non-flip’’ restraints. The largest free energy barriers in each figure are labeled by terminal peaks and wells. Error estimations shown in the figure are S.E.M. doi:10.1371/journal.pcbi.1003746.g009 different rates depending on the configurations of the E53 side chain. The highest efflux rate was observed in our ‘‘one-flip’’ simulations and the lowest rate with all four glutamic acid side chains restrained to an outward-facing conformation (Figure 8B). PMF calculations further confirmed that the energy barrier for outward conduction increased from 2.3 kcal/mol to 3.4 kcal/mol when the flipping conformation is prohibited (Figure 9C). That indicates that this flipping conformation provides direct coordina- tion for Na+ ions, which lowers the energy barrier and aids outward conduction. potential depolarizes, the probability of Na+ outward transitions increases. As a result, the inactivation probability of the channel is increased probably due to a series of conformational changes starting from the EEEE locus in the SF. A general limitation of current force fields is that the simulated linear current–voltage regime can only be achieved at higher membrane potentials compared to experimental conditions, resulting from the large electrostatic barriers in the transmem- brane region [31,32]. It should be noted that the computational electrophysiology simulations in this study were not done at constant membrane potentials (5656126 mV). This may result from the movement of the ions inside the channels and the fluctuation of the ions in the aqueous compartments (Figure 2A and B). However, a single ion permeation event under physiolog- ical conditions will also exist as a non-equilibrium process. Thus, to which extent, current simulation methods resemble ion channels’ electrophysiology needs to be further validated. In addition, inaccuracies in the interaction parameters (from the forcefield) between ions and surrounding atoms could also A simulation study published [30] after the submission of this manuscript, indicates that the SF dynamics, especially the side chain conformational changes of the EEEE locus in the SF, may lead to the conformational changes of the cavity lining helix on the ms timescale, subsequently initiating slow inactivation in NaV channels. We hypothesize that the E53 dynamics under depolar- izing potentials uncovered in this study provide further insights into slow inactivation, especially the fast slow inactivation for prokaryotic species during action potentials. PLOS Computational Biology \| www.ploscompbiol.org MD simulations D s u at o s The coordinates of NaVMs (PDB Entry: 4F4L; Resolution: 3.49 A˚ ) [7] with a conductive pore gate were used. The symmetric tetrameric structure consists of residues 8 to 94. All charged residues were treated keeping their charge states at physiological pH 7.4. In order to investigate ion conductance under two opposite membrane potentials, we used the computational electrophysiology method developed by Kutzner et al. [27] with a double-bilayer scheme. Each bilayer leaflet consists of 242 1- palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipids en- compassing the protein structure, solvated with 500 mM NaCl solution. The system was then duplicated in the Z direction (pore axis). The virtual-site model was adopted for hydrogen atoms [34]. MD simulations were performed with Gromacs version 4.5.5- dev [35,36]. Simulations were carried out with the AMBER99sb [37] all atom force field, POPC lipids parameter were taken from Berger et al. [29,38] with the TIP3P water model [39]. All covalent bonds were constrained using the LINCS algorithm [40], allowing for an integration time step of 4 fs with virtual sites. A 10 A˚ cutoff was adopted for calculating short-range electrostatic interactions and the Particle Mesh Ewald [41] summation was used for calculating long-range electrostatic interactions. The corrected monovalent ion Lennard-Jones parameters for the amber forcefield [42] were implemented in this study and the vdW interactions were calculated with a cutoff of 10 A˚ . The Nose- Hoover thermostat [43,44] and the semi-isotropic Parrinello- Rahman barostat algorithm [45] was used to maintain simulation temperature and pressure constantly at 300 K and 1 bar, respectively. The coordinates of NaVMs (PDB Entry: 4F4L; Resolution: 3.49 A˚ ) [7] with a conductive pore gate were used. The symmetric tetrameric structure consists of residues 8 to 94. All charged residues were treated keeping their charge states at physiological pH 7.4. In order to investigate ion conductance under two opposite membrane potentials, we used the computational electrophysiology method developed by Kutzner et al. [27] with a double-bilayer scheme. Each bilayer leaflet consists of 242 1- palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipids en- compassing the protein structure, solvated with 500 mM NaCl solution. The system was then duplicated in the Z direction (pore axis). The virtual-site model was adopted for hydrogen atoms [34]. E53 Dynamics Facilitate NaVMs Efflux E53 Dynamics Facilitate NaVMs Efflux two outer ones [27]. In our simulations, depolarized and hyperpolarized membrane potentials were calculated as DV = 5656126 mV (Figure 2). Harmonic restraints (1 kcal/mol/A˚ 2) were exerted on the a-carbon atoms of the TM helices (S5 and S6) throughout the simulations to maintain the open configuration in the absence of the voltage-sensing domain as suggested by Ulmschneider et al. [22]. Four times 500 ns MD simulations were performed; the first 100 ns were treated as equilibration. Simulation trajectories were saved every 100 ps; as a result, 4000 snapshots (analyzing windows) were recorded for analyzing data. Three repeated 500 ns simulations (400 ns were adopted for analysis) with only ions neutralizing the system and no ions in the SF (‘‘no salt’’) were performed as control to investigate the influence of the membrane potential on the E53 side chain dynamics. In this setup, only ions used to generate the charge imbalance and neutralize the net charge were kept. Further, four repeated 300 ns simulation (200 ns were adopted for analysis) for ‘‘non-flip’’ and ‘‘one-flip’’ configurations respectively were carried out, where the dihedral restraints were applied on the x2 dihedral of E53 for all four subunits with a force constant of 500 kcal/mol/ rad2. This allows dynamic ranges of 56610u for ‘‘one-flip’’ configuration and 288610u for ‘‘non-flip’’ configuration of the x2 angle. influence the conduction rates [33]. Thus, further structural and computational studies (including optimized strategies for ion interaction with surrounding atoms and polarizable force fields with different lipid species) will be required to further investigate the conformational changes of the SF under different electro- chemical drives and the influence of different protonation states of the EEEE locus. In addition, experimental validation is essential to further uncover the structural determinants and the importance of the protonation states of the EEEE locus on ion conductance and selectivity. Summarizing, our simulations, using applied membrane poten- tials, reveal different conduction mechanisms for ion inward and outward transitions respectively. An inward facing conformation (flip) of one glutamic acid side chain in the SF would reduce the energy barrier for ion outward transition by providing direct coordination with interacting Na+ ions. This local change can provide insights into the slow inactivation of NaV channels as suggested by Boiteux et al [30] during an action potential, when the membrane potential is depolarized. Potential of mean force (PMF) The 1-D potential of mean force profile of the ions under membrane potentials were calculated by taking the logarithm of the Na+ probability distribution along the channel axis (z) in the SF region, according to G(z) = 2kBT ln [p(Ri)], where kB is the Boltzmann constant, T is the temperature, and p(Ri) is the probability distribution of the probe ions. 100 bins were used to achieve a bin width of 0.15 A˚ depicting the details of the profile. Error bars are S.E.M. from four different simulations. Prior to MD simulations, 3000 conjugate gradient energy- minimization steps were performed, followed by 5 ns equilibration in order to fully solvate mobile water and lipids around the restrained protein with a force constant of 1000 kJ/mol/nm2 on all heavy atoms. Hereafter, an equal number of Na+ ions and a net difference of 4 e of Cl2 across each lipid bilayer between the central electrolyte bath and the two outer ones were sustained during the simulation by a swapping mechanism [27]. In this scheme, a new form of Poisson equation [46] was adopted to derive the potential profile as a function of system length (z). The well-defined transmembrane voltage across each lipid patch was directly assessed by twice integration of this sustained charge density differences between the central electrolyte bath and the Ionic binding modes The total number of ions (i) which completed their conduction in the SF were analyzed (i = 158, from four inward simulations; and i = 79 from four outward simulations). All snapshots of the probe ions (yellow) and coupling ions in the SF (blue) were rendered for five different interaction sites (SEX, SHFS, SBAR, SCEN and SIN). For sites SHFS, SBAR and SCEN, the side chain isomerization states were separated into flipped and non-flipped categories and analyzed. For flipped ones, all four protein chains and the relative ion positions were aligned to chain A to achieve a better representation of the ionic binding patterns. Two proba- bility parameters Pi and Pt were calculated to characterize the influence of E53 dynamics on ion conduction for these three sites. Pi = (Fi/i)100%, where Fi denotes the number of ions which generated at least one E53 flipping event during their permeation through each site. Pt = Ft/Tt100%, where Ft denotes the number of snapshots where E53 flipped when the probe ions traversed each site and Tt denotes the total number of snapshots when the probe ions traversed each site. ) p y g [ ] MD simulations were performed with Gromacs version 4.5.5- dev [35,36]. Simulations were carried out with the AMBER99sb [37] all atom force field, POPC lipids parameter were taken from Berger et al. [29,38] with the TIP3P water model [39]. All covalent bonds were constrained using the LINCS algorithm [40], allowing for an integration time step of 4 fs with virtual sites. A 10 A˚ cutoff was adopted for calculating short-range electrostatic interactions and the Particle Mesh Ewald [41] summation was used for calculating long-range electrostatic interactions. The corrected monovalent ion Lennard-Jones parameters for the amber forcefield [42] were implemented in this study and the vdW interactions were calculated with a cutoff of 10 A˚ . The Nose- Hoover thermostat [43,44] and the semi-isotropic Parrinello- Rahman barostat algorithm [45] was used to maintain simulation temperature and pressure constantly at 300 K and 1 bar, respectively. Discussion When the membrane PLOS Computational Biology \| www.ploscompbiol.org July 2014 \| Volume 10 \| Issue 7 \| e1003746 July 2014 \| Volume 10 \| Issue 7 \| e1003746 10 PLOS Computational Biology \| www.ploscompbiol.org References 1. 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Eisenman G, Horn R (1983) Ionic selectivity revisited: The role of kinetic and equilibrium processes in ion permeation through channels. J Membr Biol 76: 197–225. doi:10.1007/BF01870364. 26. Cuello LG, Jogini V, Cortes DM, Perozo E (2010) Structural mechanism of C- type inactivation in K+ channels. Nature 466: 203–208. 13. Carnevale V, Treptow W, Klein ML (2011) Sodium Ion Binding Sites and Hydration in the Lumen of a Bacterial Ion Channel from Molecular Dynamics Simulations. J Phys Chem Lett 2: 2504–2508. doi:10.1021/jz2011379. 27. Kutzner C, Grubmu¨ller H, de Groot BL, Zachariae U (2011) Computational Electrophysiology: The Molecular Dynamics of Ion Channel Permeation and Selectivity in Atomistic Detail. Biophys J 101: 809–817. 14. (TIFF) Figure S4 Cumulative ion flux counts with flip counts as a function of time from simulation 4 without dihedral restrains of E53. A) & C) Ion flux counts through the SF and flipping counts of E53 over 400 ns trajectory of inward simulation (color: blue). B) & D) Ion flux counts through the SF and flipping counts of E53 in over 400 ns trajectory of outward simulation (color: red). Author Contributions Figure S5 Hydration shell of the simulations without dihedral restrains of E53. (A) Oxygen coordination numbers in the first hydration shell for inward sodium conduction (Oxygen atoms closer than 3.0 A˚ to Na+ were considered coordinating Conceived and designed the experiments: SK ASW. Performed the experiments: SK. Analyzed the data: SK ENT ASW. Contributed reagents/materials/analysis tools: ENT. Wrote the paper: SK ASW. Conceived and designed the experiments: SK ASW. Performed the experiments: SK. Analyzed the data: SK ENT ASW. Contributed reagents/materials/analysis tools: ENT. Wrote the paper: SK ASW. E53 Dynamics Facilitate NaVMs Efflux E53 Dynamics Facilitate NaVMs Efflux atoms): the total coordination number is depicted in grey; water oxygens are depicted as blue lines. Protein backbone oxygens are shown in green. E53 side chain oxygens are colored red. B) Coordination oxygen atoms numbers for outward sodium conduction. (TIFF) Figure S2 Cumulative ion flux counts with flip counts as a function of time from simulation 2 without dihedral restrains of E53. A) & C) Ion flux counts through the SF and flipping counts of E53 over 400 ns trajectory of inward simulation (color: blue). B) & D) Ion flux counts through the SF and flipping counts of E53 in over 400 ns trajectory of outward simulation (color: red). (TIFF) Movie S1 The movie shows the double bilayer simula- tion scheme (400 ns). (MP4) Supporting Information Figure S1 Cumulative ion flux counts with flip counts as a function of time from simulation 1 without dihedral restrains of E53. A) & C) Ion flux counts through the SF and flipping counts of E53 over 400 ns trajectory of inward simulation (color: blue). B) & D) Ion flux counts through the SF and flipping counts of E53 in over 400 ns trajectory of outward simulation (color: red). July 2014 \| Volume 10 \| Issue 7 \| e1003746 PLOS Computational Biology \| www.ploscompbiol.org 11 Acknowledgments We thank Tobias Linder and Eva-Maria Zangerl for helpful discussions and critical reading of the manuscript. The computational results presented have been achieved using the Vienna Scientific Cluster (VSC). We thank Tobias Linder and Eva-Maria Zangerl for helpful discussions and critical reading of the manuscript. The computational results presented have been achieved using the Vienna Scientific Cluster (VSC). Movie S1 The movie shows the double bilayer simula- tion scheme (400 ns). (MP4) Figure S3 Cumulative ion flux counts with flip counts as a function of time from simulation 3 without dihedral restrains of E53. A) & C) Ion flux counts through the SF and flipping counts of E53 over 400 ns trajectory of inward simulation (color: blue). B) & D) Ion flux counts through the SF and flipping counts of E53 in over 400 ns trajectory of outward simulation (color: red). (TIFF) Movie S2 A ‘‘flipped’’ trajectory clip showing one example conduction event from an outward simulation. Two opposite subunits are shown as cyan sticks for clarity. The E53 side chains are highlighted in yellow. Sodium ions are shown in yellow, except for Na+ ions in the SF, which are colored in different colors. The water molecules within 3 A˚ of these ions are represented as sticks to depict the hydration shell. (MP4) E53 Dynamics Facilitate NaVMs Efflux 38. Berger O, Edholm O, Jahnig F (1997) Molecular dynamics simulations of a fluid bilayer of dipalmitoylphosphatidylcholine at full hydration, constant pressure, and constant temperature. Biophys J 72: 2002–2013. 29. Cordomı´ A, Caltabiano G, Pardo L (2012) Membrane Protein Simulations Using AMBER Force Field and Berger Lipid Parameters. J Chem Theory Comput 8: 948–958. doi:10.1021/ct200491c. p p y J 39. Jorgensen WL, Chandrasekhar J, Madura JD, Impey RW, Klein ML (1983) Comparison of simple potential functions for simulating liquid water. J Chem Phys 79: 926–935. doi:10.1063/1.445869. p 30. Boiteux C, Vorobyov I, Allen TW (2014) Ion conduction and conformational flexibility of a bacterial voltage-gated sodium channel. Proc Natl Acad Sci 111: 3454–3459. doi:10.1073/pnas.1320907111. p 31. Bockmann RA, Hac A, Heimburg T, Grubmuller H (2003) Effect of Sodium Chloride on a Lipid Bilayer. Biophys J 85: 1647–1655. 40. Hess B, Bekker H, Berendsen HJC, Fraaije JGEM (1997) LINCS: A linear constraint solver for molecular simulations. J Comput Chem 18: 1463–1472. doi:10.1002/(SICI)1096-987X(199709)18:12,1463::AID-JCC4.3.0.CO;2-H. 32. Jensen MØ, Jogini V, Eastwood MP, Shaw DE (2013) Atomic-level simulation of current–voltage relationships in single-file ion channels. J Gen Physiol 141: 619–632. doi:10.1085/jgp.201210820. ( ) ( ) J ; 41. Darden T, York D, Pedersen L (1993) Particle mesh Ewald: An N log (N) method for Ewald sums in large systems. J Chem Phys 98: 10089–10089. 33. Luo Y, Roux B (2010) Simulation of Osmotic Pressure in Concentrated Aqueous Salt Solutions. J Phys Chem Lett 1: 183–189. doi:10.1021/jz900079w. 42. Joung IS, Cheatham III TE (2008) Determination of alkali and halide monovalent ion parameters for use in explicitly solvated biomolecular simulations. J Phys Chem B 112: 9020–9041. 34. Feenstra KA, Hess B, Berendsen HJC (1999) Improving efficiency of large time- scale molecular dynamics simulations of hydrogen-rich systems. J Comput Chem 20: 786–798. doi:10.1002/(SICI)1096-987X(199906)20:8,786::AID- JCC5.3.0.CO;2-B. 43. Nose S, Nose S (1984) A unified formulation of the constant temperature molecular-dynamics methods. J Chem Phys 81: 511–519. doi:10.1063/ 1.447334. 44. Hoover, William G., Hoover WG (1985) Canonical dynamics: Equilibrium phase-space distributions. Phys Rev A 31: 1695–1697. doi:10.1103/Phys- RevA.31.1695. 35. Hess B, Kutzner C, Van Der Spoel D, Lindahl E (2008) GROMACS 4: Algorithms for highly efficient, load-balanced, and scalable molecular simula- tion. J Chem Theory Comput 4: 435–447. J y p 36. Van Der Spoel D, Lindahl E, Hess B, Groenhof G, Mark AE, et al. (2005) GROMACS: fast, flexible, and free. J Comput Chem 26: 1701. 45. References Corry B, Thomas M (2011) Mechanism of Ion Permeation and Selectivity in a Voltage Gated Sodium Channel. J Am Chem Soc 134: 1840–1846. doi:10.1021/ja210020h. 28. Amaral C, Carnevale V, Klein ML, Treptow W (2012) Exploring conforma- tional states of the bacterial voltage-gated sodium channel NavAb via molecular dynamics simulations. Proc Natl Acad Sci 109: 21336–21341. doi:10.1073/ pnas.1218087109. j 15. Furini S, Domene C (2012) On Conduction in a Bacterial Sodium Channel. PLoS Comput Biol 8: e1002476. doi:10.1371/journal.pcbi.1002476. July 2014 \| Volume 10 \| Issue 7 \| e1003746 July 2014 \| Volume 10 \| Issue 7 \| e1003746 July 2014 \| Volume 10 \| Issue 7 \| e1003746 12 PLOS Computational Biology \| www.ploscompbiol.org E53 Dynamics Facilitate NaVMs Efflux Martonˇa´k R, Laio A, Parrinello M (2003) Predicting crystal structures: The Parrinello-Rahman method revisited. Phys Rev Lett 90: 75503. J y p . Van Der Spoel D, Lindahl E, Hess B, Groenhof G, Mark AE 46. Sachs JN, Crozier PS, Woolf TB (2004) Atomistic simulations of biologically realistic transmembrane potential gradients. J Chem Phys 121: 10847–10851. doi:10.1063/1.1826056. 37. Hornak V, Abel R, Okur A, Strockbine B, Roitberg A, et al. (2006) Comparison of multiple Amber force fields and development of improved protein backbone parameters. Proteins 65: 712–725. doi:10.1002/prot.21123. PLOS Computational Biology \| www.ploscompbiol.org July 2014 \| Volume 10 \| Issue 7 \| e1003746 PLOS Computational Biology \| www.ploscompbiol.org 13
https://openalex.org/W2120225004	https://pubs.asahq.org/anesthesiology/article-pdf/116/2/415/256822/0000542-201202000-00027.pdf	English	null	Effects of Regional and Whole-body Hypothermic Treatment before and after Median Nerve Injury on Neuropathic Pain and Glial Activation in Rat Cuneate Nucleus	Anesthesiology	2,012	cc-by	16,481	* Associate Professor, School of Medicine, College of Medicine, Fu Jen Catholic University, Taipei, Taiwan. † Lecturer, Department of In- ternal Medicine and Traumatology, National Taiwan University Hospi- tal, Taipei, Taiwan. ‡ Associate Professor, Division of Allergy and Im- munology, Department of Internal Medicine, Cathay General Hospital, Taipei, Taiwan. § Associate Professor, Department of Neurology, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan. What This Article Tells Us That Is New Methods: Sprague-Dawley rats (n 246) that underwent median nerve ligature at the elbow received various degrees of regional and whole-body hypothermia 15 min before CCI and 5 h, 1, 3, and 5 days after CCI. Hypothermia was main- tained for 4 h. Seven days after CCI, behavioral and electro- physiological testings were conducted. Immunohistochemis- try, immunoblotting, and enzyme-linked immunosorbent assay were used for qualitative and quantitative analysis of glial activation and measuring pro-inflammatory cytokines, respectively. • In rats, regional and whole body hypothermia at the time of nerve injury reduced behavioral signs of neuropathic hyper- sensitivity as well as concomitant glial activation in the cuneate nucleus • In rats, regional and whole body hypothermia at the time of nerve injury reduced behavioral signs of neuropathic hyper- sensitivity as well as concomitant glial activation in the cuneate nucleus • These data suggest that regional or whole body hypothermia during surgery might reduce the incidence of chronic pain hypothermia preinjury and 5 h postinjury provided a similar therapeutic effect. However, whole-body hypothermia, but not regional hypothermia, applied 1, 3, and 5 days postinjury attenuated glial activation and neuropathic pain. Similarly, on days 1, 3, and 5 postinjury, only whole-body hypothermia was effective in decreasing proinflammatory cytokine levels. The increase in injury discharge observed after CCI could be suppressed by regional or whole-body hypothermia at differ- ent stages of nerve injury. p Results: Mild (32°C) and deep (28°C) regional hypothermia administered preinjury and 5 h postinjury attenuated neuro- pathic pain and glial activation. Application of whole-body Conclusions: Attheearlystagefollowingnerveinjury,regional and whole-body hypothermia suppresses ectopic discharges, and consequently inhibits glial activation and neuropathic pain. At the later stage, pain processing is mediated mainly by cyto- kines released from activated microglia; therefore, only whole- body hypothermia is effective in modulating pain. Received from the School of Medicine, College of Medicine, Fu Jen Catholic University, Taipei, Taiwan. Submitted for publication May 31, 2011. Accepted for publication October 26, 2011. Supported by grant nos. NSC 96–2320-B-030–007-MY2 and NSC 98–2320-B- 030–002-MY3 from the National Science Council, Taipei, Taiwan. N bl N EUROPATHIC pain afflicts millions of people worldwide and is one of the most significant health problems.1,2 Neuropathic pain may be associated with pe- ripheral nerve injury, inflammation, or infection.3–5 Periph- Address correspondence to Dr. Tsai: School of Medicine, College of Medicine, Fu Jen Catholic University, # 510 Chung- Cheng Road, Hsin-Chuang, Taipei, Taiwan 24205, R.O.C. med0056@mail.fju.edu.tw. PAIN MEDICINE PAIN MEDICINE Copyright © 2012, the American Society of Anesthesiologists, Inc. Lippincott Williams & Wilkins. Anesthesiology 2012; 116:415–31 Effects of Regional and Whole-body Hypothermic Treatment before and after Median Nerve Injury on Neuropathic Pain and Glial Activation in Rat Cuneate Nucleus Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/116/2/415/256822/0000542-201202000-00027 Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/116/2/415/256822/0000542-201202000-00027.pdf by guest on 24 October 2024 Yi-Ju Tsai, Ph.D.,* Chun-Ta Huang, M.D.,† Shih-Chang Lin, M.D., Ph.D.,‡ Jiann-Horng Yeh, M.D.,§ Yi-Ju Tsai, Ph.D.,* Chun-Ta Huang, M.D.,† Shih-Chang Lin, M.D., Ph.D.,‡ Jiann-H What We Already Know about This Topic • Hypothermia reduces neural degeneration after peripheral nerve injury, but its effect on nerve injury-induced neuropathic pain has not been examined • Hypothermia reduces neural degeneration after peripheral nerve injury, but its effect on nerve injury-induced neuropathic pain has not been examined Background: Neuroprotective effects of hypothermia on pe- ripheral nerve injury remain uncertain. This study investi- gated the efficacy of hypothermia in attenuating neuropathic pain and glial activation in the cuneate nucleus in a median nerve chronic constriction injury (CCI) model. ABSTRACT pubs.asahq.org/anesthesiology/article-pdf/116/2/415/256822/0000542-201202000-00027.pdf by guest on 24 October 2024 What This Article Tells Us That Is New Information on purchasing reprints may be found at www.anesthesiology.org or on the masthead page at the beginning of this issue. ANESTHESIOLOGY’s articles are made freely accessible to all readers, for personal use only, 6 months from the cover date of the issue. This article is accompanied by an Editorial View. Please see: Todd MM: Another intriguing application of hypothermia: And some words of caution. ANESTHESIOLOGY 2012; 116:246–7. This article is accompanied by an Editorial View. Please see: Todd MM: Another intriguing application of hypothermia: And some words of caution. ANESTHESIOLOGY 2012; 116:246–7. 415 Received from the School of Medicine, College of Medicine, Fu Jen Catholic University, Taipei, Taiwan. Submitted for publication May 31, 2011. Accepted for publication October 26, 2011. Supported by grant nos. NSC 96–2320-B-030–007-MY2 and NSC 98–2320-B- 030–002-MY3 from the National Science Council, Taipei, Taiwan. This article is accompanied by an Editorial View. Please see: Todd MM: Another intriguing application of hypothermia: And some words of caution. ANESTHESIOLOGY 2012; 116:246–7. Animal Preparation These experiments were reviewed and approved by the Na- tional Science Council Committee as well as the Animal Center Committee at the College of Medicine of Fu Jen Catholic University, Taipei, Taiwan. The ethical guidelines of the International Association for the Study of Pain23 for animal experimentation were followed. All efforts were made to minimize animal suffering and reduce the number of ex- perimental animals used. Animals used in this study were male Sprague-Dawley rats weighing 180–250 g, which were housed under approved conditions with a 12-h light-dark cycle and food and water available ad libitum. Anesthesiology, V 116 • No 2 February 2012 Effect of Hypothermia on Neuropathic Pain Hypothermia Procedure R i l H th i U Regional Hypothermia. Under general anesthesia, a temper- ature therapy pad (Model TP3E; Gaymar Industries, Inc., Orchard Park, NY) was wrapped around the right forelimb to manipulate the temperature of the limb. The tubing of the temperature therapy pad was connected to a water-cooling circulator (Model CL-300R; TAITEC Co., Tokyo, Japan). The limb temperature was monitored by a digital electronic thermometer (Model TH-8; Physitemp Instruments, Inc., Clifton, NJ) with a 0.15-mm diameter temperature probe (Model ICT-4; Physitemp Instruments, Inc.) attached to the surface of the pronator teres muscle. The temperature of the right forelimb was maintained at 37°C (normothermia), 32°C (mild hypothermia), or 28°C (deep hypothermia). Deep rectal temperature, measured by inserting a rectal probe (Model RET-2; Physitemp Instruments, Inc.) 7 cm into the rectum, was also monitored, and was maintained at 37°C using a servo-controlled infrared lamp. The target tem- perature was achieved within 15 min. The experimental animals (n 123) were divided into six groups according to the timing of induction of regional hy- pothermia. In the first group (preinjury group), the temper- ature of the right forelimb was maintained at 37°C (n 7), 32°C (n 7), or 28°C (n 7) 15 min before ligation of the median nerve. In the second group (5 h postinjury group), the temperature of the right forelimb was maintained at 37°C (n 7), 32°C (n 7), or 28°C (n 7) 5 h after In the present study, we aimed to evaluate the efficacy of hypothermia in relieving neuropathic pain and attenuating glial activation responses in a median nerve CCI model of neuropathic pain. We manipulated the degree of hypother- mia (28°C or 32°C), initiation time of hypothermia (prein- jury; 5 h, 1, 3, and 5 days postinjury), and the location of hypothermia application (regional or whole-body). Materials and Methods Animal Preparation Materials and Methods eral nerve injury evokes a barrage of injury discharges origi- nating from the injured nerve fibers and their dorsal root ganglia; subsequently, these ectopic discharges facilitate the release of excitatory amino acids from damaged primary af- ferents, which cause central sensitization and contribute to the development of neuropathic pain.6–8 Neuropathic pain manifests as spontaneous pain, allodynia (pain evoked by a normally innocuous stimulus), or hyperalgesia (enhanced pain evoked by a noxious stimulus). Tactile allodynia in par- ticular is a cardinal symptom of neuropathic pain.3 Neuro- pathic pain can last for months or even years after the pri- mary tissue damage has healed. Current treatments for this type of pain are often of limited efficacy and are beneficial in only a few patients.3,9 Therefore, developing effective treat- ments for neuropathic pain is an urgent matter. Nerve Injury Surgery Th l d Recently, hypothermia has been established as an effective neuroprotective treatment for peripheral nerve injury in- duced by multiple arterial ligatures. Several mechanisms of hypothermic neuroprotection have been proposed, includ- ing suppression of the inflammatory response,10 reduction of fiber degeneration and demyelination,11–13 and ameliora- tion of motor dysfunction.14 Electrophysiological studies have demonstrated that peripheral nerve ischemia-in- duced sensory nerve dysfunction can be improved by hy- pothermic treatment.11,12 However, the effects of hypo- thermic treatment on preventing neuropathic pain have not yet been investigated. The experimental model of median nerve CCI was based on the methods described by Bennett and Xie.6 Briefly, anesthe- sia was induced by an intraperitoneal injection of 7% chloral hydrate (0.45 ml/100 g body weight). Under a dissecting microscope, the right median nerve was separated from the surrounding tissue at the elbow level, immediately proximal to the entry between two heads of the pronator teres muscle. Four loose ligatures (4.0 chromic gut) were made around the nerve,19–22 and the incision was then sutured. For the sham surgeries, the median nerve was exposed in the same area of the right forelimb without ligation. Accumulating evidence suggests that following peripheral nerve injury, glia are involved in a cascade of events leading to the development of neuropathic pain.15,16 Further, the ma- jority of human peripheral nerve injuries occur in the upper limbs, and median nerve injury is a common form of periph- eral neuropathy caused by laceration, fracture-associated stretch and contusion, compression, and injection inju- ries.17,18 Accordingly, we have developed an experimental model of chronic constriction injury (CCI) on the median nerve based on the methods described by Bennett and Xie6 to induce neuropathic pain in the forelimbs of rats.19 –22 In this model, activation of microglia and astrocytes was observed in the cuneate nucleus (CN) after CCI of the median nerve.19,21 Furthermore, inhibition of microglial activation by minocycline prevented the development of neuropathic pain.21 Microinjection of the astrocytic toxin fluorocitrate also reversed nerve injury-induced mechani- cal allodynia.19 These data support an important role for microglia and astrocytes in the development of neuro- pathic pain. Behavioral Testing Mechanical Allodynia Behavioral Testing Mechanical Allodynia. Rats were placed in individual plexi- glass chambers (25 40 18 cm) with wire mesh bottoms and were allowed to acclimatize to the environment for 30 min. The mechanical withdrawal threshold of the rat fore- paws was determined using a series of von Frey filaments (Semmes-Weinstein Monofilaments, North Coast Medical, Inc., Gilroy, CA; bending force: 0.16, 0.4, 0.6, 1.0, 1.4, 2.0, 4.0, 6.0, 8.0, 10.0, 15.0, and 26.0 g). The measurement was performed according to the method described by Tal and Bennett.24 Quantitative mechanical stimuli were applied to the medial plantar surface of each forepaw in an ascending order to evaluate the withdrawal threshold. Each von Frey filament was applied five times. When rats showed at least two withdrawal responses to a filament, the bending force of the filament was defined as the withdrawal threshold. Whole-body Hypothermia. Under general anesthesia, the temperature therapy pad (Model TP22C; Gaymar Indus- tries, Inc.) was wrapped around the whole body of the ani- mal, and the tubing of the temperature therapy pad was connected to a water-cooling circulator. Rectal temperature was monitored using a digital electronic thermometer with a rectal probe inserted into the rectum. The rat’s rectal tem- perature was maintained at 37°C, 32°C, or 28°C. Thermal Hyperalgesia. To measure the thermal withdrawal latency of the forepaws, the plantar test (Ugo Basile, Com- erio, Italy) was utilized. Briefly, rats were individually placed in one of the three plexiglass containers (22 17 14 cm) located on the increased floor of a clear glass plate (3 mm thick) and allowed to habituate to the apparatus for 30 min. A radiant heat source was positioned under the glass plate directly beneath the plantar surface of the forepaw. The with- drawal latency was automatically measured as the time elapsed from the onset of radiant heat stimulation to the withdrawal of the forepaw. In order to avoid tissue damage, the maximum thermal stimulus duration was 20 s. A more detailed description of the apparatus has been published pre- viously.25 Each forepaw was alternately tested five times with a minimal interval of 10 min between measurements, and readings were recorded to the nearest 0.1 s. Five latency values per side were averaged. Animals were tested at least 5 h after von Frey filament testing. Behavioral Testing Mechanical Allodynia p 37 , 3 , The experimental animals (n 123) were divided into six groups according to the timing of induction of whole-body hypothermia. In the first group (preinjury group), the whole- body temperature was maintained at 37°C (n 7), 32°C (n 7), or 28°C (n 7) 15 min before ligation of the median nerve. In the second group (5 h postinjury group), the whole-body temperature was maintained at 37°C (n 7), 32°C (n 7), or 28°C (n 7) 5 h after median nerve injury. In the third group (1 day postinjury group), the whole-body temperature was maintained at 37°C (n 7), 32°C (n 7), or 28°C (n 7) 1 day after median nerve injury. In the fourth group (3 days postinjury group), the whole-body temperature was maintained at 37°C (n 7), 32°C (n 7), or 28°C (n 7) 3 days after median nerve injury. In the fifth group (5 days postinjury group), the whole-body temperature was maintained at 37°C (n 7), 32°C (n 7), or 28°C (n 7) 5 days after median nerve injury. In the sixth group (sham-operated group), the whole- body temperature was maintained at 37°C (n 6), 32°C (n 6), or 28°C (n 6) 1 day after sham operation. The whole-body temperature was maintained at the target tem- perature for 4 h in all rats. All studies were performed in a randomized, blinded manner to avoid expectation bias. In brief, all animals were selected in a random order for behavioral testing, and an- other investigator, who was blinded to the experimental sta- tus of the rats, made the observations. Electrophysiology T h f h Two hours after the end of behavioral testing, all animals were reanesthetized, and the right median nerve was carefully reexposed and dissected free. The nerve was placed on a pair of platinum hook electrodes just proximal to the site of nerve ligation or at the same site in sham-operated rats to record discharge activity of the nerve. Paraffin oil was applied around the exposed segment of the nerve to prevent drying. The electrodes were connected to the Xction View II Data Acquisition System (Model XD-04 II; Singa, Taoyuan, Tai- wan), and the discharge signals were amplified 2000-fold, After regional or whole-body hypothermic treatment, rats were returned to their home cages, and the limb or body temperature was allowed to rise back to normal naturally. The animals survived for 7 days after median nerve injury, and then the behavioral testing and electrophysiological studies were conducted on these animals. Afterward, the rats were randomly assigned to one of the two experiments. In one experiment, the animals were processed for immunohis- tochemistry, and in the other experiment, the animals were Anesthesiology 2012; 116:415–31 416 Tsai et al. PAIN MEDICINE median nerve injury. In the third group (1 day postinjury group), the temperature of the right forelimb was maintained at 37°C (n 7), 32°C (n 7), or 28°C (n 7) 1 day after median nerve injury. In the fourth group (3 days postinjury group), the temperature of the right forelimb was maintained at 37°C (n 7), 32°C (n 7), or 28°C (n 7) 3 days after median nerve injury. In the fifth group (5 days postinjury group), the temperature of the right forelimb was maintained at 37°C (n 7), 32°C (n 7), or 28°C (n 7) 5 days after median nerve injury. In the sixth group (sham-operated group), the temperature of the right forelimb was maintained at 37°C (n 6), 32°C (n 6), or 28°C (n 6) 1 day after the sham operation (median nerve exposure without liga- tion). The temperature of the right forelimb was maintained at the target temperature for 4 h in all rats. processed for Western blotting and enzyme-linked immu- nosorbent assay. Immunohistochemistry The rats were deeply anesthetized and perfused with 4% paraformaldehyde per 0.1 M phosphate buffer, pH 7.4. Me- dulla tissue blocks that contained the CN were resected and stored overnight in phosphate buffer with 30% sucrose, and 30-m slices were transversely cut with a cryostat (Leica, Nussloch, Germany). Anesthesiology 2012; 116:415–31 417 Tsai et al. Effect of Hypothermia on Neuropathic Pain recorded for 10 min, and analyzed using Xction View II software by an investigator blinded to the treatment group. Peroxidase activity was detected by incubating the mem- brane with the enhanced chemiluminescence Western blot- ting detection reagents (GE Healthcare) and exposing it to hyperfilm (GE Healthcare). The blots were then incubated in stripping buffer (67.5 mM Tris, pH 6.8, 2% SDS, and 0.7% -mercaptoethanol) and reprobed with mouse anti-- actin monoclonal antibody (1:5,000; Sigma-Aldrich) as loading controls. Enzyme-linked Immunosorbent Assay y The immunohistochemical procedure was performed as described previously.19,21 In brief, floating medulla sections were collected and treated with 1% H2O2, blocked with 2% normal goat serum (GibcoBRL, Gel Company, San Fran- cisco, CA), and incubated in either mouse monoclonal anti- OX-42 (CD11b, 1:200; Serotec, Indianapolis, IN) or mouse monoclonal anti-glial fibrillary acidic protein (GFAP) (1: 800; Sigma-Aldrich, St. Louis, MO) antibody for 48 h at 4°C. After rinsing with phosphate-buffered saline, the sec- tions were incubated in 1:200 biotinylated antimouse IgG (Vector, Burlingame, CA), and processed with avidin-bio- tin-horseradish peroxidase complex (Vector). Peroxidase ac- tivity was subsequently visualized by treating with Vector SG Substrate Kit. Finally, the floating sections were mounted onto gelatinized slides and examined under a light microscope (Zeiss Axiophot, Jena, Germany). For negative controls, in which immunolabeling was not present, sections were incubated in normal mouse serum in lieu of the above- mentioned primary antibodies, or with the omission of the same antibodies. The concentration of proinflammatory cytokines in the CN was determined by the enzyme-linked immunosorbent assay. The sample was prepared as described above. The superna- tant was collected and the levels of tumor necrosis factor- (TNF-) and interleukin-1 (IL-1) were quantified using TNF- Immunoassay Kit (R&D Systems, Inc., Minneapo- lis, MN) and IL-1 Immunoassay Kit (R&D Systems, Inc.), respectively. All procedures were performed following manu- facturer’s instructions; microplates were read using a microplate reader (Molecular Devices Corporation, Sunnyvale, CA). Sample Preparation and Western Blot Analysis After rats were sacrificed by decapitation, the CN was micro- dissected according to the method described by Palkovits and Brownstein.26 The punched tissue was homogenized in 100 l lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid, 1% NP-40, 0.5% de- oxycholic acid, 0.1% SDS, 1 mM Na3VO4, 20 g/ml apro- tinin, 20 g/ml leupeptin, 20 g/ml pepstatin A) with a grinder on ice. The homogenate was centrifuged at 10,000 g for 20 min at 4°C, and the supernatant was harvested. Data Presentation and Statistical Analysis For Western blot analysis, the optical density of specific OX-42 and GFAP bands was measured with a computer- assisted image analysis system (Gel-Pro Analyzer software, Media Cybernetics, Inc., Bethesda, MD). All results are pre- sented as the mean SD. Mean values were analyzed using two-way ANOVA followed by Tukey post hoc tests. There were no missing data needing adjustment. All P values were two-tailed and a P value of less than 0.05 was considered to be statistically significant. All statistical analyses were per- formed using SPSS 19.0 software (SPSS Inc., Chicago, IL). Results In addition, deep regional hypothermia administered preinjury and 5 h postinjury was more effective in ameliorating mechanical allodynia (A) and thermal hyperalgesia (B) than mild regional hypothermia (#P 0.05, by Tukey test). In 1 day, 3 days, and 5 days postinjury groups, there were no significant differences in the mechanical withdrawal threshold (A) and thermal withdrawal latency (B) among CCI rats treated with regional normothermia, mild regional hypothermia, or deep regional hypothermia (P 0.05, by Tukey test). Similarly, a significant improvement in both mechanical allodynia (C) and thermal hyperalgesia (D) was achieved after applying whole-body hypothermia (P 0.05, by two-way ANOVA). In preinjury, 5 h, 1 day, and 3 days postinjury groups, mechanical allodynia (C) and thermal hyperalgesia (D) were significantly suppressed in CCI rats treated with mild or deep whole-body hypothermia compared with those treated with whole-body normothermia (P 0.05, by Tukey test). In the 5 days postinjury group, deep whole-body hypothermia, but not mild whole-body hypothermia, significantly attenuated mechanical allodynia (C) and thermal hyperalgesia (D) compared with whole-body normothermia (P 0.05, by Tukey test). In all five groups, deep whole-body hypothermia was more effective in ameliorating mechanical allodynia (C) and thermal hyperalgesia (D) than mild whole-body hypothermia (#P 0.05, by Tukey test). Regional and whole-body normothermia had similar effects on mechanical allodynia and thermal hyperalgesia (P 0.05, by two-way ANOVA). In contrast, mild and deep whole-body hypothermia were more effective in attenuating mechanical allodynia and thermal hyperalgesia than mild and deep regional hypothermia, respectively (P 0.05, by two-way ANOVA). Error bars represent mean SD; n 6 or 7 rats per treatment. no differences in mechanical withdrawal threshold and ther- mal withdrawal latency among CCI rats treated with regional normothermia, mild regional hypothermia, and deep re- gional hypothermia (figs. 1A and B). Furthermore, in the preinjury and 5 h postinjury groups, the mechanical with- drawal threshold and thermal withdrawal latency were sig- nificantly higher in CCI rats receiving deep regional hypo- thermia than in those receiving mild regional hypothermia (figs. 1A and B). hypothermia had similar mechanical withdrawal thresholds and thermal withdrawal latencies (figs. 1C and D). In the preinjury, 5 h, 1 day, and 3 days postinjury groups, the mechanical withdrawal thresholds and thermal withdrawal latencies were significantly increased in CCI rats treated with either mild or deep whole-body hypothermia compared with those treated with whole-body normothermia (figs. Results Effect of Regional or Whole-body Hypothermic Treatment on Behavior on Day 7 after Median Nerve CCI g y yp Treatment on Behavior on Day 7 after Median Nerve CCI Behavioral testing showed that sham-operated rats treated with regional normothermia (37°C), mild regional hypo- thermia (32°C), or deep regional hypothermia (28°C) had similar mechanical withdrawal thresholds and thermal with- drawal latencies (fig. 1A-B). In CCI rats pretreated with re- gional normothermia, the mechanical withdrawal threshold and thermal withdrawal latency were significantly decreased compared with sham-operated rats (figs. 1A and B). How- ever, a significant increase in the mechanical withdrawal threshold and thermal withdrawal latency was observed in CCI rats pretreated with mild or deep regional hypothermia compared with rats pretreated with regional normothermia (figs. 1A and B). Similarly, in the 5 h postinjury group, there was a significant increase in the mechanical withdrawal threshold and thermal withdrawal latency in CCI rats treated with mild or deep regional hypothermia compared with those treated with regional normothermia (figs. 1A and B). In the 1 day, 3 days, and 5 days postinjury groups, there were Western blot analysis was performed as described previ- ously.19,21 In brief, protein concentration was measured us- ing a detergent-compatible protein assay with a bovine serum albumin standard (BioRad, Hercules, CA). Proteins (20 g) were separated on 10% SDS-PAGE and transferred to nitro- cellulose membrane (GE Healthcare, Piscataway, NJ). The membranes were blocked in 5% nonfat dry milk in phos- phate-buffered saline with 0.1% Tween-20, and then incu- bated with either mouse monoclonal anti-OX-42 (CD11b, 1:1000; BD Biosciences, San Jose, CA) or mouse monoclo- nal anti-GFAP (1:2,000) antibody for 18 h at 4°C. The membranes were washed in phosphate buffered saline with 0.1% Tween-20 and incubated with horseradish peroxidase- conjugated sheep antimouse IgG (1:5,000; GE Healthcare). Anesthesiology 2012; 116:415–31 Tsai et al. 418 PAIN MEDICINE Fig. 1. Effects of regional and whole-body hypothermic treatment on nerve injury-induced mechanical allodynia and thermal hyperalgesia. A significant amelioration of both mechanical allodynia (A) and thermal hyperalgesia (B) was observed after applying regional hypothermia (P 0.05, by two-way ANOVA). In preinjury and 5 h postinjury groups, mechanical allodynia (A) and thermal hyperalgesia (B) were significantly suppressed in chronic constriction injury (CCI) rats treated with mild or deep regional hypothermia compared with those treated with regional normothermia (P 0.05, by Tukey test). Results In addition, deep regional hypothermia administered preinjury and 5 h postinjury was more effective in ameliorating mechanical allodynia (A) and thermal hyperalgesia (B) than mild regional hypothermia (#P 0.05, by Tukey test). In 1 day, 3 days, and 5 days postinjury groups, there were no significant differences in the mechanical withdrawal threshold (A) and thermal withdrawal latency (B) among CCI rats treated with regional normothermia, mild regional hypothermia, or deep regional hypothermia (P 0.05, by Tukey test). Similarly, a significant improvement in both mechanical allodynia (C) and thermal hyperalgesia (D) was achieved after applying whole-body hypothermia (P 0.05, by two-way ANOVA). In preinjury, 5 h, 1 day, and 3 days postinjury groups, mechanical allodynia (C) and thermal hyperalgesia (D) were significantly suppressed in CCI rats treated with mild or deep whole-body hypothermia compared with those treated with whole-body normothermia (P 0.05, by Tukey test). In the 5 days postinjury group, deep whole-body hypothermia, but not mild whole-body hypothermia, significantly attenuated mechanical allodynia (C) and thermal hyperalgesia (D) compared with whole-body normothermia (P 0.05, by Tukey test). In all five groups, deep whole-body hypothermia was more effective in ameliorating mechanical allodynia (C) and thermal hyperalgesia (D) than mild whole-body hypothermia (#P 0.05, by Tukey test). Regional and whole-body normothermia had similar effects on mechanical allodynia and thermal hyperalgesia (P 0.05, by two-way ANOVA). In contrast, mild and deep whole-body hypothermia were more effective in attenuating mechanical allodynia and thermal hyperalgesia than mild and deep regional hypothermia, respectively (P 0.05, by two-way ANOVA). Error bars represent mean SD; n 6 or 7 rats per treatment. Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/116/2/415/256822/0000542-201202000-00027. Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/116/2/415/256822/0000542-201202000-00027.pdf by guest on 24 October 2024 pubs.asahq.org/anesthesiology/article-pdf/116/2/415/256822/0000542-201202000-00027.pdf by guest on 24 October 2024 Fig. 1. Effects of regional and whole-body hypothermic treatment on nerve injury-induced mechanical allodynia and thermal hyperalgesia. A significant amelioration of both mechanical allodynia (A) and thermal hyperalgesia (B) was observed after applying regional hypothermia (P 0.05, by two-way ANOVA). In preinjury and 5 h postinjury groups, mechanical allodynia (A) and thermal hyperalgesia (B) were significantly suppressed in chronic constriction injury (CCI) rats treated with mild or deep regional hypothermia compared with those treated with regional normothermia (P 0.05, by Tukey test). Anesthesiology 2012; 116:415–31 Results Regional and whole-body hypothermia, either mild or deep, had similar effects on the nerve discharge rate (P 0.05, by two-way ANOVA). Error bars represent mean SD; n 6 or 7 rats per treatment. Fig. 2. Effects of regional or whole-body hypothermic treatment on the discharge activity of the injured median nerve. A low frequency of spikes was observed in sham-operated rats treated with regional normothermia (Aa). In contrast, high rates of nerve discharge were seen in chronic constriction injury (CCI) rats pretreated with regional normothermia (Ab). In CCI rats pretreated with mild (Ac) or deep regional hypothermia (Ad), the discharge rate was decreased. (B) A significant decrease in the rate of nerve discharge was noted after application of regional hypothermia (P 0.05, by two-way ANOVA). In preinjury, 5 h, 1 day, and 3 days postinjury groups, the discharge rates were significantly decreased in CCI rats treated with mild or deep regional hypothermia compared with those treated with regional normothermia (P 0.05, by Tukey test). On postinjury day 5, deep, but not mild, regional hypothermia significantly decreased the rate of nerve discharge compared with regional normo- thermia (P 0.05, by Tukey test). Further, the discharge rate was significantly lower in CCI rats that received deep regional hypothermia than mild regional hypothermia (#P 0.05, by Tukey test). (C) A significant decrease in the rate of nerve discharge was achieved after applying whole-body hypothermia (P 0.05, by two-way ANOVA). The discharge rate was significantly decreased in CCI rats treated with mild or deep whole-body hypothermia compared with whole-body normothermia in preinjury, 5 h, 1 day, and 3 days postinjury groups (P 0.05, by Tukey test). Deep whole-body hypothermia, but not mild whole-body hypothermia, significantly decreased the rate of discharge compared with whole-body normothermia on postinjury day 5 (P 0.05, by Tukey test). In addition, the discharge activity was significantly lower in CCI rats that received deep whole-body hypothermia than in those that received mild whole-body hypothermia (#P 0.05, by Tukey test). Regional and whole-body hypothermia, either mild or deep, had similar effects on the nerve discharge rate (P 0.05, by two-way ANOVA). Error bars represent mean SD; n 6 or 7 rats per treatment. gional normothermia, mild regional hypothermia, or deep regional hypothermia (fig. 2B). Results 1C and D). In the 5 days postinjury group, the mechanical with- drawal threshold and thermal withdrawal latency were sig- nificantly increased in CCI rats receiving deep whole-body hypothermia, but not mild whole-body hypothermia, com- no differences in mechanical withdrawal threshold and ther- mal withdrawal latency among CCI rats treated with regional normothermia, mild regional hypothermia, and deep re- gional hypothermia (figs. 1A and B). Furthermore, in the preinjury and 5 h postinjury groups, the mechanical with- drawal threshold and thermal withdrawal latency were sig- nificantly higher in CCI rats receiving deep regional hypo- thermia than in those receiving mild regional hypothermia (figs. 1A and B). The sham-operated rats treated with whole-body normo- thermia, mild whole-body hypothermia, or deep whole-body Anesthesiology 2012; 116:415–31 419 Tsai et al. Fig. 2. Effects of regional or whole-body hypothermic treatment on the discharge activity of the injured median nerve. A low frequency of spikes was observed in sham-operated rats treated with regional normothermia (Aa). In contrast, high rates of nerve discharge were seen in chronic constriction injury (CCI) rats pretreated with regional normothermia (Ab). In CCI rats pretreated with mild (Ac) or deep regional hypothermia (Ad), the discharge rate was decreased. (B) A significant decrease in the rate of nerve discharge was noted after application of regional hypothermia (P 0.05, by two-way ANOVA). In preinjury, 5 h, 1 day, and 3 days postinjury groups, the discharge rates were significantly decreased in CCI rats treated with mild or deep regional hypothermia compared with those treated with regional normothermia (P 0.05, by Tukey test). On postinjury day 5, deep, but not mild, regional hypothermia significantly decreased the rate of nerve discharge compared with regional normo- thermia (P 0.05, by Tukey test). Further, the discharge rate was significantly lower in CCI rats that received deep regional hypothermia than mild regional hypothermia (#P 0.05, by Tukey test). (C) A significant decrease in the rate of nerve discharge was achieved after applying whole-body hypothermia (P 0.05, by two-way ANOVA). The discharge rate was significantly decreased in CCI rats treated with mild or deep whole-body hypothermia compared with whole-body normothermia in preinjury, 5 h, 1 day, and 3 days postinjury groups (P 0.05, by Tukey test). Deep whole-body hypothermia, but not mild whole-body hypothermia, significantly decreased the rate of discharge compared with whole-body normothermia on postinjury day 5 (P 0.05, by Tukey test). Results In the preinjury, 5 h, 1 day, and 3 days postinjury groups of CCI rats, the rate of dis- charge was significantly decreased in those treated with either mild or deep regional hypothermia compared with those treated with regional normothermia. In the 5 days postin- jury group, the nerve discharge rate was significantly de- creased in CCI rats receiving deep, but not mild, regional hypothermia compared with those receiving regional nor- mothermia. In addition, a significantly lower nerve dis- charge rate was observed in CCI rats that underwent deep regional hypothermia than in those that underwent mild regional hypothermia. pared with those receiving whole-body normothermia (figs. 1C and D). In addition, the mechanical withdrawal thresh- old and thermal withdrawal latency were significantly higher in CCI rats that received deep whole-body hypothermia than in those that received mild whole-body hypothermia (figs. 1C and D). A more profound increase in mechanical withdrawal threshold and thermal withdrawal latency was observed in CCI rats that were administered deep whole-body hypother- mia compared with deep regional hypothermia (fig. 1). Sim- ilar results were also obtained when mild regional and whole- body hypothermia were applied. Anesthesiology 2012; 116:415–31 Results In addition, the discharge activity was significantly lower in CCI rats that received deep whole-body hypothermia than in those that received mild whole-body hypothermia (#P 0.05, by Tukey test). Regional and whole-body hypothermia, either mild or deep, had similar effects on the nerve discharge rate (P 0.05, by two-way ANOVA). Error bars represent mean SD; n 6 or 7 rats per treatment. Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/116/2/415/256822/0000542-201202000-00027 Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/116/2/415/256822/0000542-201202000-00027.pdf by guest on 24 October 2024 Fig. 2. Effects of regional or whole-body hypothermic treatment on the discharge activity of the injured median nerve. A low frequency of spikes was observed in sham-operated rats treated with regional normothermia (Aa). In contrast, high rates of nerve discharge were seen in chronic constriction injury (CCI) rats pretreated with regional normothermia (Ab). In CCI rats pretreated with mild (Ac) or deep regional hypothermia (Ad), the discharge rate was decreased. (B) A significant decrease in the rate of nerve discharge was noted after application of regional hypothermia (P 0.05, by two-way ANOVA). In preinjury, 5 h, 1 day, and 3 days postinjury groups, the discharge rates were significantly decreased in CCI rats treated with mild or deep regional hypothermia compared with those treated with regional normothermia (P 0.05, by Tukey test). On postinjury day 5, deep, but not mild, regional hypothermia significantly decreased the rate of nerve discharge compared with regional normo- thermia (P 0.05, by Tukey test). Further, the discharge rate was significantly lower in CCI rats that received deep regional hypothermia than mild regional hypothermia (#P 0.05, by Tukey test). (C) A significant decrease in the rate of nerve discharge was achieved after applying whole-body hypothermia (P 0.05, by two-way ANOVA). The discharge rate was significantly decreased in CCI rats treated with mild or deep whole-body hypothermia compared with whole-body normothermia in preinjury, 5 h, 1 day, and 3 days postinjury groups (P 0.05, by Tukey test). Deep whole-body hypothermia, but not mild whole-body hypothermia, significantly decreased the rate of discharge compared with whole-body normothermia on postinjury day 5 (P 0.05, by Tukey test). In addition, the discharge activity was significantly lower in CCI rats that received deep whole-body hypothermia than in those that received mild whole-body hypothermia (#P 0.05, by Tukey test). Effect of Regional or Whole-body Hypothermic Treatment on Injury Discharge on Day 7 after Median Nerve CCI g p Low rates of nerve discharge were observed in sham- operated rats treated with whole-body normothermia, mild whole-body hypothermia, or deep whole-body hy- pothermia (fig. 2C). In the preinjury, 5 h, 1 day, and 3 days postinjury groups of CCI rats, the discharge rate was significantly decreased in those treated with either mild or deep whole-body hypothermia compared with those treated with whole-body normothermia. In the 5 days postinjury group, the rate of nerve discharge was signifi- cantly decreased in CCI rats receiving deep whole-body hypothermia, but not mild whole-body hypothermia, compared with those receiving whole-body normother- mia. Moreover, the rate of discharge was significantly lower in CCI rats that received deep whole-body hypo- Electrophysiological studies showed that sham-operated rats treated with regional normothermia displayed low-fre- quency spikes (fig. 2Aa). In CCI rats pretreated with regional normothermia, the rate of discharge was dramatically in- creased compared with that in sham-operated rats (figs. 2Aa and Ab). However, a significantly lower nerve discharge rate was observed in CCI rats pretreated with mild or deep re- gional hypothermia than in those pretreated with regional normothermia (figs. 2Ac and Ad). Quantitative analysis revealed that low rates of nerve dis- charge were observed in sham-operated rats treated with re- Anesthesiology 2012; 116:415–31 Tsai et al. 420 PAIN MEDICINE i th i th th t i d ild h l b d h ith i l th i ild i l h 3. Photomicrographs showing OX-42 immunoreactivity in the ipsilateral cuneate nucleus (CN) 7 days a striction injury (CCI) in rats that received regional hypothermic treatment. Little OX-42 immunoreactivity was obs of sham-operated rats treated with regional normothermia (A), mild regional hypothermia (B), or deep regional h In the preinjury and 5 h postinjury groups, OX-42 immunoreactivity decreased in the CN of CCI rats that re onal hypothermia (E, H) or deep regional hypothermia (F, I), but not in those that received regional normotherm ay, 3 days, and 5 days postinjury groups, there was intense OX-42 immunoreactivity in the CN of CCI rats t onal normothermia (J, M, P), mild regional hypothermia (K, N, Q), and deep regional hypothermia (L, O, R). Bar Fig. 3. Photomicrographs showing OX-42 immunoreactivity in the ipsilateral cuneate nucleus (CN) 7 days after chronic constriction injury (CCI) in rats that received regional hypothermic treatment. Effect of Regional or Whole-body Hypothermic Treatment on Injury Discharge on Day 7 after Median Nerve CCI Effect of Regional or Whole-body Hypothermic Treatment on Glial Activation on Day 7 after Median Nerve CCI Microglial Activation Responses. Little OX-42 immunore- activity was observed in the CN of sham-operated rats treated Microglial Activation Responses. Little OX-42 immunore- activity was observed in the CN of sham-operated rats treated Microglial Activation Responses. Little OX-42 immunore- activity was observed in the CN of sham-operated rats treated Tsai et al. Effect of Regional or Whole-body Hypothermic Treatment on Injury Discharge on Day 7 after Median Nerve CCI Little OX-42 immunoreactivity was observed in the CN of sham-operated rats treated with regional normothermia (A), mild regional hypothermia (B), or deep regional hypothermia (C). In the preinjury and 5 h postinjury groups, OX-42 immunoreactivity decreased in the CN of CCI rats that received mild regional hypothermia (E, H) or deep regional hypothermia (F, I), but not in those that received regional normothermia (D, G). In 1 day, 3 days, and 5 days postinjury groups, there was intense OX-42 immunoreactivity in the CN of CCI rats treated with regional normothermia (J, M, P), mild regional hypothermia (K, N, Q), and deep regional hypothermia (L, O, R). Bar 100 m. Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/116/2/415/256822/0000542-201202000-00027 Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/116/2/415/256822/0000542-201202000-00027.pdf by guest on 24 October 2024 pubs.asahq.org/anesthesiology/article-pdf/116/2/415/256822/0000542-201202000-00027.pdf by guest on 24 October 2024 Fig. 3. Photomicrographs showing OX-42 immunoreactivity in the ipsilateral cuneate nucleus (CN) 7 days after chronic constriction injury (CCI) in rats that received regional hypothermic treatment. Little OX-42 immunoreactivity was observed in the CN of sham-operated rats treated with regional normothermia (A), mild regional hypothermia (B), or deep regional hypothermia (C). In the preinjury and 5 h postinjury groups, OX-42 immunoreactivity decreased in the CN of CCI rats that received mild regional hypothermia (E, H) or deep regional hypothermia (F, I), but not in those that received regional normothermia (D, G). In 1 day, 3 days, and 5 days postinjury groups, there was intense OX-42 immunoreactivity in the CN of CCI rats treated with regional normothermia (J, M, P), mild regional hypothermia (K, N, Q), and deep regional hypothermia (L, O, R). Bar 100 m. thermia than in those that received mild whole-body hy- pothermia. with regional normothermia, mild regional hypothermia, or deep regional hypothermia (fig. 3A-C). In the preinjury group, intense OX-42 immunoreactivity was noted in the ipsilateral CN of CCI rats pretreated with regional normo- thermia (fig. 3D). However, OX-42 immunoreactivity was markedly decreased in the ipsilateral CN of CCI rats pre- treated with either mild or deep regional hypothermia com- pared with those pretreated with regional normothermia (figs. 3E and F). In the 5 h postinjury group, OX-42 immunoreactivity in the ipsilateral CN of CCI rats treated with mild or deep regional hypothermia was decreased in When comparing between regional and whole-body hy- pothermia, there were no differences in nerve discharge rates with respect to each treatment temperature (figs. 2B and C). Anesthesiology 2012; 116:415–31 Anesthesiology 2012; 116:415–31 421 Effect of Hypothermia on Neuropathic Pain hotomicrographs showing OX-42 immunoreactivity in the ipsilateral cuneate nucleus (CN) on day 7 after chron CI) in rats that received whole-body hypothermic treatment. There was little OX-42 immunoreactivity presen erated rats treated with whole-body normothermia (A), mild whole-body hypothermia (B), or deep whole-body einjury, 5 h, 1 day, and 3 days postinjury groups, OX-42 immunoreactivity was decreased in the CN of CCI rats H, K, N) or deep whole-body hypothermia (F, I, L, O) compared with those that received whole-body normot the 5 days postinjury group, OX-42 immunoreactivity was decreased in the CN of CCI rats receiving deep mia (R), but not in those receiving whole-body normothermia (P) or mild whole-body hypothermia (Q). Bar Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/116/2/415/256822/0000542-201202000-00027 Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/116/2/415/256822/0000542-201202000-00027.pdf by guest on 24 October 2024 pubs.asahq.org/anesthesiology/article-pdf/116/2/415/256822/0000542-201202000-00027.pdf by guest on 24 October 2024 Fig. 4. Photomicrographs showing OX-42 immunoreactivity in the ipsilateral cuneate nucleus (CN) on day 7 after chronic constriction injury (CCI) in rats that received whole-body hypothermic treatment. There was little OX-42 immunoreactivity present in the CN of sham-operated rats treated with whole-body normothermia (A), mild whole-body hypothermia (B), or deep whole-body hypothermia (C). In preinjury, 5 h, 1 day, and 3 days postinjury groups, OX-42 immunoreactivity was decreased in the CN of CCI rats that received mild (E, H, K, N) or deep whole-body hypothermia (F, I, L, O) compared with those that received whole-body normothermia (D, G, J, M). In the 5 days postinjury group, OX-42 immunoreactivity was decreased in the CN of CCI rats receiving deep whole-body hypothermia (R), but not in those receiving whole-body normothermia (P) or mild whole-body hypothermia (Q). Bar 100 m. treated with whole-body normothermia (fig. 4D). However, OX-42 immunoreactivity in the ipsilateral CN of CCI rats pretreated with mild or deep whole-body hypothermia was significantly decreased in comparison with those pretreated with whole-body normothermia (figs. 4E and F). Similarly, in the 5 h, 1 day, and 3 days postinjury groups, OX-42 immunoreactivity in the ipsilateral CN of CCI rats treated with mild or deep whole-body hypothermia was decreased in comparison with those treated with whole-body normother- mia (figs. 4G–O). In the 5 days postinjury group, OX-42 immunoreactivity was decreased in CCI rats receiving deep comparison with those treated with regional normother- mia (figs. 3G–I). Anesthesiology 2012; 116:415–31 In the 1 day, 3 days, and 5 days postin- jury groups, intense OX-42 immunoreactivity was ob- served in the ipsilateral CN of CCI rats that received regional normothermia, mild regional hypothermia, or deep regional hypothermia (figs. 3J–R). In the CN of sham-operated rats treated with whole-body normothermia, mild whole-body hypothermia, or deep whole-body hypothermia, little OX-42 immunoreactivity was observed (fig. 4A-C). In contrast, intense OX-42 immu- noreactivity was seen in the ipsilateral CN of CCI rats pre- Anesthesiology 2012; 116:415–31 Anesthesiology 2012; 116:415–31 5. Western blot analysis showing the levels of OX-42 in the ipsilateral cuneate nucleus (CN) 7 days after chronic constriction injury (CCI) in rats treated with regional or whole-body hypothermia. (A) OX-42 levels significantly decreased after applying regional hypothermia (P 0.05, by two-way ANOVA). There was a significant decrease in OX-42 levels in rats pretreated with mild or deep regional hypothermia, compared with those pretreated with regional normothermia (P 0.05, by Tukey test). Similarly, in the 5 h postinjury group, OX-42 levels in the CN were significantly decreased in CCI rats that received mild or deep regional hypothermia compared with those that received regional normothermia (P 0.05, by Tukey test). Furthermore, when administered preinjury and 5 h postinjury, deep regional hypothermia was more effective in suppressing OX-42 levels than mild regional hypothermia (#P 0.05, by Tukey test). However, in 1 day, 3 days, and 5 days postinjury groups, the levels of OX-42 in the CN did not differ significantly among rats that received regional normothermia, mild regional hypothermia, or deep regional hypothermia (P 0.05, by Tukey test). (B) A significant decrease in OX-42 levels was noted after applying whole-body hypothermia (P 0.05, by two-way ANOVA). Compared with whole-body normothermia, mild and deep whole-body hypo- thermia administered preinjury, 5 h, 1 day, and 3 days postinjury were effective in decreasing OX-42 levels in the CN of CCI rats (P 0.05, by Tukey test). On day 5 postinjury, a significant decrease in OX-42 levels was observed in CCI rats treated with deep whole-body hypothermia, but not mild whole-body hypothermia, compared with those treated with whole-body normo- thermia (P 0.05, by Tukey test). In addition, lower levels of OX-42 were observed in rats treated with deep whole-body hypothermia than in those treated with mild whole-body hypothermia (#P 0.05, by Tukey test). Regional and whole-body normothermia resulted in a similar increase in OX-42 levels (P 0.05, by two-way ANOVA). Whole-body hypothermia, either mild or deep, contributed to lower OX-42 levels than the corresponding regional hypothermia (P 0.05, by two-way ANOVA). -actin was used as a loading control. Error bars represent mean SD; n 5 rats per treatment. Fig. 5. Western blot analysis showing the levels of OX-42 in the ipsilateral cuneate nucleus (CN) 7 days after chronic constriction injury (CCI) in rats treated with regional or whole-body hypothermia. Anesthesiology 2012; 116:415–31 422 Tsai et al. Fig. 5. Western blot analysis showing the levels of OX-42 in the ipsilateral cuneate nucleus (CN) 7 days after chronic constriction injury (CCI) in rats treated with regional or whole-body hypothermia. (A) OX-42 levels significantly decreased after applying regional hypothermia (P 0.05, by two-way ANOVA). There was a significant decrease in OX-42 levels in rats pretreated with mild or deep regional hypothermia, compared with those pretreated with regional normothermia (P 0.05, by Tukey test). Similarly, in the 5 h postinjury group, OX-42 levels in the CN were significantly decreased in CCI rats that received mild or deep regional hypothermia compared with those that received regional normothermia (P 0.05, by Tukey test). Furthermore, when administered preinjury and 5 h postinjury, deep regional hypothermia was more effective in suppressing OX-42 levels than mild regional hypothermia (#P 0.05, by Tukey test). However, in 1 day, 3 days, and 5 days postinjury groups, the levels of OX-42 in the CN did not differ significantly among rats that received regional normothermia, mild regional hypothermia, or deep regional hypothermia (P 0.05, by Tukey test). (B) A significant decrease in OX-42 levels was noted after applying whole-body hypothermia (P 0.05, by two-way ANOVA). Compared with whole-body normothermia, mild and deep whole-body hypo- thermia administered preinjury, 5 h, 1 day, and 3 days postinjury were effective in decreasing OX-42 levels in the CN of CCI rats (P 0.05, by Tukey test). On day 5 postinjury, a significant decrease in OX-42 levels was observed in CCI rats treated with deep whole-body hypothermia, but not mild whole-body hypothermia, compared with those treated with whole-body normo- thermia (P 0.05, by Tukey test). In addition, lower levels of OX-42 were observed in rats treated with deep whole-body hypothermia than in those treated with mild whole-body hypothermia (#P 0.05, by Tukey test). Regional and whole-body normothermia resulted in a similar increase in OX-42 levels (P 0.05, by two-way ANOVA). Whole-body hypothermia, either mild or deep, contributed to lower OX-42 levels than the corresponding regional hypothermia (P 0.05, by two-way ANOVA). -actin was used as a loading control. Error bars represent mean SD; n 5 rats per treatment. Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/116/2/415/256822/0000542-201202000-00027.pdf by guest on 24 October 2024 Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/116/2/415/256822/0000542-201202000-00027.pdf by guest on 24 October 2024 Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/116/2/415/256822/0000542-201202000-00027. /pubs.asahq.org/anesthesiology/article-pdf/116/2/415/256822/0000542-201202000-00027.pdf by guest on 24 October 2024 Fig. Anesthesiology 2012; 116:415–31 (A) OX-42 levels significantly decreased after applying regional hypothermia (P 0.05, by two-way ANOVA). There was a significant decrease in OX-42 levels in rats pretreated with mild or deep regional hypothermia, compared with those pretreated with regional normothermia (P 0.05, by Tukey test). Similarly, in the 5 h postinjury group, OX-42 levels in the CN were significantly decreased in CCI rats that received mild or deep regional hypothermia compared with those that received regional normothermia (P 0.05, by Tukey test). Furthermore, when administered preinjury and 5 h postinjury, deep regional hypothermia was more effective in suppressing OX-42 levels than mild regional hypothermia (#P 0.05, by Tukey test). However, in 1 day, 3 days, and 5 days postinjury groups, the levels of OX-42 in the CN did not differ significantly among rats that received regional normothermia, mild regional hypothermia, or deep regional hypothermia (P 0.05, by Tukey test). (B) A significant decrease in OX-42 levels was noted after applying whole-body hypothermia (P 0.05, by two-way ANOVA). Compared with whole-body normothermia, mild and deep whole-body hypo- thermia administered preinjury, 5 h, 1 day, and 3 days postinjury were effective in decreasing OX-42 levels in the CN of CCI rats (P 0.05, by Tukey test). On day 5 postinjury, a significant decrease in OX-42 levels was observed in CCI rats treated with deep whole-body hypothermia, but not mild whole-body hypothermia, compared with those treated with whole-body normo- thermia (P 0.05, by Tukey test). In addition, lower levels of OX-42 were observed in rats treated with deep whole-body hypothermia than in those treated with mild whole-body hypothermia (#P 0.05, by Tukey test). Regional and whole-body normothermia resulted in a similar increase in OX-42 levels (P 0.05, by two-way ANOVA). Whole-body hypothermia, either mild or deep, contributed to lower OX-42 levels than the corresponding regional hypothermia (P 0.05, by two-way ANOVA). -actin was used as a loading control. Error bars represent mean SD; n 5 rats per treatment. jury, 5 h, 1 day, and 3 days postinjury groups, mild and deep whole-body hypothermia significantly decreased OX-42 lev- els in the CN of CCI rats than whole-body normothermia (fig. 5B). Deep whole-body hypothermia, but not mild whole-body hypothermia, significantly decreased the levels of OX-42 in CCI rats compared with whole-body normo- thermia in the 5 days postinjury group (fig. 5B). Anesthesiology 2012; 116:415–31 Photomicrographs showing glial fibrillary acidic protein (GFAP) immunoreactivity in the ipsilateral cuneate nucleus (CN) on day 7 days after chronic constriction injury (CCI) in rats that received regional hypothermic treatment. Little GFAP immunoreactivity was observed in the CN of sham-operated rats treated with regional normothermia (A), mild regional hypothermia (B), or deep regional hypothermia (C). In preinjury and 5 h postinjury groups, GFAP immunoreactivity in the CN was decreased in CCI rats that received mild (E, H) or deep regional hypothermia (F, I) compared with those that received regional normothermia (D, G). In 1 day, 3 days, and 5 days postinjury groups, intense GFAP immunoreactivity was observed in the CN of CCI rats treated with regional normothermia (J, M, P), mild regional hypothermia (K, N, Q), or deep regional hypothermia (L, O, R). Bar 100 m. Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/116/2/415/256822/0000542-201202000-00027 Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/116/2/415/256822/0000542-201202000-00027.pdf by guest on 24 October 2024 pubs.asahq.org/anesthesiology/article-pdf/116/2/415/256822/0000542-201202000-00027.pdf by guest on 24 October 2024 Fig. 6. Photomicrographs showing glial fibrillary acidic protein (GFAP) immunoreactivity in the ipsilateral cuneate nucleus (CN) on day 7 days after chronic constriction injury (CCI) in rats that received regional hypothermic treatment. Little GFAP immunoreactivity was observed in the CN of sham-operated rats treated with regional normothermia (A), mild regional hypothermia (B), or deep regional hypothermia (C). In preinjury and 5 h postinjury groups, GFAP immunoreactivity in the CN was decreased in CCI rats that received mild (E, H) or deep regional hypothermia (F, I) compared with those that received regional normothermia (D, G). In 1 day, 3 days, and 5 days postinjury groups, intense GFAP immunoreactivity was observed in the CN of CCI rats treated with regional normothermia (J, M, P), mild regional hypothermia (K, N, Q), or deep regional hypothermia (L, O, R). Bar 100 m. treated with regional normothermia, mild regional hypo- thermia, or deep regional hypothermia (figs. 6A–C). In con- trast, intense GFAP immunoreactivity was present in the ipsilateral CN of CCI rats pretreated with regional normo- thermia (fig. 6D). However, GFAP immunoreactivity was significantly decreased in the ipsilateral CN of CCI rats pre- treated with either mild or deep regional hypothermia com- pared with CCI rats pretreated with regional normothermia (figs. 6E and F). In the 5 h postinjury group, GFAP immu- noreactivity in the ipsilateral CN of CCI rats treated with mild or deep regional hypothermia was decreased compared with those treated with regional normothermia (figs. 6G–I). Anesthesiology 2012; 116:415–31 More- over, in all five groups, lower levels of OX-42 were ob- served in CCI rats treated with deep whole-body hypothermia than in those treated with mild whole-body hypothermia (fig. 5B). Compared with mild and deep regional hypothermia, significantly decreased OX-42 lev- els were noted in CCI rats receiving mild and deep whole- body hypothermia, respectively (fig. 5). jury, 5 h, 1 day, and 3 days postinjury groups, mild and deep whole-body hypothermia significantly decreased OX-42 lev- els in the CN of CCI rats than whole-body normothermia (fig. 5B). Deep whole-body hypothermia, but not mild whole-body hypothermia, significantly decreased the levels of OX-42 in CCI rats compared with whole-body normo- thermia in the 5 days postinjury group (fig. 5B). More- over, in all five groups, lower levels of OX-42 were ob- served in CCI rats treated with deep whole-body hypothermia than in those treated with mild whole-body hypothermia (fig. 5B). Compared with mild and deep regional hypothermia, significantly decreased OX-42 lev- els were noted in CCI rats receiving mild and deep whole- body hypothermia, respectively (fig. 5). whole-body hypothermia, but not mild whole-body hypo- thermia, compared with those receiving whole-body normo- thermia (figs. 4P–R). Western blot analysis showed a significant decrease in the levels of OX-42 in the CN of CCI rats pretreated with mild or deep regional hypothermia compared with those pre- treated with regional normothermia (fig. 5A). Similarly, in the 5 h postinjury group, mild or deep regional hypothermia, but not regional normothermia, attenuated OX-42 levels in the CN of CCI rats (fig. 5A). In the 1 day, 3 days, and 5 days postinjury groups, regional normothermia, mild regional hy- pothermia, and deep regional hypothermia were ineffective in decreasing OX-42 levels (fig. 5A). Furthermore, in the preinjury and 5 h postinjury groups, deep regional hypother- mia was more effective in suppressing OX-42 levels in CCI rats than mild regional hypothermia (fig. 5A). In the prein- y yp p y g Astrocyte Activation Responses. Little immunoreactivity for GFAP was observed in the CN of sham-operated rats y yp p y g Astrocyte Activation Responses. Little immunoreactivity for GFAP was observed in the CN of sham-operated rats Anesthesiology 2012; 116:415–31 423 Tsai et al. Effect of Hypothermia on Neuropathic Pain Fig. 6. Anesthesiology 2012; 116:415–31 In the 1 day, 3 days, and 5 days postinjury groups, intense GFAP immunoreactivitywasobservedintheipsilateralCNofCCIrats that received regional normothermia, mild regional hypother- mia, or deep regional hypothermia (figs. 6J–R). Little GFAP immunoreactivity was present in the CN of sham-operated rats treated with whole-body normothermia, mild whole-body hypothermia, or deep whole-body hypo- thermia (figs. 7A–C). In the preinjury group, intense GFAP immunoreactivity was observed in the ipsilateral CN of CCI rats pretreated with whole-body normothermia (fig. 7D). In Anesthesiology 2012; 116:415–31 424 Tsai et al. Tsai et al. PAIN MEDICINE Fig. 7. Photomicrographs showing glial fibrillary acidic protein (GFAP) immunoreactivity in the ipsilateral cuneate nucleus (CN) 7 days after chronic constriction injury (CCI) in rats that received whole-body hypothermic treatment. Little GFAP immunore- activity was present in the CN of sham-operated rats treated with whole-body normothermia (A), mild whole-body hypothermia (B), or deep whole-body hypothermia (C). In preinjury, 5 h, 1 day, and 3 days postinjury groups, GFAP immunoreactivity in the CN of CCI rats that received mild (E, H, K, N) or deep whole-body hypothermia (F, I, L, O) was decreased compared with those that received whole-body normothermia (D, G, J, M). On day 5 postinjury, GFAP immunoreactivity was decreased in the CN of CCI rats receiving deep whole-body hypothermia (R), but not in those receiving whole-body normothermia (P) or mild whole-body hypothermia (Q). Bar 100 m. Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/116/2/415/256822/0000542-201202000-00027 Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/116/2/415/256822/0000542-201202000-00027.pdf by guest on 24 October 2024 pubs.asahq.org/anesthesiology/article-pdf/116/2/415/256822/0000542-201202000-00027.pdf by guest on 24 October 2024 Fig. 7. Photomicrographs showing glial fibrillary acidic protein (GFAP) immunoreactivity in the ipsilateral cuneate nucleus (CN) 7 days after chronic constriction injury (CCI) in rats that received whole-body hypothermic treatment. Little GFAP immunore- activity was present in the CN of sham-operated rats treated with whole-body normothermia (A), mild whole-body hypothermia (B), or deep whole-body hypothermia (C). In preinjury, 5 h, 1 day, and 3 days postinjury groups, GFAP immunoreactivity in the CN of CCI rats that received mild (E, H, K, N) or deep whole-body hypothermia (F, I, L, O) was decreased compared with those that received whole-body normothermia (D, G, J, M). On day 5 postinjury, GFAP immunoreactivity was decreased in the CN of CCI rats receiving deep whole-body hypothermia (R), but not in those receiving whole-body normothermia (P) or mild whole-body hypothermia (Q). Bar 100 m. Anesthesiology 2012; 116:415–31 hypothermia, but not mild whole-body hypothermia, dis- played decreased GFAP immunoreactivity compared with those receiving whole-body normothermia (figs. 7P–R). contrast, GFAP immunoreactivity in the ipsilateral CN of CCI rats pretreated with either mild or deep whole-body hypothermia was significantly decreased in comparison with CCI rats pretreated with whole-body normothermia (figs. 7E and F). Similarly, in the 5 h, 1 day, and 3 days postinjury groups, GFAP immunoreactivity in the ipsilateral CN of CCI rats treated with mild or deep whole-body hypothermia was decreased compared with those treated with whole-body normothermia (figs. 7G–O). In the 5 days postinjury group, the ipsilateral CN of CCI rats receiving deep whole-body hypothermia, but not mild whole-body hypothermia, dis- played decreased GFAP immunoreactivity compared with those receiving whole-body normothermia (figs. 7P–R). Western blot analysis showed a significant decrease in the levels of GFAP in the CN of CCI rats pretreated with either mild or deep regional hypothermia compared with those pretreated with regional normothermia (fig. 8A). Similarly, in the 5 h postinjury group, mild or deep regional hypother- mia, but not regional normothermia, attenuated GFAP lev- els in the CN of CCI rats (fig. 8A). In the 1 day, 3 days, and Western blot analysis showed a significant decrease in the levels of GFAP in the CN of CCI rats pretreated with either mild or deep regional hypothermia compared with those pretreated with regional normothermia (fig. 8A). Similarly, in the 5 h postinjury group, mild or deep regional hypother- mia, but not regional normothermia, attenuated GFAP lev- els in the CN of CCI rats (fig. 8A). In the 1 day, 3 days, and 425 Anesthesiology 2012; 116:415–31 (A) Application of regional hypothermia led to a significant decrease in GFAP levels (P 0.05, by two-way ANOVA). In rats pretreated with mild or deep regional hypothermia, there was a significant decrease in GFAP levels compared with those pretreated with regional normothermia (P 0.05, by Tukey test). In the 5 h postinjury group, GFAP levels in the CN were also significantly decreased in CCI rats treated with mild or deep regional hypothermia compared with those treated with regional normothermia (P 0.05, by Tukey test). Furthermore, when administered preinjury and 5 h postinjury, deep regional hypothermia was more effective in suppressing GFAP levels than mild regional hypothermia (#P 0.05, by Tukey test). However, in 1 day, 3 days, and 5 days postinjury groups, no significant differences in the levels of GFAP in the CN were observed among rats that received regional normothermia, mild regional hypothermia, or deep regional hypothermia (P 0.05, by Tukey test). (B) A significant decrease in GFAP levels was noted after applying whole-body hypothermia (P 0.05, by two-way ANOVA). In preinjury, 5 h, 1 day and 3 days postinjury groups, mild and deep whole-body hypothermia were effective in decreasing GFAP levels in the CN of CCI rats compared with whole-body normothermia (P 0.05, by Tukey test). On day 5 postinjury, deep whole-body hypothermia, but not mild whole-body hypothermia, resulted in a significant decrease in the levels of GFAP compared with whole-body normothermia (P 0.05, by Tukey test). In addition, deep whole-body hypothermia was more effective in suppressing GFAP levels than mild whole-body hypothermia (#P 0.05, by Tukey test). Whole-body hypothermia, either mild or deep, resulted in lower GFAP levels than the corresponding regional hypothermia (P 0.05, by two-way ANOVA). Regional and whole-body normothermia contributed to a similar increase in GFAP levels (P 0.05, by two-way ANOVA). -actin was used as a loading control. Error bars represent mean SD; n 5 rats per treatment. 8B). Mild and deep whole-body hypothermia were more effective in suppressing GFAP levels in CCI rats than mild and deep regional hypothermia, respectively (fig. 8). 5 days postinjury groups, neither regional normothermia nor regional hypothermia was effective in decreasing GFAP lev- els (fig. 8A). Furthermore, in both preinjury and 5 h postin- jury groups, deep regional hypothermia was more effective in suppressing GFAP levels than mild regional hypothermia (fig. 8A). Anesthesiology 2012; 116:415–31 Western blot analysis showing the levels of glial fibrillary acidic protein (GFAP) in the ipsilateral cuneate nucleus (CN) on day 7 after chronic constriction injury (CCI) in rats treated with regional or whole-body hypothermia. (A) Application of regional hypothermia led to a significant decrease in GFAP levels (P 0.05, by two-way ANOVA). In rats pretreated with mild or deep regional hypothermia, there was a significant decrease in GFAP levels compared with those pretreated with regional normothermia (P 0.05, by Tukey test). In the 5 h postinjury group, GFAP levels in the CN were also significantly decreased in CCI rats treated with mild or deep regional hypothermia compared with those treated with regional normothermia (P 0.05, by Tukey test). Furthermore, when administered preinjury and 5 h postinjury, deep regional hypothermia was more effective in suppressing GFAP levels than mild regional hypothermia (#P 0.05, by Tukey test). However, in 1 day, 3 days, and 5 days postinjury groups, no significant differences in the levels of GFAP in the CN were observed among rats that received regional normothermia, mild regional hypothermia, or deep regional hypothermia (P 0.05, by Tukey test). (B) A significant decrease in GFAP levels was noted after applying whole-body hypothermia (P 0.05, by two-way ANOVA). In preinjury, 5 h, 1 day and 3 days postinjury groups, mild and deep whole-body hypothermia were effective in decreasing GFAP levels in the CN of CCI rats compared with whole-body normothermia (P 0.05, by Tukey test). On day 5 postinjury, deep whole-body hypothermia, but not mild whole-body hypothermia, resulted in a significant decrease in the levels of GFAP compared with whole-body normothermia (P 0.05, by Tukey test). In addition, deep whole-body hypothermia was more effective in suppressing GFAP levels than mild whole-body hypothermia (#P 0.05, by Tukey test). Whole-body hypothermia, either mild or deep, resulted in lower GFAP levels than the corresponding regional hypothermia (P 0.05, by two-way ANOVA). Regional and whole-body normothermia contributed to a similar increase in GFAP levels (P 0.05, by two-way ANOVA). -actin was used as a loading control. Error bars represent mean SD; n 5 rats per treatment. Fig. 8. Western blot analysis showing the levels of glial fibrillary acidic protein (GFAP) in the ipsilateral cuneate nucleus (CN) on day 7 after chronic constriction injury (CCI) in rats treated with regional or whole-body hypothermia. Anesthesiology 2012; 116:415–31 Tsai et al. Effect of Hypothermia on Neuropathic Pain Fig. 8. Western blot analysis showing the levels of glial fibrillary acidic protein (GFAP) in the ipsilateral cuneate nucleus (CN) on day 7 after chronic constriction injury (CCI) in rats treated with regional or whole-body hypothermia. (A) Application of regional hypothermia led to a significant decrease in GFAP levels (P 0.05, by two-way ANOVA). In rats pretreated with mild or deep regional hypothermia, there was a significant decrease in GFAP levels compared with those pretreated with regional normothermia (P 0.05, by Tukey test). In the 5 h postinjury group, GFAP levels in the CN were also significantly decreased in CCI rats treated with mild or deep regional hypothermia compared with those treated with regional normothermia (P 0.05, by Tukey test). Furthermore, when administered preinjury and 5 h postinjury, deep regional hypothermia was more effective in suppressing GFAP levels than mild regional hypothermia (#P 0.05, by Tukey test). However, in 1 day, 3 days, and 5 days postinjury groups, no significant differences in the levels of GFAP in the CN were observed among rats that received regional normothermia, mild regional hypothermia, or deep regional hypothermia (P 0.05, by Tukey test). (B) A significant decrease in GFAP levels was noted after applying whole-body hypothermia (P 0.05, by two-way ANOVA). In preinjury, 5 h, 1 day and 3 days postinjury groups, mild and deep whole-body hypothermia were effective in decreasing GFAP levels in the CN of CCI rats compared with whole-body normothermia (P 0.05, by Tukey test). On day 5 postinjury, deep whole-body hypothermia, but not mild whole-body hypothermia, resulted in a significant decrease in the levels of GFAP compared with whole-body normothermia (P 0.05, by Tukey test). In addition, deep whole-body hypothermia was more effective in suppressing GFAP levels than mild whole-body hypothermia (#P 0.05, by Tukey test). Whole-body hypothermia, either mild or deep, resulted in lower GFAP levels than the corresponding regional hypothermia (P 0.05, by two-way ANOVA). Regional and whole-body normothermia contributed to a similar increase in GFAP levels (P 0.05, by two-way ANOVA). -actin was used as a loading control. Error bars represent mean SD; n 5 rats per treatment. Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/116/2/415/256822/0000542-201202000-00027.pdf by guest on 24 October 2024 Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/116/2/415/256822/0000542-201202000-00027. /pubs.asahq.org/anesthesiology/article-pdf/116/2/415/256822/0000542-201202000-00027.pdf by guest on 24 October 2024 Fig. 8. Anesthesiology 2012; 116:415–31 Similarly, in the 5 h postinjury group, TNF- (A) and IL-1 (B) levels in the CN were significantly decreased in CCI rats that received mild or deep regional hypothermia compared with those that received regional normothermia (P 0.05, by Tukey test). In addition, deep regional hypothermia administered preinjury and 5 h postinjury more effectively suppressed TNF- (A) and IL-1 (B) levels than did mild regional hypothermia (#P 0.05, by Tukey test). However, in 1 day, 3 days, and 5 days postinjury groups, the levels of TNF- (A) and IL-1 (B) in the CN did not differ significantly among rats that received regional normothermia, mild regional hypothermia, or deep regional hypothermia (P 0.05, by Tukey test). Levels of TNF- (C) and IL-1 (D) decreased significantly in response to application of whole-body hypothermia (P 0.05, by two-way ANOVA). Compared with whole-body normothermia, mild and deep whole-body hypothermia was effective in decreasing TNF- (C) and IL-1 (D) levels in the CN of CCI rats in preinjury, 5 h, 1 day, and 3 days postinjury groups (P 0.05, by Tukey test). In the 5 days postinjury group, a significant decrease in levels of TNF- (C) and IL-1 (D) in CCI rats receiving deep whole-body hypothermia, but not mild whole-body hypothermia, compared with those receiving whole-body normothermia (P 0.05, by Tukey test). Lower levels of TNF- (C) and IL-1 (D) were observed in rats treated with deep whole-body hypothermia than in those treated with mild whole-body hypothermia (#P 0.05, by Tukey test). Mild and deep whole-body hypothermia more effectively decreased TNF- and IL-1 levels than mild and deep regional hypothermia, respectively (P 0.05, by two-way ANOVA). Regional and whole-body normothermia had similar effects on levels of TNF- and IL-1 (P 0.05, by two-way ANOVA). Error bars represent mean SD; n 5 rats per treatment. and B). Similarly, in the 5 h postinjury group, there was a significant decrease in the levels of TNF- and IL-1 in the ipsilateral CN of CCI rats treated with mild or deep regional hypothermia compared with those treated with regional nor- mothermia (figs. 9A and B). Anesthesiology 2012; 116:415–31 In the preinjury, 5 h, 1 day, and 3 days postinjury groups of CCI rats, GFAP levels were significantly decreased in the CN in rats treated with mild or deep whole-body hypothermia compared with rats treated with whole-body normothermia (fig. 8B). In the 5 days postinjury group, lev- els of GFAP significantly decreased in CCI rats receiving deep whole-body hypothermia, but not mild whole-body hypothermia, compared with those receiving whole-body normothermia (fig. 8B). In addition, deep whole-body hy- pothermia was more effective in suppressing GFAP levels than mild whole-body hypothermia in all five groups (fig. Effect of Regional or Whole-body Hypothermic Treatment on Proinflammatory Cytokines on Day 7 after Median Nerve CCI The CN of sham-operated rats treated with regional normo- thermia, mild regional hypothermia, or deep regional hypo- thermia showed low levels of TNF- and IL-1 (figs. 9A and B). The levels of TNF- and IL-1 in the ipsilateral CN of CCI rats pretreated with regional normothermia were mark- edly increased compared with those of sham-operated rats (figs. 9A and B). However, TNF- and IL-1 levels in the ipsilateral CN of CCI rats pretreated with mild or deep re- gional hypothermia showed significant decreases compared with those pretreated with regional normothermia (figs. 9A Anesthesiology 2012; 116:415–31 426 Tsai et al. Fig. 9. Histograms showing the proinflammatory cytokine expression in the ipsilateral cuneate nucleus (CN) on day 7 after chronic constriction injury (CCI) in rats treated with regional or whole-body hypothermia. A significant decrease in levels of tumor necrosis factor (TNF)- (A) and interleukin (IL)-1 (B) was observed after applying regional hypothermia (P 0.05, by two-way ANOVA). In rats pretreated with mild or deep regional hypothermia, there was a significant decrease in TNF- (A) and IL-1 (B) levels compared with those pretreated with regional normothermia (P 0.05, by Tukey test). Similarly, in the 5 h postinjury group, TNF- (A) and IL-1 (B) levels in the CN were significantly decreased in CCI rats that received mild or deep regional hypothermia compared with those that received regional normothermia (P 0.05, by Tukey test). In addition, deep regional hypothermia administered preinjury and 5 h postinjury more effectively suppressed TNF- (A) and IL-1 (B) levels than did mild regional hypothermia (#P 0.05, by Tukey test). Anesthesiology 2012; 116:415–31 However, in 1 day, 3 days, and 5 days postinjury groups, the levels of TNF- (A) and IL-1 (B) in the CN did not differ significantly among rats that received regional normothermia, mild regional hypothermia, or deep regional hypothermia (P 0.05, by Tukey test). Levels of TNF- (C) and IL-1 (D) decreased significantly in response to application of whole-body hypothermia (P 0.05, by two-way ANOVA). Compared with whole-body normothermia, mild and deep whole-body hypothermia was effective in decreasing TNF- (C) and IL-1 (D) levels in the CN of CCI rats in preinjury, 5 h, 1 day, and 3 days postinjury groups (P 0.05, by Tukey test). In the 5 days postinjury group, a significant decrease in levels of TNF- (C) and IL-1 (D) in CCI rats receiving deep whole-body hypothermia, but not mild whole-body hypothermia, compared with those receiving whole-body normothermia (P 0.05, by Tukey test). Lower levels of TNF- (C) and IL-1 (D) were observed in rats treated with deep whole-body hypothermia than in those treated with mild whole-body hypothermia (#P 0.05, by Tukey test). Mild and deep whole-body hypothermia more effectively decreased TNF- and IL-1 levels than mild and deep regional hypothermia, respectively (P 0.05, by two-way ANOVA). Regional and whole-body normothermia had similar effects on levels of TNF- and IL-1 (P 0.05, by two-way ANOVA) Error bars represent mean SD; n 5 rats per treatment PAIN MEDICINE PAIN MEDICINE PAIN MEDICINE Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/116/2/415/256822/0000542-201202000-00027 Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/116/2/415/256822/0000542-201202000-00027.pdf by guest on 24 October 2024 /pubs.asahq.org/anesthesiology/article-pdf/116/2/415/256822/0000542-201202000-00027.pdf by guest on 24 October 2024 Fig. 9. Histograms showing the proinflammatory cytokine expression in the ipsilateral cuneate nucleus (CN) on day 7 after chronic constriction injury (CCI) in rats treated with regional or whole-body hypothermia. A significant decrease in levels of tumor necrosis factor (TNF)- (A) and interleukin (IL)-1 (B) was observed after applying regional hypothermia (P 0.05, by two-way ANOVA). In rats pretreated with mild or deep regional hypothermia, there was a significant decrease in TNF- (A) and IL-1 (B) levels compared with those pretreated with regional normothermia (P 0.05, by Tukey test). Similarly, in the 5 h postinjury group, TNF- (A) and IL-1 (B) levels in the CN were significantly decreased in CCI rats that received mild or deep regional hypothermia compared with those that received regional normothermia (P 0.05, by Tukey test). Anesthesiology 2012; 116:415–31 In addition, deep regional hypothermia administered preinjury and 5 h postinjury more effectively suppressed TNF- (A) and IL-1 (B) levels than did mild regional hypothermia (#P 0.05, by Tukey test). However, in 1 day, 3 days, and 5 days postinjury groups, the levels of TNF- (A) and IL-1 (B) in the CN did not differ significantly among rats that received regional normothermia, mild regional hypothermia, or deep regional hypothermia (P 0.05, by Tukey test). Levels of TNF- (C) and IL-1 (D) decreased significantly in response to application of whole-body hypothermia (P 0.05, by two-way ANOVA). Compared with whole-body normothermia, mild and deep whole-body hypothermia was effective in decreasing TNF- (C) and IL-1 (D) levels in the CN of CCI rats in preinjury, 5 h, 1 day, and 3 days postinjury groups (P 0.05, by Tukey test). In the 5 days postinjury group, a significant decrease in levels of TNF- (C) and IL-1 (D) in CCI rats receiving deep whole-body hypothermia, but not mild whole-body hypothermia, compared with those receiving whole-body normothermia (P 0.05, by Tukey test). Lower levels of TNF- (C) and IL-1 (D) were observed in rats treated with deep whole-body hypothermia than in those treated with mild whole-body hypothermia (#P 0.05, by Tukey test). Mild and deep whole-body hypothermia more effectively decreased TNF- and IL-1 levels than mild and deep regional hypothermia, respectively (P 0.05, by two-way ANOVA). Regional and whole-body normothermia had similar effects on levels of TNF- and IL-1 (P 0.05, by two-way ANOVA). Error bars represent mean SD; n 5 rats per treatment. Fig. 9. Histograms showing the proinflammatory cytokine expression in the ipsilateral cuneate nucleus (CN) on day 7 after chronic constriction injury (CCI) in rats treated with regional or whole-body hypothermia. A significant decrease in levels of tumor necrosis factor (TNF)- (A) and interleukin (IL)-1 (B) was observed after applying regional hypothermia (P 0.05, by two-way ANOVA). In rats pretreated with mild or deep regional hypothermia, there was a significant decrease in TNF- (A) and IL-1 (B) levels compared with those pretreated with regional normothermia (P 0.05, by Tukey test). Anesthesiology 2012; 116:415–31 In the 1 day, 3 days, and 5 days postinjury groups, there were no differences in the TNF- and IL-1 levels in the ipsilateral CN of CCI rats treated with mild or deep regional hypothermia compared with those of rats treated with regional normothermia (figs. 9A and B). Furthermore, in preinjury and 5 h postinjury groups, TNF- and IL-1 levels were significantly lower in CCI rats treated with deep regional hypothermia than in those treated with mild regional hypothermia (figs. 9A and B). We observed low levels of TNF- and IL-1 in the CN of sham-operated rats treated with whole-body normothermia, mild whole-body hypothermia, or deep whole-body hypo- Anesthesiology 2012; 116:415–31 427 Tsai et al. Effect of Hypothermia on Neuropathic Pain injury in humans or animal models have not been reported. Several studies have demonstrated that hypothermic treat- ment can inhibit the elevation of excitatory amino acids in ischemic regions of the brain,33,34 as well as microglial acti- vation in the gray matter of the spinal cord after spinal cord compression injury14 and posttraumatic inflammatory cas- cades in brain tissue, such as IL-1, inducible nitric oxide synthase, and superoxide.35 In the present study, preinjury treatment with regional or whole-body hypothermia de- creased glial activation in the CN and ameliorated nerve injury-induced neuropathic pain. Furthermore, regional or whole-body hypothermic treatment before median nerve in- jury attenuated the discharge activity of the injured nerve. Electrophysiological studies have shown enhanced expres- sion of persistently activated sodium channels in nerve fibers following nerve injury, which subsequently contributes to the generation of ectopic discharges and the release of gluta- mate.36,37 Pharmacological studies have demonstrated that the activated phenotype of cultured glia is observed after exposure to excitatory amino acids, such as glutamate and aspartate.38–40 Based on these observations, it is thought that using preinjury hypothermia to block injury-induced ectopic discharges in the median nerve would reduce glutamate re- lease and thus inhibit the activation of glia and the develop- ment of neuropathic pain. thermia (figs. 9C and D). In the preinjury, 5 h, 1 day, and 3 days postinjury groups, TNF- and IL-1 levels were signif- icantly decreased in the ipsilateral CN of CCI rats treated with mild or deep whole-body hypothermia compared with those treated with whole-body normothermia (figs. 9C and D). Discussion This study demonstrates that various degrees of regional hy- pothermia administered preinjury or 5 h postinjury attenu- ated the development of neuropathic pain behaviors and glial activation responses in the CN following median nerve CCI; however, regional hypothermia applied 1, 3, and 5 days postinjury failed to suppress neuropathic pain and glial acti- vation. Further, various degrees of whole-body hypothermia could ameliorate nerve injury-induced neuropathic pain and decrease glial activation in the CN when applied in a wider time frame, from preinjury to day 3 postinjury. On day 5 postinjury, only deep whole-body hypothermia was capable of modulating neuropathic pain and glial activation. Deep hypothermia, either regional or whole-body, was more effec- tive in attenuating neuropathic pain and glial activation than mild hypothermia. p p Mitsui et al.12 reported that early postischemic treatment (ischemia for 1 h), but not late postischemic treatment (isch- emia for 3 h), with mild or deep regional hypothermia re- duced fiber degeneration and endoneurial edema in a model of ischemic neuropathy. This is consistent with the findings of Kawamura et al. that inflammatory responses after periph- eral nerve ischemia were attenuated in rats given mild or deep regional hypothermia immediately or 1 h postischemia, but not 3 h postischemia.11 The present study showed that mild or deep regional hypothermia administered 5 h postinjury could reduce glial activation and suppress neuropathic pain following CCI of the median nerve; however, regional hypo- thermia applied 1, 3, and 5 days postinjury had no such effects. In previous studies, we examined the glial activation responses in the CN after median nerve CCI at different time-points, and found that the levels of OX-42 and GFAP evidently increased as early as 1 day and 3 days after nerve injury, respectively.19,21 When regional hypothermia was ad- ministered 1, 3, and 5 days after CCI, microglia in the CN had already been activated. Conversely, microglia were still inactive when regional hypothermia was applied 5 h after CCI. It was speculated that regional hypothermia could in- hibit microglial activation, but could confer no effects on already-activated microglia, even though postinjury admin- istration of regional hypothermia remained effective in sup- pressing the generation of ectopic discharges following nerve injury in the electrophysiological studies. Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/116/2/415/256822/0000542-201202000-00027.pdf by guest on 24 October 2024 TNF- and IL-1 levels decreased more profoundly in CCI rats receiving deep whole-body hypothermia than in those receiving deep regional hypothermia (fig. 9). A similar finding was also observed when CCI rats were applied with mild regional and whole-body hypothermia. Anesthesiology 2012; 116:415–31 Anesthesiology 2012; 116:415–31 In the 5 days postinjury group, the levels of TNF- and IL-1 showed a significant decrease in CCI rats receiving deep whole-body hypothermia, but not mild whole-body hypothermia, compared with those receiving whole-body normothermia (figs. 9C and D). Moreover, the levels of TNF- and IL-1 were significantly lower in CCI rats that received deep whole-body hypothermia than in those that received mild whole-body hypothermia (figs. 9C and D). Discussion is evidence that activated microglia produce cytotoxic mole- cules, including reactive oxygen species, nitric oxide, prosta- glandins, and a variety of proinflammatory cytokines.41 An electrophysiological study of superficial dorsal horn neurons showed that TNF- enhances excitatory synaptic transmis- sion by increasing the frequency of spontaneous excitatory postsynaptic currents, and IL-1 can both enhance excit- atory synaptic transmission and reduce inhibitory synaptic transmission.29,42 The results suggest a pivotal role of proin- flammatory cytokines in modulating synaptic plasticity and neuronal excitability, which subsequently cause central sen- sitization and contribute to the development of neuropathic pain. In addition, Gao and Ji29 reported that in response to nerve injury, microglia produced TNF-, which activated astrocytes via c-Jun N-terminal kinase. Therefore, regional hypothermia may not have an effect on already-activated microglia; consequently, the release of proinflammatory cy- tokines by activated microglia contributes to the activation of astrocytes and the development of neuropathic pain. is evidence that activated microglia produce cytotoxic mole- cules, including reactive oxygen species, nitric oxide, prosta- glandins, and a variety of proinflammatory cytokines.41 An electrophysiological study of superficial dorsal horn neurons showed that TNF- enhances excitatory synaptic transmis- sion by increasing the frequency of spontaneous excitatory postsynaptic currents, and IL-1 can both enhance excit- atory synaptic transmission and reduce inhibitory synaptic transmission.29,42 The results suggest a pivotal role of proin- flammatory cytokines in modulating synaptic plasticity and neuronal excitability, which subsequently cause central sen- sitization and contribute to the development of neuropathic pain. In addition, Gao and Ji29 reported that in response to nerve injury, microglia produced TNF-, which activated astrocytes via c-Jun N-terminal kinase. Therefore, regional hypothermia may not have an effect on already-activated microglia; consequently, the release of proinflammatory cy- tokines by activated microglia contributes to the activation of astrocytes and the development of neuropathic pain. y p f p p Several studies have shown that whole-body hypothermia could prevent secondary neuronal damage, which was initi- ated through proinflammatory cytokine release from acti- vated microglia following traumatic brain injury.43,44 Schmitt et al.45 reported that hypothermic treatment dimin- ished release of TNF- and nitric oxide from activated mi- croglial cells in an in vitro cell culture model. The present study showed that whole-body hypothermia applied after nerve injury was effective in attenuating neuropathic pain behaviors and glial activation responses in the CN. The authors thank Ya-Ting Jhan, B.S., Research Technologist, School of Medicine, College of Medicine, Fu Jen Catholic University, Taipei, Taiwan, for technical assistance. Discussion The exact mechanism underlying these phenomena remains uncertain; however, it is thought that nerve injury initiates a signaling cascade in the nervous system and whole-body hypothermia plays a significant role in several aspects of the cascade. At an early stage, or before ectopic discharge-triggered microglial activation, whole-body hypothermia could suppress injury- induced ectopic discharges and consequently inhibit the ac- tivation of glia and the development of neuropathic pain. At a later stage, or after the initiation of microglial activation, whole-body hypothermia could inhibit proinflammatory cy- tokine release from activated microglia and remain effective in attenuating neuropathic pain and glial activation. How- ever, a diminishing effect of whole-body hypothermia on neuropathic pain modulation and glial activation was ob- served when hypothermic treatment was given at an even later time. We speculate that the interactions between neu- rons, glia, cytokines, and central pain processing become increasingly complex and widespread during this time, and whole-body hypothermia alone is no longer able to tackle all these mechanisms of neuropathic pain. In conclusion, the present study demonstrates that in the early stage after nerve injury, ectopic discharges from injured nerves play an important role in pathologic pain states, and the suppression of ectopic discharges effectively attenuates glial ac- tivation and neuropathic pain. Once microglia are activated, a variety of cytokines are released, which subsequently contribute to paracrine activation of the surrounding glial cells and the development of neuropathic pain. Hence, it is mandatory to modulate the cytokines involved in subsequent pain processing to achieve optimum pain relief. This study offers evidence to corroborate the concept and importance of choosing an appro- priate therapeutic modality based on the stage of nerve injury when applying therapeutic hypothermia for neuropathic pain. Discussion Further, high levels of proinflammatory cytokines, such as TNF- and IL-1, were detected in the CN of CCI rats treated with regional hypothermia 1, 3, and 5 days, but not 5 h, postinjury. There Glial activation in the central nervous system is a com- mon phenomenon following peripheral nerve injury27,28 that may contribute to the development of neuropathic pain.29 The activation of microglia and astrocytes is accom- panied by up-regulation of the surface antigen, complement receptor 3, and the intermediate filament protein, GFAP, respectively.30 We assessed the glial activation response in the CN following median nerve CCI using immunocytochemi- cal detection of OX-42 monoclonal antibody, which recog- nizes complement receptor 3, and GFAP monoclonal anti- body. Previous studies showed that the activation of microglia and astrocytes reaches a peak and that mechanical hypersensitivity is most pronounced on day 7 after median nerve injury.19,21 Therefore, we chose day 7 as the time-point for experimental observations in the present study. During the past decade, several clinical studies have pro- vided evidence that systemic or focal hypothermia could con- fer protection against the pathologic consequences of tissue damage after central nervous system injury.31,32 However, the protective effects of hypothermia on peripheral nerve Tsai et al. 428 PAIN MEDICINE central nervous system injury in real patients because lower tem- peratures could result in potentially life-threatening complica- tions, including infection, arrhythmia, hypokalemia, coagu- lopathy,andheartfailure.50 Toinvestigateitstherapeuticeffects in animal models, whole-body hypothermia has been induced to as low as 21°C.51,52 Therefore, it is speculated that the adap- tation to and tolerance of low body temperature varies among different species. Our study revealed that after nerve injury, whole-bodyhypothermiawasmoreeffectiveinattenuatingneu- ropathic pain and glial activation than regional hypothermia. 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J Neurosurg 2000; 92:91–9 Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/116/2/415/256822/0000542-201202000-00027.pdf by guest on 24 October 2024 ANESTHESIOLOGY REFLECTIONS pubs.asahq.org/anesthesiology/article-pdf/116/2/415/256822/0000542-201202000-00027.pdf by guest on 24 October 2024 Analgine by H. K. Mulford of Philadelphia y p Promoting themselves as “Factors of Reliable Tablets” in their advertisements, the Philadelphia pharma- ceutical firm of H. K. Mulford compounded pain-relieving Analgine. Stacked in corked cylinders of glass (above), these five-grain compressed tablets were targeted at relieving migraine, neuralgia, and rheumatic pains. Marketed by 1896 as “a most reliable analgesic” that would “not depress the heart,” Analgine became surprisingly popular with the public. Perhaps people enjoyed the proprietary blending of “Acet- anilid” with a salicylate and caffeine. Or maybe some customers appreciated the extract of hyoscyamus. Or did the quarter-grain of cannabis per tablet have something to do with Analgine’s success? (Copyright © the American Society of Anesthesiologists, Inc. This image also appears in the Anesthesiology Reflec- tions online collection available at www.anesthesiology.org.) George S. Bause, M.D., M.P.H., Honorary Curator, ASA’s Wood Library-Museum of Anesthesi- ology, Park Ridge, Illinois, and Clinical Associate Professor, Case Western Reserve University, Cleveland, Ohio. UJYC@aol.com. Promoting themselves as “Factors of Reliable Tablets” in their advertisements, the Philadelphia pharma- ceutical firm of H. K. Mulford compounded pain-relieving Analgine. Anesthesiology 2012; 116:415–31 Stacked in corked cylinders of glass (above), these five-grain compressed tablets were targeted at relieving migraine, neuralgia, and rheumatic pains. Marketed by 1896 as “a most reliable analgesic” that would “not depress the heart,” Analgine became surprisingly popular with the public. Perhaps people enjoyed the proprietary blending of “Acet- anilid” with a salicylate and caffeine. Or maybe some customers appreciated the extract of hyoscyamus. Or did the quarter-grain of cannabis per tablet have something to do with Analgine’s success? (Copyright © the American Society of Anesthesiologists, Inc. This image also appears in the Anesthesiology Reflec- tions online collection available at www.anesthesiology.org.) George S. Bause, M.D., M.P.H., Honorary Curator, ASA’s Wood Library-Museum of Anesthesi- ology, Park Ridge, Illinois, and Clinical Associate Professor, Case Western Reserve University, Cleveland, Ohio. UJYC@aol.com. 431 Tsai et al. Anesthesiology 2012; 116:415–31 Anesthesiology 2012; 116:415–31

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